U.S. patent application number 12/542792 was filed with the patent office on 2011-09-29 for methods for protein expression in mammalian cells in serum-free medium.
This patent application is currently assigned to IMMUNOMEDICS, INC.. Invention is credited to Chien-Hsing Chang, David M. Goldenberg, Diane Nordstrom, Edmund A. Rossi.
Application Number | 20110236404 12/542792 |
Document ID | / |
Family ID | 46331815 |
Filed Date | 2011-09-29 |
United States Patent
Application |
20110236404 |
Kind Code |
A1 |
Goldenberg; David M. ; et
al. |
September 29, 2011 |
Methods for Protein Expression in Mammalian Cells in Serum-Free
Medium
Abstract
Disclosed are compositions and methods for increasing the
longevity of a cell culture and permitting the increased production
of proteins, preferably recombinant proteins, such as antibodies,
peptides, enzymes, growth factors, interleukins, interferons,
hormones, and vaccines. Cells transfected with an
apoptosis-inhibiting gene or vector, such as a triple mutant Bcl-2
gene, can survive longer in culture, resulting in extension of the
state and yield of protein biosynthesis. Such transfected cells
exhibit maximal cell densities that equal or exceed the maximal
density achieved by the parent cell lines. Transfected cells can
also be pre-adapted for growth in serum-free medium, greatly
decreasing the time required to obtain protein production in
serum-free medium. In certain methods, the pre-adapted cells can be
used for protein production following transfection under serum-free
conditions. In preferred embodiments, the cells of use are SpESF or
SpESF-X cells.
Inventors: |
Goldenberg; David M.;
(Mendham, NJ) ; Chang; Chien-Hsing; (Downingtown,
PA) ; Rossi; Edmund A.; (Woodland Park, NJ) ;
Nordstrom; Diane; (Rockaway, NJ) |
Assignee: |
IMMUNOMEDICS, INC.
Morris Plains
NJ
|
Family ID: |
46331815 |
Appl. No.: |
12/542792 |
Filed: |
August 18, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11877728 |
Oct 24, 2007 |
7608425 |
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12542792 |
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11487215 |
Jul 14, 2006 |
7537930 |
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11877728 |
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11187863 |
Jul 25, 2005 |
7531327 |
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11487215 |
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60590349 |
Jul 23, 2004 |
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Current U.S.
Class: |
424/184.1 ;
435/183; 435/325; 435/326; 435/328; 530/350; 530/351; 530/387.1;
530/387.3; 530/397; 530/399 |
Current CPC
Class: |
C12N 2510/02 20130101;
C12P 21/02 20130101; C12N 5/0694 20130101; A61K 2039/505 20130101;
C07K 14/4747 20130101; C12N 2501/48 20130101; C07K 16/3007
20130101; C12N 2740/16043 20130101; C07K 16/00 20130101; C07K 16/18
20130101; C12N 2500/90 20130101; C07K 2317/31 20130101; C07K
2317/24 20130101; A61P 37/04 20180101; C07K 16/2803 20130101 |
Class at
Publication: |
424/184.1 ;
435/325; 435/326; 435/328; 435/183; 530/387.3; 530/387.1; 530/350;
530/351; 530/399; 530/397 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 5/10 20060101 C12N005/10; C12N 9/00 20060101
C12N009/00; C12P 21/00 20060101 C12P021/00; C07K 2/00 20060101
C07K002/00; C07K 14/54 20060101 C07K014/54; C07K 14/555 20060101
C07K014/555; C07K 14/575 20060101 C07K014/575; C07K 14/52 20060101
C07K014/52; C07K 14/475 20060101 C07K014/475; C07K 14/535 20060101
C07K014/535; C07K 14/505 20060101 C07K014/505; C07K 14/545 20060101
C07K014/545; C07K 14/55 20060101 C07K014/55; C07K 14/56 20060101
C07K014/56; C07K 14/565 20060101 C07K014/565; C07K 14/57 20060101
C07K014/57; C07K 14/485 20060101 C07K014/485; A61P 37/04 20060101
A61P037/04 |
Claims
1. A mammalian cell line comprising a gene encoding a mutant Bcl-2
protein, said protein comprising T69E, S70E and S87E amino acid
substitutions, the cell line pre-adapted to grow in serum-free
medium, the cell line capable of transfection under serum-free
conditions with one or more expression vectors expressing a protein
of interest and of producing said protein of interest under
serum-free conditions.
2. The cell line of claim 1, wherein said cell line is transfected
with one or more expression vectors expressing a protein of
interest.
3. The cell line of claim 2, wherein the transfected cell line is
capable of producing said protein of interest under serum-free
conditions without the need for further adaptation to serum-free
conditions after transfection.
4. The cell line of claim 2, wherein the protein of interest is
selected from the group consisting of an antibody, a humanized
antibody, a chimeric antibody, a human antibody, a bispecific
antibody, a multispecific antibody, a multivalent antibody and an
antigen-binding antibody fragment.
5. The cell line of claim 1, wherein the cell line is frozen.
6. The cell line of claim 1, wherein the pre-adapted cell line may
be frozen and thawed prior to transfection with one or more
expression vectors expressing a protein of interest.
7. The cell line of claim 2, wherein said transfected cell line
produces the protein of interest at a yield of at least 150 mg
protein/mL of growth medium.
8. The cell line of claim 7, wherein said transfected cell line
produces the protein of interest at a yield of at least 200 mg
protein/mL of growth medium.
9. The cell line of claim 2, wherein said cell line is stably
transfected with the one or more expression vectors.
10. The cell line of claim 1, wherein said cell line is a myeloma
cell line.
11. The cell line according to claim 10, wherein said myeloma cell
line is an Sp2/0 cell line or derivative thereof.
12. A protein produced by a cell line comprising a gene encoding a
mutant Bcl-2 protein, said mutant Bcl-2 protein comprising T69E,
S70E and S87E amino acid substitutions, the cell line pre-adapted
to grow in serum-free medium, the cell line capable of transfection
under serum-free conditions with one or more expression vectors
expressing a protein of interest and of producing said protein of
interest under serum-free conditions, wherein said cell line is
transfected with one or more expression vectors expressing a
protein of interest according to claim 2.
13. The protein of claim 12, wherein the protein is selected from
the group consisting of an antibody, a humanized antibody, a
chimeric antibody, a human antibody, a bispecific antibody, a
multispecific antibody, a multivalent antibody and an
antigen-binding antibody fragment.
14. The protein of claim 12, wherein the protein is selected from
the group consisting of a growth factor, a hormone, a cytokine, a
chemokine, an interleukin, an interferon, a vaccine and an
enzyme.
15. The protein of claim 14, wherein the protein is selected from
the group consisting of EPO, G-CSF, GM-CSF, EGF, VEGF,
thrombopoietin, IL-1 through IL-31, interferon-alpha,
interferon-beta and interferon-gamma.
18. The protein of claim 12, wherein the protein is produced in
serum-free medium.
19. The protein of claim 18, wherein the cell line is transfected
under serum-free conditions and the protein is produced in
serum-free medium, without further adaptation to serum-free
conditions after transfection.
20. The protein of claim 12, wherein the protein is produced by the
transfected cell line at a yield of at least 150 mg protein/mL of
growth medium.
21. The protein of claim 12, wherein the protein is produced by the
transfected cell line at a yield of at least 200 mg protein/mL of
growth medium.
22. The protein of claim 12, wherein said cell line is a myeloma
cell line.
23. The protein of claim 22, wherein said myeloma cell line is an
Sp2/0 cell line or derivative thereof.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 11/877,728, filed Oct. 24, 2007, which was
continuation-in-part of U.S. patent application Ser. No. 11/487,215
(now issued U.S. Pat. No. 7,537,930), filed Jul. 14, 2006, which
was a continuation-in-part of U.S. patent application Ser. No.
11/187,863 (now issued U.S. Pat. No. 7,531,327), filed Jul. 25,
2005, which claimed the benefit under 35 U.S.C. .sctn.119(e) of
provisional U.S. Patent Application No. 60/590,349, filed Jul. 23,
2004, each of which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] Various embodiments of the present invention concern methods
and compositions for increasing longevity and/or protein yield from
a cell line. In particular embodiments, the cell line may be a
hybridoma cell line that produces antibodies, antibody fragments or
other therapeutic proteins. In more particular embodiments, the
methods may comprise transfecting a cell line with one or more
genes, such as genes encoding E6, E7 and/or Bcl-2 or related
proteins. Such proteins are not limited to their native sequence,
but may include one or more substituted amino acids. Other
embodiments concern mammalian cell lines that are capable of
growth, transfection and protein production in serum-free medium.
Such cell lines may be used in methods of protein production, by
transfecting the cell line with an expression vector that expresses
a heterologous protein. In preferred embodiments, the cell line may
be transfected in serum-free medium, providing considerable time
savings in avoiding having to adapt the transfected cell line for
serum-free growth and protein production. A more preferred
embodiment concerns the SpESF cell line, produced by transfecting
the Sp2/0 cell line with a triple mutant Bcl-2 gene (T69E, S70E,
S87E) followed by adaptation for growth in serum-free medium to
produce SpESF. The SpESF cell line may be grown, transfected and
produces proteins in serum-free medium. SpESF may be further
adapted by exposure to stressful growth conditions to produce
highly robust, serum-free cell lines such as SpESF-X.
BACKGROUND OF THE INVENTION
[0003] In 2006, biopharmaceuticals, including monoclonal antibodies
(mAbs) and other recombinant proteins, accounted for nearly half of
all drugs in the development phase and a quarter of drugs in
preclinical and clinical trials (Walsh, 2006, Nat Biotechnol
24:769-776). As the demand for biopharmaceuticals continues to
increase, there is a commensurate need for better bioproduction
vehicles. Although non-mammalian production systems, such ds
cultured Escherichia coli, yeast, plant and insect cell lines,
often result in high yields, cultured mammalian host cell lines are
preferred for production of many humanized proteins that require
post-translational modifications to preserve their bioactivity.
