CHO EXPRESSION SYSTEM
FIELD OF THE INVENTION
The present invention is within the field of industrial protein production. The inventors
have designed and constructed a new expression system comprising an expression
vector coding for a glutamine synthetase of human or of dog origin, and a CHO cell line.
More specifically, the invention pertains to a combination of (i) a DNA vector suitable for
production of a recombinant protein, wherein said vector comprises a sequence coding
for a glutamine synthetase, and (ii) a Chinese Hamster Ovary (CHO) cell line, wherein
said GS comprises a sequence at least 94.5 % identical to the sequence of SEQ ID NO:
1 or to the sequence of SEQ ID NO: 2.
BACKGROUND OF THE INVENTION
When producing recombinant proteins at industrial scale, one must isolate clones
producing high amounts of recombinant proteins.
Introducing heterologous genes into animal host cells and screening for expression of
the added genes is a lengthy and complicated process. The process involves the
transfection and the selection of clones with stable long-term expression, and the
screening for high expression rates of the recombinant protein.
When generating clones expressing a recombinant protein from expression vectors,
host cells are usually transfected with a DNA vector encoding both the protein of
interest and the selection marker on the same vector. Such an expression vector thus
comprises a selectable marker allowing the selection of clones in which the expression
vector is present. Such a selectable marker may also lead to a co-amplification taking
place, thereby allowing the isolation of high-producer clones.
Several such selectable markers are known in the art, including e.g. G418, hygromycin,
puromycin, zeomycin, dihydrofolate reductase (DHFR), glutamine synthetase (GS) and
hypoxanthine-guanine phosphoribosyltransferase (HPRT). In particular, GS is widely
used as a selectable marker in the field of industrial recombinant protein production in
eukaryotic cells.
More specifically, WO 87/04462 describes the use of glutamine synthetase (GS) as a
selectable marker. The examples teach an expression vector comprising, as a
selectable marker, the sequence coding for a GS of Chinese hamster origin. It is further
shown that such an expression vector allows production of a recombinant protein upon
transfection of the expression vector into CHO cells, the recombinant protein being tPA.
Even though the above CHO expression system based on the use of GS as a
selectable marker was described as early as in the 80'ies, it remains a standard in the
art still today. In particular, no significant improvement to the original GS selectable
marker has been published.
Indeed, the Korean patent KR1 0-0267720 discloses the use of human GS as a
selectable marker. However, the exact sequence of the human GS used is not
disclosed. Moreover, it is also indicated the technical effect (high yield) is only linked
both with the human GS and with the specific SV40 promoter that is used (i.e. an SV40
promoter that lacks positions 128 to 270).
There is thus a need in the art for additional and/or improved expression systems
allowing the isolation of a high number of clones expressing the recombinant protein for
which production is desired, at least some of these clones exhibiting high expression
rates of the recombinant protein.
SUMMARY OF THE INVENTION
The inventors have surprisingly found that when producing recombinant proteins in
CHO cells, the use of a GS of human or of dog origin yields better results than the use
of a GS of CHO origin (see e.g. Figures 2 and 3).
In particular, it has been found that the use of a GS of human origin is especially
advantageous since it allows the isolation of more clones expressing the recombinant
proteins than when a GS of CHO origin is used, some of them expressing the
recombinant protein at higher levels than when a GS of CHO origin is used (see e.g.
Figure 3).
One embodiment of the invention provides a Chinese Hamster Ovary (CHO) cell line
comprising a deoxyribonucleic acid (DNA) expression vector, and wherein the vector
comprises a nucleotide sequence coding for a heterologous mammalian glutamine
synthetase (GS) and at least one expression cassette for expressing a recombinant
protein, wherein the GS comprises a protein sequence that is at least 94.5 % identical
to the sequence of SEQ ID NO: 1 or to the sequence of SEQ ID NO: 2; or a protein
fragment of at least 100 consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2. In
another embodiment of the invention, the GS comprises a sequence at least 94.5 %
identical to the sequence of SEQ ID NO: 1 and to the sequence of SEQ ID NO: 2. In
another embodiment of the invention, the GS comprises a sequence at least 97.5 %
identical to the sequence of SEQ ID NO: 1. In a particular embodiment of the invention,
the GS is a human GS and comprises a sequence of SEQ ID NO: 1. In another
embodiment of the invention, the GS is a dog GS and has a sequence of SEQ ID NO: 2.
In still another embodiment of the invention the CHO cell line comprises a
deoxyribonucleic acid (DNA) expression vector, and the vector comprises a nucleotide
sequence coding for a heterologous mammalian glutamine synthetase (GS) and at least
one expression cassette for expressing a recombinant protein, wherein the triplet
codons of said sequence coding for a GS have been biased for expression in CHO cells.
In another embodiment of the invention, said sequence coding for a GS comprises a
sequence at least 80 % identical to the sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In
a particular embodiment of the invention, the sequence coding for a GS comprises a
sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In another embodiment of the invention,
the sequence coding for human GS is placed under the control of a Simian vacuolating
virus 40 (SV40) promoter and the recombinant protein is a monoclonal antibody.
In still another embodiment of the invention the CHO cell line comprises a
deoxyribonucleic acid (DNA) expression vector, and the vector comprises a nucleotide
sequence coding for a heterologous mammalian glutamine synthetase (GS) and at least
one expression cassette for expressing a recombinant protein wherein the triplet
codons of said sequence coding for a GS have been biased for expression in CHO cells.
In another embodiment, the vector comprises a first expression cassette suitable for
cloning of an antibody light chain, and a second expression cassette suitable for cloning
of an antibody heavy chain. In yet another embodiment, the first and second expression
cassettes each comprise a CMV promoter and the CHO cell line is capable of growing
in serum-free medium or serum free and animal derived protein free medium.
In one embodiment of the invention, the CHO cell line is the cell line deposited under
No. CCL-61 at the ATCC or is derived from the cell line deposited under No. CCL-61 at
the ATCC. In another embodiment of the invention, the CHO cell line allows for
obtaining clones producing at least 1 mg/L of recombinant protein upon transfection of
said vector into the CHO cell line deposited under No. CCL-61 at the ATCC.
In still another embodiment of the invention, the CHO cell line comprises a
deoxyribonucleic acid (DNA) expression vector, and the vector comprises a glutamine
synthase (GS) nucleotide sequence (i.e. a nucleotide sequence coding for a glutamine
synthetase (GS)) and at least one expression cassette for expressing a recombinant
protein. In one embodiment, the vector does not contain a heterologous gene for
expression of a recombinant protein. In another embodiment of the invention, the vector
contains at least one sequence coding for a recombinant protein. In another
embodiment, the vector contains a heterologous gene encoding a recombinant protein
that is a monoclonal antibody. In another embodiment of the invention, the vector
contains a heterologous gene encoding a recombinant protein that is an immunogenic
protein for inducing an antibody response. In another embodiment of the invention, the
vector contains a heterologous gene encoding a recombinant protein that is an enzyme
for enzyme replacement therapy or for industrial use.
One embodiment of the invention provides a deoxyribonucleic acid (DNA) expression
vector, and the vector comprises a nucleotide sequence coding for a glutamine
synthetase (GS) under the control of a Simian vacuolating virus 40 (SV40) promoter
and a first expression cassette suitable for cloning of a heterologous recombinant
protein under the control of a CMV promoter. In a particular embodiment of the
invention, the GS comprises a protein sequence that is at least 94.5 % identical to the
sequence of SEQ ID NO: 1 or to the sequence of SEQ ID NO: 2; or a fragment of at
least 100 consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2.
Another embodiment of the invention provides a deoxyribonucleic acid (DNA)
expression vector, and the vector comprises a nucleotide sequence coding for a
glutamine synthetase (GS) under the control of a Simian vacuolating virus 40 (SV40)
promoter and a first expression cassette suitable for cloning of an antibody light chain
under the control of a CMV promoter, and a second expression cassette suitable for
cloning of an antibody heavy chain under the control of a CMV promoter. In a particular
embodiment of the invention, the GS comprises a protein sequence that is least 94.5 %
identical to the sequence of SEQ ID NO: 1 or to the sequence of SEQ ID NO: 2 or a
fragment of at least 100 consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2.
