U.S. patent application number 11/544125 was filed with the patent office on 2007-02-08 for selection of cells expressing heteromeric polypeptides.
This patent application is currently assigned to Immunex Corporation. Invention is credited to Allison A. Bianchi, Jeffrey T. McGrew.
Application Number | 20070031422 11/544125 |
Document ID | / |
Family ID | 23261429 |
Filed Date | 2007-02-08 |
United States Patent
Application |
20070031422 |
Kind Code |
A1 |
McGrew; Jeffrey T. ; et
al. |
February 8, 2007 |
Selection of cells expressing heteromeric polypeptides
Abstract
This invention is in the general field of recombinant expression
of polypeptides in animal cell culture. More particularly, the
invention concerns improved selection in cells of recombinantly
engineered vectors designed to express polypeptides.
Inventors: |
McGrew; Jeffrey T.;
(Seattle, WA) ; Bianchi; Allison A.; (Seattle,
WA) |
Correspondence
Address: |
IMMUNEX CORPORATION
PATENT OPERATIONS/MS 28-2-C
ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Assignee: |
Immunex Corporation
|
Family ID: |
23261429 |
Appl. No.: |
11/544125 |
Filed: |
October 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10251447 |
Sep 20, 2002 |
|
|
|
11544125 |
Oct 5, 2006 |
|
|
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60323954 |
Sep 20, 2001 |
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Current U.S.
Class: |
424/146.1 ;
424/155.1; 435/191; 435/372; 435/455; 435/5; 435/6.16; 435/69.1;
536/23.2 |
Current CPC
Class: |
C12N 15/1055 20130101;
C12N 15/62 20130101; C07K 2319/73 20130101; C07K 16/2866 20130101;
C07K 2319/00 20130101 |
Class at
Publication: |
424/146.1 ;
424/155.1; 435/006; 435/069.1; 435/191; 435/372; 435/455;
536/023.2 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12Q 1/68 20060101 C12Q001/68; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C12N 9/06 20060101
C12N009/06; C12N 5/08 20060101 C12N005/08 |
Claims
1. A method of selecting cells comprising transfecting cells with a
vector comprised of a first nucleic acid encoding a first desired
polypeptide, wherein the transcription of said first nucleic acid
is operably linked to a second nucleic acid encoding a first
subunit of a selectable marker, and further comprising a third
nucleic acid encoding a second desired polypeptide that is capable
of associating with the first desired polypeptide to form a
heteromeric complex, wherein the transcription of said third
nucleic acid is operably linked to a fourth nucleic acid which
encodes a second subunit of a selectable marker, and wherein said
second subunit is capable of associating with the first subunit of
the selectable marker, thereby providing a selectable activity
applying selection conditions to the transfected cells and
selecting for cells expressing the heteromeric complex.
2. The method of claim 1, wherein the heteromeric complex is an
antibody.
3. The method of claim 1, wherein the first nucleic acid encodes a
polypeptide selected from the group consisting of an antibody heavy
chain and an antibody light chain.
4. The method of claim 1, wherein the selectable marker is selected
from the group consisting of a drug resistance marker, a metabolic
survival marker, a color marker and a fluorescent marker.
5. The method of claim 4, wherein the selectable marker is selected
from the group consisting of dihydrofolate reductase, neomycin
resistance, hygromycin resistance, beta-galactosidase, and green
fluorescent protein.
6. The method of claim 1, wherein an internal ribosomal entry site
occurs between the first nucleic acid and the second nucleic
acid.
7. The method of claim 1, wherein an internal ribosomal entry site
occurs between the third nucleic acid and the fourth nucleic
acid.
8. The method of claim 1, wherein the selectable marker subunit is
a fusion polypeptide comprising an interaction domain.
9. The method of claim 8, wherein the interaction domain is a
dimerization sequence that is a leucine zipper from a polypeptide
selected from the group consisting of GCN4, C/EBP, c-Fos, c-Jun,
c-Myc and c-Max.
10. The method of claim 1, further encoding a different functional
selectable marker selected from the list consisting of zeomycin,
neomycin, puromycin, Blasticidin S, and GPT.
11. The host cell of claim 1, which is selected from the group
consisting of CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, a myeloma
cell line, and W138 cells.
12. A method of selecting cells comprising transfecting an
expression system comprising a first vector comprising a first
nucleic acid encoding a light chain of an antibody wherein the
transcription of said light chain is operably linked to the
transcription of a second nucleic that encodes a fusion polypeptide
of a first subunit of dihydrofolate reductase fused to a
dimerization sequence, and a second vector comprising a third
nucleic acid encoding a heavy chain of an antibody wherein the
transcription of said heavy chain is operably linked to the
transcription of a fourth nucleic acid that encodes a fusion
polypeptide of a second subunit of a dihydrofolate reductase fused
to a dimerization sequence wherein each subunit of dihydrofolate
reductase does not have selectable activity when expressed alone
and co-expression of the first dihydrofolate reductase subunit with
the second dihydrofolate reductase subunit provides dihydrofolate
reductase activity applying selection conditions to the cells, and
selecting for cells expressing the heteromeric complex.
13. The expression system of claim 12, wherein one subunit of
dihydrofolate reductase is amino acids 1 to 105 of SEQ ID NO:5 and
the other subunit of dihydrofolate reductase is amino acids 106 to
187 of SEQ ID NO:5.
14. The expression system of claim 13, wherein the dimerization
sequence fused to the dihydrofolate reductase subunit is derived
from the GCN4 leucine zipper sequence.
15. A host cell transfected with the expression system of any of
claims 12, 13, or 14.
16. An isolated nucleic acid molecule comprising a first nucleic
acid encoding a polypeptide, wherein said first nucleic acid is
operably linked to a second nucleic acid encoding a subunit of a
selectable marker, and wherein said subunit or subunits is capable
of interacting with a different subunit of the selectable marker
thereby providing a selectable activity.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/251,447, filed Sep. 20, 2002, which claims benefit to U.S.
Application Ser. No. 60/323,954, filed Sep. 20, 2001, and are
hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the general field of recombinant
expression of polypeptides in animal cell culture. More
particularly, the invention concerns improved selection in cells of
recombinantly engineered vectors designed to express
polypeptides.
BACKGROUND OF THE INVENTION
[0003] Many commercially important proteins are produced in
recombinantly engineered cells that have been adapted for long term
growth in culture. Frequently, the proteins are expressed as a
single polypeptide chain. Also expressed in these cells are
multiple heterologous polypeptides that can associate to form
heteromeric complexes, such as for example, an antibody, which is
formed by the expression of equal parts of heavy chains and light
chains.
[0004] One difficulty that can be encountered when expressing
heteromeric complexes in cells is obtaining appropriate amounts of
each of the recombinant polypeptides that form a component of the
complex. For example, in the expression of an antibody frequently
either the heavy chain or the light chain are expressed to
relatively high levels with respect to the corresponding partner;
however, obtaining a cell line expressing both chains to high
levels and in roughly equal amounts is difficult.