[0004] Culturing cells in vitro, especially in large bioreactors,
has been the basis of the production of numerous biotechnology
products, and involves the elaboration by these cells of protein
products into the support medium, from which these products are
isolated and further processed prior to use clinically. The
quantity of protein production over time from the cells growing in
culture depends on a number of factors, such as, for example, cell
density, cell cycle phase, cellular biosynthesis rates of the
proteins, condition of the medium used to support cell viability
and growth, and the longevity of the cells in culture (i.e., how
long before they succumb to programmed cell death, or apoptosis).
Various methods of improving the viability and lifespan of the
cells in culture have been developed, together with methods of
increasing productivity of a desired protein by, for example,
controlling nutrients, cell density, oxygen and carbon dioxide
content, lactate dehydrogenase, pH, osmolarity, catabolites, etc.
For example, increasing cell density can make the process more
productive, but can also reduce the lifespan of the cells in
culture. Therefore, it may be desirous to reduce the rate of
proliferation of such cells in culture when the maximal density is
achieved, so as to maintain the cell population in its most
productive state as long as possible. This results in increasing or
extending the bioreactor cycle at its production peak, elaborating
the desired protein products for a longer period, and this results
in a higher yield from the bioreactor cycle.
[0005] Many different approaches have been pursued to increase the
bioreactor cycle time, such as adjusting the medium supporting cell
proliferation, addition of certain growth-promoting factors, as
well as inhibiting cell proliferation without affecting protein
synthesis. One particular approach aims to increase the lifespan of
cultured cells via controlling the cell cycle by use of genes or
antisense oligonucleotides to affect cell cycle targets, whereby a
cell is induced into a pseudo-senescence stage by transfecting,
transforming, or infecting with a vector that prevents cell cycle
progression and induces a so-called pseudo-senescent state that
blocks further cell division and expands the protein synthesis
capacity of the cells in culture; in other words, the
pseudo-senescent state can be induced by transfecting the cells
with a vector expressing a cell cycle inhibitor (Bucciarelli et
al., U.S. Patent Appl. 2002/0160450 A1; WO 02/16590 A2): The latter
method, by inhibiting cell duplication, seeks to force cells into a
state that may have prolonged cell culture lifetimes, as described
by Goldstein and Singal (Exp Cell Res 88, 359-64, 1974; Brenner et
al., Oncogene 17:199-205, 1998), and may be resistant to apoptosis
(Chang et al., Proc Natl Acad Sci USA 97, 4291-6, 2000; Javeland et
al., Oncogene 19, 61-8, 2000).
[0006] Still another approach involves establishing primary,
diploid human cells or their derivatives with unlimited
proliferation following transfection with the adenovirus E1 genes.
The new cell lines, one of which is PER.C6 (ECACC deposit number
96022940), which expresses functional Ad5 E1A and E1B gene
products, can produce recombinant adenoviruses, as well as other
viruses (e.g., influenza, herpes simplex, rotavirus, measles)
designed for gene therapy and vaccines, as well as for the
production of recombinant therapeutic proteins, such as human
growth factors and human antibodies (Vogels et al., WO 02/40665
A2).
[0007] Other approaches have focused on the use of caspase
inhibitors for preventing or delaying apoptosis in cells. See, for
example, U.S. Pat. No. 6,586,206. Still other approaches have tried
to use apoptosis inhibitors such as members of the Bcl-2 family for
preventing or delaying apoptosis in cells. See Arden et al.,
Bioprocessing Journal, 3:23-28 (2004). These approaches have
yielded unpredictable results. For example, in one study,
expression of Bcl-2 increased cell viability but did not increase
protein production. (See Tey et al., Biotechnol. Bioeng. 68:31-43,
2000.) Another example disclosed overexpression of Bcl-2 proteins
to delay apoptosis in CHO cells, but Bcl-xL increased protein
production whereas Bcl-2 decreased protein production (see
WO03/083093). A further example disclosed experiments using
expression of Bcl-2 proteins to prolong the survival of Sp2/0-Ag14
(ATCC #CRL-1581, hereafter referred to as Sp2/0) cells in cultures.
However, the cell density of the Bcl-2 expressing clones were 20 to
50% lower than that of their parental cultures, raising concerns
for their practical application in biopharmaceutical industry (see
WO03/040374; U.S. Pat. No. 6,964,199).
[0008] It is apparent, therefore, that improved host cells for high
level expression of recombinant proteins and methods for reliably
increasing recombinant protein production, in particular the
production of antibodies and antibody fragments, multispecific
antibodies, fragments and single-chain constructs, peptides,
enzymes, growth factors, hormones, interleukins, interferons, and
vaccines, in host cells are needed in the art. A need also exists
for cell lines that are pre-adapted to grow in serum-free or
serum-depleted medium, that can be transfected with expression
vectors under serum free conditions and used for protein production
without going through a lengthy adaptation period before serum-free
growth and protein production.
SUMMARY OF THE INVENTION
[0009] The present invention fulfills unresolved needs in the art
by providing improved host cells and methods to increase the
longevity and/or recombinant protein yields of a cell culture. In
some embodiments, the methods involve introducing into cells agents
that inhibit senescence or that promote cell survival, e.g.,
anti-apoptotic agents. The use of such agents preferentially
increases the lifespan and viability of cells in culture used for
the production of a desired recombinant protein, concomitantly
increasing the productivity of such cells in culture, and thereby
the optimal yield of the desired protein. Preferably, the apoptosis
inhibitors used in the method of the present invention include but
are not limited to Bcl-2 and its family members. Alternately, the
longevity and recombinant protein yields of a cell clone can be
improved by introducing into the cell agents that down-regulate the
level of intracellular pro-apoptotic proteins, such as p53 and Rb,
or up-regulate intracellular anti-apoptotic proteins, such as
Bcl-2.
[0010] Preferably, the regulatory agents used in the claimed
methods include, but are not limited to, human papillomavirus type
16 (HPV-16) oncoproteins E6 and E7, anti-apoptosis protein Bcl-2
and combinations thereof. Additionally, caspase inhibitors, as
described herein, may also contribute to blocking or reducing
apoptosis, thus increasing cell survival and increasing the
production of recombinant proteins by said cells in culture. A
further class of anti-apoptotic agents that can be used in these
cultures to enhance production of recombinant proteins includes
certain members of the cytokine type I superfamily, such as
erythropoietin (EPO). EPO, as a prototype molecule of this class,
is a major modifier of apoptosis of multiple cell types, not just
erythrocytes, and thus has more general cytoprotective functions,
such as in endothelial cells, myocardial cells, tubular epithelial
cells of the kidney, skin, and neurons [cf. review by P. Ghezzi and
M. Brines, Cell Death and Differentiation 11 (suppl. 1), s37-s44,
July 2004]. In alternative embodiments, host cell lines may be
transfected with expression vectors comprising EPO and/or EPOR,
instead of supplying EPO externally. (See, e.g., Levin et al., FEBS
Lett. 427:164-70, 1998.)
[0011] In various embodiments, the cell lines that have been
transfected with one or more regulatory agents, such as HPV-16, E6,
E7 and/or Bcl-2 may be pre-adapted for growth in serum-free medium.
Such pre-adapted cell lines, including but not limited to the SpESF
cell line (see Examples below), are able to undergo further
transfection, under serum-free conditions, with one or more
expression vectors, thus allowing expression and protein production
under serum-free conditions without extensive time required for
adaption to serum-free growth. This surprising result allows
protein production under serum free or low serum conditions,
providing significant savings on medium cost. At the same time,
transfection and protein production under serum-free conditions
saves substantial time needed for serum-free adaptation that is
required when using standard mammalian cell lines, which are only
transfectable under serum-rich conditions and require an additional
6 to 12 months to adapt to serum-free protein production. Certain
pre-adapted cell lines, such as SpESF, may be "banked" or stored
frozen and then thawed before transfection with an expression
vector to produce recombinant proteins. The ability to bank such
pre-adapted cell lines provides significant savings in time, cost
and efficiency of protein production.
[0012] In other embodiments, transfected and pre-adapted cell
lines, such as SpESF, may be further adapted by exposure to
stressful growth conditions, such as over-growing the cells until
viability is reduced to about 50-75%, followed by full recovery.
Such stressful conditions favor the growth of highly robust, high
productivity cell lines such as the SpESF-X cell line (see Examples
below). These robust, high-productivity cell lines can achieve
protein production levels that are substantially higher than the
parent cell lines.
[0013] The claimed cell culture methods incorporating novel
combinations of factors including, but not limited to, transfection
vectors, screening and selection of cell clones with desired
properties, cell culture media, growth conditions, bioreactor
configurations, and cell types to create cell culture conditions in
which the longevity of the cell culture is increased and/or made
optimal and the yield of a desired recombinant protein is
increased. These cell culture methods include suspension,
perfusion, and fed-batch methods of production. See Tey et al., J.
Biotechnol. 79: 147-159 (2000); Zhang et al., J. Chem. Technol.
Biotechnol. 79: 171-181 (2004); Zhou et al., Biotechnol. Bioeng.
55: 783-792 (1997).
[0014] Unless otherwise defined, all technical and scientific terms
used herein have their plain and ordinary meaning. In addition, the
contents of all patents and other references cited herein are
incorporated by reference in their entirety.
BRIEF DESCRIPTION OF DRAWINGS/FIGURES
[0015] FIG. 1. Immunoblot analysis of SP2/0 transgenic clones for
Bcl-2-EEE expression. For 40 clones, total protein was resolved by
SDS-PAGE under denaturing conditions and transferred to PVDF
membranes. Approximately 10K cells were loaded per lane. Blots were
probed with anti-hBcl-2 (C-2) for detection of Bcl-2-EEE and
anti-.beta. actin for loading control. The three highest-expressing
clones (7, 25 and 87) are identified. Lanes representing a
transgenic Sp2/0 clone expressing wild-type hBcl-2 are indicated
(wt).
