Note: Descriptions are shown in the official language in which they were submitted.
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81844fft.208
Novel neomycin-phosphotransferase-genes and methods for the selection of
recombinant cells producing high levels of a desired Gene product
Scope of the Invention
The invention relates to new modified neomycin-phosphotransferase genes
and their use in selection methods for high-producing recombinant cells.
1 o Accordingly, the present invention also relates to new expression vectors
which contain a modified neomycin-phosphotransferase gene, preferably
combined with a gene of interest functionally linked to a heterologous
promoter. The invention further relates to methods of preparing heterologous
gene products using the corresponding high-producing recombinant cells.
Background to the Invention
Mammalian cells are the preferred host cells for the production of complex
biopharmaceutical proteins as the modifications carried out post-
2 o translationally are compatible with humans both functionally and
pharmacokinetically. The main relevant cell types are hybridoma, myeloma
CHO (Chinese Hamster Ovary) cells and BHK (Baby Hamster Kidney) cells.
The cultivation of the host cells is increasingly carried out under serum- and
protein-free production conditions. The reasons for these are the
2 5 concomitant cost reduction, the reduced interference in the purification
of the
recombinant protein and the reduction in the potential for the introduction of
pathogens (e.g. prions and viruses). The use of CHO cells as host cells is
becoming more widespread as these cells adapt to suspension growth in
serum- and protein-free medium and are also regarded and accepted as safe
3 o production cells by the regulatory authorities.
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In order to produce a stable mammalian cell line which expresses a
heterologous gene of interest (GOI), the heterologous gene is generally
inserted in the desired cell line together with a selectable marker gene such
as e.g. neomycin phosphotransferase (NPT) by transfection. The
s heterologous gene and the selectable marker gene can be expressed in a
host cell starting from one individual or separate cotransfected vectors. Two
to three days after transfection the transfected cells are transferred into
medium containing a selective agent, e.g. G418 when using neomycin
phosphotransferase-gene (NPT gene), and cultivated for some weeks under
1 o these selective conditions. The emerging resistance cells which have
integrated the exogenous DNA can be isolated and investigated for
expression of the desired gene product (of the GOI).
A major problem in establishing cell lines with a high expression of the
15 desired proteins arises from the random and undirected integration of the
recombinant vector into transcriptionally -active or -inactive loci in the
host
cell genome. As a result a population of cells is obtained which have
completely different expression rates of the heterologous gene, the
productivity of the cells generally following normal distribution. In order to
2 o identify cell clones which have a very high expression of the heterologous
gene of interest it is therefore necessary to examine and test a large number
of clones, which is time consuming, labour intensive and expensive.
Improvements to the vector system used for transfection therefore set out to
increase the proportion of high producers in the transfected cell population
by
2 s suitable selection strategies and thereby reduce the expenditure and work
involved in clone identification. The development of such an expression
system is the subject of the present invention.
The amino glycoside-3'-phosphotransferase II enzyme (neomycin-
3 o phosphotransferase) (EC27195) the gene of which is transposon 5-
associated in Escherichia coli is used as a selectable marker in a number of
organisms (e.g. bacteria, yeasts, plants and mammalian cells). This enzyme
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confers resistance to various aminoglycoside antibiotics such as neomycin,
kanamycin and 6418, by inactivating the antibiotics by transferring the
terminal phosphate from ATP to the 3' hydroxyl group of the aminohexose
ring I. In addition to the wild-type neomycin phosphotransferase some
s mutants are known which have reduced phosphotransferase activity and
hence reduced resistance to aminoglycoside antibiotics in bacteria (Blazquez
et al., 1991; Kocabiyik et al., 1992; Yenofsky et al., 1990) and in slices of
leaf
from tobacco (Yenofsky et al., 1990).
to One of these mutants (GIu182Asp) was used as a marker for selecting
embryonic stem cells, the neomycin phosphotransferase gene being
integrated into the c-myc gene by targeted homologous recombination (gene
targeting) (Hanson et al., 1995). The authors restrict themselves to the use
of the modified enzyme for gene targeting.
Patent application WO 99/53046 describes the expression of a modified
neomycin phosphotransferase gene (Asp261 Asn) in production-relevant
mammalian cells. The authors describe a non-cloning method for expression
of a gene of interest in mammalian cells. By cotransfection of the cells with
2 o three individual DNA fragments which code for a promoter element, a gene
of
interest and a selectable marker coupled with an IRES ("Internal ribosomal
entry site") element, it is possible to deliberately grow cells, under
selection
pressure, in which all three DNA fragments are combined as a functional
bicistronic transcription unit (promoter gene of interest-IRES-neomycin-
phosphotransferase gene). The arrangement of the elements only occurs in
the transfected cell, so that only a few cells show the correct arrangement of
the elements. Moreover, after gene amplification, using an amplifiable
selectable marker, no high producing clones can be generated. After
repeated selection and gene amplification the cells generated exhibited at
3 o most 6 pg of protein per cell per day (6pglcell/day).
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None of the publications discloses modified neomycin phosphotransferase
genes with particular suitability for the preparation of a high expression
vector
system for mammalian cells which makes it possible to develop high
producing cells in order to prepare recombinant biopharmaceutical proteins
which contain one or more complete functional transcription units both for one
or more genes of interest and also for a modified neomycin
phosphotransferase gene with reduced antibiotic resistance. The DNA
construct described in WO 99/53046 contains only a promoter-less neomycin
gene functionally linked to the gene for dihydrofolate reductase (DHFR).
to
There is therefore a need to make suitable modified neomycin
phosphotransferase genes available, particularly for the development of
corresponding high expression vector systems for biopharmaceutical
processes. The problem of the present invention was therefore to provide
corresponding new modified neomycin phosphotransferase genes,
expression vectors which contain a modified neomycin phosphotransferase
gene and a gene of interest functionally linked to a heterologous promoter, a
method of selection for high producing recombinant cells, preferably for
mammalian cells, and a process for producing heterologous gene products.
Surprisingly, within the scope of the present invention, it has been possible
to
produce and identify new modified highly selective neomycin
phosphotransferase genes which are characterised by their particular
suitability for the selection of high producing cells.
Summary of the Invention
The present invention provides new modified neomycin phosphotransferase
genes. Surprisingly, it has been found that an enrichment of transfected
3 o mammalian cells with high expression rates of the co-integrated gene of
interest could be achieved by using the modified neomycin
phosphotransferase genes described hereinafter as selectable markers.
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Compared with the use of the wild-type neomycin phosphotransferase as
selectable marker, after transfection with one of the new neomycin
phosphotransferase genes according to the invention the cells exhibited a
productivity of a protein (an antibody) which was increased by a factor 1.4 to
14.6.
The modified neomycin phosphotransferase genes according to the invention
are preferably mutants which code for a different amino acid from the wild-
type gene at amino acid position 91, 182, 198, 227, 240 or 261. In a
io preferred embodiment the neomycin phosphotransferase gene according to
the invention is the mutant GIu182G1y, GIu182Asp, Trp91Ala, Va1198G1y,
Asp227A1a, Asp227Va1, Asp227G1y, Asp261Asn, Asp261G1y or Phe24011e.
For selecting high producing mammalian cells it has proved particularly
suitable to use the mutants Trp91Ala, Asp227Va1, Asp261Asn, Asp261 Gly
and Phe24011e, while the mutants Asp227Va1 and Asp261 Gly in turn gave cell
clones with the highest productivity and are therefore particularly preferred.
The high-producing cells were obtained by the use of a eukaryotic expression
vector which contains a heterologous gene of interest functionally linked to a
2 o heterologous promoter and a modified neomycin phosphotransferase gene
according to the invention. The expression vector preferably contains other
regulatory elements, e.g, one or more enhancers functionally linked to the
promoter or promoters. Expression vectors are also preferred which
additionally contain a gene for a fluorescent protein which is functionally
2 5 linked to the gene of interest and the heterologous promoter, preferably
via
an internal ribosomal entry site (IRES), which enables bicistronic expression
of the gene which codes for a fluorescent protein and of the gene which
codes for a protein/product of interest, under the control of the heterologous
promoter. Particularly suitable are expression vectors in which the
3 o heterologous gene of interest is under the control of the ubiquitin/S27a
promoter.
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The invention also relates to expression vectors which instead of the gene of
interest contain a multiple cloning site for incorporating such a gene, i.e. a
sequence section with multiple recognition sequences for restriction
endonucleases.
In another aspect the invention relates to recombinant mammalian cells
which contain one of the abovementioned modified neomycin
phosphotransferase genes according to the invention. In addition the present
invention relates to recombinant mammalian cells which have been
1o transfected with one of the expression vectors according to the invention.
These are preferably recombinant rodent cells, of which recombinant hamster
cells such as e.g. CHO cells or BHK cells are particularly preferred. In
another preferred embodiment the said recombinant cells are additionally
transfected with the gene for an amplifiable selectable marker, e.g. with the
gene of dihydrofolate reductase (DHFR).
The invention also relates to a process for enriching recombinant mammalian
cells which express a modified neomycin phosphotransferase gene,
characterised in that (i) a pool of mammalian cells is transfected with a gene
2 o for a modified neomycin phosphotransferase, which has only 1 to 80%,
preferably only 1 to 60%, more preferably only 1.5 to 30%, most preferably
only 1.5 to 26% of the activity and/or one of the modifications described
above; (ii) the mammalian cells are cultivated under conditions which allow
expression of the modified neomycin phosphotransferase gene; and (iii) the
2 s mammalian cells are cultivated in the presence of at least one selecting
agent
which acts selectively on the growth of mammalian cells, and gives
preference to the growth of those cells which express the neomycin
phosphotransferase gene.
3 o The invention also relates to a process for the expression of at least one
gene of interest in recombinant mammalian cells, characterised in that (i) a
pool of mammalian cells is transfected with at least one gene of interest and
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one gene for a modified neomycin phosphotransferase which exhibits only 1
to 80%, preferably only 1 to 60°!°, more preferably only 1.5 to
30%, most
preferably only 1.5 to 26% of the activity and/or one of the modifications
described above; (ii) the cells are cultivated under conditions which allow
expression of the gene or genes of interest and the modified neomycin
phosphotransferase gene; (iii) the mammalian cells are cultivated in the
presence of at least one selecting agent which acts selectively on the growth
of mammalian cells and gives preference to the growth of those cells which
express the neomycin phosphotransferase gene; and (iv) the protein or
1 o proteins of interest is or are obtained from the mammalian cells or from
the
culture supernatant.
The present invention further relates to a process for obtaining and selecting
recombinant mammalian cells which express at least one heterologous gene
of interest, which is characterised in that (i) recombinant mammalian cells
are
transfected with an expression vector according to the invention which in
addition to the gene of interest and the modified neomycin
phosphotransferase gene codes for a fluorescent protein; (ii) the mammalian
cells are cultivated under conditions which allow expression of the gene or
2 o genes of interest, the gene which codes for a fluorescent protein and the
modified neomycin phosphotransferase gene; (iii) the mammalian cells are
cultivated in the presence of at least one selecting agent which acts
selectively on the growth of mammalian cells and gives preference to the
growth of those cells which express the neomycin phosphotransferase gene;
2 5 and (iv) the mammalian cells are sorted by flow-cytometric analysis.
If the mammalian cells have additionally been transfected with a gene for an
amplifiable selectable marker gene, e.g. the DHFR gene, it is possible to
cultivate the mammalian cells under conditions in which the amplifiable
3 o selectable marker gene is also expressed, and to add to the culture medium
a selecting agent which results in amplification of the amplifiable selectable
marker gene.
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Preferably, the processes according to the invention are carried out with
mammalian cells which are adapted to growth in suspension, i.e. with
mammalian cells which are cultivated in a suspension culture. Other
s embodiments relate to processes in which the mammalian cells, preferably
those which are adapted to growth in suspension, are cultivated under
serum-free conditions.
Description of the Figures
io
Figure 1 shows a diagrammatic representation of the base vectors used to
express the recombinant proteins in CHO-DG44 cells. "PlE" is a combination
of CMV enhancer and hamster-ubiquitin/S27a promoter, "P" on its own
indicates a promoter element and "T" is a termination signal for
transcription,
which is needed for the polyadenylation of the transcribed mRNA. The
position and direction of transcription initiation within each transcription
unit is
indicated by an arrow. For cloning the heterologous genes a sequence
region with multiple cutting sites for restriction endonucleases (multiple
cloning sites - MCS) is inserted after the promoter element. The amplifiable
2o selectable markerdihydrofolate reductase is abbreviated to "dhfr" and the
selectable marker neomycin phosphotransferase is abbreviated to "npt" (npt
wild-type or npt mutant). The "IRES" element coming from the
encephalomyocarditic virus acts an internal ribosomal entry site within the
bicistronic transcription unit and enables translation of the following green
25 fluorescent protein "GFP".
Figure 2 shows a diagrammatic view of the eukaryotic expression vectors
which code for a single-chain protein (Figure 2A) or for a subunit of a
monoclonal antibody (Figure 2B) and are used to transfect CHO-DG44 cells.
30 "P/E" is a combination of CMV enhancer and hamster ubiquitin/S27a
promoter, "P" on its own is a promoter element and "T" is a termination signal
for the transcription which is needed for the polyadenylation of the
transcribed
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mRNA. The position and direction of transcription initiation within each
transcription unit is indicated by an arrow. The amplifiable selectable marker
dihydrofolate reductase is abbreviated to "dhfr" and the selectable marker
neomycin phosphotransferase is abbreviated to "npt". The NPT mutants
E182G (SEQ ID N0:3), E182D (SEQ ID N0:19), W91A (SEQ ID N0:5),
D190G (SEQ ID N0:23), V198G (SEQ ID N0:7), D208G (SEQ ID N0:25),
D227A (SEQ ID N0:9), D227V (SEQ 1D N0:11 ), D227G (SEQ ID N0:21 ),
D261G (SEQ ID N0:13), D261N (SEQ ID N0:15) and F2401 (SEQ ID N0:17)
contain a point mutation which results in a modified amino acid (given in 1-
to letter code) at the position indicated. The IRES element originating from
the
encephalomyocarditis virus acts as an internal ribosomal entry site within the
bicistronic transcription unit and permits translation of the following green
fluorescent protein "GFP". "MCP-1" codes for the Monocyte Chemoattractant
Protein-1, whereas "HC" and "LC" code for the heavy and light chains,
respectively, of a humanised monoclonal IgG2 antibody.
Figure 3 shows the part of the sequence of the neomycin
phosphotransferase (npt) gene in which the point mutations have been
inserted by PCR with mutagenic primers. The capital letters indicate the
2 o nucleotide sequence of the npt coding region whereas the small letters
indicate the flanking non-coding nucleotide sequences. The amino acid
sequence predicted from the nucleotide sequence (3-letter code) is shown
above the coding nucleotide sequence. Arrows indicate the direction, length
and position of the primers used, the arrows with solid lines indicating the
2 s mutagenic forward primers, the broken lines indicating the mutagenic
reverse
primers, the dotted lines indicating the primers Neofor5 (SEQ ID N0:27) or
Neofor2 (SEQ ID N0:29) located upstream of the npt gene or the mutation
site, respectively, and the dot-dash line indicating the primers Neorev5 (SEQ
ID N0:28) or IC49 (SEQ ID N0:30) located downstream of the npt gene or
3 o the mutation site, respectively. The nucleotides exchanged with respect to
the wild-type sequence are emphasised above and below the arrows.
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Figure 4 shows conserved domains and the position of the inserted NPT
mutations within the NPT amino acid sequence. On the basis of sequence
homologies between different aminoglycoside-modified enzymes, different
conserved domains were identified within the NPT protein sequence (shown
in grey) The three motifs in the C-terminal region of the enzyme obviously
have special functions. Motifs 1 and 2 are presumably involved in the
catalytic transfer of the terminal phosphate in the ATP catalysis or the
nucleotide binding, whereas motif 3 is thought to have a function in the ATP
hydrolysis and/or the change in conformation in the enzyme-aminoglycoside
to complex. Amino acids which occur in at least 70% of the aminoglycoside-
modifying enzymes are emphasised in bold type. The singly underlined
amino acids are assigned to the same group on the basis of their similarity
and occur in at least 70% of the aminoglycoside-modifying enzymes. Amino
acids marked with an asterisk indicate the position of the mutation sites.
Abbildung 5 shows the influence of the NPT mutations on the selection of
stably transfected MCP-1-expressing cells. For this, CHO-DG44 cells were
transfected with the vectors pBIN-MCP1, pKS-N5-MCP1 and pKS-N8-MCP1
(Fig. 2A), which contained as selectable marker either the NPT wild-type
(WT) or the NPT mutants G1u182Asp and Asp227G1y. For selecting stably
transfected cells, 400 pg/mL or 800 Ng/mL of 6418 was added to the medium
as a selective agent. The concentration in the cell culture supernatant of the
recombinant protein MCP-1 produced was determined by ELISA and the
specific productivity per cell and per day was calculated. The bars represent
2 5 the averages of the specific productivity or of the titre of 18 pools from
6
cultivation in 6-well dishes.
In Figure 6 the influence of the NPT mutations on the selection of stably
transfected mAb expressing cells was investigated. For this, CHO-DG44 cells
3 o were transfected with the plasmid combinations pBIDG-HC/pBIN-LC (NPT-
wild-type), pBIDG-HC/pKS-N5-LC (NPT mutant GIu182Asp) or pBIDG-
HC/pKS-N8-LC (NPT mutant Asp227G1y) (Fig. 6A) or with the combinations
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pBIDG-HC/pBIN-LC (NPT- wild-type), pBIDG-HC/pBIN1-LC (NPT mutant
GIu182G1y), pBIDG-HC/pBIN2-LC (NPT mutant Trp91Ala), pBIDG-
HC/pBIN3-LC (NPT mutant Va1198G), pBIDG-HC/pBIN4-LC (NPT mutant
Asp227A1a), pBIDG-HC/pBINS-LC (NPT mutant Asp227Va1), pBIDG-
HC/pBIN6-LC (NPT mutant Asp261G1y), pBIDG-HC/pBIN7-LC (NPT mutant
Asp261Asn) or pBIDG-HC/pBINB-LC (NPT mutant Phe24011e) (Fig. 6B) which
differ from one another only in the NPT gene (wild-type or mutant) used as a
selectable marker. The concentration in the cell culture supernatant of the
recombinant monoclonal IgG2 antibody produced was determined by ELISA
z o and the specific productivity per cell and per day was calculated. In all,
5 to 9
pools were set up for each vector combination. The bars represent the
averages of the specific productivity or of the titre of all the pools in the
Test
from 6 cultivation runs in 75cm2 flasks. To calculate the relative titres or
the
relative specific productivities the averages of the pools selected with the
NPT wild-type gene were taken as 1.
Figure 7 shows the enrichment of cells with a higher GFP expression in
transfected cell pools by using the NPT mutants according to the invention as
selectable markers. For this, CHO-DG44 cells were transfected with the
2 o plasmid combinations pBIDG-HCIpBIN-LC (NPT-wild-type), pBIDG-HC/pKS-
N5-LC (NPT mutant GIu182Asp), pBIDG-HCIpKS-N8-LC (NPT mutant
Asp227G1y), pBIDG-HC/pBIN1-LC (NPT mutant GIu182G1y),pBIDG-
HC/pBIN1-LC (NPT mutant GIu182G1y), pBIDG-HC/pBIN2-LC (NPT mutant
Trp91Ala), pBIDG-HC/pBIN3-LC (NPT mutant Va1198G), pBIDG-HC/pBIN4-
LC (NPT mutant Asp227A1a), pBIDG-HC/pBINS-LC (NPT mutant Asp227Va1),
pBIDG-HC/pBIN6-LC (NPT mutant Asp261G1y), pBIDG-HC/pBIN7-LC (NPT
mutant Asp261Asn) or pBIDG-HC/pBINB-LC (NPT mutant Phe24011e) (5 to 9
pools in each case), which differ from one another only in the NPT gene
(wild-type or mutant) used as the selectable marker. Moreover, the pBIDG
3 o vectors also contained the GFP as marker gene. After 2 to 3 weeks'
selection of the transfected cell pools in HT-free medium with the addition of
6418, the GFP fluorescence was measured by FACS analysis. Every graph,
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with the exception of the non-transfected CHO-DG44 cells used as a
negative control, represents the average GFP fluorescence from the pools
which had been transfected with the same plasmid combination.