[0005] These difficulties result in additional steps and also
repetition of steps in the process of generating cell lines
expressing recombinant polypeptides resulting in delays which also
substantially increase costs associated with recombinant expression
of the polypeptides. Thus, there is a need in the art for simpler
methods of selecting for high level expression of polypeptides in
cell cultures so as to increase production of the polypeptides
thereby reducing the cost and time investment necessary for
selection of cells expressing the polypeptides. The invention
fulfills this need by providing an improved method for selecting
cells expressing polypeptides.
SUMMARY OF THE INVENTION
[0006] The invention is based, in part, on the premise that the
efficient production of recombinant heteromeric complexes in cells
is improved if each component of the complex is expressed in
proportional amounts. As such, the present invention provides
methods and compositions to select for recombinantly engineered
cells that express more than one polypeptide, where the
polypeptides are expressed in proportional quantities such that the
polypeptides can efficiently associate to form a heteromeric
complex and higher expression is achieved.
[0007] In one embodiment, the invention comprises two vectors,
where each vector comprises at least two open reading frames
encoding two different polypeptides. In this embodiment, a first
vector encodes a first polypeptide that can associate with a
corresponding first polypeptide encoded by the second vector to
form a heteromeric complex. In addition, the first vector encodes a
second polypeptide that can associate with a corresponding second
polypeptide encoded by the second vector to form a heteromeric
complex having a selectable activity.
[0008] In a particular embodiment, the invention contemplates an
isolated nucleic acid molecule comprising a first nucleic acid
encoding a polypeptide, wherein said first nucleic acid is operably
linked to a second nucleic acid encoding a subunit of a selectable
marker, and wherein said subunit or subunits is capable of
interacting with a different subunit of the selectable marker
thereby providing a selectable activity.
[0009] In another embodiment the invention contemplates an isolated
nucleic acid molecule comprising a first nucleic acid encoding a
polypeptide, wherein said first nucleic acid is operably linked to
a second nucleic acid encoding a subunit of a selectable marker,
and wherein said subunit or subunits is capable of interacting with
a different subunit of the selectable marker thereby providing a
selectable activity, and further comprising a third nucleic acid
encoding a polypeptide that is capable of associating with the
polypeptide encoded by the first nucleic acid to form a heteromeric
complex, wherein said third nucleic acid is operably linked to a
fourth nucleic acid encoding at least one subunit of a selectable
marker, and wherein said subunit or subunits are capable of
associating with the polypeptide selectable marker subunit encoded
by the second nucleic acid, thereby providing a selectable
activity.
[0010] In another particular embodiment, the heteromeric complex
described above is an antibody, and the selectable marker described
above is selected from the group consisting of a drug resistance
marker, a metabolic survival marker, a color marker and a
fluorescent marker.
[0011] The invention further provides methods for constructing the
nucleic acid molecules of the invention, methods for making host
cells expressing nucleic acids of the invention, host cell lines
expressing the nucleic acids of the invention, and methods for
producing and isolating heteromeric complexes recombinantly
expressed from the nucleic acids in host cells.
BRIEF DESCRIPTION OF THE FIGURE
[0012] FIG. 1. A schematic representation of the nucleic acid
constructs utilized in the examples, each comprising a subunit of a
selectable marker and expressing different polypeptides, which can
associate to form a heteromeric complex in a cell. The
abbreviations are as follows: EASE, expression augmenting sequence
element; CMV, cytomegalovirus promoter; HC, Heavy Chain; LC, Light
Chain; IRES, internal ribosomal entry site; DHFR, dihydrofolate
reductase; and pA, polyadenylation signal.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Efficient production of recombinant heteromeric complexes in
cells is improved if each component of the complex is expressed in
proportional and high amounts. The present invention provides
methods and compositions to select for recombinantly engineered
cells, which express more than one heterologous polypeptide in
proportional quantities such that the polypeptides can efficiently
associate to form a heteromeric complex at higher expression levels
than traditionally prepared heteromeric complexes. The present
invention is also advantageous in that it decreases the time
required to select for cells expressing high levels of a desired
recombinant heteromeric polypeptide complex.
[0014] The invention utilizes selectable markers that can exist as
two or more subunits that when expressed together will interact,
thereby providing a selectable activity. The individual subunits do
not have significant selectable activity alone, but do provide
selectable activity when co-expressed with their counterpart
subunit. The optimal activity of the subunits can depend upon their
interaction, and as such can be facilitated by interaction domains.
Such interaction domains can be endogenous to the subunit or it can
be heterologous to the subunit.
[0015] Nucleic acid molecules are constructed that encode a
polypeptide and a subunit of the selectable marker, arranged in
such a way that expression of the subunit correlates with
expression of the polypeptide. Thus, when the nucleic acid
molecules encoding both subunits are transfected into cells and
selective conditions applied, approximately equal and high levels
of expression of each of the subunits will provide the highest
selectable activity. In addition, the operably linked polypeptides
will be expressed in nearly equal and high amounts, therefore there
is optimization of selection of cells expressing equal and high
levels of the desired polypeptides.
[0016] In one non-limiting embodiment, the invention entails the
use of two subunits of a selectable marker, each expressed as a
fusion protein to an interaction domain. When expressed, the
interaction domain promotes association or dimerization of the two
subunits thereby allowing the subunits to function and providing a
selectable activity (e.g., but not limited to, that described by
Pelletier et al. (1998), Proc. Natl. Acad. Sci.,
95:12141-12146).
[0017] In an alternative embodiment, the invention entails the use
of three subunits of a selectable marker, each expressed as a
fusion protein to an interaction domain, thereby enhancing
association to provide a selectable activity. In this embodiment,
there are three components of the heteromeric complex. In the
expressed vector(s) coding sequences for each are operably linked
to coding sequences for each of the respective subunits of the
selectable marker, for example, a bispecific antibody expressing a
single heavy chain and two different light chains, wherein the two
light chains are both capable of associating with the heavy chain.
The invention also encompasses use of selectable markers known or
yet to be disclosed that have four or even more subunits.
[0018] As will be shown below in the examples, it has been
discovered that the methods and compositions of the invention
reduce the amount of time necessary to select for the desired cells
expressing high levels of a single polypeptide. Thus, in yet
another embodiment, the invention encompasses selecting for cells
expressing high levels of a recombinant polypeptides.
[0019] In some embodiments, the nucleic acids encoding the
selectable marker subunits are fused in frame to a nucleic acid
encoding a linker, which is then fused in frame to a nucleic acid
encoding an interaction domain. Linkers can include any relatively
short, flexible sequence that allows the interaction domain to
interact and for the subunits to function to provide a selectable
activity. Examples of linkers are abundant in the relevant art and
can comprise GGPGG, GPGGG, where in single letter amino acid codes,
G is glycine and P is proline. In one embodiment, the linker is
GGGGSGGGGGS (Curtis et al. (1991), Proc Natl Acad Sci
88(13):5809-5813).