[0016] FIG. 2. Flow cytometry analysis of Bcl-2-EEE expression in
selected sub-clones. Permeabilized cells were stained with
anti-hBcl-2-PE and analyzed by flow cytometry using a Guava PCA and
Guava Express software.
[0017] FIG. 3. Comparison of the level of Bcl-2 expression of
subclones #87-29 and #7-16 with, Raji, Daudi, and Sp2/0 cells. A.
Permeabilized cells were stained with anti-hBcl-2-PE and analyzed
by flow cytometry using a Guava PCA and Guava Express software. B.
Anti-hBcl-2 immunoblot analysis. Total protein was resolved by
SDS-PAGE under denaturing conditions and transferred to PVDF
membranes. Blots were probed with anti-hBcl-2 (C-2) for detection
of Bcl-2-EEE and anti-.beta. actin for loading control. The cell
equivalent/lane is indicated. The positions of Bcl-2-EEE and
.beta.-actin are indicated with arrows. Sp2/0-Bcl-2(wt) is a
transgenic line that over-expresses wild-type hBcl-2 at a high
level. C. Immunoblot analysis using a MAb that recognizes both
mouse and human Bcl-2.
[0018] FIG. 4. Growth (A) and viability (B) curves of the five
highest Bcl-2-EEE expressing clones compared to Sp2/0. T25 flasks
were seeded at 5.times.10.sup.4 cells/ml in media containing 10%
FBS. Viable cell density and viability were measured with a Guava
PCA over two weeks.
[0019] FIG. 5. Growth and viability curves comparing SpEEE
subclones, #87-29 and #7-16, to Sp2/0 in culture media containing
(A & B) 10% FBS, (C & D) 1% FBS and (E & F) 0% FBS.
Cells were seeded at 2.times.10.sup.5 cells/well in T25 flasks.
Viable cell density and viability were measured with a Guava PCA
over 12 days.
[0020] FIG. 6. (A) Growth and (B) viability curves comparing SpEEE
subclones in T-25 flasks over five days of culture. Cells were
seeded at 3.times.10.sup.5 cells/ml in serum-free media. Viable
cell density and viability were measured with a Guava PCA
[0021] FIG. 7. Growth (A) and viability (B) curves comparing SpESF,
Sp2/0 and SpEEE cell lines. T25 flasks were seeded at
1.times.10.sup.5 cells/ml in media containing 10% FBS for SpEEE and
Sp2/0 cells or media without FBS for SpESF cells. Viable cell
density and viability were measured with a Guava PCA.
[0022] FIG. 8. Growth and viability curves comparing SpESF-X
subclones #1-8 (A and B) and #9-14 (C and D) cell lines. T25 flasks
were seeded at 1.times.10.sup.5 cells/ml in 0% H-SFM. Viable cell
density and viability were measured with a Guava PCA.
[0023] FIG. 9. Growth and viability curves comparing 5 SpESF-X
subclones to parental SpESF-X, SpEEE and Sp2/0 cell lines.
[0024] FIG. 10. Flow cytometry analysis of Bcl-2-EEE expression in
select cell lines maintained in the absence or presence of zeocin.
Permeabilized cells were stained with anti-hBcl-2-PE and analyzed
by flow cytometry using Guava PCA and Guava Express software.
[0025] FIG. 11 shows the map of the pdHL2 vector used to transfect
Sp2/0 cells to obtain the 665.289 clone with humanized antibody
sequences and the SV40 promoter and enhancer sequences.
[0026] FIG. 12 shows the map of DNA plasmid with incorporated Bcl-2
gene, used for transfection of clone 665.2B9
DETAILED DESCRIPTION OF THE INVENTION
[0027] As used herein, "a" or "an" may mean one or more than one of
an item.
[0028] As used herein, the terms "and" and "or" may be used to mean
either the conjunctive or disjunctive. That is, both terms should
be understood as equivalent to "and/or" unless otherwise
stated.
[0029] As used herein, the term "about" means plus or minus ten
percent. I.e., "about 100" means a number between 90 and 110.
[0030] An "antibody," as described herein, refers to a full-length
(i.e., naturally occurring or formed by normal immunoglobulin gene
fragment recombinatorial processes) immunoglobulin molecule (e.g.,
an IgG antibody) or an immunologically active (i.e., specifically
binding) portion or analog of an immunoglobulin molecule, like an
antibody fragment.
[0031] A "naked" antibody or fragment thereof refers to an antibody
or fragment that is not conjugated to any therapeutic or diagnostic
agent. A "conjugated" antibody or fragment thereof is used
interchangeably with "immunoconjugate" to refer to an antibody or
fragment thereof that is conjugated to at least one therapeutic or
diagnostic agent.
[0032] An antibody fragment is a portion of an antibody such as
F(ab).sub.2, F(ab').sub.2, Fab, Fv, sFv and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody. The term "antibody fragment"
also includes any synthetic or genetically engineered protein that
acts like an antibody by binding to a specific antigen to form a
complex. For example, antibody fragments include isolated fragments
consisting of the variable regions, such as the "Fv" fragments
consisting of the variable regions of the heavy and light chains,
recombinant single chain polypeptide molecules in which light and
heavy variable regions are connected by a peptide linker ("scFv
proteins"), and minimal recognition units (CDR) consisting of the
amino acid residues that mimic the hypervariable region.
[0033] As used herein, the term antibody fusion protein refers to a
recombinantly produced antigen-binding molecule in which two or
more of the same or different scFv or antibody fragments with the
same or different specificities are linked. Valency of the fusion
protein indicates how many binding arms or sites the fusion protein
has to a single antigen or epitope; i.e., monovalent, bivalent,
trivalent or multivalent. The multivalency of the antibody fusion
protein means that it can take advantage of multiple interactions
in binding to an antigen, thus increasing the avidity of binding to
the antigen. Specificity indicates how many antigens or epitopes an
antibody fusion protein is able to bind; i.e., monospecific,
bispecific, trispecific, multispecific. Using these definitions, a
natural antibody, e.g., an IgG, is bivalent because it has two
binding arms but is monospecific because it binds to one epitope.
Monospecific, multivalent fusion proteins have more than one
binding site for an epitope but only binds to one such epitope, for
example a diabody with two binding site reactive with the same
antigen. The fusion protein may comprise a single antibody
component, a multivalent or multispecific combination of different
antibody components, or multiple copies of the same antibody
component. The fusion protein may additionally comprise an antibody
or an antibody fragment and a therapeutic agent. Examples of
therapeutic agents suitable for such fusion proteins include
immunomodulators ("antibody-immunomodulator fusion protein") and
toxins ("antibody-toxin fusion protein"). One preferred toxin
comprises a ribonuclease (RNase), preferably a recombinant
RNase.
Optimizing Protein Production in Cell Culture
[0034] Various approaches have been employed to augment the maximal
cell density and extend survival of mammalian cell cultures, which
in turn results in increased protein yield. Most strategies include
optimization of media formula and feeding routines to increase
total cell number. One method to increase the lifespan of cells in
culture is transfection of oncogenes, as exemplified by PER.C6,
which is a human embryonic retinal cell line that has been
immortalized with the adenovirus E1 gene (Jones, et al., 2003,
Biotechnol Prog 19:163-168).
[0035] Optimizing media conditions and attempting to increase the
culture lifespan may delay apoptosis. However, nutrient and oxygen
depletion as well as metabolite accumulation are inevitable, and
apoptosis will ultimately follow, which has been determined to be
the primary mechanism of cell death in cell culture systems
(Dickson, 1998, Trends Biotechnol 16:339-342; Fussenegger and
Bailey, 1998, Biotechnol Prog 14:807-833; Singh and Al-Rubeai,
1998, Adv Biochem Eng Biotechnol 62:167-184).
[0036] Thus, eliminating premature death by averting or delaying
apoptosis in cell culture systems is another promising approach, as
exemplified by the introduction of anti-apoptotic genes, such as
the Bcl-2 family members, including bcl-2 and bcl-x.sub.L, into
antibody-producing cell lines. Nevertheless, exogenous expression
of wild-type bcl-2 and bcl/-x.sub.L genes has resulted in limited
protection from cell death and little or no improvement in antibody
yields (Mastrangelo et al., 2000, Biotechnol Bioeng 67:555-564;
Meents, et al., 1996, J Exp Med 183:2219-2226; Tey et al., 2000,
Biotechnol Bioeng 68:31-43). More recent efforts have involved the
use of mutant forms of these proteins. In one study, a Bcl-x.sub.L
variant lacking most of the non-conserved unstructured loop domain
was found to be effective in protecting CHO cells from apoptosis in
response to serum deprivation than the wild-type Bcl-x.sub.L
(Figueroa et al., 2003, Metab Eng 5:230-245). In another study,
Deng et al. (2004, PNAS 101:153-158) demonstrated that
over-expression of a mutant Bcl-2 possessing three point mutations
(T69E, S70E and S87E), which mimics phosphorylation, exhibited
significantly higher anti-apoptotic activity compared to wild-type
Bcl-2.
[0037] In the present disclosure, described in the Examples below,
we have stably transfected the murine myeloma cell line,
Sp2/0-Ag14, with a similar Bcl-2 triple mutant to obtain a new host
cell line (SpEEE) that exhibits enhanced survival and adaptability
to growth in serum-free conditions. After one round of subcloning,
a population of cells that exhibited robust growth was identified
and named SpESF.
[0038] In a further attempt to improve the robustness of the cell
line, SpESF cells were subject to iterative rounds of environmental
insult by allowing the cells to overgrow until cell viability
dropped to .about.50-75%. The resulting subclones were designated
SpESF-X (subclones #1-14). Each of the described cell lines has
shown successful transfection and expression of appreciable levels
of mAbs, making them suitable host cell lines for expression of
mAbs and other recombinant proteins.
Cell Lines
[0039] Various embodiments of the present invention concern
improved compositions, including host cell lines, and methods for
enhanced production of recombinant proteins in such cell lines.