Figure 8 shows the increase in the mAb productivity achieved by dhfr-
mediated gene amplification taking as its example a cell pool which was
obtained from the transfection of CHO-DG44 with the vector combination
pBIDG-HC/pBIN-LC (NPT-wild-type) or pBIDG-HC/pBINS-LC (NPT mutant
D227V). After the first selection in hypoxanthine/thymidine-free CHO-S-SFMII
to medium in the presence of 6418 a dhfr-mediated gene amplification was
carried out by the addition of 100 nM and then 500 nM of MTX to the culture
medium. The concentration of the mAb in the cell culture supernatant of the
pools was determined by ELISA and the specific productivity per cell and per
day (pg/cell*day) was calculated. Each data point represents the average of
15 six cultivation runs in 75cm2 flasks.
Figure 9 shows the enzyme activity of the NPT mutants according to the
invention compared with the NPT wild-type, in a dot assay. For this, cell
extracts were prepared from two different cell pools (pool 1 and 2) expressing
2 o mAb, which had been transfected and selected either with the NPT wild-type
gene (SEQ ID N0:1 ) or with the NPT mutants E182G (SEQ ID N0:3), E182D
(SEQ ID N0:19), W91A (SEQ ID N0:5), V198G (SEQ ID N0:7), D227A
(SEQ ID N0:9), D227V (SEQ ID N0:11 ), D227G (SEQ ID N0:21 ), D261 G
(SEQ ID N0:13), D261 N (SEQ ID N0:15) and F2401 (SEQ ID
2s N0:17)GIu182Asp or Asp227G1y. Non-transfected CHO-DG44 cells were
used as negative control. 6418 was used as the substrate in the
phosphorylation assay. The extracts were filtered through a sandwich of P81
phosphocellulose and nitrocellulose membrane in a 96 well vacuum manifold.
Proteins phosphorylated by protein kinases and also non-phosphorylated
3 o proteins bind to the nitrocellulose, whereas phosphorylated and non-
phosphorylated 6418 passes through the nitrocellulose and binds to the
phosphocellulose (Fig. 9A). The radioactive signals were detected and
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quantified using a phosphoimager. The signals which had been obtained
with 5 pg of extract were used to calculate the percentage enzyme activity.
The percentage enzyme activities denote the average of the NPT mutants
from 2 cell pools expressing mAb, the enzyme activity of wild-type NPT being
s taken as 100% (Fig. 9B).
Figure 10 shows the Northern Blot analysis of NPT expression and the
number of NPT gene copies in the transfected cell pools. For this, total RNA
was prepared from two different cell pools expressing mAb which were
to transfected and selected either with the NPT wild-type gene (SEQ ID N0:1 )
or with the NPT mutants E182G (SEQ ID N0:3), E182D (SEQ ID N0:19),
W91A (SEQ ID N0:5), V198G (SEQ ID N0:7), D227A (SEQ ID N0:9),
D227V (SEQ ID N0:11 ), D227G (SEQ ID N0:21 ), D261 G (SEQ ID N0:13),
D261 N (SEQ ID N0:15) and F2401 (SEQ ID NO:17). Untransfected CHO-
15 DG44 cells were used as the negative control. 30 :g of RNA was hybridised
with a FITC-dUTP-labelled PCR product which comprised the coding region
of the NPT gene. In all the transfected cells a specific singular NPT
transcript of about 1.3 kb was detected. In order to determine the npt gene
copy number, in a dot blot analysis, genomic DNA was isolated from the
2 o abovementioned cell pools expressing mAb.
pg, 5 Ng, 2.5 Ng, 1.25 pg, 0.63 Ng and 0.32 pg of genomic DNA was
hybridised with an FITC-dUTP-labelled PCRproduct which included the
coding region of the NPT gene. Untransfected CHO-DG44 cells were used
as the negative control. The plasmid pBIN-LC was used as the standard
2 5 (320 pg, 160 pg, 80 pg, 40 pg, 20 pg, 10 pg, 5 pg, 2.5 pg). The copy
number
of the npt genes in the cell pools was calculated using the standard series
which had been determined from the signal intensities measured for the
titrated plasmid-DNA.
3 o Figure 11 shows the isolation of high-expressing mAb cell pools by a GFP-
based selection using FACS taking as the example two cell pools (cell pool 5
and 8). These cell pools, obtained from the co-transfection with the vectors
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pBID-HC and pBING-LC, were subjected to sequential GFP-based FACS
sorting. The concentration of the IgG2 antibody in the cell culture
supernatant
of the pools was determined by ELISA after each sorting step and the specific
productivity per cell and per day (pglcell*day) was calculated. In all 6 sorts
were done, and in each case the 5% of cells with the highest GFP
fluorescence were sorted out. Each data point represents the average of at
least six cultivation runs in 75cm2 flasks.
Figure 12 shows the increases in mAb productivity achieved by combining a
to GFP-based selection with an MTX amplification step taking as the example
cell pools 5 and 8 (cf. Fig. 11 ). Two weeks after the co-transfection of CHO-
DG44 with the vectors pBID-HC and pBING-LC the 5% of cells with the
highest GFP fluorescence were sorted out from pools 5 and 8. Then dhfr-
mediated gene amplification was carried out by adding 100 nM of
methotrexate (MTX) to the culture medium. The concentration of the mAb in
the cell culture supernatant of the pools was determined by ELISA and the
specific productivity per cell and per day (pg/cell*day) was calculated. Each
data point represents the average of at least six cultivation runs in 75cm2
flasks.
Figure 13 shows the correlation between the antibody productivity and the
GFP fluorescence taking as the example cell pools 5 and 8 (cf. Fig. 11 ).
These cell pools were obtained by transfecting CHO-DG44 with the vector
combination pBID-HC and pBING-LC. They were subjected to sequential
2 s GFP-based FACS sorting, and the 5% of cells with the highest GFP
fluorescence were sorted out. The concentration of the IgG2 antibody in the
cell culture supernatant of the pools was determined by ELISA after each
sorting step and the specific productivity per cell and per day (pg/cell*day)
was calculated. Each data point represents the average of at least six
3 o cultivation runs in 75cm2 flasks.
Detailed Description of the Invention and Preferred Embodiments
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The following information on the amino acid positions relates in each case to
the position of the amino acid as coded by the wild-type neomycin
phosphotrasferase gene with SEQ ID N0:1. By a "modified neomycin
phosphotransferase gene" is meant a nucleic acid which codes for a
polypeptide with neomycin phosphotransferase activity, the polypeptide
having a different amino acid from the wild-type protein at at least one of
the
amino acid positions described more fully in the specification which are
homologous to the wild-type protein with SEQ ID N0:2. In this context the
1 o term "homologous" means that the sequence region carrying the mutation
can be brought into correspondence with a reference sequence, in this case
the sequence of the wild-type neomycin phosphotransferase according to
SEQ ID N0:2, using so-called standard "alignment" algorithms, such as for
example "BLAST" (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman,
D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410;
Gish, W. & States, D.J. (1993) "Identification of protein coding regions by
database similarity search." Nature Genet. 3:266-272; Madden, T.L.,
Tatusov, R.L. & Zhang, J. (1996) "Applications of network BLAST server"
Meth. Enzymol. 266:131-141; Zhang, J. & Madden, T.L. (1997)
2 0 "PowerBLAST: A new network BLAST application for interactive or
automated sequence analysis and annotation." Genome Res. 7:649-656;
Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui
Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped
BLAST and PSI-BLAST: a new generation of protein database search
2 5 programs", Nucleic Acids Res. 25:3389-3402). Sequences are in
correspondence when they correspond in their sequence order and can be
identified using the standard "alignment" algorithms.
The present invention provides new modified neomycin phosphotransferase
3 o genes and methods of preparing and selecting mammalian cell lines which
allow a high expression of heterologous gene products, preferably
biopharmaceutically relevant polypeptides or proteins The processes
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according to the invention are based primarily on the selection of cells which
in addition to the gene of interest express a neomycin phosphotransferase
gene according to the invention which gives the transfected cells a selective
advantage over non-transfected cells. Surprisingly, it has been found that the
s use of the modified neomycin phosphotransferase genes (mNPT genes)
according to the invention described herein has a substantial selective
advantage over the wild-type neomycin phosphotransferase gene (wtNPT
gene). This particularly relates to the use of mutants which have a lower
enzyme activity compared with wtNPT.
to
Modified Neomycin Phosphotransferase Genes According To The Invention
1t has proved particularly suitable to use modified NPT genes which code for
an NPT having only 1 to 80%, preferably only 1 to 60% of the enzyme activity
15 of wtNPT. Preferred NPT mutants are those which have only 1 to 30% of the
enzyme activity of wtNPT, while those which have only 1.5 to 26% of the
enzyme activity of wtNPT are particularly preferred. The enzyme activity of
an NPT can be determined for example in a dot assay as described in
Example 4 and given as Method 5.
The term wild-type neomycin phosphotransferase refers to a neomycin
phosphotransferase gene which codes for the aminoglycoside-3'-
phosphotransferase II enzyme (EC 2.7.1.95) the gene of which is naturally
transposon 5-associated in Escherichia coli, and contains for example the
2s amino acid sequence given in SEQ ID N0:2 or is coded by the nucleotide
sequence given in SEQ ID N0:1. This enzyme gives resistance to various
aminoglycoside antibiotics such as neomycin, kanamycin and 6418, by
inactivating the antibiotics by the transfer of the terminal phosphate of ATP
to
the 3' hydroxyl group of the amino hexose ring 1. The term wtNPT also refers
3 o to all NPTs which have a comparable enzyme activity to the NPT coded by
SEQ lD N0:1. This includes in particular those NPTs in which the
enzymatically active centre which catalyses the transfer of a terminal
CA 02507664 2005-05-27
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phosphate from ATP to a substrate is present in an identical or nearly
identical conformation (Shaw et al., 1993; Hon et al; 1997; Burk et al., 2001
)
and thus has a comparable enzyme activity to an enzyme which contains the
amino acid sequence of SEQ ID N0:2. A wtNPT has a comparable enzyme
s activity if it exhibits about 81 to 150%, preferably 90 to 120% of the
enzyme
activity displayed by an NPT defined by SEQ ID N0:2, while the activity can
be determined in the dot assay described in Example 4 and referred to as
Method 5.
to Fundamentally preferred are mutants wherein the reduction in the enzyme
activity compared with wtNPT is based on a modification of the amino acid
sequence, e.g. on the substitution, insertion or deletion of at least one or
more amino acids. Deletion, insertion and substitution mutants can be
produced by "site-specific mutagenesis" and/or "PCR-based mutagenesis
15 techniques". Suitable methods are described for example by Lottspeich and
Zorbas (1998)(Chapter 36.1 with other references).
Surprisingly, it has been found that if neomycin phosphotransferase mutants
are used as selectable markers in which at least the amino acid tryptophan at
2 o amino acid position 91, the amino acid glutamic acid at amino acid
position
182, the amino acid valine at amino acid position 198, the amino acid aspartic
acid at amino acid position 227, the amino acid aspartic acid at amino acid
position 261 or the amino acid phenylalanine at amino acid position 240 has
been altered compared with wtNPT, it is possible to achieve particularly
2 5 effective enrichment of transfected mammalian cells with a high expression
rate for the co-integrated gene of interest. Accordingly, mutants which affect
the amino acids at positions 91, 182, 198, 227 and/or 240 are preferred.
Particularly advantageous are substitution mutants, i.e. mutants in which the
amino acid occurring at this location in the wild-type has been replaced by
3 o another amino acid. Even more preferred are corresponding substitution
mutants in which a change in the corresponding amino acid leads to a
reduction in the enzyme activity compared with wt-NPT to 1 to 80%,
CA 02507664 2005-05-27
- 1$ -
preferably to 1 to 60%, more preferably to 1.5 to 30%, most preferably to
1.5% to 26%. Particularly preferred are modified NPT genes in which the
amino acid 91, 227, 261 and/or 240 has been modified accordingly so that
the enzyme activity compared with the wt-NPT is only 1 to 80%, preferably
s only 1 to 60%, more preferably only 1.5 to 30%, most preferably only 1.5% to
26%. Most preferred is a substitution mutant in which the amino acid at
amino acid position 227 has been modified in the form such that the enzyme
activity of the modified NPT is less than 26%, preferably between 1 and 20%,
more preferably between 1 and 16% compared with the wt-NPT.
According to another embodiment of the present invention, advantageous
mutants are those which, by comparison with wtNPT, code for glycine,
alanine, valine, leucine, isoleucine, phenylalanine or tyrosine at amino acid
positions 91, 182 or 227. Moreover, the glutamic acid at amino acid position
1s 182 may also be replaced by aspartic acid, asparagine, glutamine or any
other preferably negatively charged amino acid. Also preferred are modified
NPT genes which, by comparison with wtNPT, code for glycine, alanine,
leucine, isoleucine, phenylalanine, tyrosine or tryptophan at amino acid
position 198. Also preferred are modified NPT genes which, by comparison
2o with wtNPT, code for glycine, alanine, leucine, isoleucine, phenylalanine,
tyrosine, tryptophan, asparagine, glutamine or aspartic acid at amino acid
position 261. In particular, it has been found that with the mutants
GIu182G1y, GIu182Asp, Trp91Ala, Va1198G1y, Asp227A1a, Asp227Va1,
Asp227G1y, Asp261 Gly, Asp261Asn and Phe24011e as selectable markers it
2 s was possible to achieve an enrichment of transfected mammalian cells with
high expression rates of the co-integrated gene of interest, with the result
that
these mutants are particularly preferred. Still more preferred are the mutants
Asp227Va1, Asp227G1y, Asp261G1y, Asp261Asn, Phe24011e and Trp91Ala, as
the best enrichment rates are achieved using them. The utant Asp227Va1 is
3 o particularly preferred.
CA 02507664 2005-05-27
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By contrast, the Asp190G1y and Asp208G1y mutants proved to be unsuitable
markers for the selection of transfected CHO-DG44 cells under serum-free
culture conditions. As a result of the presumably greatly reduced enzyme
function of these mutants (Asp190G1y, Asp208G1y), only a few cells were
s obtained after the selection phase, which were moreover severely impaired in
their growth and vitality.
The amino acids at positions 182 and 227 based on the wild-type are non-
conserved amino acids which are located outside the three conserved motifs
1 o in the C-terminal region of the aminoglycoside-3'-phosphotransferases. The
amino acid at position 91 also belongs to the non-conserved amino acids and
is located outside one of the conserved motifs in the N-terminal region of the
aminoglycoside-3'-phosphotransferases. By contrast the amino acids at
positions 198 and 240 are conserved amino acids in the C-terminal region of
is the NPT, but are nevertheless outside the conserved motifs. By contrast,
the
amino acid at position 261 is a conserved amino acid in the third conserved
motif of the C-terminal region (Shaw et al., 1993; Hon et al., 1997; Burk et
al., 2001 ).
2 o Compared with the use of wtNPT as selectable marker the cells in the case
of
the Gfu182G1y, GIu182Asp and Va1198G1y mutant showed a productivity
increased by a factor of 1.4 - 2.4, in the case of the Asp227G1y mutant
productivity was increased by a factor of 1.6 - 4.1, in the case of the
Asp227A1a or Trp91Ala mutant productivity was increased by a factor of 2.2
25 or 4, in the case of the Phe24011e or Asp261Asn mutant productivity was
increased by a factor of 5.7 or 7.3 and in the case of the Asp261 Gly or
Asp227Va1 mutant it was even increased by a factor of 9.3 or 14.6. To
express the multi-chained protein (an antibody), co-transfection was carried
out. The two protein chains were each expressed by their own vector, one
3 o vector additionally coding for the NPT gene while the other vector coded
for
the amplifiable selectable dihydrofolate reductase gene.
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The present invention thus relates to a process for enriching for recombinant
mammalian cells which express a modified neomycin phosphotransferase
gene, characterised in that (i) a pool of mammalian cells is transfected with
a
gene for a modified neomycin phosphotransferase which has only 1 to 80%,
preferably 1 to 60°l°, more preferably 1.5 to 30%, most
preferably 1.5 to 26%
of the activity of wild-type neomycin phosphotransferase and/or one of the
modifications described herein; (ii) the mammalian cells are cultivated under
conditions which allow expression of the modified neomycin
phosphotransferase gene; and (iii) the mammalian cells are cultivated in the
1 o presence of at least one selecting agent which acts selectively on the
growth
of mammalian cells, and gives preference to the growth of those cells which
express the neomycin phosphotransferase gene.
Particularly preferred is a corresponding process which uses a modified NPT
s5 gene described in more detail in this application, particularly if the
modified
NPT gene used codes for a modified NPT which, by comparison with the
wild-type gene, codes for alanine at amino acid position 91, for glycine or
aspartic acid at amino acid position 182, for glycine at amino acid position
198, for alanine, glycine or valine at amino acid position 227, for glycine or
2 o asparagine at amino acid position 261 or for isoleucine at amino acid
position
240. Still more preferred are NPT genes which, by comparison with the wild-
type gene, code for valine at amino acid position 227 and /or for glycine
and/or asparagine at amino acid position 261. Particularly preferred are
those NPT genes which, by comparison with the wild-type gene, code at
2 s amino acid position 240 for an isoleucine or at amino acid position 227
for a
valine, while an NPT gene which codes for valine at amino acid position 227
compared with the wild-type gene is particularly preferred. Naturally, the
present invention also includes modified NPT genes and the use of modified
NPT genes according to the invention which comprise a combination of the
3 o corresponding amino acid exchanges.
CA 02507664 2005-05-27
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The present invention further relates to eukaryotic expression vectors which
contain (i) a heterologous gene of interest functionally linked to a
heterologous promoter and (ii) a modified neomycin phosphotransferase
gene according to the invention which codes for a neomycin
s phosphotransferase which has low enzyme activity compared with wild-type
neomycin phosphotransferase. By a "low" or "lower" enzyme activity for the
purposes of the invention is meant an enzyme activity which corresponds to
at most 80%, preferably 1 to 80%, more preferably only 1 to 60% of the
enzyme activity of wtNPT. According to one embodiment of the present
1 o invention "lower enzyme activity" denotes an enzyme activity of 1 to 30%,
preferably 1.5 to 26% compared with wild-type neomycin
phosphotransferase.
A preferred expression vector contains a modified NPT gene which codes for
is a modified NPT which has only 1 to 80%, preferably only 1 to
60°l° of the
enzyme activity of wtNPT. Also preferred are expression vectors with
modified NPT genes which code for mutants having only 1 to 30% of the
enzyme activity of wtNPT. Particularly preferred are those expression
vectors which contain a modified NPT gene which code for mutants having
2 0 only 1.5 to 26% of the enzyme activity of wtNPT, the activity being
determined in the dot assay described in Example 4 and referred to as
method 5.
In another embodiment of the invention the expression vectors contain genes
25 of modified NPT which have been modified, compared with wtNPT, at amino
acid position Trp91, GIu182, Va1198, Asp227, Phe240 or at position Asp261.