[0020] An interaction domain is a domain, including but not limited
to, polypeptides capable of facilitating the interaction or
association of two or more homologous or heterologous polypeptides.
As used herein, the terms "associating" or "interacting" are meant
to describe a relationship between at least two molecules wherein
one molecule binds to the others and/or affects the activity of the
others. Interaction can include the direct or indirect binding of
two polypeptides (or polypeptide and nucleic acid), or the
functional activation or inhibition of a molecule's activity by
another molecule.
[0021] In one embodiment, the interaction domain is a dimerization
domain. A dimerization domain can be a polypeptide capable of
inducing interaction or association of two polypeptides. There are
two types of dimers, those capable of forming homodimers (with the
same sequence), or heterodimers (with another sequence).
[0022] In one illustrative but non-limiting embodiment, the
interaction domain is a leucine zipper coiled coil polypeptide. A
leucine zipper typically comprises about 35 amino acids containing
a characteristic seven residue repeat with hydrophobic residues at
the first and fourth residues of the repeat (Harbury et al. (1993),
Science 262:1401). Thus a leucine zipper is amenable to fusion to a
polypeptide for the purpose of oligomerizing the polypeptide as it
is a small molecule and is less likely to disrupt the polypeptides
normal function than would a larger interaction domain. Examples of
leucine zippers include but are not limited leucine zipper domains
from polypeptides such as GCN4, C/EBP, c-Fos, c-Jun, c-Myc and
c-Max.
[0023] Additional examples of dimerization domains include
helix-loop-helix domains (Murre et al. (1989), Cell 58:537-544).
The retinoic acid receptor, thyroid hormone receptor, other nuclear
hormone receptors (Kurokawa et al. (1993), Genes Dev. 7:1423-1435)
and yeast transcription factors GAL4 and HAP1 (Marmonstein et al.
(1992), Nature 356:408-414; Zhang et al. (1993), Proc. Natl. Acad.
Sci. USA 90:2851-2855; U.S. Pat. No. 5,624,818) all have
dimerization domains with this motif.
[0024] In yet another embodiment, the interaction domain is a
tetramerization domain, which is a polypeptide capable of binding
three other tetramerization domains to form a tetrameric complex.
Examples of proteins containing tetramerization domains include but
are not limited to the E. coli lactose repressor (amino acids
46-360; Chakerian et al. (1991), J. Biol. Chem. 266:1371; Alberti
et al. (1993), EMBO J. 12:3227; and Lewis et al. (1996), Nature
271:1247), and the p53 tetramerization domain at residues 322-355
(Clore et al. (1994), Science 265:386; Harbury et al. (1993),
Science 262:1401; U.S. Pat. No. 5,573,925).
[0025] In one embodiment, the two subunits are expressed from two
vectors, wherein the first vector comprises a first nucleic acid
encoding a first polypeptide, and wherein the first nucleic acid is
operably linked to a second nucleic acid encoding a subunit of a
selectable marker. The second vector comprises a third nucleic acid
encoding a polypeptide that is capable of associating with the
polypeptide encoded by the first nucleic acid, wherein the third
nucleic acid is operably linked to a fourth nucleic acid encoding a
different subunit of the selectable marker. Thus, both vectors are
simultaneously transfected into a cell population and selection for
expression of the selectable marker (comprised of two subunits) is
applied.
[0026] In another embodiment, the invention further comprises a
nucleic acid encoding a different functional selectable marker, in
addition to a subunit of a selectable marker and a polypeptide of a
heteromeric complex. For purposes herein, a "different functional
selectable marker" is not a subunit of a selectable marker, but is
a protein with fully functional selectable activity. Well known
markers such as zeomycin, neomycin, puromycin, Blasticidin S, or
GPT which confers resistance to mycophenolic acid, etc., can be
used as different functional selectable markers. In this
embodiment, the invention comprises two vectors, wherein each of
the vectors comprises a first nucleic acid encoding a polypeptide
that can form a heteromeric complex operably linked to a second
nucleic acid encoding at least one subunit of a selectable marker,
as well as also a nucleic acid encoding a different, functional
selectable marker. Further, the respective polypeptides encoded by
the first nucleic acid of each vector can associate to form a
complex, and the subunit or subunits encoded by the second nucleic
acids of each vector can associate to provide a selectable activity
and the polypeptides encoded by the third nucleic acids provide
selectable activities different than the selectable activity of the
subunits encoded by the second nucleic acids. For example, the
first vector can encode resistance to neomycin and the second
vector can encode resistance to zeomycin or only one vector can
contain the additional different functional selectable marker.
Thus, one vector is transfected into a cell line and selection is
applied (i.e., the drug G418 is added to neomycin resistant cells).
After selection, conventional methods can be used to determine the
presence of the vector and the expression level of the polypeptides
encoded by the nucleic acids on the vector, for example by PCR,
Southern blot, ELISA, western blot, and the like. Once high level
expression has been obtained, the second vector is transfected into
the cell line. While maintaining selection for the first vector,
selection is applied for the second selectable marker (i.e.,
zeomycin resistance) and the presence of the second vector and
expression of the respective vector encoded proteins are assessed.
In this embodiment, once it has been determined that both vectors
are present, selection is applied for expression of the subunits
that have associated in the cell to provide a selectable activity,
e.g., dihydrofolate reductase (DHFR), as described above.
[0027] In an alternative embodiment, both the nucleic acids of the
invention encoding independent selectable activities are
transfected simultaneously and selection is applied at the same
time. Once it has been determined that both vectors are present,
selection is applied for expression of the subunits that have
associated in the cell to provide a selectable activity, e.g.,
dihydrofolate reductase (DHFR), as described above.
[0028] In yet another embodiment, the vectors of the invention
encoding independent selectable activities are each transfected
into separate cell lines. Once selection is applied and clones have
been identified that express high levels of the proteins encoded by
each desired vector, the cells are fused as described in Hori et
al. (U.S. Pat. No. 5,916,771). Once fusion is complete, selection
is applied for the selectable activity provided by the
subunits.
[0029] In yet another embodiment, nucleic acids of the invention
optionally not containing an independent selectable activity are
transfected simultaneously with a third vector. The third vector
encodes for a separate selectable activity, such as for example,
neomycin resistance or beta galactosidase that can allow for a
preliminary selection of cells that were successfully transfected.
Once this preliminary selection has been performed, selection can
be applied for the selectable activity of the subunits, e.g., DHFR.
In this embodiment, equal quantities of the two expression vectors
are transfected while the third vector is transfected at one-third
the concentration of the first two vectors (e.g., a ratio of 3:3:1
or 6:6:1 or the like). One of skill in the art will recognize that
variations in the ratios are within the scope of the invention.
[0030] The nucleic acids encoding a component of the desired
heteromeric complex can be obtained as a cDNA or as a genomic DNA
by methods known in the art. For example, messenger RNA coding for
a desired component can be isolated from a suitable source
employing standard techniques of RNA isolation, and the use of
oligo-dT cellulose chromatography to segregate the poly-A mRNA.