Cell lines have been created that constitutively express one or
more anti-apoptotic genes and that can be transfected with an
expression construct encoding a protein or peptide of interest,
where expression of the anti-apoptotic gene(s) prolongs survival of
the transfected cell in culture and provides for enhanced yields of
the protein or peptide of interest.
[0040] Specific embodiments concern derivatives of the Sp2/0
myeloma cell line that provide novel cell lines, referred to as
Sp-E26, SpEEE, SpESF and SpESF-X, which show enhanced survival in
batch culture. Sp-E26 constitutively expresses the E6 and E7
proteins of HPV-16. SpEEE, SpESF and SpESF-X constitutively express
a Bcl-2 mutant, referred to as Bcl-2-EEE. In addition, recombinant
protein production, and particularly production of recombinant
antibodies and antibody fragments, can be improved upon
transfecting Sp-E26, SpEEE, SpESF or SpESF-X with an expression
vector for the recombinant protein of interest. The E6/E7 or
Bcl-2-EEE proteins delay induction of apoptosis in the host cells
and permit enhanced recombinant protein production in the host
cells. Protein production can be boosted still further by addition
of one or more caspase inhibitors (e.g., caspase 1 and/or 3
inhibitors) (Bin Yang et al. Nephron Experimental Nephrology
2004;96:e39-e51), and/or by addition of one or more members of the
cytokine type I superfamily, such as erythropoietin (EPO), into the
growth medium of the cells. A pan-caspase inhibitor is particularly
effective in this regard.
[0041] Further, the SpEEE cell line can be pre-adapted for growth
and protein production in serum-free or low-serum conditions,
resulting in serum-free pre-adapted cell lines such as SpESF or
SpESF-X. The SpESF, SP-ESF-X and similar cell lines may be
transfected with one or more expression vectors encoding a protein
of interest, such as an antibody, antibody fragment, bispecific
antibody, etc. The use of serum-free conditions for transfection,
which is unique among mammalian cell lines available for
transfection and protein production, saves a significant amount of
time required for adaptation to serum-free growth.
[0042] In certain embodiments, such pre-adapted cell lines as SpESF
or SpESF-X may be stored frozen and thawed prior to transfecting
with one or more expression vectors encoding a protein of interest.
This ability to "bank" frozen cell lines that are pre-adapted for
growth, transfection and/or protein production in low serum or
serum-free medium is unexpected and provides a substantial
advantage over cell lines known in the prior art for cost, ease of
use, rapidity of protein production and relative productivity of
expressed proteins.
[0043] Production of recombinant proteins, such as antibodies or
antibody fragments, can be significantly enhanced in the host cell
by co-expression of an apoptosis inhibitor, such as Bcl-2. In
particular, protein production is significantly enhanced in a
myeloma cell line, such as Sp2/0, that is stably transfected with
an expression vector encoding an antibody or antibody fragment and
that is co-transfected with an expression vector encoding an
apoptosis inhibitor, such as Bcl-2. Increased production of
antibody can also be obtained from a host cell transfected with the
E6/E7 gene. Recombinant protein production can be boosted still
further by addition of one or more caspase inhibitors into the
growth medium of the cells. A pan-caspase inhibitor is particularly
effective in this regard. Also, recombinant protein production can
be enhanced by feeding EPO, or another anti-apoptotic cytokine,
into the medium of the cell culture.
[0044] Physiological, or programmed, cell death, referred to as
apoptosis (Kerr et al., Br J Cancer., 26:239-257, 1972), is
essential for proper tissue development and maintenance and is
controlled by an intrinsic genetic program that has been conserved
in evolution (Ellis et al., Annu Rev Cell Biol, 7, 663-698, 1991).
Hence, when cells grow in artificial environments, such as ex vivo
cultures, this genetic endowment results in a finite lifespan.
Therefore, the utility of such cell cultures for the production of
proteins used in medicine and industry, as well as research, is
dependent on maintaining such cultures for extended lifespan, or
cycles, before they die according to apoptotic mechanisms.
[0045] Methods and agents have been discovered that act
independently on cell proliferation and cell death events, by
differentiating cell cycle from apoptotic effects. Bcl-2, a
well-known intracellular regulator of apoptosis (Vaux et al.,
Nature 335, 440-2, 1988), is a proto-oncogene that has been found
to have an anti-apototic effect that is genetically different from
its inhibitory influence on cell cycle entry (Huang et al., EMBO J
16, 4628-38, 1997). Two homologues of Bcl-2, Bcl-x.sub.L and Bcl-w,
also extend cell survival, but other members of the Bcl-2 family,
such as Bax and Bak, are pro-apoptotic (Oltvai et al., Cell 74,
609-19, 1993; Chittenden et al., Nature 374, 733-6, 1995; Farrow et
al., Nature 374, 731-3, 1995; Kiefer et al., Nature 374, 736-9,
1995). Other anti-apoptotic genes include Bcl-6 and Mcl-1.
[0046] Thus, Bcl-2 and certain of its family members exert
protection against apoptosis, and it may be used in a method to
increase the lifespan of certain host cells in culture that are
used for the production of proteins, thereby enhancing the amount
of proteins produced and isolated. Over-expression of an
anti-apoptotic Bcl-2 family member, such as Bcl-2, BCl-x.sub.L,
Bcl-w or mutant varieties of these proteins, inhibits apoptosis,
resulting in increased cell density and longer culture survival.
Hence, transfection of anti-apoptotic Bcl-2 family genes avoids the
necessity to prolong the cell culture by interfering with the cell
cycle per se, as others have proposed (ibid.). Similarly,
transfection of fibroblasts with genes for Bcl-2 results in
over-expression of Bcl-2 in these cells, resulting in an antagonism
of apoptosis and increasing the lifespan of these cells, with a
concomitant increase in the production and isolation of recombinant
proteins. It has also been observed that upon cytokine withdrawal,
interleukin-6 (IL-6)-dependent murine myeloma cells expire as if
they undergo apoptosis. It was also found that IL-6-receptors in
such cells could be regulated by Bcl-2 or Bcl-x.sub.L in extending
apoptosis (Schwarz et al., Cancer Res 55:2262-5, 1995).
[0047] It has been reported that a mutant Bcl-2 possessing three
point mutations (T69E, S70E and S87E) exhibited significantly more
anti-apoptotic activity compared to wild type or single point
mutants (Deng et al., PNAS (101) 153-158, 2004). Thus, various
embodiments concern the construction of an expression vector for a
Bcl-2-EEE triple mutant, which was then used to transfect Sp2/0
cells to create SpEEE clones and subclones that show improved
longevity and recombinant protein production.
[0048] Other agents, such as oncogenic viruses, can also oppose
apoptosis as part of their eliciting cellular immortalization and
ultimately complete malignant transformation, such as high-risk
type HPV oncoproteins E6 and E7 (Finzer et al., Cancer Lett 188,
15-24, 2002). For example, the viral E6 protein effectively blocks
the epidermal apoptotic response to ultraviolet light (Storey,
Trends Mol Med 8, 417-21, 2002). It has also been suggested, from
indirect evidence, that the human papillomavirus may cause reduced
apoptosis in squamous (but not basal cell) carcinoma (Jackson et
al., Br J Cancer 87, 319-23, 2002). However, not all papillomavirus
oncoproteins have anti-apoptotic effects. For example, other
studies have reported that the papillomavirus E6 protein of bovine
species sensitizes cells to apoptosis (Liu et al., Virology 295,
230-7, 2002), which is in contrast to other studies showing that
HPV-16 E7 gene protects astrocytes against apoptosis induced by
certain stimuli (Lee et al., Yonsei Med J 42, 471-9, 2001). By use
of E6-binding peptide aptamers, direct experimental evidence was
obtained that HPV E6 oncoprotein has anti-apoptotic activity in
HPV-positive tumor cells (Butz et al., Proc Natl Acad Sci USA 97,
6693-7, 2000). However, other HPV oncoproteins can have the
opposite effect. The E2 protein induces apoptosis in the absence of
other HPV proteins (Webster et al., J Biol Chem 275, 87-94,
2000).
[0049] Continuous expression of both the E6 and E7 proteins is
known to be required for optimal proliferation of cervical cancer
cells and the two viral proteins exert distinct effects on cell
survival (DeFilippis et al., J Virol 77, 1551-63, 2003). The
primary intracellular target attributed to HPV-16 E6 is p53. E6
forms a ternary complex with p53 and a cellular ubiquitin ligase,
E6AP, resulting in the ubiquitination and degradation of p53
through the proteosome pathway and inactivation of p53. On the
other hand, HPV-16 E7 protein interacts and destabilizes the tumor
suppressor protein Rb. Moreover, levels of a variety of other
intracellular proteins involved in apoptosis and cell cycle
pathways were reported to be regulated by E6 and E7 transformation,
such as Bcl-2, Bcl-x.sub.L, p73, MDM2, p21, cyclins and cdc, cdk
proteins, etc. Changes in the expression of these proteins will
greatly influence the physiological properties of the cell. The
present inventors therefore hypothesized that transfection of cells
in culture by HPV-16E6 and E7 would be effective in generating
genetically modified clones that are resistant to
aging-culture-condition induced apoptosis and, therefore, prolong
the lifespan of the cell culture. It was also postulated that
introduction into a cell of either HPV-16 oncoprotein E7 or E6
alone might be sufficient to generate genetically modified clones
with improved resistance to aging-culture-condition induced
apoptosis. When the cell is a recombinant protein-producing clone,
the improved physiological properties would in turn translate into
enhanced overall protein productivity.
Generation of New Host Cells Expressing Viral Anti-Apoptotic
Genes
[0050] Host cells, such as myeloma host cells, can be generated
that constitutively express viral anti-apoptotic genes, such as
HPV-16 E6 and E7 proteins. These host cells can be transfected with
an expression vector that encodes a recombinant protein of interest
and co-expression of the anti-apoptotic genes results in
significantly increased production of the recombinant protein.