In this context, NPT mutants are preferred which are modified at position
Trp91, GIu182, Va1198, Asp227, Phe240 or Asp261 and have only 1 to 80%,
preferably only 1 to 60%, more preferably only 1.5 to 30%, and most
3 o preferably only 1.5 to 26% of the enzyme activity of wtNPT. Preferably the
amino acids Tryp91, GIu182 or Asp227 may each be replaced by glycine,
alanine, valine, leucine, isoleucine, phenylalanine or tyrosine at the
CA 02507664 2005-05-27
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corresponding position. Preferably the glutamic acid at position 182 may also
be replaced by aspartic acid, asparagine, glutamine or another preferably
negatively charged amino acid. Also preferred are modified NPT genes
which code for glycine, alanine, leucine, isoleucine, phenylalanine, tyrosine
or
tryptophan at amino acid position 198 compared with wtNPT. In addition,
modified NPT genes are preferred which code for glycine, alanine, valine,
isoleucine, tyrosine or tryptophan at amino acid position 240 compared with
wtNPT. Also preferred are modified NPT genes which code for glycine,
alanine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, asparagine,
1 o glutamine or aspartic acid at amino acid 261 compared with wtNPT. It is
particularly preferred to use a mutant wherein the aspartic acid at position
227 is replaced by glycine, alanine, valine, leucine or isoleucine, the
aspartic
acid at position 261 is replaced by an alanine, valine, leucine, isoleucine or
glutamine, particularly by glycine or asparagine.
Particularly preferred are expression vectors which contain modified NPT
genes which code for a GIu182G1y, GIu182Asp, Trp91Ala, Va1198G1y,
Asp227A1a, Asp227Va1, Asp227G1y, Asp261 Gly, Asp261 Asn or Phe24011e
mutant, which in the case of the GIu182G1y mutant contains the amino acid
2o sequence of SEQ ID N0:4, in the case of the GIu182Asp mutant contains the
amino acid sequence of SEQ ID N0:20, in the case of the Trp91Ala mutant
contains the amino acid sequence of SEQ ID N0:6, in the case of the
Va1198G1y mutant contains the amino acid sequence of SEQ ID N0:8, in the
case of the Asp227A1a mutant contains the amino acid sequence of SEQ ID
2 s N0:10, in the case of the Asp227Va1 mutant contains the amino acid
sequence of SEQ ID N0:12, in the case of the Asp227G1y mutant contains
the amino acid sequence of SEQ ID N0:22, in the case of the Asp261 Gly
mutant contains the amino acid sequence of SEQ ID N0:14, in the case of
the Asp261Asn mutant contains the amino acid sequence of SEQ ID N0:16
3 o and in the case of the Phe24011e mutant contains the amino acid sequence
of
SEQ ID N0:18. Most preferred is an expression vector using an Asp227Va1,
Asp227G1y, Asp261G1y, Asp261Asn, Phe24011e or Trp91Ala mutant,
CA 02507664 2005-05-27
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particularly if it contains the amino acid sequence given in SEQ ID N0:12,
SEQ ID N0:22, SEQ ID N0:14, SEQ ID N0:16, SEQ ID N0:18 or SEQ ID
N0:6 or also if it is coded by the nucleic acid sequence given in SEQ ID
N0:11, SEQ ID N0:21, SEQ ID N0:13, SEQ ID N0:15, SEQ ID N0:17 or
s SEQ ID N0:5 or contains it.
In addition the present invention provides for the first time modified
neomycin
phosphotransferase genes and the gene products thereof which compared
with wtNPT code for a different amino acid than the wt amino acid at amino
1 o acid position Trp91, Val198 or Phe240. The present invention particularly
provides for the first time Trp91, Va1198 or Phe240 mutants which have a
reduced enzyme activity compared with wtNPT. The modified NPTs
described here and made available within the scope of the invention
preferably code for alanine at amino acid position 91, for glycine at position
15 198 and for isoleucine at position 240. Furthermore, the present invention
provides for the first time NPT mutants which, compared with wtNPT, code
for glycine at position 182, for alanine or valine at position 227 and for
glycine at position 261. Both the genes and the gene products (enzymes) are
provided for the first time within the scope of the invention. In this context
the
2 o present invention provides for the first time modified NPT with the amino
acid
sequences according to SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:8, SEQ ID
N0:10, SEQ 1D N0:12, SEQ ID N0:14 and SEQ ID N0:18. Moreover, the
present invention provides modified NPT genes with the DNA sequences
according to SEQ ID N0:3, SEQ ID N0:5, SEQ ID N0:7, SEQ ID N0:9, SEQ
2 s ID N0:11, SEQ ID N0:13, and SEQ ID N0:17.
Gene of Interest
The gene of interest contained in the expression vector according to the
3 o invention comprises a nucleotide sequence of any length which codes for a
product of interest. The gene product or "product of interest" is generally a
protein, polypeptide, peptide or fragment or derivative thereof. However, it
CA 02507664 2005-05-27
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may also be RNA or antisense RNA. The gene of interest may be present in
its full length, in shortened form, as a fusion gene or as a labelled gene. It
may be genomic DNA or preferably cDNA or corresponding fragments of
fusions. The gene of interest may be the native gene sequence, or it may be
mutated or otherwise modified. Such modifications include codon
optimisations for adapting to a particular host cell and humanisation. The
gene of interest may, for example, code for a secreted, cytoplasmic, nuclear-
located, membrane-bound or cell surface-bound polypeptide.
1 o The term "nucleotide sequence" or "nucleic acid sequence" indicates an
oligonucleotide, nucleotides, polynucleotides and fragments thereof as well
as DNA or RNA of genomic or synthetic origin which occur as single or
double strands and can represent the coding or non-coding strand of a gene.
Nucleic acid sequences may be modified using standard techniques such as
15 site-specific mutagenesis or PCR-mediated mutagenesis (e.g. described in
Sambrook et al., 1989 or Ausubel et al., 1994).
By "coding" is meant the property or capacity of a specific sequence of
nucleotides in a nucleic acid, for example a gene in a chromosome or an
2 o mRNA, to act as a matrix for the synthesis of other polymers and
macromolecules such as for example rRNA, tRNA, mRNA, other RNA
molecules, cDNA or polypeptides in a biological process. Accordingly, a
gene codes for a protein if the desired protein is produced in a cell or
another
biological system by transcription and subsequent translation of the mRNA.
2 s Both the coding strand whose nucleotide sequence is identical to the mRNA
sequence and is normally also given in sequence databanks, e.g. EMBL or
GenBank, and also the non-coding strand of a gene or cDNA which acts as
the matrix for transcription may be referred to as coding for a product or
protein. A nucleic acid which codes for a protein also includes nucleic acids
3 o which have a different order of nucleotide sequence on the basis of the
degenerate genetic code but result in the same amino acid sequence of the
CA 02507664 2005-05-27
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protein. Nucleic acid sequences which code for proteins may also contain
introns.
The term cDNA denotes deoxyribonucleic acids which are prepared by
reverse transcription and synthesis of the second DNA strand from a mRNA
or other RNA produced from a gene. If the cDNA is present as a double
stranded DNA molecule it contains both a coding and a non-coding strand.
The term intron denotes non-coding nucleotide sequences of any length.
to They occur naturally in numerous eukaryotic genes and are eliminated from a
previously transcribed mRNA precursor by a process known as splicing. This
requires precise excision of the intron at the 5' and 3' ends and correct
joining
of the resulting mRNA ends so as to produce a mature processed mRNA with
the correct reading frame for successful protein synthesis. Many of the splice
donor and splice acceptor sites involved in this splicing process, i.e. the
sequences located directly at the exon-intron or intron-exon intertaces, have
been characterised by now. For an overview see Ohshima et al., 1987.
Protein/Product of Interest
Proteins/polypeptides with a biopharmaceutical significance include for
example antibodies, enzymes, cytokines, lymphokines, adhesion molecules,
receptors and the derivatives or fragments thereof, but are not restricted
thereto. Generally, all polypeptides which act as agonists or antagonists
and/or have therapeutic or diagnostic applications are of value.
The term "polypeptides" is used for amino acid sequences or proteins and
refers to polymers of amino acids of any length. This term also includes
proteins which have been modified post-translationally by reactions such as
3 o glycosylation, phosphorylation, acetylation or protein processing. The
structure of the polypeptide may be modified, for example, by substitutions,
deletions or insertions of amino acids and fusion with other proteins while
CA 02507664 2005-05-27
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retaining its biological activity. The term "polypeptides" thus also includes,
for
example, fusion proteins consisting of an immunoglobulin component, e.g.
the Fc component, and a growth factor, e.g. an interleukin.
Examples of therapeutic proteins are insulin, insulin-like growth factor,
human
growth hormone (hGH) and other growth factors, tissue plasminogen
activator (tPA), erythropoietin (EPO), cytokines, e.g. interleukines (IL) such
as
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-
13, IL-
14, IL-15, IL-16, IL-17, IL-18 interferon (IFN)-alpha, -beta, -gamma, -omega
to or -tau, tumour necrosis factor (TNF) such as TNF-alpha, beta or gamma,
TRAIL, G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF. Other examples are
monoclonal, polyclonal, multispecific and single chain antibodies and
fragments thereof such as for example Fab, Fab', F(ab')2, Fc and Fc'
fragments, light (L) and heavy (H) immunoglobulin chains and the constant,
variable or hypervariable regions thereof as well as Fv and Fd fragments
(Chamov et al., 1999). The antibodies may be of human or non-human
origin. Humanised and chimeric antibodies are also possible.
Fab fragments (fragment antigen binding = Fab) consist of the variable
2 o regions of both chains which are held together by the adjacent constant
regions. They may be produced for example from conventional antibodies by
treating with a protease such as papain or by DNA cloning. Other antibody
fragments are F(ab')2 fragments which can be produced by proteolytic
digestion with pepsin.
30
By gene cloning it is also possible to prepare shortened antibody fragments
which consist only of the variable regions of the heavy (VH) and light chain
(VL). These are known as Fv fragments (fragment variable = fragment of the
variable part). As covalent binding via the cystein groups of the constant
chains is not possible in these Fv fragments, they are often stabilised by
some other method. For this purpose the variable region of the heavy and
light chains are often joined together by means of a short peptide fragment of
CA 02507664 2005-05-27
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about 10 to 30 amino acids, preferably 15 amino acids. This produces a
single polypeptide chain in which VH and VL are joined together by a peptide
linker. Such antibody fragments are also referred to as single chain Fv
fragments (scFv). Examples of scFv antibodies are known and described, cf.
s for example Huston et al., 1988.
In past years various strategies have been developed for producing
multimeric scFv derivatives. The intention is to produce recombinant
antibodies with improved pharmacokinetic properties and increased binding
to avidity. In order to achieve the multimerisation of the scFv fragments they
are produced as fusion proteins with multimerisation domains. The
multimerisation domains may be, for example, the CH3 region of an IgG or
helix structures ("coiled coil structures") such as the Leucine Zipper
domains.
In other strategies the interactions between the VH and VL regions of the
15 scFv fragment are used for multimerisation (e.g. dia, tri- and
pentabodies).
The term diabody is used in the art to denote a bivalent homodimeric scFv
derivative. Shortening the peptide linker in the scFv molecule to 5 to 10
amino acids results in the formation of homodimers by superimposing VH/VL
2 o chains. The diabodies may additionally be stabilised by inserted
disulphite
bridges. Examples of diabodies can be found in the literature, e.g. in Perisic
et al., 1994.
The term minibody is used in the art to denote a bivalent homodimeric scFv
2 s derivative. It consists of a fusion protein which contains the CH3 region
of an
immunoglobulin, preferably IgG, most preferably IgG1, as dimerisation
region. This connects the scFv fragments by means of a hinge region, also
of IgG, and a linker region. Examples of such minibodies are described by
Hu et al., 1996.
CA 02507664 2005-05-27
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The term triabody is used in the art to denote a trivalent homotrimeric scFv
derivative (Kortt et al., 1997). The direct fusion of VH VL without the use of
a
linker sequence leads to the formation of trimers.
s The fragments known in the art as mini antibodies which have a b-i, tri- or
tetravalent structure are also derivatives of scFv fragments. The
multimerisation is achieved by means of di, tri- or tetrameric coiled coil
structures (Pack et al., 1993 and 1995; Lovejoy et al., 1993).
1 o Gene which codes for a fluorescent protein
In another embodiment the expression vector according to the invention
contains a gene coding for a fluorescent protein, preferably functionally
linked
to the gene of interest. Preferably, both genes are transcribed under the
15 control of a single heterologous promoter so that the protein/ product of
interest and the fluorescent protein are coded by a bicistronic mRNA. This
makes it possible to identify cells which produce the protein/product of
interest in large amounts, by means of the expression rate of the fluorescent
protein.
The fluorescent protein may be, for example, a green, bluish-green, blue,
yellow or other coloured fluorescent protein. One particular example is green
fluorescent protein (GFP) obtained from Aequorea victoria or Renilla
reniformis and mutants developed from them; cf. for example Bennet et al.,
2 s 1998; Chalfie et al., 1994; WO 01 /04306 and the literature cited therein.
Other fluorescent proteins and genes coding for them are described in WO
00/34318, WO 00/34326, WO 00/34526 and WO 01/27150 which are
incorporated herein by reference. These fluorescent proteins are
3 o fluorophores of non-bioluminescent organisms of the species Anthozoa, for
example Anemonia majano, Clavularia sp., Zoanthus sp. I, Zoanthus sp. II,
CA 02507664 2005-05-27
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Discosoma striafa, Discosoma sp. "red'; Discosoma sp. "green'; Discosoma
sp. "Magenta'; Anemonia sulcafa.
The fluorescent proteins used according to the invention contain in addition
to
s the wild-type proteins natural or genetically engineered mutants and
variants,
fragments, derivatives or variants thereof which have for example been fused
with other proteins or peptides. The mutations used may for example alter
the excitation or emission spectrum, the formation of chromophores, the
extinction coefficient or the stability of the protein. Moreover, the
expression
to in mammalian cells or other species can be improved by codon optimisation.
According to the invention the fluorescent protein may also be used in fusion
with a selectable marker, preferably an amplifiable selectable marker such as
dihydrofolate reductase (DHFR).
15 The fluorescence emitted by the fluorescent proteins makes it possible to
detect the proteins, e.g. by throughflow cytometry with a fluorescence-
activated cell sorter (FACS) or by fluorescence microscopy.
CA 02507664 2005-05-27
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Other reaulatory elements
The expression vector contains at least one heterologous promoter which
allows expression of the gene of interest and preferably also of the
fluorescent protein.
The term promoter denotes a polynucleotide sequence which allows and
controls the transcription of the genes or sequences functionally connected
therewith. A promoter contains recognition sequences for binding RNA
to polymerase and the initiation site for transcription (transcription
initiation site).
In order to express a desired sequence in a certain cell type or a host cell a
suitable functional promoter must be chosen. The skilled man will be familiar
with a variety of promoters from various sources, including constitutive,
inducible and repressible promoters. They are deposited in databanks such
as GenBank, for example, and may be obtained as separate elements or
elements cloned within polynucleotide sequences from commercial or
individual sources. In inducible promoters the activity of the promoter may be
reduced or increased in response to a signal. One example of an inducible
promoter is the tetracycline (tet) promoter. This contains tetracycline
2 0 operator sequences (tet0) which can be induced by a tetracycline-regulated
transactivator protein (tTA). In the presence of tetracycline the binding of
tTA
to tet0 is inhibited. Examples of other inducible promoters are the jun, fos,
metallothionein and heat shock promoter (see also Sambrook et al., 1989;
Gossen et al., 1994).
Of the promoters which are particularly suitable for high expression in
eukaryotes, there are for example the ubiquitin/S27a promoter of the hamster
(VllO 97/15664), SV 40 early promoter, adenovirus major late promoter,
mouse metallothionein-I promoter, the long terminal repeat region of Rous
3 o Sarcoma Virus, the early promoter of human Cytomegalovirus. Examples of
other heterologous mammalian promoters are the actin, immunoglobulin or
heat shock promoter(s).
CA 02507664 2005-05-27
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A corresponding heterologous promoter can be functionally connected to
other regulatory sequences in order to increase/regulate the transcription
activity in an expression cassette.
For example, the promoter may be functionally linked to enhancer sequences
in order to increase the transcriptional activity. For this, one or more
enhancers and/or several copies of an enhancer sequence may be used, e.g.
a CMV or SV40 enhancer. Accordingly, an expression vector according to
to the invention, in another embodiment, contains one or more enhancers/
enhancer sequences, preferably a CMV or SV40 enhancer.
The term enhancer denotes a polynucleotide sequence which in the cis
location acts on the activity of a promoter and thus stimulates the
transcription of a gene functionally connected to this promoter. Unlike
promoters the effect of enhancers is independent of position and orientation
and they can therefore be positioned in front of or behind a transcription
unit,
within an intron or even within the coding region. The enhancer may be
located both in the immediate vicinity of the transcription unit and at a
2 o considerable distance from the promoter. It is also possible to have a
physical and functional overlap with the promoter. The skilled man will be
aware of a number of enhancers from various sources (and deposited in
databanks such as GenBank, e.g. SV40 enhancers, CMV enhancers,
polyoma enhancers, adenovirus enhancers) which are available as
independent elements or elements cloned within polynucleotide sequences
(e.g. deposited at the ATCC or from commercial and individual sources). A
number of promoter sequences also contain enhancer sequences such as
the frequently used CMV promoter. The human CMV enhancer is one of the
strongest enhancers identified hitherto. One example of an inducible
3 o enhancer is the metallothionein enhancer, which can be stimulated by
glucocorticoids or heavy metals.
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Another possible modification is, for example, the introduction of multiple
Sp1
binding sites. The promoter sequences may also be combined with
regulatory sequences which allow control/regulation of the transcription
activity. Thus, the promoter can be made repressible/ inducible. This can be
done for example by linking to sequences which are binding sites for up- or
down-regulating transcription factors. The above mentioned transcription
factor Sp1, for example, has a positive effect on the transcription activity.
Another example is the binding site for the activator protein AP1, which may
act both positively and negatively on transcription. The activity of AP1 can
be
~.o controlled by all kinds of factors such as, for example, growth factors,
cytokines and serum (Faisst et al., 1992 and references therein). The
transcription efficiency can also be increased by changing the promoter
sequence by the mutation (substitution, insertion or deletion) of one, two,
three or more bases and then determining, in a reporter gene assay, whether
this has increased the promoter activity.
Basically, the additional regulatory elements include heterologous promoters,
enhancers, termination and polyadenylation signals and other expression
control elements. Both inducible and constitutively regulatory sequences are
2 o known for the various cell types.
"Transcription-regulatory elements" generally comprise a promoter upstream
of the gene sequence to be expressed, transcription initiation and termination
sites and a polyadenylation signal.
The term "transcription initiation site" refers to a nucleic acid in the
construct
which corresponds to the first nucleic acid which is incorporated in the
primary transcript, i.e. the mRNA precursor. The transcription initiation site
may overlap with the promoter sequences.
The term "transcription termination site" refers to a nucleotide sequence
which is normally at the 3' end of the gene of interest or of the gene section
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which is to be transcribed, and which brings about the termination of
transcription by RNA polymerase.
The "polyadenylation signal" is a signal sequence which causes cleavage at
a specific site at the 3' end of the eukaryotic mRNA and post-transcriptional
incorporation of a sequence of about 100-200 adenine nucleotides (polyA tail)
at the cleaved 3' end. The polyadenylation signal comprises the sequence
AATAAA about 10-30 nucleotides upstream of the cleavage site and a
sequence located downstream. Various polyadenylation elements are known
1 o such as tk polyA, SV40 late and early polyA or BGH polyA (described for
example in US 5,122,458).
In a preferred embodiment of the present invention each transcription unit
has a promoter or a promoter/enhancer element, a gene of interest andlor a
marker gene as well as a transcription termination element. In another
preferred embodiment the transcription unit contains two further translation
regulatory units.