When the heteromeric complex to be expressed is an antibody,
suitable sources of desired nucleic acids can be isolated from
mature B cells or a hybridoma culture. In addition, the nucleic
acids for use in the invention can be obtained by chemical
synthesis.
[0031] The term "heteromeric complex" is meant to include a
molecular complex formed by the association of at least two
different molecules. The association can be non-covalent
interaction or covalent attachment, e.g., disulfide bonds. The two
different molecules are typically two different polypeptides,
however, the invention contemplates heteromeric complexes between
polypeptides and nucleic acids and between different nucleic acids.
In one embodiment, the heteromeric complex provides a functional
activity, such as, the ability to bind a substrate (e.g., an
immunoglobulin capable of binding a corresponding antigen),
enzymatic activity or the like. In one embodiment, the heteromeric
complex of the invention is secreted into the culture medium of the
host cell in which it is being produced.
[0032] In a particular embodiment, the heteromeric complex is an
immunoglobulin molecule. The immunoglobulin in vertebrate systems
is an antibody comprised of two identical light chains and two
identical heavy chains. The four chains are joined together by
disulfide bonds, such that each light chain is joined with a heavy
chain and the heavy chains are connected across their tails
altogether forming a Y-shaped heteromeric complex. Numerous
techniques are known by which DNA encoding immunoglobulin molecules
can be manipulated to yield DNAs capable of encoding recombinant
proteins such as antibodies with enhanced affinity, or other
antibody-based polypeptides (see, for example, Larrick et al.
(1989), Biotechnology 7:934-938; Reichmann et al. (1988), Nature
332:323-327; Roberts et al. (1987), Nature 328:731-734; Verhoeyen
et al. (1988), Science 239:1534-1536; Chaudhary et al. (1989),
Nature 339:394-397).
[0033] Recombinant cells producing fully human antibodies (such as
are prepared using antibody libraries, and/or transgenic animals,
and optionally further modified in vitro), as well as humanized
antibodies can also be used in the invention. See, e.g., Cabilly et
al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No.
0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,
European Patent No. 0,120,694 B1; Neuberger et al., WO 86/01533;
Neuberger et al., European Patent No. 0,194,276 B1; Winter, U.S.
Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen
et al., European Patent No. 0,451,216 B1; and Padlan et al.,
European Patent No. 0,519,596 A1. For example, the invention can be
used to induce the expression of human and/or humanized antibodies
that immunospecifically recognize specific cellular targets, e.g.,
the human EGF receptor, the her-2/neu antigen, the CEA antigen,
Prostate Specific Membrane Antigen (PSMA), CD5, CD11a, CD18, NGF,
CD20, CD45, Ep-cam, other cancer cell surface molecules, TNF-alpha,
TGF-b 1, VEGF, other cytokines, alpha 4 beta 7 integrin, IgEs,
viral proteins (for example, cytomegalovirus), etc., to name just a
few.
[0034] Examples of heteromeric complexes, in addition to
immunoglobulins, include but are not limited to any heterodimeric
or hetero-oligomeric protein, e.g., BMP2/BMP7, osteogenic protein,
interleukin 1 converting enzyme (ICE), various interleukin
receptors (e.g., the IL-18 receptor, IL-13 receptor, IL-4 receptor
and IL-7 receptor), receptors of the nucleus such as retinoid
receptors, T-cell receptors, integrins such as cell adhesion
molecules, betal-integrins, tumor necrosis factor receptor and
soluble and membrane bound forms of class I and class II major
histocompatibility complex proteins (MHC). For heteromeric
complexes that are receptors, the invention encompasses both
soluble and membrane bound forms of the polypeptides. Descriptions
of additional heteromeric proteins that can be produced according
to the invention can be found in, for example, Human Cytokines:
Handbook for Basic and Clinical Research, Vol. II (Aggarwal and
Gutterman, eds. Blackwell Sciences, Cambridge Mass., 1998); Growth
Factors: A Practical Approach (McKay and Leigh, Eds. Oxford
University Press Inc., New York, 1993) and The Cytokine Handbook (A
W Thompson, ed.; Academic Press, San Diego Calif.; 1991).
[0035] As used herein, the term "fusion protein" refers to a
protein, or domain of a protein (e.g., a soluble extracellular
domain) fused to a heterologous protein or peptide. Examples of
such fusion proteins include proteins expressed as a fusion with a
portion of an immunoglobulin molecule, proteins expressed as fusion
proteins with a zipper moiety, and novel polyfunctional proteins
such as fusion proteins of cytokines and growth factors (i.e.,
GM-CSF and IL-3, MGF and IL-3). WO 93/08207 and WO 96/40918
describe the preparation of various soluble oligomeric forms of a
molecule referred to as CD40L, including an immunoglobulin fusion
protein and a zipper fusion protein, respectively; the techniques
discussed therein are applicable to other proteins. Any of the
molecules herein described can be expressed as a fusion protein
including but not limited to the extracellular domain of a cellular
receptor molecule, an enzyme, a hormone, a cytokine, a portion of
an immunoglobulin molecule, a zipper domain, and an epitope.
[0036] The invention finds particular utility in improving the
production of heteromeric complexes via cell culture processes. The
cell lines used in the invention can be genetically engineered to
express a protein of commercial or scientific interest. By
"genetically engineered" is meant that the cell line has been
transfected, transformed or transduced with a recombinant
polynucleotide molecule, so as to cause the cell to express a
desired protein. Methods and vectors for genetically engineering
cells and/or cell lines to express a protein of interest are well
known to those of skill in the art; for example, various techniques
are illustrated in Current Protocols in Molecular Biology, Ausubel
et al., eds. (Wiley & Sons, New York, 1988, and quarterly
updates) and Sambrook et al., Molecular Cloning: A Laboratory
Manual (Cold Spring Laboratory Press, 1989).
[0037] In addition to the nucleic acid encoding the desired
component of the heteromeric complex, vector constructs can include
additional components to facilitate replication in prokaryotic
and/or eukaryotic cells, integration of the construct into a
eukaryotic chromosome, and markers to aid in selection of and/or
screening for cells containing the construct. Vectors of the
invention are recombinant DNA vectors including, but not limited
to, plasmids, phages, phagemids, cosmids, viruses, retroviruses,
and the like, which insert a desired nucleic acid into a cell.
[0038] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid. More
specifically, operably linked means that two different nucleic
acids encoding different polypeptides have transcription induced
simultaneously. Operably linked is also intended to mean that the
linked nucleic acids can be contiguous in a single transcriptional
unit, while translation is directed from one or more ribosomal
start sites (e.g., internal ribosomal start site).