One embodiment of the invention provides a vector as defined in Figure 1.
One embodiment of the invention provides an in vitro method of producing a
recombinant protein comprising the steps of providing a CHO cell line; culturing the
CHO cell line obtained under conditions suitable for production of the recombinant
protein; and isolating and/or purifying said recombinant protein. Another embodiment
provides a further step of formulating the recombinant protein into a pharmaceutical
composition.
One embodiment of the invention pertains to a combination of:
i) a eukaryotic cell line (e.g. a Chinese Hamster Ovary (CHO) cell line); and
ii) a DNA vector suitable for production of a recombinant protein, wherein
said vector comprises a sequence coding for a heterologous mammalian
glutamine synthetase (GS) (e.g. a GS comprising a sequence at least
94.5 % identical to the sequence of SEQ ID NO: 1 or to the sequence of
SEQ ID NO: 2).
Another aspect of the present invention is directed to a kit comprising the above
combination.
Still another aspect of the invention is directed to the DNA vector as such.
Still another aspect of the invention is directed to a CHO cell line comprising the DNA
vector.
In still another aspect, the invention pertains to an in vitro method of producing a
recombinant protein comprising the steps of:
a) providing a vector as defined hereabove;
b) transfecting a cell line with said vector;
c) culturing the transfected cell line obtained at step (b) under conditions suitable
for production of the recombinant protein; and
d) isolating and/or purifying said recombinant protein.
Still another aspect of the invention pertains to the use of such a combination, or of
such a vector, or of such cell line, for producing a recombinant protein in vitro.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a scheme of the vectors used in the Examples (pBH3695, pBH3700,
pBH3694, pBH3699, pBH3698, pBH3697 and pBH3623).
Figure 2 shows the number of occupied wells and productivities achieved with a CHO
cell line transformed with vectors pBH3695, pBH3700, pBH3694, pBH3699, pBH3698,
pBH3697 and pBH3623.
Figure 3 shows the productivities achieved by the clones obtained during the
experiment shown in Figure 2, for vectors pBH3695, pBH3700 and pBH3623. Each bar
represents a clone.
Figure 4 shows the results of an experiment carried out in the 9E4 cell line.
Productivities and the number of clones obtained are shown. The number "0" on the
horizontal axis indicates that no clone was obtained (which is the case for the pBH3700,
pBH3694, pBH3697, pBH3698 and pBH3699 vectors).
Figure 5 shows a sequence alignment between the human GS of SEQ ID NO: 1, the
dog GS of SEQ ID NO: 2, and the CHO GS of SEQ ID NO: 3, which was made using
the "CLUSTAL 2.1 multiple sequence alignment" program. Residues that are different in
human and in dog GS, as compared with CHO GS, are indicated with a black arrows.
These residues correspond to residues 12, 16, 18, 19, 33, 49, 80, 82, 9 1, 116, 191 , 269,
282, 350, 355 and 356 of SEQ ID NO: 1 and of SEQ ID NO: 2 (the amino acid
variations corresponding to 12G, 16V, 18M, 19S, 33I, 49S, 80V, 82A, 9 1K, 116T, 191A,
269Y, 282Q, 350S, 355L and 356I, respectively). Residues that are different in human
GS, as compared to dog and CHO GS, are indicated with a grey arrow. These residues
correspond to residues at position 2, 68, 98, 107, 169, 213 of SEQ ID NO: 1 (the amino
acid variations corresponding to 2T, 68L, 98L, 107R, 169R and 2 13S, respectively).
Figure 6 shows the antibody concentration obtained after transient transfection of CHOS
cells with control vectors (Control 1 and Control 2), and with the pBH3695 and
pBH3772 vectors, respectively expressing the 13C3 and anti-CD38 antibodies.
DETAILED DESCRIPTION OF THE INVENTION
Another aspect of the invention is directed to a combination of:
i) a eukaryotic cell line; and
ii) a DNA (deoxyribonucleic acid) vector suitable for production of a
recombinant protein, wherein said vector comprises a sequence coding for
a hererologous mammalian glutamine synthetase (GS).
The eukaryotic cell line may for instance be a yeast cell line (e.g. a Saccharomyces
cerevisiae or a Yarrowia lipolytica cell line), a fungal cell line (e.g. an Aspergillus niger
cell line), an insect cell line or a mammalian cell line (including but not limited to CHO
cell lines, human cell lines such as HEK293 or PERC.6, mouse cell lines such as NS0,
and monkey cell lines). In a specific embodiment, the eukaryotic cell line is a CHO cell
line. The GS encoded by the DNA vector originates from a heterologous mammalian
species, and may for instance originate from human or dog.
In a specific embodiment, said heterologous mammalian GS comprises or consists of a
sequence:
- at least 94.5 % identical to at least one of SEQ ID NO: 1 or SEQ ID NO: 2,
and/or
consisting of a fragment of at least 100, 150, 200, 250, 300 or 350 consecutive
amino acids of SEQ ID NO: 1 or SEQ ID NO: 2.
Such a combination, further referred to as "combination according to the invention",
constitutes an expression system.
More specifically, one aspect of the invention is directed to a combination of:
i) a DNA vector suitable for production of a recombinant protein, wherein
said vector comprises a sequence coding for a glutamine synthetase (GS);
and
ii) a Chinese Hamster Ovary (CHO) cell line;
wherein said GS comprises a sequence:
at least 94.5 % identical to the sequence of SEQ ID NO: 1 or to the sequence of
SEQ ID NO: 2; or
- consisting of a fragment of at least 100, 150, 200, 250, 300 or 350 consecutive
amino acids of SEQ ID NO: 1 or SEQ ID NO: 2.
The combination according to the invention may for example be provided under the
form of a kit, e.g. with one vial comprising the DNA vector, and another vial comprising
the cell line.
When the expression system is used for producing a recombinant protein, the vector is
introduced into the cell line (it may for example be stably or transiently transfected into
the cell line).
The present invention thus encompasses:
a combination wherein the vector is present within the cell line on the one hand,
and
a combination wherein the vector is isolated from the cell line on the other hand.
1. Vector according to the invention
The DNA vector for use in the combination according to the invention (further referred to
as "vector according to the invention") is suitable for the production of a recombinant
protein, and comprises a sequence coding for a glutamine synthetase (GS).
As used herein, the term "glutamine synthetase " or "GS" refers to a polypeptide capable
of catalyzing the condensation of glutamate and ammonia to form glutamine, as
represented by the following biochemical reaction:
ATP + L-glutamate + NH3 <=> ADP + phosphate + L-glutamine.
Such a polypeptide is classified under Enzyme Commission (EC) number 6.3.1 .2.
Polypeptides capable of catalyzing the above reaction exhibit "GS activity " .
The GS that is used in the frame of the present invention (further referred to as "GS
according to the invention") may comprise or consist of a sequence at least 94.5 %,
95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% identical to
at least one of SEQ ID NO: 1 or SEQ ID NO: 2. Indeed, it has been found that such a
GS is advantageous for use as a selectable marker in CHO cells (see Example 1) . It
may also comprise or consist of a fragment of at least 100, 150, 200, 250, 300 or 350
consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, provided the protein
retains its GS activity.
In a specific embodiment, the GS according to the invention comprises or consists of a
sequence at least 94.5 %, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%,
99.5% or 100% identical both to the sequence of SEQ ID NO: 1 and to the sequence of
SEQ ID NO: 2.
In a specific embodiment, the GS according to the invention comprises or consists of a
sequence at least 97.5%, 98%, 98.5%, 99%, 99.5% or 100% identical to the sequence
of SEQ ID NO: 1. Such a GS is particularly advantageous for use as a selectable
marker in CHO cells (see Example 1) , in particular in the E94 CHO cell line (see
Example 2).