"Translation regulatory elements" comprise a translation initiation site
(AUG),
2 o a stop codon and a polyA signal for each polypeptide to be expressed. For
optimum expression it may be advisable to remove, add or change 5'- and/or
3'-untranslated regions of the nucleic acid sequence which is to be
expressed, in order to eliminate any potentially unsuitable additional
translation initiation codons or other sequences which might affect expression
at the transcription or expression level. In order to promote expression,
ribosomal consensus binding sites may alternatively be inserted immediately
upstream of the start codon. In order to produce a secreted polypeptide the
gene of interest usually contains a signal sequence which codes for a signal
precursor peptide which transports the synthesised polypeptide to and
3 o through the ER membrane. The signal sequence is often but not always
located at the amino terminus of the secreted protein and is cleaved by signal
peptidases after the protein has been filtered through the ER membrane.
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The gene sequence will usually but not necessarily contain its own signal
sequence. If the native signal sequence is not present a heterologous signal
sequence may be introduced in known manner. Numerous signal sequences
of this kind are known to the skilled man and deposited in sequence
databanks such as GenBank and EMBL.
One important regulatory element according to the invention is the internal
ribosomal entry site (IRES). The IRES element comprises a sequence which
functionally activates the translation initiation independently of a 5'-
terminal
to methylguanosinium cap (CAP structure) and the upstream gene and in an
animal cell allows the translation of two cistrons (open reading frames) from
a
single transcript. The IRES element provides an independent ribosomal entry
site for the translation of the open reading frame located immediately
downstream. In contrast to bacterial mRNA which may be multicistronic, i.e.
15 it may code for numerous different polypeptides or products which are
translated one after the other by the mRNA, the majority of mRNAs from
animal cells are monocistronic and code for only one protein or product. In
the case of a multicistronic transcript in a eukaryotic cell the translation
would
be initiated from the translation initiation site which was closest upstream
and
2 o would be stopped by the first stop codon, after which the transcript would
be
released from the ribosome. Thus, only the first polypeptide or product coded
by the mRNA would be produced during translation. By contrast, a
multicistronic transcript with an IRES element which is functionally linked to
the second or subsequent open reading frame in the transcript allows
2 s subsequent translation of the open reading frame located downstream
thereof, so that two or more polypeptides or products coded by the same
transcript are produced in the eukaryotic cell.
The IRES element may be of various lengths and various origins and may
3 0 originate, for example, from the encephalomyocarditis virus (EMCV) or
other
Picorna viruses. Various IRES sequences and their use in the construction of
vectors are described in the literature, cf. for example Pelletier et al.,
1988;
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Jang et al., 1989; Davies et al., 1992; Adam et al., 1991; Morgan et al.,
1992;
Sugimoto et al., 1994; Ramesh et al., 1996; Mosser et al., 1997.
The gene sequence located downstream is functionally linked to the 3' end of
the IRES element, i.e. the spacing is selected so that the expression of the
gene is unaffected or only marginally affected or has sufficient expression
for
the intended purpose. The optimum permissible distance between the IRES
element and the start colon of the gene located downstream thereof for
sufficient expression can be determined by simple experiments by varying the
1 o spacing and determining the expression rate as a function of the spacing
using reporter gene assays.
By the measures described it is possible to obtain an optimum expression
cassette which is of great value for the expression of heterologous gene
products. An expression cassette obtained by means of one or more such
measures is therefore a further subject of the invention.
Hamster-Ubiauitin/S27a Promoter
2 o In another embodiment the expression vector according to the invention
contains the ubiquitin/S27a promoter of the hamster, preferably functionally
linked to the gene of interest and even more preferably functionally linked to
the gene of interest and the gene which codes for a fluorescent protein.
The ubiquitin/S27a promoter of the hamster is a powerful homologous
promoter which is described in WO 97/15664. Such a promoter preferably
has at least one of the following features: GC-rich sequence area, Sp1
binding site, polypyrimidine element, absence of a TATA box. Particularly
preferred is a promoter which has an Sp1 binding site but no TATA box. Also
3 o preferred is a promoter which is constitutively activated and in
particular is
equally active under serum-containing, low-serum and serum-free cell culture
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conditions. In another embodiment it is an inducible promoter, particularly a
promoter which is activated by the removal of serum.
A particularly advantageous embodiment is a promoter with a nucleotide
sequence as contained in Fig. 5 of WO 97/15664. Particularly preferred are
promoter sequences which contain the sequence from position -161 to -45 of
Fig. 5.
The promoters used in the examples of the present patent specification each
1 o contain a DNA molecule with the sequence from position 1923 to 2406 of
SEQ ID N0:55 of the attached sequence listing. This sequence corresponds
to the fragment -372 to +111 from Fig. 5 of WO 97/15664 and represents the
preferred promoter, i.e a preferred promoter should incorporate this sequence
region. Another suitable promoter fragment contains the sequence from
position 2134 to 2406 (corresponding to -161 to +111 in Fig. 5 of WO
97/15664). A promoter which contains only the sequence from position 2251
to 2406 is no longer functional (corresponds to position -45 to +111 in Fig. 5
of WO 9/15664). It is possible to extend the promoter sequence in the 5'
direction starting from position 2134.
It is also possible to use functional subfragments of the complete hamster
ubiquitin/S27a promoter sequence as well as functional mutants/variants of
the complete sequence of subfragments thereof which have been modified,
for example, by substitution, insertion or deletion. Corresponding
2 5 subfragments, mutants or variants are hereinafter also referred to as
"modified promoters".
A modified promoter, optionally combined with other regulatory elements,
preferably has a transcription activity which corresponds to that of the
3 o promoter fragment from position 1923 to 2406 of the nucleotide sequence
given in SEQ ID N0:55 (-372 to +111 from Fig. 5 of WO 97/15664). A
modified promoter proves to be useful for the purposes of the invention if it
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has a transcription activity which has at least 50%, preferably at least 80%,
more preferably at least 90% and most preferably at least 100% of the activity
of the 1923 to 2406 fragment (-372 to +111 fragment) in a comparative
reporter gene assay. Particularly preferred are modified promoters which
s have a minimum sequence homology to the wild-type sequence SEQ ID
N0:55 of the hamster ubiquitin/ S27a promoter of at least 80%, preferably at
least 85%, preferably at least 90%, more preferably at least 95% and most
preferably at least 97% and have a corresponding promoter activity in a
comparative reporter gene assay.
to
In a corresponding comparative reporter gene assay the promoter fragments
to be tested including the reference sequence are cloned in front of a
promoterfess reporter gene which codes, for example for luciferase, secreted
alkaline phosphotase or green fluorescent protein (GFP). These constructs
15 (promoter sequence + reporter gene) are subsequently introduced into the
test cells, e.g. CHO-DG44, by transfection and the induction of the reporter
gene expression by the promoter fragment in question is determined by
measuring the protein content of the reporter gene. A corresponding test is
found for example in Ausubel et al., Current Protocols in Molecular Biology,
2 0 1994, updated.
The promoter sequence of the hamster ubiquitin/S27a promoter and the
modified promoters, which may also include, for example, the 5' untranslated
region or selected fragments thereof, and the coding region as well as the 3'-
2 s untranslated region of the ubiquitin/S27a gene or selected fragments
thereof,
may be obtained by a skilled man with a knowledge of the sequence
described in WO 97/15664 using various standard methods as described for
example in Sambrook et al., 1989; Ausubel et al., 1994. Starting from the
sequence described in WO 97/15664 a suitable fragment may be selected,
3 o for example, and an oligonucleotide probe containing the sequence of this
fraction may be chemically synthesised. A probe of this kind may be used for
example to clone the ubiquitinlS27a gene or the 5' untranslated region or
CA 02507664 2005-05-27
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other fragments thereof, for example by hybridisation from a library of the
hamster genome. Using the reporter gene assay described above the skilled
man is in a position to identify promoter-active fragments without any great
effort and use them for the purposes of the present invention. The 5'
untranslated region or special fragments thereof can easily be obtained by
PCR amplification with corresponding primers from genomic DNA or a
genomic library. Fragments of the 5' untranslated region may also be
obtained by limited exonuclease III digestion from larger DNA fragments.
Such DNA molecules may also be chemically synthesised or produced from
1 o chemically synthesised fragments by ligation.
Deletion, insertion and substitution mutants may be produced by "site-specific
mutagenesis" and/or "PCR-based mutagenesis techniques". Corresponding
methods are mentioned for example in Lottspeich and Zorbas 1998 Chapter
36.1 with further references.
By cross-hybridisation with probes from the 5' untranslated region of the
hamster ubiquitin/S27a gene or from the S27a part of the hamster ubiquitin
S27a gene or the 3'-untranslated region it is also possible to identify and
2 o isolate suitable promoter sequences from corresponding homologous genes
of other, preferably mammalian species. Suitable techniques are described
by way of example in Lottspeich and Zorbas 1998 Chapter 23. Genes are
"homologous" for the purposes of the invention if their nucleotide sequence
exhibits at least 70%, preferably at least 80%, preferably at least 90%, more
preferably at least 95% and most preferably at least 97% conformity to the
nucleotide sequence of the gene with which it is homologous.
Using the measures described above it is possible to obtain an optimised
expression cassette which is highly valuable for the expression of
3 o heterologous gene products. An expression cassette obtained by one or
more such measures is therefore a further object of the invention.
CA 02507664 2005-05-27
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Preparation of expression vectors according to the invention
The expression vector according to the invention may theoretically be
prepared by conventional methods known in the art, as described by
s Sambrook et al. (1989), for example. Sambrook also describes the functional
components of a vector, e.g. suitable promoters (in addition to the hamster
ubiquitinlS27a promoter), enhancers, termination and polyadenylation
signals, antibiotic resistance genes, selectable markers, replication starting
points and splicing signals. Conventional cloning vectors may be used to
1 o produce them, e.g. plasmids, bacteriophages, phagemids, cosmids or viral
vectors such as baculovirus, retroviruses, adenoviruses, adeno-associated
viruses and herpes simplex virus, as well as artificial chromosomes/mini
chromosomes. The eukaryotic expression vectors typically also contain
prokaryotic sequences such as, for example, replication origin and antibiotic
15 resistance genes which allow replication and selection of the vector in
bacteria. A number of eukaryotic expression vectors which contain multiple
cloning sites for the introduction of a polynucleotide sequence are known and
some may be obtained commercially from various companies such as
Stratagene, La Jolla, CA, USA; Invitrogen, Carlsbad, CA, USA; Promega,
2 o Madison, WI, USA or BD Biosciences Clontech, Palo Alto, CA, USA.
The heterologous promoter, the gene of interest and the modified neomycin
phosphotransferase gene and optionally the gene coding for a fluorescent
protein, additional regulatory elements such as the internal ribosomal entry
2s site (IRES), enhancers or a polyadenylation signal are introduced into the
expression vector in a manner familiar to those skilled in the art. An
expression vector according to the invention contains, at the minimum, a
heterologous promoter, the gene of interest and a modified neomycin
phosphotransferase gene. Preferably, the expression vector also contains a
3 o gene coding for a fluorescent protein. It is particularly preferred
according to
the invention to use a ubiquitin/S27a promoter as heterologous promoter.
Particularly preferred is an expression vector in which the heterologous
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promoter, preferably a ubiquitin/S27a promoter, the gene of interest and the
gene which codes for a fluorescent protein are functionally linked together or
are functionally linked and the neomycin phosphotransferase gene is located
in the same or in a separate transcription unit.
Within the scope of the present description the term "functional linking" or
"functionally linked" refers to two or more nucleic acid sequences or partial
sequences which are positioned so that they can perform their intended
function. For example, a promoter/enhancer is functionally linked to a coding
1 o gene sequence if it is able to control or modulate the transcription of
the
linked gene sequence in the cis position. Generally, but not necessarily,
functionally linked DNA sequences are close together and, if two coding gene
sequences are linked or in the case of a secretion signal sequence, in the
same reading frame. Although a functionally finked promoter is generally
located upstream of the coding gene sequence it does not necessarily have
to be close to it. Enhancers need not be close by either, provided that they
assist the transcription of the gene sequence. For this purpose they may be
both upstream and downstream of the gene sequence, possibly at some
distance from it. A polyadenylation site is functionally linked to a gene
2 o sequence if it is positioned at the 3' end of the gene sequence in such a
way
that the transcription progresses via the coding sequence to the
polyadenylation signal. Linking may take place according to conventional
recombinant methods, e.g. by the PCR technique, by ligation at suitable
restriction cutting sites or by splicing. If no suitable restriction cutting
sites
2 5 are available synthetic oligonucleotide linkers or adaptors may be used in
a
manner known per se. According to the invention the functional linking
preferably does not take place via intron sequences.
In one of the embodiments described, the heterologous promoter, preferably
3o a ubiquitin/S27a promoter, the gene of interest and the gene coding for a
fluorescent protein are functionally linked together. This means for example
CA 02507664 2005-05-27
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that both the gene of interest and the gene coding for a fluorescent protein
are expressed starting from the same heterologous promoter.
In a particularly preferred embodiment the functional linking takes place via
an IRES element, so that a bicistronic mRNA is synthesised from both genes.
The expression vector according to the invention may additionally contain
enhancer elements which act functionally on one or more promoters.
Particularly preferred is an expression vector in which the heterologous
promoter, preferably the ubiquitin/S27a promoter or a modified form thereof,
1 o is linked to an enhancer element, e.g. an SV40 enhancer or a CMV enhancer
element.
Fundamentally, the expression of the genes within an expression vector may
take place starting from one or more transcription units. The term
transcription unit is defined as a region which contains one or more genes to
be transcribed. The genes within a transcription unit are functionally linked
to
one another in such a way that all the genes within such a unit are under the
transcriptional control of the same promoter or promoter/ enhancer. As a
result of this transcriptional linking of genes, more than one protein or
product
2 o can be transcribed from a transcription unit and thus expressed. Each
transcription unit contains the regulatory elements which are necessary for
the transcription and translation of the gene sequences contained therein.
Each transcription unit may contain the same or different regulatory elements.
IRES elements or introns may be used for the functional linking of the genes
2 5 within a transcription unit.
The expression vector may contain a single transcription unit for expressing
the gene of interest, the modified NPT gene and optionally the gene which
codes for the fluorescent protein. Alternatively, these genes may also be
3 o arranged in two or more transcription units. Various combinations of the
genes within a transcription unit are possible. In another embodiment of the
present invention more than one expression vector consisting of one, two or
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more transcription units may be inserted in a host cell by cotransfection or
in
successive transfections in any desired order. Any combination of regulatory
elements and genes on each vector can be selected provided that adequate
expression of the transcription units is ensured. If necessary, other
regulatory elements and genes, e.g. additional genes of interest or selectable
markers, may be positioned on the expression vectors.
Accordingly, an expression vector according to the invention containing a
gene of interest and a gene which codes for a modified neomycin
to phosphotransferase may contain both genes in one or in two separate
transcription units. Each transcription unit can transcribe and express one or
more gene products. If both genes are contained in one transcription unit
they are under the control of the same promoter or promoter/ enhancer, while
preferably an IRES element is used to ensure the functional linking of all the
components. If the gene which codes for modified neomycin
phosphotransferase and the gene of interest are contained in two separate
transcription units, they may be under the control of the same or different
promoterslenhancers. However, preferably, a weaker heterologous
promoter, e.g. SV40 early promoter, is used for the modified NPT gene and
2 o preferably no enhancer is used. Expression vectors with two separate
transcription units are preferred within the scope of the invention. One
(bicistronic) transcription unit contains the gene of interest and optionally
a
gene coding for a fluorescent protein, while the other transcription unit
contains the modified NPT gene. Preferably, each transcription unit is limited
2 5 at the 3' end by a sequence which codes for a polyA signal, preferably BGH
polyA or SV40 polyA.
Also preferred according to the invention are those expression vectors which
instead of the gene of interest have only a multiple cloning site which allows
3 o the cloning of the gene of interest via recognition sequences for
restriction
endonucleases. Numerous recognition sequences for all kinds of restriction
endonucleases as well as the associated restriction endonucleases are
CA 02507664 2005-05-27
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known from the prior art. Preferably, sequences are used which consist of at
least six nucleotides as recognition sequence. A list of suitable recognition
sequences can be found for example in Sambrook et al., 1989.
Host Cells
For transfection with the expression vector according to the invention
eukaryotic host cells are used, preferably mammalian cells and more
particularly rodent cells such as mouse, rat and hamster cell lines. The
1 o successful transfection of the corresponding cells with an expression
vector
according to the invention results in transformed, genetically modified,
recombinant or transgenic cells, which are also the subject of the present
invention.
Preferred host cells for the purposes of the invention are hamster cells such
as BHK21, BHK TK- CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1 and CHO-
DG44 cells or derivatives/descendants of these cell lines. Particularly
preferred are CHO-DG44, CHO-DUKX, CHO-K1 and BHK21 cells,
particularly CHO-DG44 and CHO-DUKX cells. Also suitable are myeloma
2 o cells from the mouse, preferably NSO and Sp2/0 cells and
derivatives/descendants of these cell lines.
Examples of hamster and mouse cells which can be used according to the
invention are given in Table 1 that follows. However, derivatives and
descendants of these cells, other mammalian cells including but not restricted
to cell lines of humans, mice, rats, monkeys, rodents, or eukaryotic cells,
including but not restricted to yeast, insect and plant cells, may also be
used
as host cells for the production of biopharmaceutical proteins.
3 o Table 1: Hamster and Mouse Production Cell Lines
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Cell line Accession Number
NSO ECASS No. 85110503
Sp2/0-Ag14 ATCC CRL-1581
BHK21 ATCC CCL-10
BHK TK- ECACC No. 85011423
HaK ATCC CCL-15
2254-62.2 ATCC CRL-8544
(BHK-21-derivative)
CHO ECACC No. 8505302
CHO-K1 ATCC CCL-61
CHO-DUKX ATCC CRL-9096
(= CHO duk- CHO/dhf~ )
CHO-DUKX B1 ATCC CRL-9010
CHO-DG44 Urlaub et al;
Cell 32[2], 405-412, 1983
CHO Pro-5 ATCC CRL-1781
V79 ATCC CCC-93
B14AF28-G3 ATCC CCL-14
CHL ECACC No. 87111906
The transfection of the eukaryotic host cells with a polynucleotide or one of
the expression vectors according to the invention is carried out by
conventional methods (Sambrook et al., 1989; Ausubel et al., 1994). Suitable
methods of transfection include for example lyposome-mediated transfection,
calcium phosphate co precipitation, electroporation, polycation- (e.g. DEAE
dextran)-mediated transfection, protoplast fusion, microinjection and viral
infections. According to the invention stable transfection is preferably
carried
out in which the constructs are either integrated into the genome of the host
to cell or an artificial chromosome/minichromosome, or are episomally
contained in stable manner in the host cell. The transfection method which
gives the optimum transfection frequency and expression of the heterologous
CA 02507664 2005-05-27
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gene in the host cell in question is preferred. By definition, every sequence
or every gene inserted in a host cell is referred to as a "heterologous
sequence" or "heterologous gene" in relation to the host cell. This applies
even if the sequence to be introduced or the gene to be introduced is
identical to an endogenous sequence or an endogenous gene of the host
cell. For example, a hamster actin gene introduced into a hamster host cell is
by definition a heterologous gene.
According to the invention, recombinant mammalian cells, preferably rodent
to cells, most preferably hamster cells such as CHO or BHK cells which have
been transfected with one of the expression vectors according to the
invention described herein are preferred.