[0039] The methods of the invention also can be used in combination
with known or yet to be discovered methods of inducing the
production of recombinant proteins. By "inducing conditions" is
meant a technique to increase the relative production per cell of a
desired recombinant protein. Such techniques include cold
temperature shift, and additions of chemicals, and combinations of
any known or yet to be discovered techniques, to name just a few
examples, as well as any yet to be described and/or discovered
induction techniques. Typically, a batch or a perfusion culture of
cells at high density is induced to produce the recombinant
protein. Often, other cell processes (such as growth and division)
are inhibited so as to direct most of the cells' energy into
recombinant protein production.
[0040] Any selectable marker having complementing subunits can be
used in the methods and compositions of the invention. As used
herein, the term "subunit" when referring to a selectable marker
refers to a portion of a selectable marker. Further, a first
subunit of a selectable marker can be expressed with a second
different subunit of the same selectable marker to provide a level
of selectable activity not present in either subunit alone. A
subunit can also refer to a polypeptide having mutations that are
complemented by another mutated polypeptide that is also a
different subunit of the selectable marker.
[0041] Selectable markers that confer resistance to particular
drugs that are ordinarily toxic to an animal cell can be used in
the methods and compositions of the invention. For example, the
following are non-limiting examples of resistance selectable
markers: zeomycin (zeo); puromycin (PAC); Blasticidin S (BlaS),
GPT, which confers resistance to mycophenolic acid (Mulligan &
Berg (1981), Proc. Natl. Acad. Sci. USA 78:2072); the neomycin
resistance gene, which confers resistance to the aminoglycoside
G-418 (Colberre-Garapin et al. (1981), J. Mol. Biol. 150:1); and
hygro, which confers resistance to hygromycin (Santerre et al.
(1984), Gene 30:147).
[0042] Metabolic enzymes that confer cell survival or induce cell
death under prescribed conditions can also be used in the methods
and compositions of the inventions. Examples include but are not
limited to: dihydrofolate reductase (DHFR); herpes simplex virus
thymidine kinase (TK) (Wigler et al. (1977), Cell 11:223),
hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (Szybalska
& Szybalski (1962), Proc. Natl. Acad. Sci. USA 48:2026), and
adenine phosphoribosyltransferase (APRT) (Lowy et al. (1980), Cell
22:817), which are genes which can be employed in cells lacking TK,
HGPRT or APRT, respectively.
[0043] In a particular embodiment, dihydrofolate reductase (DHFR)
is the selectable marker used in the methods and compositions of
the present invention. DHFR can also be used for antimetabolite
resistance to methotrexate (Wigler et al. (1980), Natl. Acad. Sci.
USA 77:3567; O'Hare et al. (1981), Proc. Natl. Acad. Sci. USA
78:1527). More particularly, as used in the invention, DHFR is
divided into two subunits, F[1,2] and F[3] (from amino acids 1-105
and 106-187) and association of the subunits in a cell is promoted
by interaction domains attached to the respective subunits (see
Examples below; Pelletier et al. (1998), PNAS, 95:12141-12146).
During the selection process, cells lack DHFR activity such that
they will not grow in selection media (-GHT) without the DHFR
activity. Growth is restored upon association of the DHFR
fragments. Alternatively, cells expressing endogenous DHFR can be
used and transfectants can be selected by conferring increased
resistance to toxic levels of methotrexate.
[0044] Methotrexate can also be used in accordance with the
invention to amplify recombinant nucleic acids after selection of
-GHT sensitive cells. Selection is commonly at a concentration of
25 nM, more preferably 50 nM, even more preferably 150 nM and most
preferably 300 nM of methotrexate. The skilled artisan will
recognize that methotrexate concentrations can be as high as 500 nM
or higher to amplify recombinant nucleic acids that give resistance
to the drug, such as those described herein. Amplification using
the vectors and methods of the invention is particularly
advantageous because it has been found that in the case of
expressing a heavy and light chain, both chains are amplified in
roughly equal levels.
[0045] Selectable markers that are based on color selection can
also be used in the methods and compositions of the invention. In a
particular example, beta-galactosidase can be used (Blau et al., WO
98/44350). Fluorescence markers can also be used in the methods of
the present invention, for example, GFP has been used for clonal
selection of cells to measure protein interactions in
protein-fragment complementation assays (Remy and Michnick (1999),
Proc. Natl. Acad. Sci., 96:5394-5399). Similarly
fluorescein-conjugated methotrexate can be used to detect cells
expressing complementing DHFR fragments (Remy and Michnick (2001),
Proc. Natl. Acad. Sci., 98:7678-83). An advantage for fluorescent
markers is that this selection can be done in any animal cell type
and is not restricted to those having a deficiency in a metabolic
pathway, e.g., as with DHFR selection, or does not require a drug
sensitivity, e.g., to neomycin.
[0046] As used herein, the term "polypeptide" includes naturally
occurring or recombinantly expressed proteins, including pre- and
post-translational processing, or fragments thereof, which
typically retain secondary structure. Proteins are large molecules
with high molecular weights (from about 10,000 for small ones [of
50-100 amino acids] to more than 1,000,000 for certain forms); they
are composed of varying amounts of the same 20 amino acids, which
in the intact protein are united through covalent chemical linkages
called peptide bonds. The amino acids, linked together, form linear
unbranched polymeric structures called polypeptide chains; such
chains can contain hundreds of amino acid residues; these are
arranged in specific order for a given species of protein. The term
"peptide" includes short fragments of polypeptides or proteins, of
typically less than 20 amino acids in length.
[0047] The term "cell culture" is meant to include the growth and
propagation of cells outside of a multicellular organism or tissue.
Typically, cell culture is performed under sterile, controlled
temperature and atmospheric conditions in tissue culture plates
(e.g., 10-cm plates, 96 well plates, etc.), or other adherent
culture (e.g., on microcarrier beads) or in suspension culture such
as in roller bottles. Cultures can be grown in shake flasks, small
scale bioreactors, and/or large-scale bioreactors. A bioreactor is
a device used to culture cells in which environmental conditions
such as temperature, atmosphere, agitation, and/or pH can be
monitored and adjusted. A number of companies (e.g., ABS Inc.,
Wilmington, Del.; Cell Trends, Inc., Middletown, Md.) as well as
university and/or government-sponsored organizations (e.g., The
Cell Culture Center, Minneapolis, Minn.) offer cell culture
services on a contract basis.
[0048] Optimal periods for which the cultures are in contact with
agents that select for the selectable activity are for longer than
the typical period for one normal growth cycle (e.g., for Chinese
hamster ovary cells (CHO cells), where one growth cycle has been
reported to be approximately 20-22 hours (Rasmussen et al. (1998),
Cytotechnology, 28:31-42)). As such, in one embodiment, the
cultures comprise selectable conditions, e.g., drugs, metabolites,
or color substrates, preferably for at least about one day, more
preferably for at least about 3 days, and even more preferably for
at least about 7 days.