[0051] The host cell can be essentially any host cell suitable for
recombinant protein production that can be stably transfected with
the viral anti-apoptosis genes. For many recombinant proteins, host
cells such as COS cells are advantageous, while for other proteins,
such as antibodies, host cells such as myeloma cells are the common
choices. Other examples of useful host cell lines are VERO and HeLa
cells, W138, BHK, COS-7, 293, HepG2, 3T3, NSO, NS1, RIN and MDCK
cell lines. Cell lines of use may be obtained from commercial
sources, such as the COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC
CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), P3X3Ag8.653 (ATCC
CRL-1580) and BSC-1 (e.g., ATCC CRL-26) cell lines. In preferred
embodiments, the host cell is a mammalian cell line other than the
CHO cell line. The viral (e.g., E6/E78) and/or eukaryotic genes can
be introduced into the host cell by any suitable method that
results in constitutive or inducible expression of the genes, i.e.,
any method that permits stable integration of the genes into the
host cell chromosome while permitting expression of the genes.
Methods for stable transfection of host cells with a gene of
interest are well known in the art. A particularly advantageous
method is to use a retroviral vector that encodes the viral
anti-apoptosis genes. Suitable vectors include the LSXN vector
(Miller et al. Biotechniques 7, 980-90, 1989). However, any
alternative methods known in the art, such as electroporation or
cell fusion, may be utilized.
[0052] In preferred embodiments, the Sp2/0 cell line is used as the
host cell. More preferably, Sp2/0 is transfected with the triple
mutant Bcl-2 gene to form the SpEEE host cell line. Even more
preferably, the SpEEE cell line is pre-adapted to growth,
transfection and protein production in serum-free medium to form a
pre-adapted cell line such as SpESF. Most preferably, the SpESF
cell line is grown under stressful growth conditions to form a
highly robust, high productivity host cell line such as SpESF-X.
SpESF-X may be grown, transfected with an expression vector and
produce expressed proteins under serum-free conditions. Protein
production from SpESF-X is surprisingly higher than known prior art
cell lines, such as CHO cells.
[0053] Advantageously, the vector used to transfect the host cell
contains a selectable marker that permits selection of cells
containing the vector. Suitable selection markers, such as enzymes
that confer antibiotic resistance on transfected cells, are well
known in the art. After transfection, cells are maintained in a
medium containing the selection agent, such as an antibiotic, and
screened for resistance to the marker. Cells can be selected and
cloned by limiting dilution using conventional methods.
[0054] The ability of the viral anti-apoptosis genes to increase
cell viability can be tested by challenging the cells with an agent
that induces apoptosis, such as cycloheximide (CHX). Cells that do
not express the viral anti-apoptosis genes tend to demonstrate
significant onset of apoptosis, whereas cells expressing the genes
exhibit drastically reduced apoptotic activity. Methods of
detecting apoptosis are well known in the art and include, for
example, cell surface FITC-Annexin V binding assay, DNA laddering
assay and TUNEL assay.
[0055] Upon selection of suitable cells expressing the viral
anti-apoptosis genes, the cells can be transfected with an
expression vector encoding the recombinant protein of choice. The
expression vector can be a vector suitable for transient expression
or, advantageously, can be an episomal vector containing a
eukaryotic origin of replication, or an amplifiable vector that
permits stable integration and subsequent gene amplification of the
expression cassette. Suitable vectors are well known in the art and
include, for example, the pdHL2 vector, which is particularly
suited for production of antibodies and antibody fragments. When an
amplifiable expression cassette is used, it advantageously contains
a selectable marker that is different from the selectable marker
used in the retroviral vector, to allow selection of transfected
cells. Once again, suitably transfected cells can be selected and
then cloned by limiting dilution.
[0056] Upon selection of suitable clones, the cells can be placed
in a suitable medium and cultured to produce the desired protein of
interest. The medium can contain serum or, preferably, be
serum-free. In addition, cell longevity and protein production also
can be increased by adding one or more caspase inhibitors (e.g.,
caspase 1 or 3) to the culture medium. Preferably the caspase
inhibitor acts to inhibit one or more of caspase 3, caspase 9
and/or caspase 12. A cell-penetrating caspase inhibitor
advantageously is used, and a pan-caspase inhibitor is particularly
advantageous. Suitable inhibitors such as Z-VAD-fmk and Ac-DEVD-cho
(SEQ ID NO: 7) are well known in the art. Alternatively, the cell
line can be further transfected to express a caspase inhibitor,
such as Aven or XIAP, to enhance its growth properties by affecting
apoptosis. In this regard, certain members of the cytokine type I
superfamily, such as EPO, can also increase cell survival by having
anti-apoptotic and cytoprotective actions.
[0057] The methods described above generate a cell line that can be
used for transfection with essentially any desired gene. However,
the skilled artisan will recognize that established cell lines that
constitutively express a desired protein, and particularly a
recombinant protein, can be subsequently transfected with a
suitable vector encoding the viral or Bcl-2 family anti-apoptosis
genes. See Example 2 below.
Proteins and Peptides of Interest
[0058] The protein of interest can be essentially any protein that
can be produced in detectable quantities in the host cell. Examples
include traditional IgG type antibodies, Fab', Fab, F(ab).sub.2 or
F(ab).sub.2 fragments, scFv, diabody, IgG-scFv or Fab-scFv fusion
antibodies, IgG- or Fab-peptide toxin fusion proteins, or vaccines
[e.g., including not limited to, Hepatitis A, B or C; HIV,
influenza viruses, respiratory syncytial virus, papilloma viruses,
Herpes viruses, Hantaan virus, Ebola viruses, Rota virus,
Cytomegalovirus, Leishmania RNA viruses, SARS, malaria,
tuberculosis (Mycobacteria), Anthrax, Smallpox, Tularemia, and
others listed in the vaccines.org website, incorporated herein by
reference in its entirety]. The host cells described herein are
particularly suitable for highly efficient production of antibodies
and antibody fragments in myeloma cell lines as described in
Examples 1 and 2, as well as recombinant growth factors (e.g., EPO,
G-CSF, GM-CSF, EGF, VEGF, thrombopoietin), hormones, interleukins
(e.g., IL-1 through IL-31), interferons (e.g., alpha, beta, gamma,
and consensus), and enzymes. These methods could be applied to any
number of cell lines that are used for production of recombinant
proteins, including other myeloma cell lines, such as murine NSO or
rat YB2/0; epithelial lines, such as HEK 293; mesenchymal cell
lines, such as fibroblast lines COS-1 or COS-7; and neuronal cells,
such as retinal cells, as well as glial and glioma cells.
[0059] The skilled artisan will realize that a wide variety of
nucleic acid sequences encoding potential proteins or peptides of
interest are known in the art and any such known nucleic acid or
known proteins or peptides may be utilized in or produced by the
disclosed methods and compositions. In particular, the well-known
GenBank database contains thousands of protein-encoding nucleic
acid sequences, any one of which could potentially be used.
[0060] Exemplary proteins or peptides of interest that may be
produced are discussed herein. The skilled artisan will realize
that these are preferred embodiments only and do not limit the
scope of the claimed subject matter. For example, MIF is a pivotal
cytokine of the innate immune system and plays an important part in
the control of inflammatory responses. Originally described as a T
lymphocyte-derived factor that inhibited the random migration of
macrophages, the protein known as macrophage migration inhibitory
factor (MIF) was an enigmatic cytokine for almost 3 decades. In
recent years, the discovery of MIF as a product of the anterior
pituitary gland and the cloning and expression of bioactive,
recombinant MIF protein have led to the definition of its critical
biological role in vivo. MIF has the unique property of being
released from macrophages and T lymphocytes that have been
stimulated by glucocorticoids. Once released, MW overcomes the
inhibitory effects of glucocorticoids on TNF-.alpha., IL-1 beta,
IL-6, and IL-8 production by LPS-stimulated monocytes in vitro and
suppresses the protective effects of steroids against lethal
endotoxemia in vivo. MIF also antagonizes glucocorticoid inhibition
of T-cell proliferation in vitro by restoring IL-2 and WN-gamma
production. MIF is the first mediator to be identified that can
counter-regulate the inhibitory effects of glucocorticoids and thus
plays a critical role in the host control of inflammation and
immunity. MW is particularly useful in treating cancer,
pathological angiogenesis, and sepsis or septic shock.
[0061] HMGB-1, a DNA binding nuclear and cytosolic protein, is a
proinflammatory cytokine released by monocytes and macrophages that
have been activated by IL-1.beta., TNF, or LPS. Via its B box
domain, it induces phenotypic maturation of DCs. It also causes
increased secretion of the proinflammatory cytokines IL-1 alpha,
IL-6, IL-8, IL-12, TNF-.alpha. and RANTES. HMGB-1 released by
necrotic cells may be a signal of tissue or cellular injury that,
when sensed by DCs, induces and/or enhances an immune reaction.
Palumbo et al. report that HMBG1 induces mesoangioblast migration
and proliferation (J Cell Biol, 164:441-449 (2004)). HMBG-1 may be
useful in treating sepsis and/or septic shock. Yang et al., PNAS
USA 101:296-301 (2004); Kokkola et al., Arthritis Rheum, 48:2052-8
(2003); Czura et al., J Infect Dis, 187 Suppl 2:S391-6 (2003);
Treutiger et al., J Intern Med, 254:375-85 (2003).
[0062] TNF-.alpha. is an important cytokine involved in systemic
inflammation and the acute phase response. TNF-.alpha. is released
by stimulated monocytes, fibroblasts, and endothelial cells.
Macrophages, T-cells and B-lymphocytes, granulocytes, smooth muscle
cells, eosinophils, chondrocytes, osteoblasts, mast cells, glial
cells, and keratinocytes also produce TNF-.alpha. after
stimulation. Its release is stimulated by several other mediators,
such as interleukin-1 and bacterial endotoxin, in the course of
damage, e.g., by infection. It has a number of actions on various
organ systems, generally together with interleukins-1 and -6. One
of the actions of TNF-.alpha. is appetite suppression.