In a specific embodiment, the GS according to the invention is a human GS, i.e., a GS
of human origin. As used herein, the term "human GS" refers to a sequence comprising
or consisting of SEQ ID NO: 1, as well as variants thereof exhibiting GS activity. Such
variants may for example correspond to variants that occur naturally in human species
(such as allelic variants or splice variants). Alternatively, such variants may correspond
to variants obtained by genetic engineering. Most preferably, such variants only differ
from the sequence of SEQ ID NO: 1 by the presence of at most 22, 20, 15, 10, 9, 8, 7, 6,
5, 4, 3, 2 or 1 amino acid variations as compared to SEQ ID NO: 1 (said variations
including substitutions, insertions and deletions).
In another specific embodiment, the GS according to the invention is a dog GS, i.e., a
GS of dog origin. As used herein, the term "dog GS" refers to a sequence comprising or
consisting of SEQ ID NO: 2, as well as variants thereof exhibiting GS activity. Such
variants may for example correspond to variants that occur naturally in dog species
(such as allelic variants or splice variants). Alternatively, such variants may correspond
to variants obtained by genetic engineering. Most preferably, such variants only differ
from the sequence of SEQ ID NO: 2 by the presence of at most 22, 20, 15, 10, 9, 8, 7, 6,
5, 4, 3, 2 or 1 amino acid variations as compared to SEQ ID NO: 2 (said variations
including substitutions, insertions and deletions).
In a specific embodiment, the GS according to the invention comprises at least 1, 2, 3, 4,
5, 6, 10, 15, 16, 20 or 22 of the following amino acids: 12G, 16V, 18M, 19S, 33I, 49S,
80V, 82A, 9 1K, 116T, 191A, 269Y, 282Q, 350S, 355L, 356I, 2T, 68L, 98L, 107R, 169R
and 213S, wherein the number indicates the position on SEQ ID NO: 1 and SEQ ID NO:
2, and the letter the nature of the amino acid (using the one-letter genetic code). In a
more specific embodiment, the GS according to the invention comprises at least 1, 2, 3,
4, 5 or 6 of the following amino acids: 2T, 68L, 98L, 107R, 169R and 213S. In another
more specific embodiment, the GS according to the invention comprises at least 1, 2, 3,
4, 5, 6, 10, 15 or 16 of the following amino acids: 12G, 16V, 18M, 19S, 33I, 49S, 80V,
82A, 9 1K, 116T, 191 A, 269Y, 282Q, 350S, 355L and 356I. The above amino acids
appear to be specific to the human and/or dog GS, as compared to the CHO GS (see
Figure 5).
By a polypeptide having an amino acid sequence at least, for example, 95% "identical "
to a query amino acid sequence of the present invention, it is intended that the amino
acid sequence of the subject polypeptide is identical to the query sequence except that
the subject polypeptide sequence may include up to five amino acid alterations per
each 100 amino acids of the query amino acid sequence. In other words, to obtain a
polypeptide having an amino acid sequence at least 95% identical to a query amino
acid sequence, up to 5% (5 of 100) of the amino acid residues in the subject sequence
may be inserted, deleted, or substituted with another amino acid.
In the frame of the present application, the percentage of identity is calculated using a
global alignment (i.e., the two sequences are compared over their entire length).
Methods for comparing the identity and homology of two or more sequences are well
known in the art. The « needle » program, which uses the Needleman-Wunsch global
alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the
optimum alignment (including gaps) of two sequences when considering their entire
length, may for example be used when performing a global alignment. This needle
program is for example available on the ebi.ac.uk world wide web site. The percentage
of identity in accordance with the invention is preferably calculated using the
EMBOSS::needle (global) program with a "Gap Open" parameter equal to 10.0, a "Gap
Extend" parameter equal to 0.5, and a Blosum62 matrix.
Variants of a reference sequence may comprise mutations such as deletions, insertions
and/or substitutions compared to the reference sequence. In case of substitutions, the
substitution preferably corresponds to a conservative substitution as indicated in the
table below.
The DNA vector according to the invention comprises a sequence coding for such a GS
according to the invention. The sequence coding for such a GS according to the
invention may be the naturally-occurring nucleotide sequence. Alternatively, the triplet
codons of the sequence coding for such a GS may be biased for expression in CHO
cells. Software and algorithms for biasing sequence in order to obtain an optimal
expression are known in the art and include, e.g., the algorithm described in Raab et al.
(2010, Syst Synth Biol. 4:215-25). This algorithm not only provides the best available
codons for expression, but also takes into account the GC content and the absence of
non desired DNA motifs.
For instance, the sequence coding for the GS according to the invention may comprise
or consist of a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to the
sequence of SEQ ID NO: 8 (i.e. a sequence coding for the human GS of SEQ ID NO: 1,
which has been designed for optimal expression in CHO cells) and/or to the sequence
of SEQ ID NO: 9 (i.e. a sequence coding for a dog GS of SEQ ID NO: 2, which has
been designed for optimal expression in CHO cells).
In a specific embodiment, the sequence coding for the GS according to the invention
comprises or consists of a sequence of SEQ ID NO: 8 or SEQ ID NO: 9.
On the DNA vector according to the invention, the sequence coding for the GS
according to the invention may be placed under the control of any promoter known to
those skilled in the art.
For instance, the sequence coding for the GS according to the invention may for
example be placed under the control of a Simian vacuolating virus 40 (SV40) promoter,
for instance the late or the early promoter of SV40. An early SV40 promoter is for
example described in Benoist and Chambon (1981 , Nature. 290:304-10) and in Moreau
et al. (1981 , Nucleic Acids Res. 9:6047-68). In particular, said SV40 promoter is a fulllength
promoter. Said SV40 promoter may also have a replication origin containing a
72bp repeat.
In a specific embodiment, said SV40 promoter is not an SV40 promoter in which
positions 128 to 270 have been removed, i.e. said SV40 promoter is not the SV40
promoter described in Korean patent No. 10-0267720 and transforming the E. coli
transformant deposited to the Gene Bank, Institute of Bioengineering, KIST on 17
December 1997 under the Deposition Number: KCTC 8860 P.
In another specific embodiment, the sequence coding for the GS according to the
invention is not placed under the control of a SV40 promoter.
DNA vectors that are suitable for the production of recombinant proteins are known to
those skilled in the art. Such DNA vectors typically correspond to expression vectors
that comprise an origin of replication and at least one expression cassette allowing the
cloning and the expression of the recombinant protein for which production is desired.
An expression cassette typically comprises a 5' untranslated region (comprising or
consisting of a promoter, and optionally an enhancer sequence), one or more restriction
sites allowing the cloning of a sequence coding for the recombinant protein, a 3'
untranslated region (e.g. a polyA signal), and optionally one or more introns. The
promoter sequence may correspond to any strong promoter well-known to the art, such
as e.g. the human CMV promoter. The vector according to the invention may for
instance have the structure depicted on Figure 1, which is explained in more details in
Example 1, provided that the heavy chain and the light chain of 13C3 may be replaced
with two other coding sequences (e.g. sequences coding for the heavy chain and the
light chain of another antibody).
The recombinant protein may correspond to any protein that is of interest to those
skilled in the art. As used herein, the term "protein" is meant to encompass peptides (i.e.
amino acid chains of less than 50 amino acids), polypeptides (i.e. amino acid chains of
at least 50 amino acids), monomeric proteins (i.e. proteins consisting of one amino acid
chain) and multimeric proteins (i.e. proteins consisting of two or more amino acid chains,
such as e.g. monoclonal antibodies).
The vector according to the invention typically comprises a number of expression
cassettes that is identical to the number of different amino acid chains that constitute
the protein (e.g. one expression cassette in case of a monomeric protein or
homodimeric protein, two in the case of a heterodimeric protein or of a monoclonal
antibody, eic).
Alternatively, the DNA vector according to the invention may comprise only one
expression cassette even when production of a heterodimeric protein or of a
monoclonal antibody is desired. In such a case, the sequence(s) coding for the other
amino acid chain(s) of the protein is (are) present on an expression separate vector,
which is co-transfected with the vector according to the invention into the CHO cell line.
In a specific embodiment, the DNA vector according to the invention may be devoid of
expression cassette. In such a case, the expression cassette(s) suitable for expression
of the recombinant protein is (are) present on a separate vector, which is co-transfected
with the vector according to the invention into the CHO cell line.