[0049] A wide variety of animal cell lines suitable for growth in
culture are available from, for example, the American Type Culture
Collection (ATCC, Manassas, Va.) and NRRL (Peoria, Ill.). Some of
the more established cell lines typically used in the industrial or
academic laboratory include CHO, VERO, BHK, HeLa, Cos, CV1, MDCK,
293, 3T3, PC12, mycloma (e.g., NSO), and WI38 cell lines, to name
but a few examples. In other embodiments, non-animal cell lines can
be used in the methods of the invention, for example, plant cell
lines, insect cell lines (e.g., sf9), yeast cells or bacterial
cells such as E. coli.
[0050] In particular embodiments, the dihydrofolate reductase
(DHFR)-deficient mutant cell line (Urlaub et al. (1980), Proc Natl
Acad Sci USA 77:4216-4220), DXB11 and DG-44, are the CHO host cell
lines of choice because the efficient DHFR selectable and
amplifiable gene expression system allows high level recombinant
protein expression in these cells (Kaufman R. J. (1990), Meth
Enzymol 185:527-566). In addition, these cells are easy to
manipulate as adherent or suspension cultures and exhibit
relatively good genetic stability. In addition, new animal cell
lines can be established using methods well known by those skilled
in the art (e.g., by transformation, viral infection, and/or
selection).
[0051] As noted above, a variety of host-expression vector systems
can be utilized to express the heteromeric complexes of the
invention. Where the heteromeric complex is soluble, the peptide or
polypeptide can be recovered from the culture, i.e., from the host
cell in cases where the heteromeric complexes are not secreted, and
from the culture media in cases where the heteromeric complexes are
secreted by the cells. However, the expression systems also
encompass engineered host cells that express the heteromeric
complexes anchored in the cell membrane.
[0052] Purification or enrichment of the heteromeric complexes from
such expression systems can be accomplished using appropriate
detergents and lipid micelles and methods well known to those
skilled in the art. However, such engineered host cells themselves
can be used in situations where it is important not only to retain
the structural and functional characteristics of the heteromeric
complexes, but also to assess biological activity, e.g., in drug
screening assays.
[0053] The protein expressed by the methods of the invention can be
collected. In addition the protein can be purified, or partially
purified, from such culture or component (e.g., from culture medium
or cell extracts or bodily fluid) using known processes. The phrase
"partially purified" means that some fractionation procedure, or
procedures, have been carried out, but that more polypeptide
species (at least 10%) than the desired protein is present. By
"purified" is meant that the protein is essentially homogeneous,
i.e., less than 1% contaminating proteins are present.
Fractionation procedures can include but are not limited to one or
more steps of filtration, centrifugation, precipitation, phase
separation, affinity purification, gel filtration, ion exchange
chromatography, size exclusion chromatography (SEC), hydrophobic
interaction chromatography (HIC; using such resins as phenyl ether,
butyl ether, or propyl ether), HPLC, or some combination of
above.
[0054] The invention also optionally encompasses further
formulating the proteins. By the term "formulating" is meant that
the proteins can be buffer exchanged, sterilized, bulk-packaged
and/or packaged for a final user. For purposes of the invention,
the term "sterile bulk form" means that a formulation is free, or
essentially free, of microbial contamination (to such an extent as
is acceptable for food and/or drug purposes), and is of defined
composition and concentration.
[0055] The term "sterile unit dose form" means a form that is
appropriate for the customer and/or patient administration or
consumption. Such compositions can comprise an effective amount of
the protein, in combination with other components such as a
physiologically acceptable diluent, carrier, or excipient. The term
"physiologically acceptable" means a non-toxic material that does
not interfere with the effectiveness of the biological activity of
the active ingredient(s).
[0056] The invention having been described, the following examples
are offered by way of illustration and not limitation.
EXAMPLES
Example 1
Construction of DHFR Complementation Vectors
[0057] Construction of recombinant vectors expressing subunits of a
selectable marker was performed as follows. Dihydrofolate reductase
(DHFR) was chosen as the selectable marker to be used in the
following experiments. Previous work has shown that due to its
modular three-dimensional structure, DHFR can be broken into two
parts and when expressed as a fusion protein having an interaction
domain, the subunits can then be reassociated in a cell providing
selectable activity. See FIG. 1 for a general overview of the order
of the various nucleic acids described in one embodiment of the
invention.
[0058] Sequential polymerase chain reaction (PCR) SOEing was
utilized to generate nucleic acids suitable for cloning into
expression vectors that encode a fusion of a leucine zipper
interaction domain fused to a linker polypeptide fused to a subunit
of DHFR. Briefly, PCR SOEing is splicing of genes by overlap
extension for recombining DNA molecules at junctions without the
use of restriction endonucleases or ligase (Methods in Molecular
Biology, Vol. 15, "PCR protocols: Current Methods and
Applications," and "Chapter 25: In Vitro Recombination," Editor. B.
A. White, 1993, Humana Press, Inc., Totowa, N.J.; and Mutagenesis
of DNA, Robert M. Horton, pp. 251-261).
[0059] Fragments from the genes that are to be recombined are
generated in separate polymerase chain reactions (PCRs). The
primers are designed so that the ends of the products contain
complementary sequences, such as a common restriction site, i.e.,
BamH1. When these PCR products are subsequently mixed, denatured,
and reannealed, the strands having the matching sequences at their
3' ends overlap and act as primers for each other (Horton et al.
(1989), Gene, 77(1):61-8).
[0060] The primers used in the present example are as follows:
TABLE-US-00001 1 JM238
5'-ATATCTCGAGATCCGTGCCATCATGTCTGACCGTATGAAAC-3' JM239
5'-GCCACCGCCGGATCCACCGCCACCCCGCTCGCCTACCAG CTTTT-3' JM240
5'-GGTGGATCCGGCGGTGGCGGCGGCTCAATGGTTCGACCA TTGAAC-3' PDHFR106
5'-ATATCAATTGTTATTCCGGTTGTTCAAT-AAGTC-3' JM242
5'-GTGGATCCGGCGGTGGCGGCGGCTCATTGGCAAGTAAAG TAGACA-3' JM244
5'-ATATCAATTGTTAGTCTTTCTTCTCGTAGAC-TT-3'
[0061] The following strategy was employed to create a nucleic acid
encoding a leucine zipper interaction domain fused in frame to a
linker fused in frame to DHFR amino acids 1-105. The first PCR
reaction amplified the yeast GCN4 leucine zipper (Lz) using primers
JM238 (SEQ ID NO:1) and JM239 (SEQ ID NO:2). All PCR reactions
utilized Roche Expand High Fidelity PCR system, which included all
of the required reagents, except for 10 mM dNTP's, which are
commercially available. Thermal cycle conditions (PCR condition 1)
were as follows:
[0062] 94 .degree. C. for 5 min
[0063] 94 .degree. C. for 30 sec - - -
[0064] 37 .degree. C. for 30 sec vertline. - - - 25 cycles
[0065] 72 .degree. C. for 30 sec - - -
[0066] 72 .degree. C. for 7 minutes
[0067] 4 .degree. C. (hold).