[0063] Coagulation factors may also be of use, particularly tissue
factor (TF) and thrombin. TF is also known also as tissue
thromboplastin, CD142, coagulation factor III, or factor III. TF is
an integral membrane receptor glycoprotein and a member of the
cytokine receptor superfamily. The ligand binding extracellular
domain of TF consists of two structural modules with features that
are consistent with the classification of TF as a member of type-2
cytokine receptors. TF is involved in the blood coagulation
protease cascade and initiates both the extrinsic and intrinsic
blood coagulation cascades by forming high affinity complexes
between the extracellular domain of TF and the circulating blood
coagulation factors, serine proteases factor VII or factor VIIa.
These enzymatically active complexes then activate factor IX and
factor X, leading to thrombin generation and clot formation.
Genetic defects in production of one or more coagulation factors
may result in hereditary anemia.
[0064] In rheumatoid arthritis, a recombinant interleukin-1
receptor antagonist, IL-1Ra or anakinra (Kineret.RTM.), has shown
activity (Cohen et al., Ann Rheum Dis 2004; 63:1062-8; Cohen, Rheum
Dis Clin North Am 2004; 30:365-80). An improvement in treatment of
these patients, which hitherto required concomitant treatment with
methotrexate, is to combine anakinra with one or more of the
anti-proinflammatory effector cytokines or anti-proinflammatory
effector chemokines. Indeed, in a review of antibody therapy for
rheumatoid arthritis, Taylor (Curr Opin Pharmacol 2003; 3:323-328)
suggests that in addition to TNF, other antibodies to such
cytokines as IL-1, IL-6, IL-8, IL-15, IL-17 and IL-18, are useful.
These and many other therapeutic proteins or peptides may be
produced using the disclosed methods and compositions.
Recombinant Antibody Expression in Cells Expressing Apoptosis
Inhibitors
[0065] Prior work has described the effects of co-expressing Bcl-2,
a naturally occurring apoptosis inhibitor, in recombinant CHO cells
producing a chimeric antibody. (See Tey et al., Biotechnol. Bioeng.
68:31-43 (2000).) Although increased cell culture life was
observed, antibody production did not increase over equivalent
cells that lacked Bcl-2 expression. Further, there was no evidence
that the expression vector was stably transfected into the CHO cell
line. However, the present inventors have found that production of
recombinant antibody from myeloma cells is significantly increased
when the cells also express Bcl-2. The Bcl-EEE transfected cell
lines described below also evidence stable transfection of the
expression vector(s), resulting in long-term protein
production.
[0066] Advantageously, the myeloma cell line is stably transfected
with an expression cassette encoding the antibody or antibody
fragment. A suitable expression cassette contains one or more
promoters that controls expression of the antibody heavy and light
chains (or single chain in the case of an scFv) together with a
selectable marker as described above. A particularly useful vector
is pdHL2, which contains a selectable marker gene comprising a
promoter operatively linked to a DNA sequence encoding a selectable
marker enzyme; a transcription unit having a promoter operatively
linked to a DNA sequence encoding the protein of interest; an
enhancer element between the selectable marker gene and the
transcription unit, which stimulates transcription of both the
selectable marker gene and the first transcription unit compared to
the transcription of both the selectable marker gene and the first
transcription unit in the absence of the first enhancer.
[0067] The vector also contains a blocking element composed of a
promoter placed between the first enhancer and the selectable
marker gene, which is potentially useful for selectively
attenuating the stimulation of transcription of the selectable
marker gene. V.sub.H and V.sub.L sequences can be ligated into
pdHL2, which is an amplifiable vector containing sequences for the
human light chain constant region, the heavy chain constant region,
and an amplifiable dhfr gene, each controlled by separate
promoters. See Leung et al., Tumor Targeting 2:184 (1996) and
Losman et al., Cancer 80:2660-2667 (1997). This vector can be
transfected into cells by, for example, electroporation. Selection
can be performed by the addition of 0.1 .mu.M or a suitable
concentration of methotrexate (MTX) into the culture media.
Amplification can be carried out in a stepwise fashion with
increasing concentration of MTX, up to 3 .mu.M or higher. Cells
stably transfected with the expression cassette and that
constitutively express the antibody of interest can therefore be
obtained and characterized using methods that are well known in the
art. See also Example 4, below. After selection and cloning, the
antibody-expressing cell line can then be transfected with an
expression vector that encodes an anti-apoptosis gene, such as
Bcl-2. For example, the vector pZeoSV (Invitrogen, Carlsbad,
Calif.) containing the Bcl-2 gene fused to an SV40 promoter is
transfected into the cell using a suitable method such as
electroporation, and selection and gene amplification can be
carried out if necessary.
[0068] Alternatively, a suitable host cell may be transfected with
an apoptosis inhibitor, such as a mutant Bcl-2 gene, then adapted
for growth in serum-free medium prior to further transfection,
preferably in serum-free medium, with an expression vector encoding
a desired protein of interest. Antibody production using the
resulting cell line can be carried out as above and compared to
production in cells that do not express an apoptosis inhibitor.
Representative examples to illustrate the present invention are
given below.
[0069] While preferred embodiments are illustrated herein by way of
cell lines transfected with one or more genes encoding inhibitors
of apoptosis known in the art, the skilled artisan will realize
that in alternative embodiments, various substitutions, deletions
or insertions may be made in the coding and/or non-coding sequence
of such genes within the scope of the claimed methods and
compositions, so long as the encoded protein exhibits the same
physiological function (anti-apoptosis) as the native protein. In
certain embodiments, the encoded protein(s) may exhibit 80% or
greater sequence identity with the native (wild-type) protein, more
preferably 85% or greater, more preferably 90% or greater, more
preferably 95% or greater, more preferably 98% or greater, more
preferably 99% or greater, most preferably 99.5% or greater
sequence identity.
Antibodies
[0070] Various embodiments may concern antibodies and/or antibody
fragments expressed from the transfected cell lines of interest.
The term "antibody" is used herein to refer to any antibody-like
molecule that has an antigen binding region, and includes antibody
fragments such as Fab', Fab, F(ab').sub.2, single domain antibodies
(DABs), Fv, scFv (single chain Fv), and the like. Techniques for
preparing and using various antibody-based constructs and fragments
are well known in the art. Means for preparing and characterizing
antibodies are also well known in the art (See, e.g., Harlowe and
Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory). Antibodies of use may also be commercially obtained
from a wide variety of known sources. For example, a variety of
antibody secreting hybridoma lines are available from the American
Type Culture Collection (ATCC, Manassas, Va.). A large number of
antibodies against various disease targets, including but not
limited to tumor-associated antigens, have been deposited at the
ATCC and are available for use in the claimed methods and
compositions. (See, for example, U.S. Pat. Nos. 7,060,802;
7,056,509; 7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293;
7,038,018; 7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976;
6,994,852; 6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981;
6,962,813; 6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020;
6,939,547; 6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078;
6,916,475; 6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466;
6,884,594; 6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006;
6,864,062; 6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549;
6,835,370; 6,824,780; 6,824,778; 6,812,206; 6,793,924; 8,783,758;
6,770,450; 6,767,711; 6,764,681; 6,764,679; 6,743,898; 6,733,981;
6,730,307; 6,720,15; 6,716,966; 6,709,653; 6,693,176; 6,692,908;
6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;
6,673,344; 6,652,852; 6,635,482; 6,630,144; 6,610,833; 6,610,294;
6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572;856;
6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058; 6,528,625;
6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930;
6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173;
6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310; 6,444,206`
6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694;
6,403,770; 6,403,091; 6,395,274; 6,383,759; 6,383,484; 6,376,654;
6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;
6,346,246; 6,344,198; 6,340,571; 6,340,459 each incorporated herein
by reference with respect to the ATCC deposit number for the
antibody-secreting hybridoma cell lines and the associated target
antigens for the antibodies or fragments thereof.) These are
exemplary only and a wide variety of other antibody-secreting
hybridomas are known in the art. The skilled artisan will realize
that antibody-secreting hybridomas against almost any
disease-associated antigen may be obtained by a simple search of
the ATCC, PubMed and/or USPTO databases for antibodies against a
selected disease-associated target of interest. The antigen binding
domains of the cloned antibodies may be amplified, excised, ligated
into an expression vector, transfected into an adapted host cell
and used for protein production, using standard techniques well
known in the art.
[0071] Production of Antibody Fragments
[0072] Some embodiments of the claimed methods and/or compositions
may concern antibody fragments. Exemplary methods for producing
antibody fragments are disclosed in U.S. Pat. No. 4,036,945; U.S.
Pat. No. 4,331,647; Nisonoff et al., 1960, Arch. Biochem. Biophys.,
89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al., 1967,
METHODS IN ENZYMOLOGY, page 422 (Academic Press), and Coligan et
al. (eds.), 1991, CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley &
Sons).
[0073] Other methods of forming antibody fragments, such as
separation of heavy chains to form monovalent light-heavy chain
fragments, further cleavage of fragments or other enzymatic,
chemical or genetic techniques also may be used, so long as the
fragments bind to the antigen that is recognized by the intact
antibody. For example, Fv fragments comprise an association of
V.sub.H and V.sub.L chains. This association can be noncovalent, as
described in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA,
69:2659. Alternatively, the variable chains may be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See Sandhu, 1992, Crit. Rev. Biotech., 12:437.
[0074] Preferably, the Fv fragments comprise V.sub.H and V.sub.L
chains connected by a peptide linker. These single-chain antigen
binding proteins (sFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains, connected by an oligonucleotides linker sequence. The
structural gene is inserted into an expression vector that is
subsequently introduced into a host cell. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing sFv's are
well-known in the art. See Whitlow et al., 1991, Methods: A
Companion to Methods in Enzymology 2:97; Bird et al., 1988,
Science, 242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993,
Bio/Technology, 11:1271, and Sandhu, 1992, Crit. Rev. Biotech.,
12:437.
[0075] 100731 Another form of an antibody fragment is a peptide
coding for a single complementarity-determining region (CDR). CDR
peptides ("minimal recognition units") can be obtained by
constructing genes encoding the CDR of an antibody of interest.