Throughout the present specification, the term "recombinant protein " refers to any
recombinant protein for which production is desired. It can for example correspond to a
therapeutic and/or a prophylactic protein, i.e. a protein intended for use as a
medicament (including vaccines). In a specific embodiment, the recombinant protein for
which production is desired is not a glutamine synthetase (GS). In another specific
embodiment, the recombinant protein for which production is desired is an antibody, for
instance a monoclonal antibody. In still another specific embodiment, the recombinant
protein for which production is desired is an antigenic protein. In still another specific
embodiment, the recombinant protein for which production is desired is not
erythropoietin (EPO).
The term "antibody " is used herein in the broadest sense and specifically covers
monoclonal antibodies (including full length monoclonal antibodies) of any isotype such
as IgG, IgM, IgA, IgD, and IgE, polyclonal antibodies, multispecific antibodies (including
bispecific antibodies), antibody fragments (such as e.g. Fv, scFv, dsFv, Fab, Fab', or
F(ab')2 fragments), and fusion proteins comprising an antibody fragment. An antibody
reactive with a specific antigen can be generated by recombinant methods such as
selection of libraries of recombinant antibodies in phage or similar vectors, or by
immunizing an animal with the antigen or an antigen-encoding nucleic acid.
A "monoclonal antibody " , as used herein, is an antibody obtained from a population of
substantially homogeneous antibodies, i.e. the antibodies forming this population are
essentially identical except for possible naturally occurring mutations which might be
present in minor amounts. These antibodies are directed against a single epitope (or a
single group of epitopes in the case of multispecific monoclonal antibodies) and are
therefore highly specific.
A typical monoclonal antibody is comprised of two identical heavy chains and two
identical light chains that are joined by disulfide bonds. Each heavy and light chain
contains a constant region and a variable region. Each variable region contains three
segments called "complementarity-determining regions" ("CDRs") or "hypervariable
regions", which are primarily responsible for binding an epitope of an antigen. They are
usually referred to as CDR1 , CDR2, and CDR3, numbered sequentially from the Nterminus
(see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition,
National Institute of Health, Bethesda, MD, 1991). The more highly conserved portions
of the variable regions are called the "framework regions".
The monoclonal antibody may for example be a murine antibody, a chimeric antibody, a
humanized antibody, or a fully human antibody.
When the recombinant protein for which production is desired is a monoclonal antibody,
the vector according to the invention may comprise a first expression cassette suitable
for cloning of the antibody light chain, and a second expression cassette suitable for
cloning of the antibody heavy chain.
In a specific embodiment, said first and second expression cassettes each comprise the
cytomegalovirus (CMV) promoter, for instance a CMV promoter from a human or a
murine CMV. More specifically, said first and second expression cassettes may
comprise:
a CMV immediate early enhancer promoter (e.g. the one having the sequence
described in Teschendorf et al., 2002, Anticancer Res. 22:3325-30); or
a IE2 promoter/enhancer region from mouse CMV (e.g. the one having the
sequence described in Chatellard et al., 2007, Biotechnol Bioeng. 96:106-17); or
- a hCMV-MIE regulatory element (e.g. the one having the sequence described in
WO 89/01036).
The term "antigenic protein " is used herein in the broadest sense and covers any
protein capable of generating an immune response, either alone or in combination with
an adjuvant. It may be intended for use either in a prophylactic vaccine or in a
therapeutic vaccine. In a specific embodiment the antigenic protein is a vaccinal protein,
1.e. a protein intended for use in a prophylactic vaccine.
In the frame of the present invention, the DNA vector might either comprise at least one
sequence coding for the recombinant protein of interest (e.g. one sequence coding for a
monomeric protein, one sequence coding for an antibody chain, or two sequences,
coding for an antibody light chain and an antibody heavy chain, respectively), or it might
be empty (i.e. devoid of such a sequence coding for the recombinant protein of interest).
In one aspect, the invention is directed to the vector according to the invention per se.
Such a vector is preferably intended for use in a CHO cell line. However, it may also be
used for expressing proteins in other eukaryotic cell lines such as yeast, fungal, insect
or mammalian (e.g. human, mouse, monkey, etc.) cell lines.
2. Cell line according to the invention
The cell line for use in the combination according to the invention (further referred to as
"cell line according to the invention") is a eukaryotic cell line, e.g. a mammalian cell line
such as a CHO cell line. CHO cell lines are commonly used for industrial protein
production, and many CHO cell lines are known to those skilled in the art. For instance,
such CHO cell lines include, e.g., the CHO-K1 cell line (ATCC Number: CCL-61), the
CHO DP-12 cell line (ATCC Nos. CRL-12444 and 12445) and the CHO 1-15 cell line
(ATCC Number CRL-9606). These strains are publically available from the American
Type Culture Collection.
In a specific embodiment, the CHO cell line according to the invention is capable of
growing in serum-free medium (e.g. a chemically-defined medium) and/or in suspension.
Such a cell line can easily be obtained by those skilled in the art by adapting the parent
cell line to grow in serum-free medium and/or in suspension (e.g. through single cell
cloning, through progressive adaptation and/or through a "starve and save" process).
The CHO cell line according to the invention may either be a GS deficient cell line, or a
cell line comprising an endogenous GS gene coding for an endogenous GS polypeptide.
In a specific embodiment, the CHO cell line is the cell line deposited under No. CCL-61
at the ATCC. As used herein, the term 'cell line deposited under No. CCL-61 at the
ATCC" encompasses the parental clone actually deposited at the ATCC on the one
hand, and clones derived therefrom, for instance through single cell cloning,
progressive adaptation and/or through a "starve and save" process, on the other hand.
More specifically, the cell line deposited under No. CCL-61 at the ATCC can be used to
obtain clones capable of growing in serum-free medium and/or in suspension.
In a specific embodiment, the combination according to the invention is characterized in
that it allows obtaining clones producing at least 1, 2, 3, 4 or 5 mg/L of recombinant
protein upon transfection of the vector into the cell line deposited under No. CCL-61 at
the ATCC.
In another aspect, the invention is directed to a CHO cell line comprising a vector
according to the invention. Preferably, said CHO cell line is transfected (stably or
transiently transfected) with said vector. Most preferably said CHO cell line comprises
said vector integrated in its genome.
More specifically, the invention is directed to CHO cell line comprising a DNA
expression vector, and wherein said vector comprises a nucleotide sequence coding for
a heterologous mammalian glutamine synthetase (GS) and at least one expression
cassette for expressing a recombinant protein, wherein said GS comprises a protein
sequence:
a) at least 94.5 % identical to the sequence of SEQ ID NO: 1 or to the sequence of
SEQ ID NO: 2; or
b) consisting of a fragment of at least 100 consecutive amino acids of SEQ ID NO:
1 or SEQ ID NO: 2.
3. Kits, methods and uses according to the invention
One aspect of the invention pertains to a kit comprising or consisting of a combination
according to the invention. In such a kit, the vector is preferably empty, since this allows
the cloning of the protein of interest for those skilled in the art. In addition, the DNA
vector is preferably isolated from the cell line in such a kit. The kit may further comprise
media suitable for cultivation of the cell line, media suitable for transfection of the vector
into the cell line, and/or instructions for use of the expression system.
Another aspect of the invention pertains to the use of the combination according to the
invention, or of the vector according to the invention, or of the cell line according to the
invention, for producing a recombinant protein in vitro.
Still another aspect of the invention pertains to an in vitro method of producing a
recombinant protein, said method comprising or consisting of the following steps:
a) providing a combination according to the invention;
b) transfecting said cell line with said DNA vector;
c) culturing the transfected cell line obtained at step (b) under conditions suitable
for production of the recombinant protein; and
d) isolating and/or purifying said recombinant protein.
As immediately apparent to those skilled in the art, the above aspect relates to a
combination according to the invention wherein the DNA vector is isolated from the cell
line at step (a).
Still another aspect of the invention pertains to an in vitro method of producing a
recombinant protein, said method comprising or consisting of the following steps:
a) providing a combination according to the invention;
b) culturing the transfected cell line under conditions suitable for production of the
recombinant protein; and
c) isolating and/or purifying said recombinant protein.