[0068] The JM238 primer has a Xho1 site at the 5' terminus and the
JM239 primer has a BamH1 site at the 5' terminus. At the same time,
primers JM240 (SEQ ID NO:3) and PDHFR106 (SEQ ID NO:4) were used to
PCR amplify the DHFR subunit encoding amino acids 1-105 of DHFR
(SEQ ID NO:5) [same as above, except with a 1 minute duration at 94
.degree. C. and at 72 .degree. C. for the 25 cycles (PCR condition
2)]. JM240 has a BamH1 site at its 5' terminus and PDHFR106 has a
Mfe1 site at its 5' terminus. Each respective PCR product was gel
purified using standard gel purification techniques, and a second
PCR reaction was performed using PCR condition 2. The resulting
product was then cloned into a pGEM-T vector (Promega) and
sequenced.
[0069] A similar strategy was employed to create a nucleic acid
encoding a leucine zipper fused in frame to a linker fused in frame
to DHFR amino acids 106-187. The first PCR reaction amplified the
yeast GCN4 leucine zipper using primers JM238 (SEQ ID NO:1) and
JM239 (SEQ ID NO:2) using PCR condition 1. The JM238 primer has a
Xho1 site at the 5' terminus and the JN239 primer has a BamH1 site
at the 5' terminus. At the same time, primers JM242 (SEQ ID NO:6)
and JM244 (SEQ ID NO:7) were used to PCR amplify the DHFR subunit
encoding amino acids 106-187 of DHFR (SEQ ID NO:5) using PCR
Condition 1. JM242 has a BamH1 site at its 5' terminus and JM244
has a Mfe1 site at its 5' terminus. Each respective PCR product was
gel purified using standard gel purification conditions, and a
second PCR reaction was performed using PCR Condition 1. The
resulting product was then cloned into a pGEM-T vector (Promega)
and sequenced.
[0070] Once the correct sequences were verified, the Lz-linker-DHFR
1-105 (363 bp) and Lz-linker-DHFR 106-187 (343 bp) fragments were
cut from the pGEM-T vector with Xho1 and Mfe1 and the nucleic acids
were gel purified. The vector pDC317 was digested with Not1 and
Xho1 and the 558 bp internal ribosomal entry site (IRES) element
was recovered by gel purification. Since Xho1 is not a unique site
on pDC317, a triple ligation between the Not1/Xho1 IRES element,
the Xho1/Mfe1 Lz-linker-DHFR 1-105 and Lz-linker-DHFR 106-187 was
performed in pDC317 and isolates were tested and confirmed to have
successful ligation by restriction digest.
[0071] The antibody (Ab) heavy and light chain genes, encoding an
antibody which specifically recognizes the murine interleukin
4-receptor (IL4R), were each cloned into the vectors prepared as
described above. Anti-IL4R heavy chain (HC) was digested with Not1
and Sal1. From this digestion, a 1413 bp fragment was isolated by
gel purification. Likewise, light chain (LC) of the anti-IL4R
antibody, was cut from a vector with the same enzymes and the 736
bp light chain fragment was gel purified. The Lz-LINKER-DHFR-pDC317
vectors (both 1-105 and 106-187) were also cut with Not1 and Sal1
and the heavy and light chains were cloned into the corresponding
expression vectors. The following combinations were obtained:
[0072] IL4R Ab HC: Lz-linker-DHFR 1-105 pDC317
[0073] IL4R Ab LC: Lz-linker-DHFR 106-187 pDC317
[0074] IL4R Ab LC: Lz-linker-DHFR 1-105 pDC317
[0075] IL4R Ab HC: Lz-linker-DHFR 106-187 pDC317
Example 2
Construction of a Second Set of DHFR Complementation Vectors
[0076] Construction of a second set of recombinant vectors
expressing subunits of a selectable marker was performed as
follows. Bicistronic vectors containing the internal ribosomal
entry site (IRES) are based on pED4 (Kaufman (1991), Nuc Acids Res.
19(16):4485-4490). The base vector, pDC318, is a derivative of
pG2.1 (Aldrich (1998), Cytotechnology, 28:9-17) containing a
truncated 600 base pair portion of the expression augmenting
sequence element (EASE). pDC317 is a similar vector which contains
the larger 3.6 kilobase EASE. PCR was used to fuse a GCN4 leucine
zipper (LZ) and flexible linker to two separate fragments of the
selectable marker dihydrofolate reductase (DHFR). The first
fragment extends from amino acids 1-105 and the second fragment
includes amino acids 106-187. The final PCR products were then
cloned into pDC317 or pDC318 just downstream of the IRES
element.
[0077] The IRES element was modified based on the pED3 vector
created by Davies et al., to enhance translation of the
LZ-linker-DHFR fragments (Davies (1992), J. Virol.,
66(4):1924-1932). This change was incorporated into the IRES
LZ-linker-DHFR fragments in pDC317 via PCR using the primer JM256
(5'-GATAATATGGCCACAACCATGTCTGACCGTATGAAACA-3'). The underlined ATG
marks the transition from the pED3 IRES to the LZ. The fragments
were subsequently subcloned into pGEM-T (Invitrogen) containing the
full length IRES sequence. The pED3 IRES LZ-linker-DHFR 1-105 and
106-187 fragments were then cloned into pDC318 in order to create
pDC321 and pDC322 or pDC317 to create pDC323 or pDC324,
respectively.
[0078] The murine anti-IL4R antibody chains were cloned into the
multiple cloning sites of pDC321 and pDC322, just upstream of the
pED3 IRES to create pDC321 LC, pDC321 HC, pDC322 LC, and pDC322 HC.
Similarly, the heavy and light chains were cloned into the multiple
cloning sites of pDC323 and pDC324 to create pDC323 LC, pDC323 HC,
pDC324 LC, and pDC324 HC.
Example 3
Transfection and Selection
[0079] Transfection of the above vectors was performed into DHFR
deficient CHO cell line. Standard transfection protocols were used.
Cells were incubated at 37 .degree. C. until in log phase, and
transfected with an appropriate concentration of purified plasmids
with 150 uL Lipofectamine (Gibco BRL) as recommended by the
manufacturer. The Lipofectamine (Invitrogen) transfections were
performed with a 6:6:1 ratio of either pDC321 LC:pDC322 HC:pCDNA3
(Invitrogen), pDC321 HC:pDC322 LC:pCDNA3, pDC323 LC:pDC323
HC:pCDNA3, or pDC324 HC:pDC324 LC:pCDNA3.
[0080] Initial selection was performed in shake flasks in non-DHFR
selection media plus G418 with recovery of up to 70% viability,
followed by selection in DHFR selection media lacking glycine,
hypoxanthine and thymidine (-GHT) with recovery of up to 90%
viability. Pools established following G418 and -GHT selection were
exposed to 25 nM methotrexate in an attempt to amplify the antibody
chains and thereby enhance antibody production in the pools. Both
the unamplified and amplified pools demonstrate stable production
of antibody during this time period.