In the recombinant production of heteromeric proteins such as e.g.
monoclonal antibodies (mAb), the transfection of suitable host cells can
theoretically be carried out by two different methods. mAb's of this kind are
composed of a number of subunits, the heavy and light chains. Genes coding
for these subunits may be accommodated in independent or multicistronic
transcription units on a single plasmid with which the host cell is then
2 o transfected. This is intended to secure the stoichiometric representation
of
the genes after integration into the genome of the host cell. However, in the
case of independent transcriptional units it must hereby be ensured that the
mRNAs which encode the different proteins display the same stability and
transcriptional and translational efficiency. In the second case, the
expression
of the genes take place within a multicistronic transcription unit by means of
a
single promoter and only one transcript is formed. By using IRES elements,
a highly efficient internal translation initiation of the genes is obtained in
the
second and subsequent cistrons. However, the expression rates for these
cistrons are lower than that of the first cistron, the translation initiation
of
3 o which, by means of a so-called "cap"-dependent pre-initiation complex, is
substantially more efficient than IRES-dependent translation initiation. In
order to achieve a truly equimolar expression of the cistrons, additional
inter-
CA 02507664 2005-05-27
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cistronic elements may be introduced, for example, which ensure uniform
expression rates in conjunction with the IRES elements (WO 94/05785).
Another possible way of simultaneously producing a number of heterologous
proteins, which is preferred according to the invention, is cotransfectian, in
which the genes are separately integrated in different expression vectors.
This has the advantage that certain proportions of genes and gene products
with one another can be adjusted, thereby balancing out any differences in
the mRNA stability and in the efficiency of transcription and translation. In
1 o addition, the expression vectors are more stable because of their small
size
and are easier to handle both during cloning and during transfection.
In one particular embodiment of the invention, therefore, the host cells are
additionally transfected, preferably cotransfected, with one or more vectors
having genes which code for one or more other proteins of interest. The
other vector or vectors used for the cotransfection code, for example, for the
other protein or proteins of interest under the control of the same
promoter/enhancer combination and for at least one other selectable marker,
e.g. dihydrofolate reductase.
According to the invention the host cells are preferably established, adapted
and cultivated under serum-free conditions, optionally in media which are free
from animal proteins/peptides. Examples of commercially obtainable media
include Ham's F12 (Sigma, Deisenhofen, DE), RPMI-1640 (Sigma),
2s Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimal Essential
Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM;
Sigma), CD-CHO (Invitrogen, Carlsbad, CA, USA), CHO-S-SFMII
(Invitrogen), serum-free CHO-Medium (Sigma) and protein-free CHO-Medium
(Sigma). Each of these media may optionally be supplemented with various
3 o compounds, e.g. hormones and/or other growth factors (e.g. insulin,
transferrin, epidermal growth factor, insulin-like growth factor), salts (e.g.
sodium chloride, calcium, magnesium, phosphate), buffers (e.g. HEPES),
CA 02507664 2005-05-27
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nucleosides (e.g. adenosine, thymidine), glutamine, glucose or other
equivalent nutrients, antibiotics and/or trace elements. Although serum-free
media are preferred according to the invention, the host cells may also be
cultivated and protein subsequently produced using media which have been
s mixed with a suitable amount of serum. In order to select genetically
modified cells which express one or more selectable marker genes, one or
more selecting agents are added to the medium.
The term "selecting agent" refers to a substance which affects the growth or
1 o survival of host cells with a deficiency for the selectable marker gene in
question. Within the scope of the present invention, geneticin (G418) is
preferably used as the medium additive for the selection of heterologous host
cells which carry a modified neomycin phosphotransferase gene. Preferably,
6418 concentrations of between 100 and 800 :g/ml of medium are used,
is most preferably 300 to 400 :g/ml of medium. If the host cells are to be
transfected with a number of expression vectors, e.g. if several genes of
interest are to be separately introduced into the host cell, they generally
have
different selectable marker genes.
2 o A selectable marker gene is a gene which allows the specific selection of
cells which contain this gene by the addition of a corresponding selecting
agent to the cultivation medium. As an illustration, an antibiotic resistance
gene may be used as a positive selectable marker. Only cells which have
been transformed with this gene are able to grow in the presence of the
25 corresponding antibiotic and are thus selected. Untransformed cells, on the
other hand, are unable to grow or survive under these selection conditions.
There are positive, negative and bifunctional selectable markers. Positive
selectable markers permit the selection and hence enrichment of transformed
cells by conferring resistance to the selecting agent or by compensating for a
3 o metabolic or catabolic defect in the host cell. By contrast, cells which
have
received the gene for the selectable marker can be selectively eliminated by
negative selectable markers. An example of this is the thymidine kinase
CA 02507664 2005-05-27
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gene of the Herpes Simplex virus, the expression of which in cells with the
simultaneous addition of acyclovir or gancyclovir leads to the elimination
thereof. The selectable markers used in this invention, including the
amplifiable selectable markers, include genetically modified mutants and
variants, fragments, functional equivalence, derivatives, homologues and
fusions with other proteins or peptides, provided that the selectable marker
retains its selective qualities. Such derivatives display considerable
homology in the amino acid sequence in the regions or domains which are
deemed to be selective. The literature describes a large number of
1 o selectable marker genes including bifunctional (positive/negative) markers
(see for example WO 92/08796 and WO 94/28143). Examples of selectable
markers which are usually used in eukaryotic cells include the genes for
aminoglycoside phosphotransferase (APH), hygromycine
phosphostransferase (HYG), dihydrofolate reductase (DHFR), thymidine
1s kinase (TK), glutamine synthetase, asparagin synthetase and genes which
confer resistance to neomycin (G418), puromycin, histidinol D, belomycin,
phleomycin and zeocin.
It is also possible to select transformed cells by fluorescence-activated cell
2 o sorting (FACS). For this, bacterial 3-galactosidase, cell surface markers
or
fluorescent proteins may be used (e.g. green fluorescent protein (GFP) and
the variants thereof from Aequorea victoria and Renilla reniformis or other
species; red fluorescent proteins and proteins which fluoresce in other
colours and their variants from non-bioluminescent organisms such as e.g.
25 Discosoma sp., Anemonia sp., Clavularia sp., Zoanthus sp.) for the
selection
of transformed cells.
Gene expression and selection of high-producing host cells
3 o The term gene expression relates to the transcription and/or translation
of a
heterologous gene sequence in a host cell. The expression rate can be
generally determined, either on the basis of the quantity of corresponding
CA 02507664 2005-05-27
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mRNA which is present in the host cell or on the basis of the quantity of gene
product produced which is encoded by the gene of interest. The quantity of
mRNA produced by transcription of a selected nucleotide sequence can be
determined for example by northern blot hybridisation, ribonuclease-RNA-
protection, in situ hybrisation of cellular RNA or by PCR methods (Sambrook
et al., 1989; Ausubel et al., 1994). Proteins which are encoded by a selected
nucleotide sequence can also be determined by various methods such as, for
example, ELISA, western blot, radioimmunoassay, immunoprecipitation,
detection of the biological activity of the protein or by immune staining of
the
1 o protein followed by FACS analysis (Sambrook et al., 1989; Ausubel et al.,
1994).
The terms "high expression level (or rate), high expression, increased
expression or high productivity" refer to the long-lasting and sufficiently
high
15 expression or synthesis of a heterologous sequence introduced into a host
cell, e.g. of a gene coding for a therapeutic protein. Increased or high
expression or a high expression level or rate or a high productivity are
present if a cell according to the invention is cultivated by one of the
methods
according to the invention described here, without gene amplification, and if
2 o this cell produces at least more than roughly 0.5 pg of the desired gene
product per day (0.5 pg/cell/day). Increased or high expression or a high
expression or rate or a high productivity are also present if the cell
according
to the invention without prior gene amplification produces at least more than
roughly 1.0 pg of the desired gene produce per day (1.0 pg/cell/day).
25 Increased or high expression or a high expression level or rate or high
productivity are present in particular if the cell according to the invention
without prior gene amplification produces at least more than roughly 1.5 pg of
the desired gene product per day (1.5 pg/ cell/day). Increased or high
expression or a high expression level or rate or high productivity are present
3 o in particular if the cell according to the invention without prior gene
amplification produces at least more than roughly 2.0 pg of the desired gene
product per day (2.0 pg/cell/day). Particularly increased or high expression
or
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a particularly high expression level or rate or particularly high productivity
are
present if the cell according to the invention without prior gene
amplification
produces at least more than roughly 3.0 pg of the desired gene product per
day (3.0 pg/cell/day). By means of a simple gene amplification step, e.g.
using the DHFR/MTX amplification system as described hereinafter the
productivities can be increased by a factor of at least 2 to 10, so that the
terms "high expression", increased expression" or high productivity" are used
in relation to a cell which has been subjected to a gene amplification step if
this cell produces at least more than roughly 5 pg of the desired gene product
to per day (5 pg/cell/day), preferably at least more than roughly 10
pg/cell/day,
more preferably at least more than roughly 15 pg/cell/day , still more
preferably at least more than roughly 20 pg/celllday or at least more than
roughly 30 pg/cell/day.
High or increased expression, high productivity or a high expression level or
rate can be achieved both by using one of the expression vectors according
to the invention and by the use of one of the processes according to the
invention.
2 o For example, by coexpression of the gene of interest and a modified NPT
gene it is possible to select and identify cells which express the
heterologous
gene to a high degree. Compared with wtNPT, modified NPT allows more
efficient selection of stably transfected host cells with high expression of
the
heterologous gene of interest.
The present invention thus also relates to a process for expressing at least
one gene of interest in recombinant mammalian cells, characterised in that (i)
a pool of mammalian cells is transfected with at least one gene of interest
and one gene for a modified neomycin phosphotransferase which compared
3 o with the wild-type neomycin phosphotransferase has only 1 to 80% of the
activity, preferably only 1 to 60%, more preferably only 1.5 to 30%, most
preferably only 1.5 to 26°!°; (ii) the cells are cultivated
under conditions which
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allow expression of the gene or genes of interest and the modified neomycin
phosphotransferase gene; (iii) the mammalian cells are cultivated in the
presence of at least one selecting agent, preferably 6418, which acts
selectively on the growth of the mammalian cells, and gives preference to the
growth of those cells which express the modified neomycin
phosphotransferase gene; and (iv) the protein or proteins of interest is or
are
obtained from the mammalian cells or the culture supernatant. Preferably
recombinant mammalian cells are used which have been transfected with an
expression vector according to the invention.
to
The invention also relates to a process for selecting recombinant mammalian
cells which express at least one gene of interest, wherein (i) a pool of
mammalian cells is transfected with at least one gene of interest and a gene
for a modified neomycin phosphotransferase which by comparison with wild-
type neomycin phosphotransferase has only 1 to 80% of the activity,
preferably only 1 to 60%, more preferably only 1.5 to 30%, most preferably
only 1.5 to 26%; (ii) the mammalian cells are cultivated under conditions
which allow expression of the gene or genes of interest and the modified
neomycin phosphotransferase gene; and (iii) the mammalian cells are
2 o cultivated in the presence of at least one selecting agent, preferably
6418,
which acts selectively on the growth of the mammalian cells and gives
preference to the growth of those cells which express the modified neomycin
phosphotransferase gene.
Particularly preferred are processes for expressing at least one gene of
interest and for selecting recombinant cells which express a corresponding
gene of interest if a modified NPT gene described in more detail in this
application is used, particularly if a modified NPT gene is used which by
comparison with the wild-type gene codes for glycine or aspartic acid at
3 o amino acid position 182, for alanine at amino acid position 91, for
glycine at
amino acid position 198, for alanine, glycine or valine at amino acid position
227, for glycine or asparagine at amino acid position 261 or for isoleucine at
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amino acid position 240. It is particularly preferred to use the Asp227Va1,
Asp227G1y, Asp261G1y, Asp261Asn, Phe24011e or Trp91Ala mutant.
Generally, all the modified neomycin phosphotransferase genes according to
the invention mentioned in this patent specification are suitable for such a
process. For the preferred neomycin phosphotransferase genes see the
section on modified neomycin phosphotransferase genes.
The selection of the cells which express a gene of interest and a modified
NPT gene is carried out for example by adding 6418 as selecting agent.
1 o However, it is also possible to use other aminoglycoside antibiotics such
as
neomycin or kanamycin. The cells according to the invention are preferably
cultivated and selected in 200 to 800 :g of 6418 per mL of culture medium. It
has proved particularly preferable to add 300 to 700 :g of 6418 per mL of
culture medium. The addition of roughly 400 :g of 6418 per mL of culture
i5 medium is the most preferred embodiment. Using such a method it is
possible to select recombinant cells with a particularly high expression rate.
By comparison with the use of wtNPT, after selection with 400 Ng G418 per
ml of culture medium as selectable marker, the cells exhibited a productivity
increased by a factor of 1.4 - 2.4 in the case of the GIu182G1y, GIu182Asp
2 o and Va1198G1y mutant, by a factor of 1.6 to 4.1 in the case of the
Asp227G1y
mutant, by a factor of 2.2 or 4 in the case of the Asp227A1a or Trp91 Ala
mutant, by a factor of 5.7 or 7.3 in the case of the Phe24011e or Asp261Asn
mutant and even by a factor of 9.3 or 14.6 in the case of the Asp261 Gly or
Asp227Va1 mutant. The specific producitivities for the various modified NPT
25 genes are shown in Fig. 6.
The corresponding processes may be combined with a FACS-assisted
selection of recombinant host cells which contain, as additional selectable
marker, one or more fluorescent proteins (e.g. GFP) or a cell surface marker.
3 o Other methods of obtaining increased expression, and a combination of
different methods may also be used, are based for example on the use of
(artificial) transcription factors, treatment of the cells with natural or
synthetic
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agents for up-regulating endogenous or heterologous gene expression,
improving the stability (half-life) of mRNA or the protein, improving the
initiation of mRNA translation, increasing the gene dose by the use of
episomal plasmids (based on the use of viral sequences as replication
origins, e.g. SV40, polyoma, adenovirus, EBV or BPV), the use of
amplification-promoting sequences (Hemann et al., 1994) or in vitro
amplification systems based on DNA concatemers (Monaco et al., 1996).
Coupled transcription of the gene of interest and the gene which codes for
to the fluorescent protein has proved particularly effective in conjunction
with the
use of a modified NPT gene as selectable marker. The resulting bicistronic
mRNA expresses both the protein/product of interest and the fluorescent
protein. On the basis of this coupling of the expression of the protein of
interest and the fluorescent protein it is easily possible according to the
invention to identify and isolate high-producing recombinant host cells by
means of the fluorescent protein expressed, e.g. by sorting using
fluorescence activated cell sorting equipment (FACS).
The selection of recombinant host cells which exhibit high vitality and an
2 o increased expression rate of the desired gene product is a multistage
process. The host cells which have been transfected with the expression
vector according to the invention or optionally cotransfected with another
vector, for example, are cultivated under conditions which permit the
selection of cells expressing the modified NPT, e.g. by cultivation in the
2 5 presence of a selecting agent such as 6418 in concentrations of 100, 200,
400, 600, 800 :g or more of G418/mL of culture medium. Then the
corresponding cells are investigated at least for the expression of the gene
which codes for a fluorescent protein and is coupled to the gene of interest,
in
order to identify and sort out the cells/cell population which exhibit the
highest
3 o expression rates of fluorescent protein. Preferably, only the cells which
belong to the 10-20% of cells with the highest expression rate of fluorescent
protein are sorted out and further cultivated. In practice this means that the
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brightest 10% of the fluorescent cells are sorted out and further cultivated.
Accordingly, the brightest 5%, preferably the brightest 3% or even the
brightest 1 % of the fluorescent cells of a cell mixture can also be sorted
out
and replicated. In a particularly preferred embodiment only the brightest
0.5% or the brightest 0.1 % of the fluorescent cells are sorted out and
replicated.
The selection step may be carried out on cell pools or using pre-sorted cell
pools/cell clones. One or more, preferably two or more and especially three
or more sorting steps may be carried out, while between the individual sorting
steps the cells may be cultivated and replicated for a specific length of
time,
e.g. roughly two weeks in the case of pools. Figures 11 and 12 show specific
productivities after FACS-based sorting with and without a gene amplification
step for the mutant Asp227G1y, for example.
The present invention thus relates to a process for obtaining and selecting
recombinant mammalian cells which express at least one heterologous gene
of interest, characterised in that (i) recombinant mammalian cells are
transfected with an expression vector according to the invention; (ii) the
2 o transfected cells are cultivated under conditions which allow expression
of the
gene or genes of interest, the gene coding for a fluorescent protein and the
modified neomycin phosphotransferase gene; (iii) the mammalian cells are
cultivated in the presence of at least one selecting agent which acts
selectively on the growth of mammalian cells and gives preference to the
growth of those cells which express the modified neomycin
phosphotransferase gene; and (iv) the mammalian cells which exhibit a
particularly high expression of the fluorescent gene are sorted out by flow-
cytometric analysis. If desired steps (ii) to (iv) may be repeated once or
several times with the cells obtained in step (iv).
A corresponding process is preferred which is characterised in that the sorted
mammalian cells have an average specific productivity, without an additional
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gene amplification step, of more than 0.5 pg of the desired gene product or
products per day and per cell (0.5 pg/cell/day), preferably greater than 1
pg/cell/day, more preferably greater than 2 pg/cell/day, still more preferably
greater than 3 pg/cell/day, even more preferably greater than 4 pg/cell/day,
for example greater than 5, 6, 7, 8, 9, 10, etc, greater than 15, 20,
25 pg/cell/day, etc.. As mentioned above, the productivity of these cells can
be increased by a simple gene amplification step, e.g. using the DHFR/MTX
system, by a factor of at least 2 to 10. This is shown for example in Figure
12
for selection using the NTP mutant Asp227G1y. The specific productivities
1 o were between 20 and 25 pg/cell/day.
Also preferred according to the invention is a process in which suitably
sorted
cells are replicated and used to prepare the encoded gene product of
interest. For this, the selected high producing cells are preferably
cultivated
in a serum-free culture medium and preferably in suspension culture under
conditions which allow expression of the gene of interest. The
protein/product of interest is preferably obtained from the cell culture
medium
as a secreted gene product. If the protein is expressed without a secretion
signal, however, the gene product may also be isolated from cell lysates. In
2 0 order to obtain a pure homogeneous product which is substantially free
from
other recombinant proteins and host cell proteins, conventional purification
procedures are carried out. First of all, cells and cell debris are removed
from
the culture medium or lysate. The desired gene product can then be freed
from contaminating soluble proteins, polypeptides and nucleic acids, e.g. by
2 s fractionation on immunoaffinity and ion exchange columns, ethanol
precipitation, reversed phase HPLC or chromatography on Sephadex, silica
or cation exchange resins such as DEAE. Methods which result in the
purification of a heterologous protein expressed by recombinant host cells are
known to the skilled man and described in the literature, e.g. by Harris et
al.,
3 0 1995 and Scopes 1988.
Amplifiable Selectable Marker Gene
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In addition, the cells according to the invention may optionally also be
subjected to one or more gene amplification steps in which they are cultivated
in the presence of a selecting agent which leads to amplification of an
amplifiable selectable marker gene. This step may be carried out both with
cells which express a fluorescent protein and have preferably been pre-
sorted once or several times by FACS (preferably in one of the ways
described here) and with cells which have not yet been sorted.
to The prerequisite is that the host cells are additionally transfected with a
gene
which codes for an amplifiable selectable marker. It is conceivable for the
gene which codes for an amplifiable selectable marker to be present on one
of the expression vectors according to the invention or to be introduced into
the host cell by means of another vector.
The amplifiable selectable marker gene usually codes for an enzyme which is
needed for the growth of eukaryotic cells under certain cultivation
conditions.
For example, the amplifiable selectable marker gene may code for
dihydrofolate reductase (DHFR). In this case the gene is amplified if a host
2 o cell transfected therewith is cultivated in the presence of the selecting
agent
methotrexate (MTX).
The following Table 2 gives examples of other amplifiable selectable marker
genes and the associated selecting agents which may be used according to
2 5 the invention, which are described in an overview by Kaufman, Methods in
Enzymology, 185:537-566 (1990).