As immediately apparent to those skilled in the art, the above aspect relates to a
combination according to the invention wherein the cell line comprises the DNA vector
(e.g. the cell line has previously been transected with the DNA vector) at step (a).
Yet another aspect of the invention pertains to an in vitro method of producing a
recombinant protein, comprising or consisting of the following steps:
a) providing a vector according to the invention, wherein said vector comprises at
least one sequence coding for a recombinant protein;
b) transfecting a cell line according to the invention with said vector;
c) culturing the transfected cell line obtained at step (b) under conditions suitable
for production of the recombinant protein; and
d) isolating and/or purifying said recombinant protein.
Conditions suitable for production of recombinant proteins are well-known to those
skilled in the art. The protocols described in the Examples may for instance be used.
In a specific embodiment, a GS inhibitor such as methionine sulphoximine (msx) or
phophinothricin is added when culturing the cell line according to the invention. In a
more specific embodiment, increasing concentrations of such a GS inhibitor are added
when culturing the cell line. This allows selecting clones in which the vector-derived GS
gene (and thus the sequence coding for the recombinant protein) has been amplified.
The above methods may further comprise the step of formulating the recombinant
protein into a pharmaceutical composition.
Still another aspect of the invention is directed to a method for co-amplifying a
recombinant DNA sequence which encodes a recombinant protein, comprising or
consisting of the following steps:
a) providing a vector according to the invention, wherein said vector comprises a
sequence which encodes said recombinant protein;
b) providing a cell line according to the invention;
c) transfecting said cell line with said vector; and
d) culturing said transfected cell line under conditions which allow transformants
containing an amplified number of copies of a vector-derived sequence which
encodes GS to be selected, wherein said transformants also contain an
amplified number of copies of the sequence which encodes the complete amino
acid sequence of the recombinant protein.
Step (d) of the above method may comprise culturing the transfected cell line in media
containing a GS inhibitor and selecting for transformant cells which are resistant to
progressively increased level of the GS inhibitor. The media containing the GS inhibitor
may further contain methionine, whereby the concentrations of GS inhibitor in the media
can be reduced.
The invention is also directed to a method for using a DNA vector as a dominant
selectable marker in a cotransformation process, wherein said method comprises or
consists of the following steps:
a) providing a vector according to the invention, wherein said vector comprises a
sequence which encodes a recombinant protein;
b) providing a cell line according to the invention;
c) transfecting said cell line with said vector; and
d) selecting transformant cells which are resistant to GS inhibitors, whereby
transformant cells are selected in which a vector-derived recombinant DNA
sequence which encodes GS serves as a dominant selectable and coamplifiable
marker.
In a specific embodiment of the above kits and methods, the cell line is a CHO cell line.
In a specific embodiment, the use of the combination according to the invention or of
the vector according to the invention, or of the cell line according to the invention allows
(i) to increase clones expressing the recombinant proteins, and/or (ii) to increase
production of the recombinant protein, than when a GS of CHO origin is used.
Several documents are cited throughout the text of this specification. Each of the
documents herein (including any journal article or abstract, published or unpublished
patent application, issued patent, manufacturer's specifications, instructions, etc.) are
hereby incorporated by reference. However, there is no admission that any document
cited herein is indeed prior art in respect of the present invention.
The invention will further be described by reference to the following drawings and
examples, which are illustrative only, and are not intended to limit the present invention.
Indeed, the invention is defined by the claims, which should be interpreted with the help
of the description and the drawings.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 shows the amino acid sequence of a GS of human origin.
SEQ ID NO: 2 shows the amino acid sequence of a GS of dog origin.
SEQ ID NO: 3 shows the amino acid sequence of a GS of Chinese hamster origin.
SEQ ID NO: 4 shows the amino acid sequence of a GS of yeast origin (Saccharomyces
cerevisiae).
SEQ ID NO: 5 shows the amino acid sequence of a GS originating from toad (Xenopus
laevis).
SEQ ID NO: 6 shows the amino acid sequence of a GS originating from plants
(Arabidopsis thaliana).
SEQ ID NO: 7 shows the amino acid sequence of a GS originating from insects
(Drosophila melanogaster).
SEQ ID NO: 8 shows a nucleotidic sequence coding for a GS of human origin.
SEQ ID NO: 9 shows a nucleotidic sequence coding for a GS of dog origin.
SEQ ID NO: 10 shows a nucleotidic sequence coding for a GS of Chinese hamster
origin.
SEQ ID NO: 11 shows a nucleotidic sequence coding for a GS of yeast origin
{Saccharomyces cerevisiae).
SEQ ID NO: 12 shows a nucleotidic sequence coding for a GS originating from toad
(Xenopus laevis).
SEQ ID NO: 13 shows a nucleotidic sequence coding for a GS originating from plants
(Arabidopsis thaliana).
SEQ ID NO: 14 shows a nucleotidic sequence coding for a GS originating from insects
(Drosophila melanogaster).
EXAMPLES
Example 1: Identification of a GS yielding improved results
The inventors aimed at developing new vectors for expression and production of
recombinant proteins in Chinese Hamster Ovary (CHO) cell lines. A set of seven
vectors was designed as described herebelow.
Two cDNAS coding for a humanized version of the 13C3 antibody (one cDNA coding
for the 13C3 heavy chain and another cDNA coding for the 13C3 light chain,
respectively, the combination of said chains forming the humanized 13C3 antibody)
were used as reporters for assessing the quality of the vector. The murine 13C3
antibody is an antibody that specifically binds to the protofibrillar form of the human b-
amyloid protein, as described in WO 2009/065054. As further used herein, the term
"13C3" refers to the humanized version of the murine 13C3 antibody.
The seven vectors are schematically represented on Figure 1. These eight vectors all
comprise:
- a sequence coding for a GS, placed under the control of the early SV40
promoter;
a first expression cassette, in which the sequence coding for the light chain of
the 13C3 antibody is placed under the control of the CMV promoter;
a second expression cassette, in which the sequence coding for the heavy chain
of the 13C3 antibody is placed under the control of the CMV promoter;
a prokaryotic origin of replication;
a eukaryotic origin of replication; and
a selectable marker for use in prokaryotic cells, namely a sequence coding for a
protein conferring resistance to ampicillin, placed under the control of its natural
promoter.
More specifically, the sequence coding for GS is placed under the control of the SV40
promoter, including the SV40 enhancer. Such an SV40 early promoter contains the
SV40 72-bp tandem repeat enhancers linked to the 2 1-bp non tandem repeats, and the
SV40 early leader protein sequence excluding any coding sequence. The use of this
region as a strong promoter was described by Benoist and Chambon (1981 , Nature.
290:304-10) and in Moreau et al. (1981 , Nucleic Acids Res. 9:6047-68). It is classically
used as a promoter for expression of selection markers in mammalian cells. In the
seven pBH3694 to pBH3700 vectors, the natural Hindi 11 restriction site that was
disrupted, and unique restriction sites (Sail and Xmal) were added at the 5' and the 3'
end of the promoter region, in such a way as to allow an easy swapping of the different
GS cDNAs.
The seven vectors differ from one another by the sequence coding for the GS. Indeed,
sequences coding for GSs having different origins were cloned into the vectors.
More specifically, seven cDNAs coding respectively for a GS from Chinese hamster
{Cricetulus griseus), human (Homo sapiens), dog {Canis lupus), yeast (Saccharomyces
cerevisiae), drosophila (Drosophila melanogaster), plant (Arabidopsis thaliana) and toad
(Xenopus laevis) were generated using the naturally-occuring amino acid sequences
that are available are in public databases. Starting from these sequences, the proteins
were back-translated using a matrix of the most frequent codons used in CHO.
Thereafter, the cDNAs were modified to contain proper cloning sites and the nucleotidic
sequences were optimized. Of note, while the nucleotidic sequences were optimized for
CHO expression, the amino acid sequence of encoded proteins remains identical to that
of the naturally-encoded proteins.
More specifically, the naturally-occurring coding sequences for the different GS were
picked in different public cDNA libraries. For instance, NCBI Reference No.