Such genes are prepared, for example, by using the polymerase chain
reaction to synthesize the variable region from RNA of
antibody-producing cells. See Larrick et al., 1991, Methods: A
Companion to Methods in Enzymology 2:106; Ritter et al. (eds.),
1995, MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, pages 166-179 (Cambridge University Press); Birch et
al., (eds.), 1995, MONOCLONAL ANTIBODIES: PRINCIPLES AND
APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.). Where an
antibody-secreting hybridoma cell line is publicly available, the
CDR sequences encoding antigen-binding specificity may be obtained,
incorporated into chimeric or humanized antibodies, and used.
[0076] Chimeric and Humanized Antibodies
[0077] A chimeric antibody is a recombinant protein in which the
variable regions of a human antibody have been replaced by the
variable regions of, for example, a mouse antibody, including the
complementarity-determining regions (CDRs) of the mouse antibody.
Chimeric antibodies exhibit decreased immunogenicity and increased
stability when administered to a subject. Methods for constructing
chimeric antibodies are well known in the art (e.g., Leung et al.,
1994, Hybridoma 13:469).
[0078] A chimeric monoclonal antibody may be humanized by
transferring the mouse CDRs from the heavy and light variable
chains of the mouse immunoglobulin into the corresponding variable
domains of a human antibody. The mouse framework regions (FR) in
the chimeric monoclonal antibody are also replaced with human FR
sequences. To preserve the stability and antigen specificity of the
humanized monoclonal, one or more human FR residues may be replaced
by the mouse counterpart residues. Humanized monoclonal antibodies
may be used for therapeutic treatment of subjects. The affinity of
humanized antibodies for a target may also be increased by selected
modification of the CDR sequences (WO0029584A1). Techniques for
production of humanized monoclonal antibodies are well known in the
art. (See, e.g., Jones et al., 1986, Nature, 321:522; Riechmann et
al., Nature, 1988, 332:323; Verhoeyen et al., 1988, Science,
239:1534; Carter et al., 1992, Proc. Nat'l Acad. Sci. USA, 89:4285;
Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest et al., 1991,
Biotechnology 9:266; Singer et al., J. Immunol., 1993,
150:2844.)
[0079] Other embodiments may concern non-human primate antibodies.
General techniques for raising therapeutically useful antibodies in
baboons may be found, for example, in Goldenberg et al., WO
91/11465 (1991), and in Losman et al., Int. J. Cancer 46: 310
(1990).
[0080] Human Antibodies
[0081] Methods for producing fully human antibodies using either
combinatorial approaches or transgenic animals transformed with
human immunoglobulin loci are known in the art (e.g., Mancini et
al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005,
Comb. Chem. High Throughput Screen. 8:117-26; Brekke and Loset,
2003, Curr. Opin. Phamacol. 3:544-50; each incorporated herein by
reference). Such fully human antibodies are expected to exhibit
even fewer side effects than chimeric or humanized antibodies and
to function in vivo as essentially endogenous human antibodies. In
certain embodiments, the claimed methods and procedures may utilize
human antibodies produced by such techniques.
[0082] In one alternative, the phage display technique may be used
to generate human antibodies (e.g., Dantas-Barbosa et al., 2005,
Genet. Mol. Res. 4:126-40, incorporated herein by reference). Human
antibodies may be generated from normal humans or from humans that
exhibit a particular disease state, such as cancer (Dantas-Barbosa
et al., 2005). The advantage to constructing human antibodies from
a diseased individual is that the circulating antibody repertoire
may be biased towards antibodies against disease-associated
antigens.
[0083] In one non-limiting example of this methodology,
Dantas-Barbosa et al. (2005) constructed a phage display library of
human Fab antibody fragments from osteosarcoma patients. Generally,
total RNA was obtained from circulating blood lymphocytes (Id.).
Recombinant Fab were cloned from the .mu., .gamma. and .kappa.
chain antibody repertoires and inserted into a phage display
library (Id.). RNAs were converted to cDNAs and used to make Fab
cDNA libraries using specific primers against the heavy and light
chain immunoglobulin sequences (Marks et al., 1991, J. Mol. Biol.
222:581-97, incorporated herein by reference). Library construction
was performed according to Andris-Widhopf et al. (2000, In: Phage
Display Laboratory Manual, Barbas et al. (eds), 1.sup.st edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. pp.
9.1 to 9.22, incorporated herein by reference). The final Fab
fragments were digested with restriction endonucleases and inserted
into the bacteriophage genome to make the phage display library.
Such libraries may be screened by standard phage display methods,
as known in the art. The skilled artisan will realize that this
technique is exemplary only and any known method for making and
screening human antibodies or antibody fragments by phage display
may be utilized.
[0084] In another alternative, transgenic animals that have been
genetically engineered to produce human antibodies may be used to
generate antibodies against essentially any immunogenic target,
using standard immunization protocols. A non-limiting example of
such a system is the XenoMouse.RTM. (e.g., Green et al., 1999, J.
Immunol. Methods 231:11-23, incorporated herein by reference) from
Abgenix (Fremont, Calif.). In the XenoMouse.RTM. and similar
animals, the mouse antibody genes have been inactivated and
replaced by functional human antibody genes, while the remainder of
the mouse immune system remains intact.
[0085] The XenoMouse.RTM. was transformed with germline-configured
YACs (yeast artificial chromosomes) that contained portions of the
human IgH and Igkappa loci, including the majority of the variable
region sequences, along accessory genes and regulatory sequences.
The human variable region repertoire may be used to generate
antibody producing B cells, which may be processed into hybridomas
by known techniques. A XenoMouse.RTM. immunized with a target
antigen will produce human antibodies by the normal immune
response, which may be harvested and/or produced by standard
techniques discussed above. A variety of strains of XenoMouse.RTM.
are available, each of which is capable of producing a different
class of antibody. Such human antibodies may be coupled to other
molecules by chemical cross-linking or other known methodologies.
Transgenically produced human antibodies have been shown to have
therapeutic potential, while retaining the pharmacokinetic
properties of normal human antibodies (Green et al., 1999). The
skilled artisan will realize that the claimed compositions and
methods are not limited to use of the XenoMouse.RTM. system but may
utilize any transgenic animal that has been genetically engineered
to produce human antibodies.
EXAMPLES
Example 1
Generation of Apoptosis-Resistance Cell Clones by Stable Expression
of HPV-16 E6 and E7 Genes
[0086] Selection of Cell Clones Resistant to CHX Treatment
[0087] Sp2/0 cells were transduced with an LXSN retroviral vector
containing the expression cassette of HPV-16 E6 and E7 genes at an
MOI (multiple of infection) of 10:1. After recovery for 24 h, the
infected cells were selected in G418 (1000 .mu.g/ml) for 10 days.
G418-resistant cells were cloned in 96-well cell culture plates by
limiting dilution (0.5 cells/well). Stable infectants were screened
for resistance to treatment by cycloheximide (CHX), a potent
apoptosis-inducing agent. Briefly, healthy cells (viability
>95%) were incubated in medium containing 25 .mu.g/ml of CHX and
cell morphology was examined under a microscope. While more than
50% of parent Sp2/0 cells underwent morphology change after two to
three hours of incubation and became fragmented (not shown),
several E6/E7 transfected clones showed less extent of morphology
change, indicating resistance to apoptosis. The best clone,
designated as Sp-E26, showed no apparent morphology change upon
four hours of treatment (not shown).
[0088] The MIT assay was used to access the changes in viable cell
population. After the healthy cells were incubated with or without
CHX under normal culture condition for 2-3 h, MTT dye was added to
the wells. After further incubation for two hours, the cells were
solubilized by adding a lysis buffer contain SDS and HCl. The
plates were incubated overnight at 37.degree. C. and OD reading was
performed at 590 nm using an ELISA plate reader. The viable cell
population was significantly reduced when Sp2/0 cells were treated
with CHX. By comparison, under the same treatment conditions
(concentration of CHX and length of time), Sp-E26 cells tolerated
better against CHX treatment (not shown). With this method, a large
number of clones can be screened and selected for further
analyses.
[0089] Anti-Apoptosis Property of Sp-E26
[0090] CHX-induced apoptosis in Sp-E26 and the parent Sp2/0 cells
was evaluated by Annexin V staining and DNA fragmentation assay.
After being incubated in the medium containing 25 .mu.g/ml of CHX,
the cells were harvested and stained with Guava Nexin reagent
(equivalent of Annexin V staining) and analyzed in a Guava Personal
Cell Analysis system (Guava Technologies, Inc.). More than 30% of
Sp2/0 cells became Annexin V positive when exposed to CHX treatment
for about 1.5 h, indicating apoptosis, while Sp-E26 remained
healthy, showing no increase in early apoptotic cells (not
shown).
[0091] The induction of apoptosis by CHX can be revealed by
analysis of the formation of intracellular oligonucleosomal DNA
fragments, a hallmark of apoptosis. The cellular DNA was extracted
from CHX-treated and untreated Sp-E26 and Sp2/0 cells and DNA
laddering assay was performed. In Sp2/0 cells treated with CHX,
extensive DNA fragmentation was detected (not shown). In contrast,
under identical treatment conditions, the genomic DNA of Sp-E26 was
still intact, showing no appearance of DNA fragmentation (not
shown).
[0092] Presence of HPV E6 and E7 Genes in Sp-E26 Cells
[0093] To confirm that E6 and E7 genes are stably present in the
genome of Sp-E26 cells, oligonucleotide primers specific for E6 and
E7 genes were designed and used in a PCR reaction with the genomic
DNA extracted from Sp-E26 as the template, resulting in an about
700 bp DNA fragment. The PCR product was cloned and confirmed to be
E6 and E7 genes by DNA sequencing (not shown). No E6 and E7 genes
were detected in the parent Sp2/0 cells.