Table 2: Amplifiable selectable marker genes
Amplifiable selectableAccession number Selecting agent
marker ene
dihydrofolate reductaseM19869 (hamster) methotrexate (MTX)
E00236mouse
_
metallothionein ~D10551 (hamster) cadmium
~
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M13003 (human)
M 11794 rat
CAD (carbamoylphosphateM23652 (hamster) N-phosphoacetyl-L-
synthetase : aspartateD78586 (human) aspartate
transcarbamylase:
dih droorotase
adenosine-deaminase K02567 (human) Xyl-A- or adenosine,
M10319 mouse 2'deox coform cin
AMP (adenylate)- D12775 (human) adenine, azaserin,
deaminase J02811 rat coform cin
UMP-s nthase J03626 human 6-azauridine, razofuran
IMP 5'-dehydrogenase J04209 (hamster) mycophenolic acid
J04208 (human)
M33934 mouse
xanthine-guanin- X00221 (E. coli) mycophenolic acid
with
hos horibos Itransferase limitin xanthine
mutant HGPRTase or J00060 (hamster) hypoxanthine,
mutant thymidine-kinaseM13542, K02581 aminopterine and
(human) thymidine (HAT)
J00423,
M68489(mouse)
M63983 (rat)
M36160 Her es virus
thymidylate-synthetaseD00596 (human) 5-fluorodeoxyuridine
M13019 (mouse)
L12138 rat
P-glycoprotein 170 AF016535 (human) several drugs, e.g.
(MDR1 )
J03398 (mouse) adriamycin, vincristin,
colchicine
ribonucleotide reductaseM124223, K02927 aphidicoline
mouse
glutamine-synthetase AF150961 (hamster) methionine sulphoximine
U09114, M60803 (MSX)
(mouse)
M29579 rat
asparagine-synthetase M27838 (hamster) ~-aspartylhydroxamate,
M27396 (human) albizziin, 5'azacytidine
U38940 (mouse)
U07202 rat
argininosuccinate- X01630 (human) canavanin
synthetase M31690 (mouse)
M26198 bovine
ornithine-decarboxylaseM34158 (human) a-difluoromethylornithine
J03733 (mouse)
M 16982 rat
HMG-CoA-reductase L00183,M12705 compactin
hamster
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M11058 human
N-acetylglucosaminyl- M55621 (human) tunicamycin
transferase
threon I-tRNA-s nthetaseM63180 human borrelidin
Na+K+-ATPase J05096 (human) ouabain
M14511 rat
According to the invention the amplifiable selectable marker gene used is
preferably a gene which codes for a polypeptide with the function of DHFR,
e.g. for DHFR or a fusion protein from the fluorescent protein and DHFR.
DHFR is necessary for the biosynthesis of purines. Cells which lack the
DHFR genes cannot grow in purine-deficient medium. The DHFR gene is
therefore a useful selectable marker for selecting and amplifying genes in
cells cultivated in purine-free medium. The selecting medium used in
conjunction with the DHFR gene is methotrexate (MTX).
to
The present invention therefore includes a method of preparing and selecting
recombinant mammalian cells which contains the following steps: (i)
transfection of the host cells with genes which code at least for a protein/
product of interest, a modified neomycin phosphotransferase and DHFR; (ii)
cultivation of the cells under conditions which allow expression of the
various
genes; and (iii) the amplification of the co-integrated genes by cultivating
the
cells in the presence of a selecting agent which allows the amplification of
at
least the amplifiable selectable marker gene such as methotrexate.
Preferably the transfected cells are cultivated in hypoxanthine/thymidine-free
2 o medium in the absence of serum and with the addition of increasing
concentrations of MTX. Preferably the concentration of MTX in the first
amplification step is at least 5 nM. The concentration of MTX may, however,
also be at least 20 nM or 100 nM and be increased step by step to 1 :M. In
individual cases concentrations of more than 1 :M may be used, e.g. 2 :M.
If the corresponding cells are additionally transformed with a gene for a
fluorescent protein, these cells may be identified and sorted using a
fluorescence activated cell sorting device (FACS) and then cultivated in a
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gene amplification step in the presence of at least 20, preferably in the
presence of 50 or 100 nM MTX. In this way it is possible to increase
productivities substantially to more than 20 pg of gene product per cell and
per day, preferably to more than 21, 22, 23, 24, 25, etc., 30, 35, 40, etc..
The
host cells may be subjected to one or more gene amplification steps in order
to increase the copy number of at least the gene of interest and the
amplifiable selectable marker gene. According to the invention the high
productivity which can be achieved is linked to effective pre-selection by
means of neomycin phosphotransferase- mediated resistance to
1 o aminoglycoside antibiotics such as neomycin, kanamycin and 6418. It is
therefore possible to reduce the number of gene amplification steps required
and to carry out only a single gene amplification, for example.
In a further embodiment the present invention thus also relates to processes
1 s for obtaining and selecting recombinant mammalian cells which express at
least one heterologous gene of interest and are characterised in that (i)
recombinant mammalian cells are transfected with an expression vector
according to the invention and the gene for an amplifiable selectable marker
gene; (ii) the mammalian cells are cultivated under conditions which allow
2 o expression of the gene or genes of interest, the modified neomycin
phosphotransferase gene and the gene which codes for a fluorescent protein;
(iii) the mammalian cells are cultivated in the presence of at least one
selecting agent which acts selectively on the growth of mammalian cells and
gives preference to the growth of those cells which express the neomycin
25 phosphotransferase gene; (iv) the mammalian cells which exhibit high
expression of the fluorescent protein are sorted out by flow-cytometric
analysis; (v) the sorted cells are cultivated under conditions under which the
amplifiable selectable marker gene is expressed; and (vi) a selecting agent is
added to the culture medium which results in the amplification of the
3 o amplifiable selectable marker gene.
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Particularly preferred is a corresponding process in which the modified
neomycin phosphotransferase genes described in this invention are used.
Also preferred is a process in which only one amplification step is carried
out.
Also preferred is a corresponding process which leads to recombinant
mammalian cells which exhibit an average specific productivity of more than
20 pg, preferably more than 21, 22, 23, 24, 25, etc., 30, 35, 40, etc..of the
desired gene product or products per cell and per day.
Mammalian cells, preferably mouse myeloma and hamster cells, are
1 o preferred host cells for the use of DHFR as an amplifiable selectable
marker.
The cell lines CHO-DUKX (ATCC CRL-9096) and CHO-GD44 (Urlaub et al.,
1983) are particularly preferred as they have no DHFR activity of their own,
as a result of mutation. In order to be able to use the DHFR-induced
amplification in other cell types as well which have their own endogenous
DHFR activity, it is possible to use in the transfection process a mutated
DHFR gene which codes for a protein with reduced sensitivity to
methotrexate (Simonson et al., 1983; Wigler et al., 1980; Haber et al., 1982).
The DHFR marker is particularly suitable for the selection and subsequent
2 o amplification when using DHFR negative basic cells such as CHO-DG44 or
CHO-DUKX, as these cells do not express endogenous DHFR and therefore
do not grow in purine-free medium. Consequently, the DHFR gene may be
used here as a dominant selectable marker and the transformed cells are
selected in hypoxanthine/ thymidine-free medium.
The invention is described more fully hereinafter with reference to some non-
restrictive examples.
3 o EXAMPLES
Abbreviations
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Ala (=A) alanine
AP: alkaline phosphatase
Asn (=N) asparagine
s Asp (=D):aspartic acid
bp: base pair
BSA: bovine serum albumin
CHO: Chinese Hamster Ovary
dhfr: dihydrofolate-reductase
to DMSO: dimethylsulphoxide
ELISA: enzyme-linked immunosorbent
assay
FRCS: fluorescence-activated cell
sorter
FITC: fluoresceine-isothiocyanate
GFP: green fluorescent protein
1 Glu (=E):glutamic acid
s
Gly (=G):glycine
HBSS: Hanks Balanced Salt Solution
HT: hypoxanthine/thymidine
Ile (=I):isoleucine
2o IRES: internal ribosomal entry site
kb: kilobase
mAb: monoclonal antibody
MCP-1: monocyte chemoattractant protein-1
MTX: methotrexate
2 MW: mean value
s
NPT: neomycin-phosphotransferase
PCR: polymerase chain reaction
PBS: phosphate buffered saline
Phe (=F):phenylalanine
3 Trp (=W):tryptophan
o
Val (=V):valine
WT: wild-type
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Methods
1. Cell culture and Transfection
s The cells CHO-DG44/dhfr~- (Urlaub et al., 1983) were permanently cultivated
as suspension cells in serum-free CHO-S-SFMII medium supplemented with
hypoxanthine and thymidine (Invitrogen GmbH, Karlsruhe, DE) in cell culture
flasks at 37°C in a damp atmosphere and 5% C02. The cell counts and
viability were determined with a CASY1 Cell Counter (Schaerfe System, DE)
l o or by tryptan blue staining and the cells were then seeded in a
concentration
of 1 - 3 x105/mL and run every 2 - 3 days.
Lipofectamine Plus Reagent (Invitrogen GmbH) was used for the transfection
of CHO-DG44. For each transfection mixture a total of 1 Ng of plasmid-DNA,
4 pL of lipofectamine and 6 NL of Plus reagent were mixed together
15 according to the manufacturer's instructions and added in a volume of 200
NL to 6 x105 exponentially growing CHO-DG44 cells in 0.8 mL of HT-
supplemented CHO-S-SFMII medium. After three hours' incubation at 37°C
in
a cell incubator 2 mL of HT-supplemented CHO-S-SFMII medium was added.
For the NPT-based selection the cells were transferred 2 days after
2o transfection into HT-supplemented CHO-S-SFMII medium with 6418
(Invitrogen), changing the medium every 3 to 4 days. As a rule, 400 pg/mL of
6418 were added for the selection and in some experimental series the
concentration was also lowered to 200 pg/mL or raised to 500, 600 or 800
Ng/mL. In DHFR- and NPT-based selection in the event of co-transfection, in
25 which one expression vector contained a DHFR and the other expression
vector contained a neomycin-phosphotransferase selectable marker, the cells
were transferred 2 days after transfection into CHO-S-SFMII medium without
the addition of hypoxanthine and thymidine and also 6418 (Invitrogen) was
added to the medium in a concentration of 400Ng/mL.
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A DHFR-based gene amplification of the integrated heterologous genes can
be obtained by the addition of the selecting agent MTX (Sigma, Deisenhofen,
DE) in a concentration of 5 - 2000 nM to an HT-free CHO-S-SFMII medium.
s 2. Expression vectors
To analyse the expression, eukaryotic expression vectors were used which
are based on the pAD-CMV vector (Werner et al., 1998) and mediate the
constitutive expression of a heterologous gene by the combination of CMV
enhancer/hamster ubiquitin/S27a promoter (WO 97/15664). While the base
to vector pBID contains the dhfr-minigene which acts as an amplifiable
selectable marker (cf e.g. EP-0-393-438), in the vector pBIN the dhfr-
minigene has been replaced by a neomycin-phosphotransferase resistance
gene (Fig.1 ). For this purpose the selectable marker neomycin-
phosphotransferase, including SV40 early promoter and TK-polyadenylation
1 s signal, was isolated from the commercial plasmid pBK-CMV (Stratagene, La
Jolla, CA, USA) as a 1640 by Bsu361 fragment. After a reaction to fill in the
ends of the fragment with Klenow-DNA-polymerase the fragment was ligated
with the 3750 by Bsu361/Stul fragment of the vector pBID, which was also
treated with Klenow-DNA-polymerase.
In the bicistronic base vector pBIDG (Fig. 1 ) the IRES-GFP gene region was
isolated from the vector pIRES2-EGFP (Clontech, Palo Alto, CA, USA) and
brought under the control of the CMV enhancer/promoter in the vector pBID
so that the multiple cloning site between the promoter region and IRES-
element was retained. The following procedure was used. In a PCR
mutagenesis in which the plasmid pIRES2-EGFP acted as the template, on
the one hand the Hindlll cutting site AAGCTT within the IRES sequence was
converted into the sequence ATGCTT by the use of mutagenic primers and
thus eliminated. On the other hand an Xbal cutting site was inserted by
3 o means of a primer with complementarity to the 5'end of the IRES sequence
or a Spel cutting site was introduced by means of a primer with
complementarity to the 3'end of the GFP sequence. The resulting PCR
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fragment, which contained the complete IRES and GFP sequence, was
digested with Xbal and Spel and cloned into the singular Xbal cutting site at
the 3'end of the multiple cloning site of the vector pBID. In the same way the
IRES-GFP gene region from the vector pIRES2-EGFP was brought under the
s control of the CMV enhancer/hamster ubiquitin/S27a promoter in the vector
pBIN. This produced the bicistronic base vector pBING (Fig.1 ).
Human MCP-1 cDNA (Yoshimura et al., 1989) was cloned into the
corresponding cutting sites of the vector pBIN as a 0.3 kb Hindlll/EcoRl
to fragment, resulting in the vector pBIN-MCP1 (Fig. 2A).
In order to express a monoclonal humanised IgG2 antibody the heavy chain
was cloned as a 1.5 kb BamHl/Hindlll fragment into the vector pBID or
pBIDG digested with BamHl and Hindlll, to obtain the vector pBID-HC or
15 pBIDG-HC (Fig. 2B). The light chain on the other hand was cloned as a 0.7
kb BamHl/Hindlll fragment into the corresponding cutting sites of the vector
pBIN or pBING, producing the vector pBIN-LC or pBING-LC (Fig. 2B) .
3. FA CS
2 o The flow-cytometric analyses and sorting were carried out with a Coulter
Epics Altra device. The FACS is fitted with a helium-argon laser with an
excitation wavelength of 488 nm. The fluorescence intensity is absorbed at a
wavelength suited to the fluorescence protein and process by means of the
attached software Coulter Expo32. The sorting is normally carried out at a
2 s rate of 8000 - 10000 events/second. The suspended cells can be centrifuged
(5 min at 180xg) and adjusted to a cell concentration of 1 - 1.5 x107/mL in
HBSS. Then the cells can be sorted according to their fluorescence protein
signal. The cells are taken up in test tubes already containing culture
medium, then centrifuged and, depending on the number of cells sorted,
3 o seeded into suitable culture vessels or deposited directly in microtitre
plates.
4. ELISA
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The MCP-1 titres in supernatants of stably transfected CHO-DG44 cells were
quantified by ELISA using the OptEIA Human MCP-1 Set kit in accordance
with the manufacturer's instructions (BD Biosciences Pharmingen,
Heidelberg, DE).
The IgG2 mAb in the supernatants from stably transfected CHO-DG44 cells
was quantified by ELISA according to standard procedures (Current
Protocols in Molecular Biology, Ausubel et al., 1994, updated), using on the
one hand a goat anti human IgG Fc fragment (Dianova, Hamburg, DE) and
on the other hand an AP-conjugated goat anti human kappa light chain
1 o antibody (Sigma). Purified IgG2 antibody was used as the standard.
Productivities (pg/cell/day) were calculated by the formula pg/((Ct-Co) t / In
(Ct-Co)), where Co and Ct are the cell count on seeding and harvest,
respectively, and t is the cultivation time.
5. Dot Assay for determining the NPT enzyme activity
In order to prepare a cell extract according to a method of Duch et al., 1990,
6x106 cells were washed twice with PBS and then resuspended in 600 pL of
extraction buffer (0.135 M Tris-HCI pH 6.8, 20% glycerol, 4 mM
dithiothreitol).
After four cycles of freezing and thawing in a bath of dry ice or water the
cell
2 o debris was removed by centrifuging and the supernatant was used for the
subsequent enzyme assay. The protein concentration in the cell extracts was
determined by a Bradford assay using the BIO-RAD protein assay (Bio-Rad
Laboratories GmbH, Munich, DE), with BSA as the standard protein (Current
Protocols in Molecular Biology, Ausubel et al., 1994, updated). In order to
determine the NPT enzyme activity a Dot Assay was carried out, based on
the protocol of Platt et al. 1987. For this, 5 Ng, 2.5 Ng and 1.25 Ng of
protein
were adjusted with extraction buffer to a final volume of 20 pL, topping up to
a total protein content of 5 Ng with cell extract from non-transfected CHO-
DG44 cells. After the addition of 200 NL of assay buffer (67 mM Tris-HCI pH
3 0 7.1, 42 mM MgCl2, 400 mM NH4CI) plus/minus 40 Ng/mL 6418 and
plus/minus 5 NCi [y-33P]-ATP/mL (NEN) the extracts were incubated at
27°C
for 135 minutes. Then the extracts were filtered in a 96 well vacuum manifold
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(Schleicher & Schiall, Dassel, DE) through a sandwich of one layer of
Whatman 3MM paper, P81 phosphocellulose membrane (Whatman
Laboratory Division, Maidstone, Great Britain) and nitrocellulose membrane
(Schleicher & Schull). Proteins phosphorylated by protein kinases and non-
phosphorylated proteins bind to the nitrocellulose, while phosphorylated
6418 passes through the nitrocellulose and binds to the phosphocellulose.
After washing three times with deionised H20 the membranes were removed
from the apparatus, washed again with H20 and then air-dried. The
radioactive signals were quantified using a Phospho Imager (Molecular
1 o Dynamics, Krefeld, DE).
6. Northern Blot Analysis
Total RNA was isolated from the cells with the TRIZOL reagent according to
the manufacturer's instructions (Invitrogen GmbH, Karlsruhe, DE) and the
1 s separation of 30 Ng RNA by gel electrophoresis and the transfer to a
Hybond
N+ nylon membrane (Amersham Biosciences, Freiburg, DE) were carried out
according to the standard procedure for glyoxal/DMSO-denatured RNA
(Current Protocols in Molecular Biology, Ausubel et al., 1994, updated). The
probe used for the subsequent non-radioactive hybridisation with the
2 o Genelmages CDP-Star Detection Kit (Amersham Biosciences) was a PCR
product which comprised the coding region of the NPT gene, FITC-dUTP-
labelled according to the manufacturer's instructions with the Genelmages
random prime labelling kit (Amersham Biosciences, Freiburg, DE).
2 s 7. Dot Blof Analysis
Genomic DNA was isolated from the cells using a DNA isolation kit according
to the manufacturer's instructions (DNA Isolation Kit for Cells and Tissue;
Roche Diagnostics GmbH, Mannheim, DE). Various amounts of DNA (10 Ng,
Ng, 2.5 pg, 1.25 Ng, 0.63 Ng and 0.32 Ng) were filtered by the standard
3 o method (Ausubel et al., 1994) in an alkaline buffer using a 96 well vacuum
manifold (Schleicher & Schull, Dassel, DE) onto a Hybond N+ nylon
membrane (Amersham Biosciences, Freiburg, DE). Untransfected CHO-
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DG44 cells were used as the negative control. The plasmid pBIN-LC was
used as the standard (320 pg, 160 pg, 80 pg, 40 pg, 20 pg, 10 pg, 5 pg, 2.5
pg). The probe used for the subsequent non-radioactive hybridisation with the
Genelmages CDP-Star Detection Kit (Amersham Biosciences) was a PCR
s product which comprised the coding region of the NPT gene, FITC-dUTP-
labelled according to the manufacturer's instructions with the Genelmages
random prime labelling kit (Amersham Biosciences, Freiburg, DE). The
chemiluminescence signals were quantified using an ImageMaster VDS-CL
(Amersham Biosciences). Then the copy number of the npt genes in the cells
1 o in question was determined using the standard series which had been
obtained from the signal intensities of the titrated plasmid DNA. The number
of plasmid molecules was calculated using Avogadro's constant and the DNA
content of a CHO cell was taken to be about 5 pg.