NM_002065.5 was used for human GS. NCBI Reference No. NM_001002965.1 was
used for dog GS. NCBI Reference No. NM_078568.2 was used for drosophila GS. The
sequence coding for yeast GS was found in the world wide web site available at
yeastgenome dot org (Reference No. YPR035W). The Chinese hamster GS amino acid
sequence corresponds to the one that is shown in NCBI Reference Sequence:
XP_003502909.1 (REFSEQ: accession XM_003502861 . 1 ) . Starting from the naturallyoccurring
cDNA sequences, the triplet codons of the sequence coding for such a GS
was biased for expression in CHO cells using a software developed by Wagner and
coworkers, which is based on the algorithm described in Raab et al. (2010, Syst Synth
Biol. 4:215-25). This technique not only provides the best available codons for
expression, but also takes into account the GC content and the absence of non desired
DNA motifs.
The obtained cDNAs were cloned into the backbone bearing the expression cassettes
for 13C3 antibody, thereby yielding the vectors represented on Figure 1.
The name of these vectors as well as the origin and sequence of the encoded GS is
shown in the table below.
Amino acid Nucleotidic
Name Origin of the GS
sequence of the GS sequence of the GS
pBH3695 Human SEQ ID NO: 1 SEQ ID NO: 8
pBH3700 Dog SEQ ID NO: 2 SEQ ID NO: 9
pBH3623 CHO SEQ ID NO: 3 SEQ ID NO: 10
pBH3694 Yeast SEQ ID NO: 4 SEQ ID NO: 11
pBH3699 Toad SEQ ID NO: 5 SEQ ID NO: 12
pBH3698 Plant SEQ ID NO: 6 SEQ ID NO: 13
pBH3697 Drosophila SEQ ID NO: 7 SEQ ID NO: 14
The above vectors were nucleoporated using classical conditions into a CHO cell line.
24 hours post transfection, about 2000 cells were seeded in 480 to 960 wells of 96-wellplates,
each well comprising 200 m I of CD-CHO medium containing methionine
sulphoximine (msx) at a concentration of 25 mM.
About 20 days post-seeding, the media of the wells were changed to fresh and selective
medium (the same as described above).
Four days later, the number of occupied was counted, i.e. the numbers of wells that are
containing growing clones is counted. Each supernatant from occupied wells was tested
for their 13C3 antibody productivity using an homogeneous time resolved fluorescence
(HTRF) technology developed by Cisbio Bioassays (Bagnols/Ceze, France).
The results are shown on Figures 2 and 3. It can be concluded from these figures that
two vectors, namely pBH3695 and PBH3700, give better results than the other vectors.
They allow obtaining both more clones, and a better productivity.
Percentages of identity between sequences of different GS that were tested are shown
in the three tables below. These percentages of identity were calculated using the
EMBOSS Needle program, using the following default parameters:
- Matrix: EBLOSUM62;
Gap_penalty: 10.0; and
Extend_penalty: 0.5.
Percentage of identity to the
Vector Sequence Origin of GS
human GS of SEQ ID NO: 1
pBH3695 SEQ ID NO: 1 Human 100%
pBH3700 SEQ ID NO: 2 Dog 97.3%
pBH3623 SEQ ID NO: 3 CHO 94.1%
pBH3694 SEQ ID NO: 4 Yeast 52.4%
pBH3699 SEQ ID NO: 5 Toad 85.8%
pBH3698 SEQ ID NO: 6 Plant 50.3%
pBH3697 SEQ ID NO: 7 Drosophila 62.8%
From these tables, it can be concluded that the two sequences that yield the best
results, namely human and dog GS, are characterized in that their sequence exhibit at
least 94.5% identity to the sequence of SEQ ID NO: 1 and/or 2. This feature is not true
for the sequences of the other GS that were tested, which led to less optimal results.
Example 2: Confirmation of the advantageous properties of the use of vector
encoding a human GS in a second CHO cell line.
The above experiment has been repeated with the CHO cell line referred to as "9E4",
which is suitable for industrial production of recombinant proteins.
The 9E4 cell line was established from a clone of the CHO-K1 cell line through a single
cell cloning process. The CHO-K1 cell line was obtained by Puck in 1957 and has been
deposited at the ATCC under number CCL-61 . The CHO 9E4 cell line appears to
express an endogenous and functional GS protein since this cell line can grow in the
absence of glutamine. Methionine sulphoximine (msx) should thus preferably be used
for selection of transfected clones.
The vector was introduced into the 9E4 cell line through nucleoporation. A first
experiment was performed using the six vectors constructed in Example 1 (namely
pBH3695, pBH3700, pBH3694, pBH3697, pBH3698 and pBH3699). Conditions of
selection were identical to the conditions described in Example 1 (msx added at a
concentration of 25 mM) . The number of occupied wells and the concentration of the
13C3 antibody were measured as described in Example 1. The results are shown on
Figure 4.
In the 9E4 cell line, pBH3695 is the only vector capable of generating clones producing
13C3 antibodies. This plasmid is the one bearing the cDNA coding for human GS,
where the triplet codons were biased for expression in CHO cells. The use of a vector
comprising a sequence coding for a human GS is thus particularly advantageous for
producing recombinant proteins in CHO cell lines.
Example 3: Transient expression of X14 in HEK 293 using pBH vector based on
human GS and human CMV promoter.
In this experiment, a vector containing the human GS of sequence SEQ ID NO: 1
placed under the control of the SV40 promoter, and a single expression cassette
containing a cDNA coding for the human or mouse X14 receptor (also named C-type
lectin domain family 14, member A (CLEC14A), and respectively having the NCBI
Reference Number NP_778230.1 and NP_080085.3) under the control of the human
CMV promoter and a polyadenylation site, has been used. The vector containing the
cDNA coding for the human X14 receptor is hereafter pBH4590 vector, and the vector
containing the cDNA coding for the mouse X14 receptor is hereafter called pBH4589
vector.
The pBH4590 vector, pBH4589 vector or a control vector (i.e. an unrelated plasmid
vector) were introduced by transfection with Jet PEI in HEK 293-FS cells as described
by the manufacturer Poly Plus transfection.
The cells were analysed 24h after transfection by immunofluorescence, flow cytometry
or immunocytochemistry after proper labeling for human or mouse X14 detection.
Immunofluorescence detection
For immunofluorescence detection, the transfected cells were spun down and their
supernatants were discarded. Cell pellets were resuspended in PBS buffer containing
1% Bovine Serum Albumin (W/V) and 0.1% Tween (V/V) (PBS T BSA) and saturated
for 10 minutes in this buffer. The cells were washed twice with the same buffer and
incubated with primary Serum 1 (i.e. serum obtained just before immunization of the
animal, called sera pre-immune serum that represent negative control) or Serum 2 (i.e.
serum that is the unpurified immune serum obtained after immunization of the animal)
with purified X14 human extracellular domain as described below at dilution 1/5000 in
the (PBS T BSA) buffer for 10 minutes at Room Temperature.
After washing out the unbound primary antibody, a secondary goat anti Rabbit antibody
linked to an Alexa fluorophore (Alexa 488nm Ref A 11034 form Invitrogen) is added.
Immunofluorescence was performed using a Leica fluorescence microscope set for
detecting Alexa 488 nm.
No background Alexa 488 fluorescence can be observed either with control plasmid and
serum 1 or control plasmid and serum 2, respectively. This indicates the absence of
background non-specific fluorescence. On the contrary, a strong Alexa 488
fluorescence appears at the plasma membrane of cells transfected with pBH4590 and
pBH4589 vectors. The use of a vector comprising a sequence coding for a human GS is
thus particularly advantageous for promoting transient expression of membrane bound
proteins.
Flow cytometry detection.
For flow cytometry detection, human X14 labeling was achieved by two serial
incubations with three different antibody preparations and a secondary antibody anti
Rabbit IgG Fc moiety. The two transfected cell lines were analyzed with three different
dilutions of the anti-X14 serum (1/5000, 1/1000, 1/500):
. Serum 1, obtain just before immunization of the animal, called sera pre-immune
serum that represent negative control,
. Serum 2 that is the unpurified immune serum and obtain after immunization of
the animal with purified X14 human extracellular domain,
. Serum 3 that corresponds to the immunoglobulin fraction of Serum 2, directed
against X14 human lectin.