[0081] For cloning, transfected cells were diluted and plated
directly in 96 well plates in -T growth media. No pre-selection in
G418 or -GHT media was needed.
[0082] For the pDC321 and pDC322 vectors, the unamplified pool
maintained a qP of 1.mu.g/10.sup.6 cells/day. An increase in the qP
for the amplified pool correlates to an increase in viability after
recovery of the cells from selection. The qP of the amplified pool
ranged from 8-18 mu.g/10.sup.6 cells/day, indicating an 8-18 fold
increase in antibody production compared to the unamplified pool.
Five independent pools have been evaluated and found to exhibit
similar expression levels. In addition to analysis of the pools,
two of the clones were scaled up to shake flasks, amplified with 25
nM methotrexate, and evaluated for expression. Expression was
similar to the results described for the pools.
[0083] For the pDC323 and pDC324 vectors, namely the vectors with
the 3.6 kilobase EASE element, the unamplified pool maintained a qP
of about 5.mu.g/10.sup.6 cells/day.
Example 4
Expression of Antibodies From the Complementation Vectors
[0084] Unamplified and amplified pools were then evaluated under
simulated production conditions. A shift to lower temperature,
e.g., 31 .degree. C. leads to higher titers. Induction was
performed in 20 mL shake flask cultures shifted to the lower
temperature. Antibody titers were measured by ELISA. An unamplified
pool produced 80 .mu.g/mL of antibody in 9 days, while maintaining
a final viability of 65.8%. Three independent pools were analyzed.
The amplified pools produced an average of 407.8 .mu.g/mL of
antibody in 10 days, with an average final viability of 47.2%. The
specific productivity's (qP) of the pools ranged from
10-20.mu.g/10.sup.6 cells/day.
Example 5
Western Blot Analyses of Antibodies
[0085] Antibodies expressed from the cells transfected with the
pDC321 or pDC322 vectors were isolated using standard methods,
purified and run on denaturing as well as native gels. A 4-20% Tris
Glycine gel of 1 mm, 10 well was run (Invitrogen, Cat. No. E6025)
at 125 V for about 2 hours. The samples were not heated and were
suspended in 2.times.Native Gel Tris Glycine Sample buffer
(Invitrogen, Cat. No. LC2673) with (reduced) or without
(non-reduced) 5% beta mercaptoethanol (2.5% final concentration).
The sample buffer was 1.times.SDS running buffer. The gels were
non-denaturing as there was no SDS or reducing agents in the gel
itself, only the sample buffers as indicated.
[0086] Transfer to nitrocellulose (Nitrocellulose Membrane Filter
Paper Sandwich, Invitrogen, LC2001) was performed for 45 minutes at
33 V. The membranes were blocked overnight at 4 C in 5% nonfat dry
milk in PBST (0.1% Tween 20) or "blotto" solution. The blotting
grade affinity purified goat anti-mouse IgG (H+L) HRP conjugate
antibody (Bio-Rad, Cat No. 170-6516) was diluted 1:2000 in blotto
solution and applied to the blots for 2.5 hours. The blots were
then rinsed 5.times.for 5 minutes each in PBST and developed for 30
seconds with the ECL western blotting detection reagents (Amersham
Pharmacia Biotech, Cat. No. RPN2106).
[0087] The samples were all derived from supernatants from passage
90 of the cultures. Specifically, the purified antibody was taken
from an induced, unamplified culture and purified on a protein G
column. The 0 nM supernatant was concentrated to 10.times.on a
Millipore concentrator (UFV2BCC40) at 3000 rpm, since its
concentration by Mu FC ELISA was lower than the other samples. The
non-reduced gel showed that the antibody with heavy and light chain
is present in all cases and that there is very little free light
chain or dimerized light chain in the 25 and 50 nM supernatants,
while none was apparent in the 0 nM supernatants and there is some
dimerized light chain in the purified Ab.
[0088] The heavy chains and light chains were present in all of the
supernatants in equal proportions as the purified antibody, and
significantly, there is considerably more total antibody in the
methotrexate amplified supernatants, consistent with the results of
Example 3.
[0089] Antibodies were also purified from cells transfected with
the pDC323 or pDC324 vectors. The antibodies from the supernatants
of the cells had equal proportions of heavy and light chains on the
non-reduced gel, with very little free light chain or dimerized
light chain.
Example 6
FACS Analysis of DHFR Expression
[0090] Fluorescence activated cell sorting (FACS) analysis was
employed in order to verify a concurrent amplification of DHFR
expression following methotrexate exposure. Unamplified and
amplified pools were labeled with fluorescein labeled methotrexate,
which binds DHFR, and analyzed on a FACS Calibur analyzer.
Unlabeled, untransfected CS9 cells were used as a control. Both
unamplified and amplified pools show DHFR activity, as expected. A
larger degree of fluorescence is observed in the 25 nM methotrexate
amplified pool as compared to the 0 nM methotrexate unamplified
pool. This verifies that amplification of antibody expression
correlates with an amplification of DHFR expression.
Equivalents and References
[0091] The present invention is not to be limited in scope by the
specific embodiments described herein that are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
[0092] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference.
Sequence CWU 1
1
8 1 41 DNA Artificial PCR Primer 1 atatctcgag atccgtgcca tcatgtctga
ccgtatgaaa c 41 2 44 DNA Artificial PCR Primer 2 gccaccgccg
gatccaccgc caccccgctc gcctaccagc tttt 44 3 45 DNA Artificial PCR
Primer 3 ggtggatccg gcggtggcgg cggctcaatg gttcgaccat tgaac 45 4 33
DNA Artificial PCR Primer 4 atatcaattg ttattccggt tgttcaataa gtc 33
5 187 PRT Mus musculus 5 Met Val Arg Pro Leu Asn Cys Ile Val Ala
Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Leu Pro
Trp Pro Pro Leu Arg Asn Glu Phe 20 25 30 Lys Tyr Phe Gln Arg Met
Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 40 45 Asn Leu Val Ile
Met Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 55 60 Asn Arg
Pro Leu Lys Asp Arg Ile Asn Ile Val Leu Ser Arg Glu Leu 65 70 75 80
Lys Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser Leu Asp Asp 85
90 95 Ala Leu Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys Val Asp
Met 100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala
Met Asn Gln 115 120 125 Pro Gly His Leu Arg Leu Phe Val Thr Arg Ile
Met Gln Glu Phe Glu 130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp
Leu Gly Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val
Leu Ser Glu Val Gln Glu Glu Lys Gly Ile 165 170 175 Lys Tyr Lys Phe
Glu Val Tyr Glu Lys Lys Asp 180 185 6 45 DNA Artificial PCR Primer
6 gtggatccgg cggtggcggc ggctcattgg caagtaaagt agaca 45 7 33 DNA
Artificial PCR Primer 7 atatcaattg ttagtctttc ttctcgtaga ctt 33 8
38 DNA Artificial PCR Primer 8 gataatatgg ccacaaccat gtctgaccgt
atgaaaca 38
* * * * *