Example 1: Mutagenesis of the neomycin-phosphotransferase
The base substitutions in the wild-type NPT-gene needed to prepare the NPT
mutants GIu182G1y (SEQ ID N0:3), Trp91Ala (SEQ ID N0:5), Va1198G1y
(SEQ ID N0:7), Asp227A1a (SEQ ID N0:9), Asp227Va1 (SEQ ID N0:11),
Asp261G1y (SEQ ID N0:13), Asp261Asn (SEQ ID N0:15) Phe24011e (SEQ ID
N0:17), GIu182Asp (SEQ ID N0:19), Asp227G1y (SEQ ID N0:21 ),
Asp190G1y (SEQ ID N0:23) and Asp208G1y (SEQ ID N0:25) were carried
out by PCR using mutagenic primers (Fig. 3). The vector pBIN (Fig. 1 ) or
2 s pBK-CMV (Stratagene, La Jolla, USA) was used as a template for the PCR
mutagenesis. First, the 5' or 3'sections of the mutants were prepared in
separate PCR operations. To prepare the mutants GIu182G1y, GIu182Asp,
Trp91 Ala, Asp190G1y, Va1198G1y, Asp208G1y and Asp227G1y, primer
combinations were used for the amplification which consisted of Neofor5
(SEQ ID N0:27) and the relevant mutagenic reverse (rev) primer or Neorev5
(SEQ ID N0:28) and the relevant mutagenic forward (for) primer:
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- In the case of NPT mutant GIu182G1y (SEQ ID N0:3) of Neofor5 (SEQ ID
N0:27) and E182Grev (SEQ ID N0:32) or of Neorev5 (SEQ ID N0:28)
and E182Gfor (SEQ ID N0:31 );
- in the case of the NPT mutant GIu182Asp (SEQ ID N0:19) of Neofor5
s (SEQ ID N0:27) and E182Drev (SEQ ID N0:48) or of Neorev5 (SEQ ID
N0:28) and E182Dfor (SEQ ID N0:47);
- in the case of the NPT mutant Trp91Ala (SEQ ID N0:5) of Neofor5 (SEQ
ID N0:27) and W91Arev (SEQ ID N0:34) or of Neorev5 (SEQ ID N0:28)
and W91Afor (SEQ ID N0:33);
to - in the case of the NPT mutant Va1198G1y (SEQ ID N0:7) of Neofor5 (SEQ
ID N0:27) and V198Grev (SEQ ID N0:36) or of Neorev5 (SEQ ID N0:28)
and V198Gfor (SEQ ID N0:35)
- in the case of the NPT mutant Asp190G1y (SEQ ID N0:23) of Neofor5
(SEQ ID N0:27) and D190Grev (SEQ ID N0:50) or of Neorev5 (SEQ ID
15 N0:28) and D190Gfor (SEQ ID N0:49);
- in the case of the NPT mutant Asp208G1y (SEQ ID N0:25) of Neofor5
(SEQ ID N0:27) and D208Grev (SEQ ID N0:52) or of Neorev5 (SEQ ID
N0:28) and D208Gfor (SEQ ID N0:51 );
- in the case of the NPT mutant Asp227G1y (SEQ ID N0:21 ) of Neofor5
20 (SEQ ID N0:27) and D227Grev (SEQ ID N0:54) or of Neorev5 (SEQ ID
N0:28) and D227Gfor (SEQ ID N0:52).
In order to prepare the mutants Asp227A1a, Asp227Va1, Asp261 Gly,
Asp261Asn and Phe24011e primer combinations were used for the
2s amplification which consisted of Neofor2 (SEQ ID N0:29) and the relevant
mutagenic reverse (rev) primer or of IC49 (SEQ ID N0:30) and the relevant
mutagenic forward (for) primer:
- In the case of NPT mutant Asp227A1a (SEQ ID N0:9) of Neofor2 (SEQ ID
N0:29) and D227Arev (SEQ ID N0:38) or of IC49 (SEQ ID N0:30) and
3o D227Afor (SEQ ID N0:37);
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- in the case of NPT mutant Asp227Va1 (SEQ ID N0:11 ) of Neofor2 (SEQ
ID N0:29) and D227Vrev (SEQ ID N0:40) or of IC49 (SEQ ID N0:30) and
D227Vfor (SEQ ID N0:39);
- in the case of NPT mutant Asp261 Gly (SEQ ID N0:13) of Neofor2 (SEQ
ID N0:29) and D261Grev (SEQ ID N0:42) or of IC49 (SEQ ID N0:30)
and D261 Gfor (SEQ ID N0:41 );
- in the case of NPT mutant Asp261Asn (SEQ ID N0:15) of Neofor2 (SEQ
ID N0:29) and D261 Nrev (SEQ ID N0:44) or of IC49 (SEQ ID N0:30) and
D261 Nfor (SEQ ID N0:43);
to - in the case of NPT mutant Phe24011e (SEQ ID N0:17) of Neofor2 (SEQ ID
N0:29) and F2401rev (SEQ ID N0:46) or of IC49 (SEQ ID N0:30) and
F2401for (SEQ ID N0:45).
Then the coding strand of the 5'section and the complementary strand of the
3'section of the mutants in question were combined by hybridisation in the
overlapping region formed by the mutagenic primer sequences, the single
strand regions were filled in and the entire product was amplified again in a
PCR with the primers Neofor5 (SEQ ID N0:27) and Neorev5 (SEQ ID N0:28)
or Neofor2 (SEQ ID N0:29) and IC49 (SEQ ID N0:30). These PCR products
2o were digested with Stul/Rsrll (Neofor5/Neorev5 PCR-products of the mutants
GIu182G1y, Trp91Ala and Va1198G1y), Stul/BstBl (Neofor5/Neorev5 PCR
products of the mutants GIu182Asp, Asp190G1y, Asp208G1y and Asp227G1y)
or Dralll/Rsrll (Neofor2/IC49 PCR products of the mutants Asp227A1a,
Asp227Va1, Asp261 Gly, Asp261 Asn and Phe24011e). Then in the vector
pBIN-LC (Fig. 2B) or pBK-CMV (Stratagene, La Jolla, USA) part of the wild-
type NPT sequence was eliminated by Stul/Rsrll digestion, Dralll/Rsrll
digestion or Stul/BstBl digestion and replaced by the corresponding
fragments of the PCR products. By sequence analysis of both the
complementary and the coding strand the desired base substitutions in the
3 o various mutants were verified to ensure that the remaining DNA sequence
corresponded to the wild-type NPT sequence. In this way the expression
vectors pBIN1-LC, pBIN2-LC, pBIN3-LC, pBIN4-LC, pBINS-LC, pBIN6-LC,
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pBIN7-LC and pBINB-LC were generated, which contain the NPT mutants
GIu182G1y, Trp91 Ala, Va1198G1y, Asp227A1a, Asp227Va1, Asp261 Gly,
Asp261 Asn or Phe24011e (Fig. 2B).
The remaining NPT mutants were isolated as a 1640 by Bsu361 fragment
from the modified pBK-CMV, the fragment ends were filled in with Klenow-
DNA polymerase and ligated with the 3750 by Bsu361/Stul fragment of the
vector pBID, which was also treated with Klenow-DNA polymerase. In this
way the expression vectors pKS-N5, pKS-N6, pKS-N7 and pKS-N8 were
to generated which contained the NPT mutants GIu182Asp, Asp190G1y,
Asp208G1y and Asp227G1y, respectively. The human MCP-1 cDNA was then
cloned into these expression vectors as a 0.3 kb Hindlll/EcoRl fragment (Fig.
2A) or the light chain of the humanised IgG2 antibody was cloned into these
expression vectors as a 0.7 kb Hindlll/BamHl fragment (Fig. 2B).
The mutations inserted in the neomycin phosphotransferase are on the one
hand substitutions of more (Va1198G1y, Phe24011e) or less (Trp9lAla,
GIu182G1y, GIu182Asp, Asp227A1a, Asp227Va1, Asp227G1y) conserved
amino acids which flank conserved domains, such as e.g. the motifs 1, 2 and
2 0 3 (Shaw et al., 1993) (Fig. 4). On the other hand the mutations are
located
within the conserved motif 1 (Asp190G1y), 2 (Asp208G1y) or 3 (Asp261 Gly,
Asp261Asn) and relate to a conserved amino acid.
Example 2: Influence of the NPT mutations on the selection of stably
2 5 transfected MCP-1 expressing cells
MCP was used as an example of the expression of a single chained protein
in CHO cells. For this, CHO-DG44 was transfected with pKS-N5-MCP1,
pKS-N6-MCP1, pKS-N7-MCP1, pKS-N8-MCP1 or pBIN-MCP1 (Fig. 2A).
3 o Two double preparations were carried out. Two days after transfection the
cells were seeded in a 96 well-plate (2000 cells/well) and selected with 400
pg/mL of 6418 in HT-supplemented CHO-S-SFMII medium. In the case of
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the cells transfected with pBIN-MCP1 selection was also carried out in
parallel with 800 pg/mL of 6418. The cell populations obtained were
successively transferred via 24 well plates into 6 well plates. Even during
the
selection phase differences could be detected between the various
transfection mixtures. In contrast to the cell populations in which selection
had been carried out with an NPT wild-type gene (SEQ ID N0:1 ), in those
cell populations which had been transfected with a mutated NPT, fewer cells
survived the initial selection with 6418. These cell populations therefore
could not be transferred into the 24 well plates until about four days later.
to And in the mixtures which has been transfected with pKS-N6-MCP1 and
pKS-N7-MCP1 no stably transfected cells whatever could be selected at a
concentration of 400 Ng/mL of 6418. Presumably, the enzyme function is so
severely impaired in the NPT mutants with the mutations Asp190G1y and
Asp208G1y that not enough 6418 molecules can be inactivated to allow
growth of the stably transfected cells. Admittedly, when the 6418
concentration was reduced to 200 Ng/mL, a few cells survived the first
selection phase, but they were all severely impaired in their growth and
vitality and expansion was not possible, apart from a few exceptions in the
case of the mutant Asp208G1y.
From the cells transfected with the mutants GIu182Asp and Asp227G1y or
with the NPT wild-type, 18 pools were cultivated (9 pools each of mixtures 1
and 2) over four passages in 6 well plates and the concentration of the MCP-
1 produced was measured in the cell culture supernatant by ELISA. Cell
2 5 pools in which the NPT mutants had been used as selectable markers
showed on average 50% - 57% (GIu182Asp mutant) or 57% - 65%
(Asp227G1y mutant) higher productivities than cell pools in which the
selection had been carried out with the NPT wild-type at 400 or even 800
Ng/mL of 6418 (Fig. 5). Thus, by using NPT mutants as selectable markers
3 o the proportion of high producers in the transfected cell populations could
actually be increased.
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Example 3: Influence of the NPT mutations on the selection of stably
transfected mAb expressing cells
In a co-transfection CHO-DG44 cells were transfected first with the plasmid
combination pBIDG-HC/pBIN-LC (NPT wild-type), pBIDG-HC/pKS-N5-LC
(GIu182Asp NPT mutant) or pBIDG-HC/pKS-N8-LC (Asp227G1y NPT mutant)
(Fig. 2B). In the vector confirgurations used the two protein chains of a
humanised IgG2 antibody are each expressed by their own vector which
additionally codes for a DHFR or neomycin selectable marker in a separate
to transcription unit. The expression of the product genes is mediated by a
CMV-Enhancer/Hamster Ubiquitin/S27a Promoter combination. However,
comparable data may also be obtained, for example, using a CMV
Enhancer/Promoter, an SV40 Enhancer/Hamster Ubiquitin/S27a Promoter or
other promoter combinations.
In all, four series of transfections were carried out in each case using 6
pools
per plasmid combination. By contrast to the cell populations in which
selection was carried out with an NPT wild-type gene, in those cell
populations which had been transfected with a mutated NPT, fewer cells
2 o survived the initial selection with 6418. After two to three weeks'
selection of
the transfected cell pools in HT-free CHO-S-SFMII medium with the
additional 400 Ng/mL of 6418, the antibody titre in the cell culture
supernatants was determined by ELISA over several runs (6 - 8). By
comparison with the use of an NPT wild-type gene as selectable marker, the
2s cells which has been selected with the GIu182Asp mutant showed on
average an increase in productivity and titre of 86% and 77%, respectively,
and the cells selected with the Asp227G1y mutant even showed an increase
in productivity and titre of 126% and 107%, respectively. Thus, by using an
NPT mutant with reduced enzyme activity it was possible to selectively enrich
3 o cells having a basic productivity which was up to twice as high.
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In another transfection series the influence of different concentrations of
6418 on selection was tested. 400, 500 and 600 pg/mL of 6418 were used
for the selection of the transfected cell pools, 3 pools in each case. At the
higher concentrations, significantly fewer cells survived the selection in the
s cell populations in which selection has been carried out with an NPT wild-
type
gene, the effect being greatest with the Asp227G1y mutant. The stably
transfected cell populations obtained, however, showed no deterioration in
growth and vitality. However, no significant difference could be detected
between the productivities and titres achieved within a plasmid combination
to used for transfection. But, even here, the cells selected by the NPT
mutants
again had the highest productivities on average, led by the Asp227G1y
mutant, the productivity of which was four times higher than that of the NPT
wild-type, followed by the GIu182Asp mutant with a productivity 2.4 times
higher (Fig. 6A).
Then in a co-transfection CHO-DG44 cells were transfected with the plasmid
combination pBIDG-HC/pBIN-LC (NPT wild-type), pBIDG-HC/pBIN1-LC
(GIu182G1y NPT mutant), pBIDG-HC/pBIN2-LC (Trp91Ala NPT mutant),
pBIDG-HC/pBIN3-LC (Va1198G1y NPT mutant), pBIDG-HC/pBIN4-LC
(Asp227A1a), pBIDG-HC/pBINS-LC (Asp227Va1 NPT mutant), pBIDG-
HC/pBIN6-LC (Asp261G1y NPT mutant), pBIDG-HC/pBIN7-LC (Asp261Asn
NPT mutant) or pBIDG-HC/pBINB-LC (Phe24011e NPT mutant) (Fig. 2B). In
the vector configurations used, again the two protein chains of a monoclonal
humanised IgG2 antibody were each expressed by their own vector, which
2s additionally also codes for a DHFR or neomycin selectable marker in a
separate transcription unit.
For each plasmid combination, 5 pools were transfected. In contrast to the
cell populations in which selection was done with an NPT wild-type gene,
3 o fewer cells survived the initial selection with 6418 in the cell
populations
which had been transfected with a mutated NPT. After a two- to three-week
selection of the transfected cell pools in HT-free CHO-S-SFMII medium with
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the addition of 400 Ng/mL of 6418 the antibody titre in the cell culture
supernatants was determined by ELISA over six runs. Fig. 6B shows the
averages of the titres and productivities determined from the pools in the
test.
Compared with the use of an NPT wild-type gene as the selectable marker,
s all the cell pools which had been selected with an NPT mutant showed on
average an increase in productivity and titre by a factor of 1.4 - 14.6 and
1.4
- 10.8, respectively (Fig. 6B). The best selective enrichment of cells with a
higher basic productivity could thus be obtained with the NPT mutants
Asp227Va1 and Asp261 Gly, with increases in average productivity by a factor
l o of 14.6 and 9.3, respectively.
The vector pBIDG-HC contains another selectable marker, GFP. The GFP is
transcriptionally linked to the heavy chain via an IRES element. The resulting
correlation between the expression of the target protein and the selectable
15 marker GFP therefore also makes it possible to rapidly evaluate the level
and
distribution of the expression levels in the transfected cell populations on
the
basis of the GFP fluorescence determined in FACS analyses. After two to
three weeks' selection of the transfected cell pools in HT-free CHO-S-SFMII
medium with the addition of 6418 the GFP fluorescence was measured in a
2 o FACS analysis (Fig. 7). The GFP fluorescence signals in fact correlated
with
the titre data obtained for the monoclonal IgG2 antibody. Pools selected with
the NPT mutants Asp227Va1, Asp261G1y, Asp161Asn and Phe24011e also
had the higher proportion of cells with a high GFP fluorescence, followed by
the cells selected with the NPT mutants Trp91Ala, Asp227A1a, Asp227G1y,
2s GIy182Asp, GIu182G1y and Va1198G1y.
By adding the selection agent methotrexate (MTX) to the culture medium it
was possible to increase the productivity of the cells still further by
inducing a
dhfr-mediated gene amplification. Thus, for example, after a simple gene
3 o amplification step with 100 nM MTX, the specific productivity in the cells
pools
obtained by a co-transfection with the plasmid combinations pBIDG-
HC/pBINS-LC (NPT mutant Asp227Va1), pBIDG-HC/pBIN6-LC (NPT mutant
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Asp261G1y), pBIDG-HC/pBIN7-LC (NPT mutant Asp261Asn) and pBIDG-
HC/pBINB-LC (NPT mutant Phe24011e) could be increased by a factor of 2 to
4 and, depending on the pool, productivities of between 4 and 14 pg/cell/day
could be achieved. Figure 8 shows, by way of example, on a cell pool
obtained by the co-transfection of pBIDG-HC/pBINS-LC (NPT mutant
Asp227Va1), the increases in productivity to 27 pg/cell/day achieved by the
addition of MTX (100 nM MTX followed by 500 nM MTX).
Example 4: Determining and comparing the NPT enzyme activity
In order to compare the enzyme activity of the NPT mutants with that of the
NPT wild-type a dot assay was carried out to determine the NPT activity in
cell extracts, based on the procedure of Platt et al. 1987 and shown by way of
example in Fig. 9A for the NPT mutants GIu182Asp and Asp227G1y. Cell
extracts were prepared from two different mAb-expressing cell pools which
had been transfected and selected either with the NPT wild-type gene (SEQ
ID N0:1 ) or with the NPT mutants GIu182G1y (SEQ ID N0:3), G1u182Asp
(SEQ ID N0:19), Trp91Ala (SEQ ID N0:5), Asp190G1y (SEQ ID N0:23),
Va1198G1y (SEQ ID N0:7), Asp208G1y (SEQ ID N0:25), Asp227A1a (SEQ ID
2 o N0:9), Asp227Va1 (SEQ ID N0:11 ), Asp227G1y (SEQ ID N0:21 ), Asp261 Gly
(SEQ ID N0:13), Asp261Asn (SEQ ID N0:15) or Phe24011e (SEQ ID N0:17).
Cell extracts from untransfected CHO-DG44 cells were used as the negative
control.
2 5 The enzyme activities of the NPT mutants were significantly reduced,
compared with the NPT wild-type. On average the NPT mutants had only
between 1.5% and 62% of the wild-type enzyme activity, while the NPT
mutants Asp261 Gly and Asp261 Asn with 3.1 % and 1.5% had the lowest
residual activity and the NPT mutants Va1198G1y and Trp91Ala with 61.9%
3 o and 53.2% has the highest residual activity (Fig. 9B). The signals
obtained on
the phosphocellulose were specific to the phosphorylation of 6418 caused by
the NPT enzyme activity. Without the addition of the NPT substrate 6418 to
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the assay buffer no activity could be detected on the phosphocellulose. The
signals obtained on the nitrocellulose membrane, which resulted from the
proteins phosphorylated by protein kinases within the cell extract, were used
as an internal control for identical amounts of sample applied.
The reduced enzyme activity of the NPT mutants compared with the NPT
wild-type could not be put down to reduced gene expression. On the contrary,
Northern Blot analyses on total RNA showed that cell pools which had been
transfected with the NPT wild-type and exhibited a high NPT enzyme activity
1 o expressed less RNA than cell pools transfected with NPT mutants (Fig. 10).
The only exception were cells which had been transfected with the NPT
mutant GIu182G1y and expressed comparable amounts of NPT-mRNA. In
dot blot analyses carried out on genomic DNA from these transfected cell
populations it was shown that the higher expression of the NPT mutants was
obtained by gene dose effects and/or by integration of the exogenous DNA
into more transcription-active genomic regions (Fig. 10). For example, in the
cells selected with the NPT mutant Trp91Ala, the gene dose effect is
predominant in pool1 while in pool 2 the integration effect dominates. In this
way, transfected cells in which markers with a reduced enzyme activity have
2 o been used for the selection are able to synthesise enough marker protein
to
compensate for the reduced resistance to the selective agent under identical
selection pressure.