After washing out the unbound primary antibody, a secondary goat anti Rabbit antibody
linked to an Alexa fluorophore (Alexa 488nm, Catalog Number A-1 1034, from Life
Technologies) has been added. The transfected cells were analysed using flow
cytometry set for detecting Alexa 488 with three different dilutions from Serum 1, 2 and
3 respectively.
It is worth noting that all histograms present a single fluorescence peak, indicating a
homogeneous cell population. The mid fluorescence intensity of this peak is between
102 and 103 fluorescence units. However, cells incubated with serum 2, diluted to
1/500e, present a mid-fluorescence intensity of about 1000 fluorescence units. This
minimal background intensity is suitable to study the detection at the plasma membrane.
The flow cytometer was calibrated in such way that the fluorescence observed with
control vector and serum 1 diluted to 1/5000e, was taken as background reference
fluorescence.
Cells transfected with pBH4590 vector were then analyzed. Fluorescence intensity of
human X14 transfected cell with serum 1, for each dilution, is similar to the signals of
the control cells. On the contrary, the fluorescence signals were markedly more intense
for serum 2 and purified polyclonal antibodies (Serum 3) than for pre-immune serum
(Serum 1) . In fact, mid fluorescence intensity for immune serum (Serum 2) and purified
antibody (Serum 3) on human X14 transfected cells is 104 fluorescence units. It
increases by a factor of 10-20 at the three concentrations of specific antisera tested.
These results demonstrate that in HEK 293-FS cells transfected with pBH4590 vector,
human X14 is produced at a clearly detectable level as observed by fluorescent
microscopy and flow cytometry. Moreover human X14 is being accessible to
extracellular detection indicating that it is expressed at the plasma membrane.
Example 4 : Expression of human and murine X14 receptors in CHO cells
The goal of this experiment was to test if the same vector that was used for transient
human or mouse X14 expression could be used for expression in CHO cells as stable
clones of either mouse or human X14 receptor. To do so the pBH4590 vector or
pBH4589 vector bearing human GS cDNA was transfected into CHO-9E4 cell line,
using the protocol developed by Lonza/Amaxa nucleoporation device. Two millions
CHO-9E4 cells were electroporated with 10 g of the pBH4590 vector or pBH4589
vector described hereabove. Soon After the electric shock, the cells were diluted into
2ml CD-CHO fresh medium and 24 hours later, the cells were again diluted into fresh
CD-CHO medium containing 25mM methyl sulfoximide (msx) at a concentration of 10
000 cells per ml. About ten 96 well plates were seeded at 2000 cells per well.
CHO semi clones, obtained after CHO cells transfection with pBH4590 vector or
pBH4589 vector, using the selectable marker GS, were screened. A transfection with
the reference control expressing antibody 13C3 was also performed. Said control was
used as control for analysis by flow cytometer. Thirty human X14 semi clones, forty-one
mouse X14 semi clones and twenty-nine semi clones for control vector were obtained.
After the passage of semi clones in 24-well plates, that is at the beginning of the
amplification process, the detection of presence or absence of human or mouse X14
antigen using tools described previously has been performed.
It has been observed that, for example, the human semi clone n°12 has fluorescence
intensity lower than the murine semi clone n°30. In fact, with murine semi clones, the
presence of single peak fluorescence having a maximum fluorescence intensity of 103
fluorescence units was observed, while in the case of human semi clone, peak is
spread and the mean fluorescence intensity does not exceed 500 fluorescence units.
Amplification of semi clones which were analyzed as positive by flow cytometry was
performed, thereby allowing to finally generate 10 semi clones of CHO lines stably
expressing X14 murine lectin and 4 semi clones of CHO lines stably expressing X14
human lectin.
The use of a vector comprising a sequence coding for a human GS is thus particularly
advantageous for generating CHO lines stably expressing recombinant proteins.
Example 5 : Transient transfection in CHO-S of different DNA plasmid coding for
different two different antibodies
In order to study the feasibility of using our vector bearing the human GS cDNAs for
transient transfection in CHO-S, a second vector, called, derived from the pBH3695 was
constructed by replacing the two cDNAs corresponding to the light and heavy chain of
the 13C3 antibody by the light and heavy chain of the anti-CD38 cDNAs using classical
cloning technologies and four different unique restriction sites. Consequently, the
pBH3772 vector comprises:
a sequence coding for a GS, placed under the control of the early SV40
promoter;
a first expression cassette, in which the sequence coding for the light chain of
the anti-CD38 antibody is placed under the control of the CMV promoter;
a second expression cassette, in which the sequence coding for the heavy chain
of the anti-CD38 antibody is placed under the control of the CMV promoter;
a prokaryotic origin of replication;
an eukaryotic origin of replication; and
a selectable marker for use in prokaryotic cells, namely a sequence coding for a
protein conferring resistance to ampicillin, placed under the control of its natural
promoter.
CHO-S were then transfected using the Maxcyte® apparatus with (i) two control vectors
classically used for transient transfection and containing the cDNA of light and heavy
chain of two different antibodies (i.e. called Control 1 and Control 2), and (ii) the
pBH3695 and pBH3772 vectors in the conditions described by Maxcyte® Corporation.
CHO-S cells were cultivated in CD-CHO containing 8mM Glutamine in classical CHO-S
cultivating conditions (Passage every 2-3 days at 0.3 106 cells /ml). The day before
transfection, the cells were splitted to 1.2 106 cells/ ml and transfected 24 hours later.
Temperature shift and media feeding were done according to the Maxcyte® protocol.
Culture samples were taken at day 3, 6, 7, 8 and 9, and measured by SEC-HPLC using
purified antibody as standard reference curve.
Results of such an experiment are shown on the figure 6.
It can be concluded from this figure that the pBH3695 and pBH3772 vectors are
capable of producing antibodies at level that are equivalent or better than the two
control vectors classically used for transient transfection.
In conclusion the two pBH3695 and pBH3772 vectors based on human Glutamine
Synthase are capable of producing remarkable level of antibodies above 100mg/l.
The use of a vector comprising a sequence coding for a human GS is thus particularly
advantageous for stably or transiently expressing membrane bound protein or
antibodies.
Example 6 : Expression of human erythropoietin (EPO) in CHO cells
In order to perform the expression of human EPO, the pBH4590 vector was digested
with restriction enzymes Nhel and EcoRI and two human EPO cDNA, i.e. cDNA1 or
cDNA2 bordered with Nhel and EcoRI sites, were inserted in said vector using classical
molecular biology techniques.
This allows to obtain the pBH4614 vector bearing the human EPO cDNA1 , and the
pBH4615 vector bearing the human EPO cDNA2.
The two vectors were prepared at the maxi-preparation level using a kit developed by
Qiagen corporation.
pBH4614 and pBH4615 vectors were used to transfect the three cell lines CHO-S,
CHO-9E4 and CHO 30D12 using the Lonza electroporation techniques, respectively. To
do so, the cells were splitted the day before transfection to achieve a cellular density of
1X106 cells/ml. Two millions cells were spun down and suspended in 0OmI solution V in
the presence of 1C^g of DNA, respectively for each DNA and cell line. Cells were
electroporated using program X05. Rapidly after electroporation, the cells were diluted
into 2ml of CD-CHO medium containing 6mM glutamine (Life Technologies) and
incubated for 24 hours at 37°C and 5% C02. After this incubation, the cells are diluted
into 200ml of the same medium without Glutamine and in the presence of 25mM msx
and distributed in 96 well plates using 200m I per well. After 15 (CHO-S) to 25 days
(CHO-9E4, CHO 30D12), fresh medium was changed in wells containing surviving cells.
Four to 5 days later, the surviving cells were transferred into 1ml of CD-CHO containing
25mM msx without agitation respectively for each well. For CHO-S, 24 semi-clones were
amplified, meanwhile 12 semi clones were amplified for the two other cell lines,
respectively for the two EPO cDNA. Overall 96 semi-clones were amplified, grown and
verified for their capacity to produce human erythropoietin. To do so, after 3-4 day
incubation, the 1ml were diluted into 4ml of the CD-CHO medium containing 25mM msx
and put into agitation at 37°C and 5% C02. After 3-4 days, the cultures were again
diluted with 5 ml fresh medium. After 3-4 days, the cellular density was measured and
cells were diluted at 3X1 05 cells /ml and grown for 3-4 days for a first time. Cellular
density was measured and cells were seeded at 3X1 05 cells/ml in CD-CHO medium
containing 25mM msx and 30% Feed B (Life Technologies).
Cells were grown in 10ml of the above medium (37°C 5% C02) for 10 days. Cellular
density and viability were measured after 8 and 10 days. 0.6 ml samples were equally
taken to evaluate human EPO concentration. Culture supernatants (0.6ml) were first
screened using the microfluidic Caliper technology evaluating the presence of protein at
the apparent molecular weight of human EPO.
Sixteen best clone supernatants, e.g. having the most intense EPO signal, were
submitted to an ELISA specific for human EPO detection using the kit developed by
R&D System® for in vitro diagnostic (Human Erythropoietin Quantikine® IVD ELISA Kit
For In Vitro Diagnostic Use, Catalog reference DEP00). Seven clones were shown to
have interesting productivities as measured at Day 8 and Day 10 (Table below).
The productivities of the clones were ranging from 0.3 to 2.0 g/l. Semi-clone 90 was the
best producing clone with a productivity of 1. 1 to 2.0 g/L at Day 8 and Day 10
respectively.
The viability of this clone at Day 10 was around 70% with less than one million cells per
ml (0.8 million cells/ml) rendering impossible to calculate the specific productivity as the
number of cells is diminishing between Day 8 and Day 10. It renders not possible the
calculation of a specific activity (Table below). This phenomenon is observed for most
the semi-clones except for semi-clones 2 1, 24, 92 and 93. In that case, the specific
productivity expressed in mg/106 cells/day can go up to 441 and 854 (Table below).
clone 94
These results thus shown that, both in terms of volume or specific productivity, the use
of a vector comprising a sequence coding for a human GS allow having productivity
above than 300mg of protein per million cells.
CLAIMS
1. A Chinese Hamster Ovary (CHO) cell line comprising a deoxyribonucleic acid (DNA)
expression vector, and wherein said vector comprises a nucleotide sequence coding for
a heterologous mammalian glutamine synthetase (GS) and at least one expression
cassette for expressing a recombinant protein, wherein said GS comprises a sequence:
at least 94.5 % identical to the sequence of SEQ ID NO: 1 or to the
sequence of SEQ ID NO: 2; or
consisting of a fragment of at least 100 consecutive amino acids of SEQ
ID NO: 1 or SEQ ID NO: 2.
2. The CHO cell line according to claim 1, wherein said GS comprises a sequence at
least 94.5 % identical to the sequence of SEQ ID NO: 1 and to the sequence of SEQ ID
NO: 2.
3. The CHO cell line according to claim 1 or 2, wherein said GS comprises a sequence
at least 97.5 % identical to the sequence of SEQ ID NO: 1.
4. The CHO cell line according to any one of claims 1 to 3, wherein said GS is a human
GS.
5. The CHO cell line according to claim 4, wherein said GS comprises a sequence of
SEQ ID NO: 1.
6. The CHO cell line according to clai 1 or 2, wherein said GS is a dog GS.
7. The CHO cell line according to claim 6, wherein said dog GS comprises a sequence
of SEQ ID NO: 2.
8. The CHO cell line according to any one of claims 1 to 7, wherein the triplet codons of
said sequence coding for a GS have been biased for expression in CHO cells.
9. The CHO cell line according to any one of claims 1 to 8, wherein said sequence
coding for a GS comprises a sequence at least 80 % identical to the sequence of SEQ
ID NO: 8 or SEQ ID NO: 9.
10. The CHO cell line according to claim 9, wherein said sequence coding for a GS
comprises a sequence of SEQ ID NO: 8 or SEQ ID NO: 9.
11. The CHO cell line according to any one of claims 1 to 10, wherein said sequence
coding for human GS is placed under the control of a Simian vacuolating virus 40
(SV40) promoter.
12. The CHO cell line according to any one of claims 1 to 11, wherein said recombinant
protein is a monoclonal antibody.
13. The CHO cell line according to claim 12, wherein said vector comprises a first
expression cassette suitable for cloning of an antibody light chain, and a second
expression cassette suitable for cloning of an antibody heavy chain.
14. The CHO cell line according to claim 13, wherein said first and second expression
cassettes each comprise a CMV promoter.
15. The CHO cell line according to any one of claims 1 to 14, wherein said CHO cell line
is capable of growing in serum-free medium.
16. The CHO cell line according to any one of claims 1 to 15, wherein said CHO cell
line is the cell line deposited under No. CCL-61 at the ATCC.
17. The CHO cell line according to any one of claims 1 to 16, characterized in that it
allows obtaining clones producing at least 1 mg/L of recombinant protein upon
transfection of said vector into the CHO cell line deposited under No. CCL-61 at the
ATCC.
18. The CHO cell line according to any one of claims 1 to 17, wherein said vector does
not contain a heterologous gene for expression.
19. The CHO cell line according to any one of claims 1 to 18, wherein said vector
comprises at least one sequence coding for a recombinant protein.
20. The CHO cell line according to claim 19, wherein said recombinant protein is a
monoclonal antibody.
21. The CHO cell line according to claim 19, wherein said recombinant protein is an
antigenic protein.
22. A deoxyribonucleic acid (DNA) expression vector, and wherein said vector
comprises a nucleotide sequence coding for a heterologous mammalian glutamine
synthetase (GS) under the control of a Simian vacuolating virus 40 (SV40) promoter, a
first expression cassette suitable for cloning of a heterologous recombinant protein
under the control of a CMV promoter, wherein said GS comprises a protein sequence:
a) at least 94.5 % identical to the sequence of SEQ ID NO: 1 or to the
sequence of SEQ ID NO: 2; or
b) consisting of a fragment of at least 100 consecutive amino acids of
SEQ ID NO: 1 or SEQ ID NO: 2.
23. The DNA vector according to claim 22, wherein said vector is a vector as defined in
claim 20 or 2 1.
24. A deoxyribonucleic acid (DNA) expression vector, and wherein said vector
comprises comprises a nucleotide sequence coding for a heterologous mammalian
glutamine synthetase (GS) under the control of a Simian vacuolating virus 40 (SV40)
promoter, a first expression cassette suitable for cloning of an antibody light chain under
the control of a CMV promoter, and a second expression cassette suitable for cloning of
an antibody heavy chain under the control of a CMV promoter, wherein said GS
comprises a protein sequence:
a) at least 94.5 % identical to the sequence of SEQ ID NO: 1 or to the
sequence of SEQ ID NO: 2; or
b) consisting of a fragment of at least 100 consecutive amino acids of
SEQ ID NO: 1 or SEQ ID NO: 2.
25. A combination of:
i) a Chinese Hamster Ovary (CHO) cell line; and
i) a DNA (deoxyribonucleic acid) vector suitable for production of a
recombinant protein, wherein said vector comprises a sequence coding for
a hererologous mammalian glutamine synthetase (GS), wherein said GS
comprises a sequence:
at least 94.5 % identical to the sequence of SEQ ID NO: 1 or to the sequence
of SEQ ID NO: 2; or
- consisting of a fragment of at least 100 consecutive amino acids of SEQ ID
NO: 1 or SEQ ID NO: 2.
26. A kit comprising the combination according to claim 25.
27. An in vitro method of producing a recombinant protein comprising the steps of:
a) providing a CHO cell line according to any one of claims 1 to 2 1;
b) culturing said CHO cell line obtained under conditions suitable for production of
the recombinant protein; and
c) isolating and/or purifying said recombinant protein.
28. The method according to claim 27, further comprising the step of formulating said
recombinant protein into a pharmaceutical composition.
29. Use of the CHO cell line according to any one of claims 1 to 2 1, or of the vector
according to any one of claims 22 to 24, or of the combination according to claim 25, for
producing a recombinant protein in vitro.