Example 5: Isolation of cells with high expression of an mAb by GFP-based
2 s FACS sorting
In a co-transfection CHO-DG44 cells were transfected with the plasmid
combination pBID-HC and pBING-LC, coding for a monoclonal humanised
IgG2 antibody (Fig.2B). In the vector confirgurations used the two protein
3 o chains of the antibody are each expressed by their own vector which
additionally also codes for a DHFR or modified neomycin phosphotransferase
selectable marker (Asp227G1y mutant; SEQ ID N0:21 ) in a separate
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transcription unit. In addition, the vector pBING-LC contains another
selectable marker, GFP. As a result of the transcriptional linking of the
expression of GFP and the light chain by means of an IRES element, on co-
transfection of CHO-DG44 with the vectors pBID-HC/pBING-LC it was
s possible within a short time to isolate cells with high expression of the
monoclonal antibody, purely by selecting the cells with a high GFP content by
sequential FACS sorting. In all, 8 separate cell pools were transfected from
which after a first two to three weeks selection in HT-free CHO-S-SFMII
medium with the addition of 400 Ng/mL of 6418, stably transfected cell
1 o populations were obtained. The titres and productivities of all 8 pools
were
determined by ELISA overall several runs (7 - 8). On average the titres were
about 1.4 mg/L and the productivities were about 1.3 pg/cell per day. For the
subsequent sequential FACS-based sorting the pools 5 and 8 were selected,
pool 5 having the highest productivity and pool 8 having a productivity which
15 corresponded to the average of all the pools. In each step, the 5% of cells
with the highest GFP fluorescence was sorted out by FACS and further
cultivated in the pool. This sorting was carried out up to six times in all,
with a
cultivation period of about two weeks between each sort. Astonishingly, there
was found to be a good correlation between mAb productivity and GFP
2 o fluorescence (Fig. 13), although the two protein chains were each
expressed
by their own vector and during GFP-based FACS sorting, it was only possible
to selecte for the expression of the light chain, as a result of its
transcriptional
coupling to GFP. The productivities were able to be increased to 9.5
pg/cell/day by FACS-based sorting alone (Fig. 11 ). Comparable data were
2s also obtained when the Hamster Promoter was functionally linked to the
SV40-Enhancer instead of the CMV-Enhancer. By a single subsequent MTX
amplification setp, starting from pools 5 and 8 of the first sorting step, by
adding 100 nM MTX to the selection medium, it was possible to increase the
productivity of the pools to an average of more than 20 pg/cell/day (Fig. 12).
3 o The high expression levels of the fluorescent protein had no negative
effects
whatever on cell growth and vitality. During gene amplification the growth
properties of the cells are also seriously negatively influenced by the
addition
CA 02507664 2005-05-27
_ 7g _
of MTX, particularly when it is added in higher cencentrations. However, the
pre-sorted cell pools showed considerably more robust characteristics in
response to the quiet high initial dose of 100 nM MTX. They withstood the
selection phase much better, i.e. after only 2 weeks cell populations were
obtained with high vitality and a good growth rate.
In addition, the development time for selecting high producing cells was
reduced to about 6 weeks, compared with a conventional stepwise gene
amplification strategy which generally comprises 4 amplification stages with
1 o increasing additions of MTX. This was achieved by the combined use of an
enrichment of transfected cells with increased expression of the genes of
interest, achieved by using a modified NPT-selectable marker with reduced
enzyme activity, followed by a GFP-based FACS sorting with a subsequent
gene amplification step.
CA 02507664 2005-05-27
_ 79 _
LITERATURE
Adam, M.A. et al., J Virol 1991, 65, 4985 - 4990
Altschul, S.F. et al., Nucleic Acids Res. 1997, 25, 3389 - 3402
s Altschul, S.F. et al., J Mol Biol 1990, 215, 403 - 410
Ausubel, F.M, et al., Current Protocols in molecular biology. New York
Greene Publishing Associates and Wiley-Interscience. 1994 (updated)
Blazques, J. et al., Molecular Microbiology 1991, 5(6), 1511 - 1518
Bennett, R.P. et al., BiaTechniques 1998, 24, 478 - 482
~ o Burk, D.L. et al., Biochemistry 2001, 40 (30), 8756 - 8764
Chalfie, M. et al., Science 1994, 263, 802 - 805
Chamov, S.M. et al., Antibody Fusion Proteins, Wiley-Liss Inc., 1999
Davies, M.V. et al., J Virol 1992, 66, 1924 - 1932
Faisst, S. et al., Nucleic Acids Research 1992, 20, 3 - 26
15 Gish, W. & States, D.J., Nafure Genet. 1993, 3, 266 - 272
Gossen, M. et al., Curr Opi Biotech 1994, 5, 516 - 520
Duch, M. et al., Gene 1990, 95, 285 - 288
Hanson, K.D. et al., Mol Cell Biol 1995, 15(1 ), 45 - 51
Haber, D.A. et al., Somatic Cell Genetics 1982, 8, 499 - 508
2 o Harris et al., Protein Purification : A Practical Approach, Pickwood and
Hames, eds., IRL Press, 1995
Hemann, C. et al., DNA Cell Biol 1994, 13 (4), 437 - 445
Hon, W. et al., Cell 1997, 89, 887 - 895
Hu, S. et al., Cancer Res. 1996, 56 (13), 3055 - 3061
2s Huston, C. et al., Proc Natl Acad Sci USA 1988, 85 (16), 5879 - 5883
Jang, S.K. et al., J Virol 1989, 63, 1651 - 1660
Kaufman, R.J., Methods in Enzymology 1990, 185, 537 - 566
Kocabiyik, S. et al., Biochem Biophys Res Commun 1992, 185(3), 925 - 931
Kortt, A.A. et al., Protein Engineering 1997, 10 (4), 423 - 433
3 o Lottspeich F. and Zorbas H. eds., Bioanalytic, Spektrum Akad. Verl., 1998
Lovejoy, B. et al., Science 1993, 259, 1288 - 1293
Madden, T.L. et al., Meth. Enzymol. 1996, 266, 131 - 141;
CA 02507664 2005-05-27
- 80 -
Monaco, L. et al., Gene 1996, 180, 145 - 15
Morgan, R.A. et al., Nucleic Acids Research 1992, 20, 1293 - 1299
Mosser, D.D. et al., BioTechniques 1997, 22, 150 - 161
Ohshima, Y. et al., J Mol Biol 1987, 195, 247 - 259
s Pack, P. et al., Biotechnology 1993, 11, 1271 - 1277
Pack, P. et al., J Mol Biol 1995, 246 (11 ), 28 - 34
Pelletier, J. et al., Nature 1988, 334, 320 - 325
Perisic, O. et al., Structure 1994, 2, 1217 - 1226
Platt, S.G. et al., Analyt Biochem 1987, 162, 529 - 535
1 o Ramesh, N. et al., Nucleic Acids Research 1996, 24, 2697 - 2700
Sambrook, J., Fritsch, E.F. & Maniatis, T.,Molecular Cloning: A Laboratory
Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989
Scopes, R., Protein Purification, Springer Verlag, 1988
Shaw, K.J. et al., Microbiological Reviews 1993, 57(1 ), 138 - 163
15 Simonson, C.C. et al., Proc Natl Acad Sci USA 1983, 80, 2495 - 2499
Sugimoto et al., Biotechnology 1994, 12, 694 - 698
Yenofsky, R.L. et al., Proc Natl Acad Sci USA 1990, 87, 3435 - 3439
Yoshimura, T. et al., FEBS LETTERS 1989, 244(2), 487 - 493
Urlaub, G. et al., Cell 1983, 33, 405 - 412
2 o Werner, R.G. et al., Arzneim.-Forsch.lDrug.Res. 1998, 48, 870 - 880
Wigler, M. et al., Proc Natl Acad Sci USA 1980, 77, 3567 - 3570
Zhang, J. & Madden, T.L. Genome Res. 1997, 7, 649 - 656;
CA 02507664 2005-05-27
1
SEQUENCE LISTING
<110> BOEHRINGER INGELHEIM PHARMA GMBH & CO KG
<120> NEW NEOMYCIN-PHOSPHOTRANSFERASE GENES AND METHODS FOR THE SELECTION OF
RECOMBINANT CELLS PRODUCING HIGH LEVELS OF A DESIRED GENE PRODUCT
<130> Case 1-1411
<140>
<141>
<160> 55
<170> PatentIn Ver. 3.1
<210> 1
<211> 795
<212> DNA
<213> Escherichia coli
<400> 1
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct toga 795
<210> 2
<211> 264
<212> PRT
<213> Escherichia coli
<400> 2
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
CA 02507664 2005-05-27
2
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Asp Glu Phe Phe
260
<210> 3
<211> 795
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant E182G
<400> 3
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcggggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
CA 02507664 2005-05-27
3
gacgagttct tctga 795
<210> 4
<211> 264
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant E182G
<400> 4
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Gly Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
CA 02507664 2005-05-27
4
Tyr Arg Leu Leu Asp Glu Phe Phe
260
<210> 5
<211> 795
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant W91A
<400> 5
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac gcgctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 795
<210> 6
<211> 264
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant W91A
<400> 6
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Ala Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
CA 02507664 2005-05-27
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Asp Glu Phe Phe
260
<210> 7
<211> 795
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant V198G
<400> 7
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat gggggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 795
<210> 8
<211> 264
CA 02507664 2005-05-27
6
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant V198G
<400> 8
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
Leu Pro Asn Ile Met Gly Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Asp Glu Phe Phe
260
CA 02507664 2005-05-27
7
<210> 9
<211> 795
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D227A
<400> 9
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtgc tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 795
<210> 10
<211> 264
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D227A
<400> 10
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
CA 02507664 2005-05-27
8
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Ala Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Asp Glu Phe Phe
260
<210> 11
<211> 795
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D227V
<400> 11
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtgt tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 795
<210> 12
<211> 264
<212> PRT
<213> Artificial sequence
CA 02507664 2005-05-27
9
<220>
<223> Description of the artificial sequence:
Neomycin mutant D227V
<400> 12
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Val Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Asp Glu Phe Phe
260
<210> 13
<211> 795
CA 02507664 2005-05-27
l~
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D261G
<400> 13
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
ggcgagttct tctga 795
<210> 14
<211> 264
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D261G
<400> 14
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
CA 02507664 2005-05-27
11
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Gly Glu Phe Phe
260
<210> 15
<211> 795
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D261N
<400> 15
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
aacgagttct tctga 795
<210> 16
<211> 264
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D261N
CA 02507664 2005-05-27
12
<400> 16
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Asn Glu Phe Phe
260
<210> 17
<211> 795
<212> DNA
<213> Artificial sequence
CA 02507664 2005-05-27
13
<220>
<223> Description of the artificial sequence:
Neomycin mutant F240I
<400> 17
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcatc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 795
<210> 18
<211> 264
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant F240I
<400> 18
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
CA 02507664 2005-05-27
14
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Ile
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Asp Glu Phe Phe
260
<210> 19
<211> 795
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant E182D
<400> 19
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgatgatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 7g5
<210> 20
<211> 264
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant E182D
<400> 20
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
CA 02507664 2005-05-27
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Asp Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Asp Glu Phe Phe
260
<210> 21
<211> 795
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D227G
<400> 21
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
CA 02507664 2005-05-27
16
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtgg tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 795
<210> 22
<211> 264
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D227G
<400> 22
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
CA 02507664 2005-05-27
17
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Gly Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Asp Glu Phe Phe
260
<210> 23
<211> 795
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D190G
<400> 23
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcggt gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 795
<210> 24
<211> 264
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D190G
<400> 24
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
CA 02507664 2005-05-27
18
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Gly Ala Cys
180 185 190
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Asp Glu Phe Phe
260
<210> 25
<211> 795
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D208G
<400> 25
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
CA 02507664 2005-05-27
19
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cggctgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 795
<210> 26
<211> 264
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Neomycin mutant D208G
<400> 26
Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val
1 5 10 15
Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser
20 25 30
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe
35 40 45
Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala
50 55 60
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val
65 70 75 80
Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu
85 90 95
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110
Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro
115 120 125
Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
130 135 140
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu
145 150 155 160
Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala
165 170 175
Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys
180 185 190
Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Gly
195 200 205
Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala
210 215 220
CA 02507664 2005-05-27
Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe
225 230 235 240
Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe
245 250 255
Tyr Arg Leu Leu Asp Glu Phe Phe
260
<210> 27
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide Neofor5
<400> 27
ttccagaagt agtgaggagg c 21
<210> 28
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide Neorev5
<400> 28
atggcaggtt gggcgtcgc 19
<210> 29
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide Neofor2
<400> 29
gaactgttcg ccaggctcaa g 21
<210> 30
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide IC49
<400> 30
cggcaaaatc ccttataaat ca 22
CA 02507664 2005-05-27
21
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide E182Gfor
<400> 31
gacggcgggg atctcgtcgt 20
<210> 32
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide E182Grev
<400> 32
acgacgagat ccccgccgtc 20
<210> 33
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide W9lAfor
<400> 33
gggaagggac gcgctgctat tgg 23
<210> 34
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide W9lArev
<400> 34
ccaatagcag cgcgtccctt ccc 23
<210> 35
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide V198Gfor
CA 02507664 2005-05-27
22
<400> 35
ccgaatatca tgggggaaaa tggc 24
<210> 36
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide V198Grev
<400> 36
gccattttcc cccatgatat tcgg 24
<210> 37
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D227Afor
<400> 37
ctacccgtgc tattgctgaa g 21
<210> 38
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D227Arev
<400> 38
cttcagcaat agcacgggta g 21
<210> 39
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D227Vfor
<400> 39
ctacccgtgt tattgctgaa g 21
<210> 40
<211> 21
<212> DNA
<213> Artificial sequence
CA 02507664 2005-05-27
23
<220>
<223> Description of the artificial sequence:
Oligonucleotide D227Vrev
<400> 40
cttcagcaat aacacgggta g 21
<210> 41
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D261Gfor
<400> 41
gccttcttgg cgagttcttc tgag 24
<210> 42
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D261Grev
<400> 42
ctcagaagaa ctcgccaaga aggc 24
<210> 43
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D261Nfor
<400> 43
gccttcttaa cgagttcttc tgag 24
<210> 44
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D261Nrev
<400> 44
ctcagaagaa ctcgttaaga aggc 24
CA 02507664 2005-05-27
24
<210> 45
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide F240Ifor
<400> 45
ggctgaccgc atcctcgtgc tt 22
<210> 46
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide F240Irev
<400> 46
aagcacgagg atgcggtcag cc 22
<210> 47
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotid E182Dfor
<400> 47
gacggcgatg atctcgtcgt 20
<210> 48
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide E182Drev
<400> 48
acgacgagat catcgccgtc 20
<210> 49
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D190Gfor
CA 02507664 2005-05-27
<400> 49
catggcggtg cctgcttgc 19
<210> 50
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D190Grev
<400> 50
gcaagcaggc accgccatg 19
<210> 51
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D208Gfor
<400> 51
gattcatcgg ctgtggccg 19
<210> 52
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D208Grev
<400> 52
cggccacagc cgatgaatc 19
<210> 53
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide D227Gfor
<400> 53
ctacccgtgg tattgctgaa g 21
<210> 54
<211> 21
<212> DNA
<213> Artificial sequence
CA 02507664 2005-05-27
26
<220>
<223> Description of the artificial sequence:
Oligonucleotide D227Grev
<400> 54
cttcagcaat accacgggta g 21
<210> 55
<211> 2406
<212> DNA
<213> Cricetulus griseus
<300>
<310> PCT/EP/96/04631
<311> 1996-10-24
<312> 1997-05-01
<400> 55
gatctccagg acagccatgg ctattacaca gagaaaccct gtctggaaaa acaaaaaatt 60
agtgtccatg tgtaaatgtg tggagtatgc ttgtcatgcc acatacagag gtagagggca 120
gtttatggga gtcagttcct attcttcctt tatgggggac ctggggactg aactcaggtc 180
atcaggcttg gcagaaagtg cattagctca cggagcctta tcattggcga aagctctctc 240
aagtagaaaa tcaatgtgtt tgctcatagt gcaatcatta tgtttcgaga ggggaagggt 300
acaatcgttg gggcatgtgt ggtcacatct gaatagcagt agctccctag gagaattcca 360
agttctttgg tggtgtatca atgcccttaa aggggtcaac aacttttttt ccctctgaca 420
aaactatctt cttatgtcct tgtccctcat atttgaagta ttttattctt tgcagtgttg 480
aatatcaatt ctagcacctc agacatgtta ggtaagtacc ctacaactca ggttaactaa 540
tttaatttaa ctaatttaac cccaacactt tttctttgtt tatccacatt tgtggagtgt 600
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgc 660
gcgcgcgcgc gcgctcggat cattctacct tttgtttaaa aaatgttagt ccaggggtgg 720
ggtgcactgt gaaagtctga gggtaacttg ctggggtcag ttctttccac tataggacag 780
aactccaggt gtcaactctt tactgacaga accatccaaa tagccctatc taattttagt 840
tttttattta tttatttttt gtttttcgag acagggtttc tctgtggctt tggaggctgt 900
cctggaacta gctcttgtag accaggctgg tctcgaactc agagatccac ctgcctctgc 960
ctcctgagtg ctgggattaa aggcatgcgc caccaacgct tggctctacc taattttaaa 1020
agagattgtg tgtcacaagg gtgtcatgtc gccctgcaac cacccccccc ccaaaaaaaa 1080
aaaaaaaaaa acttcactga agctgaagca cgatgatttg gttactctgg ctggccaatg 1140
agctctaggg agtctcctgt caaacagaat ctcaacaggc gcagcagtct tttttaaagt 1200
ggggttacaa cacaggtttt tgcatatcag gcattttatc taagctattt cccagccaaa 1260
aatgtgtatt ttggaggcag cagagctaat agattaaaat gagggaagag cccacacagg 1320
ttattaggaa gataagcatc ttctttatat aaaacaaaac caaaccaaac tggaggaggt 1380
ctacctttag ggatggaaga aaagacattt agagggtgca atagaaaggg cactgagttt 1440
gtgaggtgga ggactgggag agggcgcaac cgctttaact gtcctgtttt gcctattttt 1500
tggggacagc acatgttcct atttttccca ggatgggcaa tctccacgtc caaacttgcg 1560
gtcgaggact acagtcattt tgcaggtttc cttactgtat ggcttttaaa acgtgcaaag 1620
gtgaccatta accgtttcac gctgggaggg cacgtgcggc tcagatgctt cctctgactg 1680
agggccagga gggggctaca cggaagaggc cacacccgca cttgggaaga ctcgatttgg 1740
gcttcagctg gctgagacgc cccagcaggc tcctcggcta caccttcagc cccgaatgcc 1800
ttccggccca taacccttcc cttctaggca tttccggcga ggacccaccc tcgcgccaaa 1860
cattcggccc catcccccgg tcctcacctg aatctctaac tctgactcca gagtttagag 1920
actataacca gatagcccgg atgtgtggaa ctgcatcttg ggacgagtag ttttagcaaa 1980
aagaaagcga cgaaaaacta caattcccag acagacttgt gttacctctc ttctcatgct 2040
aaacaagccc cctttaaagg aaagcccctc ttagtcgcat cgactgtgta agaaaggcgt 2100
ttgaaacatt ttaatgttgg gcacaccgtt tcgaggaccg aaatgagaaa gagcataggg 2160
aaacggagcg cccgagctag tctggcactg cgttagacag ccgcggtcgt tgcagcgggc 2220
aggcacttgc gtggacgcct aaggggcggg tctttcggcc gggaagcccc gttggtccgc 2280
gcggctcttc ctttccgatc cgccatccgt ggtgagtgtg tgctgcgggc tgccgctccg 2340
gcttggggct tcccgcgtcg ctctcaccct ggtcggcggc tctaatccgt ctcttttcga 2400
atgtag 2406