[0094] Improved Growth Properties of Sp-E26
[0095] The growth properties of Sp-E26 were evaluated in T-flask
and 3L-batch bioreactor. Sp-E26 showed improved growth properties
over the parent Sp2/0 cell in batch cultures, achieving higher
maximum cell density and longer survival time (not shown).
Example 2
Generation of Apoptosis-Resistance Cell Clones by Stable
Over-Expression of HPV16 E7 Gene
[0096] The structure of the poly-cistronic HPV 16 E6 and E7 genes
integrated into the genome of clone Sp-E26 was analyzed by PCR
using the primer pair E6-N8.sup.+ (ATGTTTCAGGACCCACAGGAGCGA; SEQ ID
NO: 8) and E7-C8.sup.- (TTATGGTTTCTGAGA ACAGATGGG; SEQ ID NO: 9)
and DNA sequencing. Since the sequences of primer E6-N8.sup.+ and
E7-C8.sup.- match with the coding sequence for the N-terminal 8
amino acid residues of E6 and the complementary sequence for the
C-terminal 8 codons of E7, respectively, the amplicon of
full-length E6 and E7 is expected to be about 850 bp. However,
amplification of the genomic DNA prepared from Sp-E26 cell with
E6-N8.sup.+ and E7-C8.sup.- resulted a PCR fragment of only about
700 bp. DNA sequencing of the 700 bp PCR product revealed a
deletion of a 182 poly-nucleotide fragment from the E6 gene. The
defective E6 gene likely resulted from splicing and encodes a
truncated E6 peptide with N-terminal 43 amino acid residues.
Considering the major physiological activity attributed to E6 is
its ability to down-regulate p53 expression, the truncated E6
protein is probably not fully functional because the level of p53
expression in Sp-E26 was found to be more stable than that in
Sp2/0.
[0097] Thus, to evaluate whether HPV-16 E7 gene alone is sufficient
to have anti-apoptotic effect and to improve the growth properties
of Sp2/0 cells, transfection of Sp2/0 cell with HPV-16 E7 is
performed as follows: [0098] (i) The DNA sequence encoding E7 is
cloned from Sp-E26 cell by RT-PCR. Proper restriction sites are
introduced to facilitate the ligation of the gene into a mammalian
expression vector, pRc/CMV (Invitrogen). Transcription of the viral
gene within the vector, designated as E7pRc, is directed from CMV
promoter-enhancer sequences. The vector also contains a gene
conferring neomycin resistance, which is transcribed from the SV40
promoter. [0099] (ii) Sp2/0 cells are transfected with the
expression vector containing the expression cassette of HPV-16 E7
gene. Briefly, 5 .mu.g of E7pRc is linearized by ScaI and
transfected into the cell by electroporation. [0100] (iii) After
recovery for 24 hours, the transfected cells are selected in G418
(1000 .mu.g/ml) for 10 days. [0101] (iv) G418-resistant cells are
then cloned in 96-well cell culture plates by limiting dilution
(0.5 cells/well). Stable transfectants are selected and screened
for resistance to treatment by cycloheximide (CHX), a potent
apoptosis-inducing agent. [0102] (v) Healthy cells (viability
>95%) are incubated in medium containing 25 .mu.g/ml of CHX or
in the absence of CHX for 3-4 hours under normal culture
conditions, followed by the addition of MTT dye into the wells.
After further incubation for two hours, the cells are solubilized
by adding a lysis buffer contain SDS and HCl. The plates are
incubated overnight at 37.degree. C. and an OD reading is performed
at 590 nm using an ELISA plate reader. Cell clones showing
resistance to CHX treatment are selected and expanded for further
analyses. [0103] (vi) The anti-apoptosis property of E7-transfected
cells is evaluated by Annexin V staining and DNA fragmentation
assays. In the Annexin V assay, after being incubated in the medium
containing 25 .mu.g/ml of CHX, the cells are harvested and stained
with Guava Nexin reagent (equivalent of Annexin V staining) and
analyzed in a Guava Personal Cell Analysis system (Guava
Technologies, Inc.). In the DNA fragmentation assay, the cellular
DNA is extracted from CHX-treated and untreated E7-transfectants
and Sp2/0 cells and analyzed with agarose gel electrophoresis.
[0104] (vii) Expression of the viral oncogene in E7-transfectants
is evaluated by Southern blot (genomic level), Northern blot (mRNA
level), and immunoblot (protein level) analysis. Expression of
intracellular proteins that are involved in apoptosis processes and
affected by E7 protein are examined by immunoblotting analyses.
[0105] (viii) The growth properties of selected E7-transfectants
are evaluated in T-flask and in a 3L-batch bioreactor. The
transfectants showing improved growth properties, i.e. achieving
higher maximum cell density and longer survival time, over the
parent Sp2/0 cell in batch cultures are considered to be better
host cells.
Example 3
High-Level Expression of hLL2 IgG in Sp-E26
[0106] In this example, Sp-E26 is used as a host to generate cell
clones producing hLL2 (epratuzumab), a humanized anti-CD22 Ab
developed for treating patients with NHL and autoimmune diseases.
An hLL2-producing clone, 87-2-C9, was previously generated by using
Sp2/0 cell as a host (Losman et al., Cancer 80, 2660-2666, 1997),
in which case, only one positive clone (a frequency of about
2.5.times.10.sup.-7) was identified after transfection, and the
maximum productivity (P.sub.max), defined as the concentration of
the antibody in conditioned terminal culture medium in T-flask, of
the only hLL2-producing clone, before amplification, was 1.4 mg/L.
Transfection of Sp-E26 cell with the same hLL2pdHL2 vector and by
using similar procedures as described by Losman et al. (Cancer 80,
2660-2666, 1997) resulted in more than 200 stable hLL2-producing
clones, a frequency of >10.sup.-4). The P.sub.max of 12 randomly
selected clones was evaluated and found to be between 13 and 170
mg/L, with a mean of 50 mg/L. The productivities of these clones
can be further enhanced by gene amplification with MTX. This
example demonstrated the advantage of using Sp-E26 over its parent
Sp2/0 cell as a host for the development of cell clones producing
recombinant proteins.
Example 4
Improvement of Ab-Producing Cell Lines by Stable Expression of
HPV16 E6 and E7 Genes
[0107] 607-3u-8 cells were originally generated from Sp2/0 by
transfection to produce a humanized monoclonal Ab. The clone was
developed by gene amplification (with MTX) and subcloning to
enhance the maximum (Ab) productivity up to 150 mg/L, which
decreased to about 100 mg/L following weaning off serum supplement
in the culture medium. To obtain higher antibody productivity under
serum-free conditions, E6/E7 genes of HPV-16 were introduced into
607-3u-8 and the effect of E6/E7 on Ab-productivity was evaluated
as follows.
[0108] 607-3u-8 cells maintained in HSFM supplemented with 10% FBS
and 3 .mu.M MTX were transduced with an LXSN retroviral vector
containing the expression cassette of HPV-16 E6 and E7 genes at an
MOI of 10:1. After recovery for 24 h, stably transfected cells were
selected in G418 (400 .mu.g/ml) for 10 days. G418-resistant cells
were subcloned in 96-well cell culture plates by limiting dilution
(0.5 cells/well). A surviving clone, designated as 607E1C12, was
obtained for evaluation. Two subclones, designated as 607-3u-8-7G7
and 607-3u-8-2D10, of 607-3u-8 without E6/E7 transfection were also
selected. The P.sub.max of these three clones were determined and
there were no significant difference (Table 1).
[0109] These results suggest that introducing E6/E7 genes into the
cell does not alter the ability of cells producing Ab. Next,
607E1C12, 607-3u-8-7G7 and 607-3u-8-2D10 were adapted to grow in
serum-free medium and the productivities of these clones were
determined. All cells were growing well in serum-free medium. The
final antibody productivity of clone 607E1C12 was maintained at 150
mg/L, while the two clones without E6/E7 were substantially
reduced. In addition, the productivity of 607E1C12 was stable after
a freeze (for cryopreservation) and thaw cycle (Table 1).
TABLE-US-00001 TABLE 1 The productivities of Ab-producing clones
P.sub.max(mg/L).sup.a Clone With serum Serum-free 607-3u-8-7G7 127
.+-. 16 (3).sup.b 74 .+-. 10 (4) 607-3u-8-2D10 140 .+-. 4 (3) 35
.+-. 2 (2) 607E1C12 154 (1) 142 .+-. 13 (6) 607E1C12 (Cryo).sup.c
145 .+-. 17 (5) .sup.aDetermined by protein purification of IgG
from terminal culture supernatants. .sup.bThe number in parenthesis
indicates the sample size. .sup.cCells had been frozen for
cryopreservation.
Example 5
Improvement of Ab-Producing Cell Survival in Stationary Batch
Culture by Stable Expression of a Human Bcl-2 Gene
[0110] Generation of a Bcl-2-Transfected Cell Clone
[0111] A cell clone 665.2B9 was originally generated from Sp2/0 by
transfection to produce a humanized monoclonal anti-CEA Ab, or
hMN-14 [labetuzumab] (Qu et al., unpublished results). A vector,
designated hMN14pdHL2, was used to transfect Sp2/0 cells to obtain
the cell clone 665.2B9. The pdHL2 vector was first described by
Gillies et al., and had an amplifiable murine dhfr gene that allows
subsequent selection and amplification by methotrexate treatment
(Gillies et al., J. Immunol. Methods 125:191 (1989)). Generally,
the pdHL2 vector provides expression of both IgG heavy and light
chain genes that are independently controlled by two
metallothionine promoters and IgH enhancers. A diagram of the
hMN14pdHL2 vector is shown in FIG. 11. SEQ ID NO. 1 shows the
sequence of the vector. SEQ ID NO. 2 shows the 72 by sequence
defined as the enhancer sequence; the promoter sequence corresponds
to nt2908-2979 of hMN14pdHL2.
[0112] Sp2/0 cells can be generally transfected by electroporation
with linearized pdHL2 vectors such as the hMN14pdHL2 vector used in
this instance. Selection can be initiated 48 hours after
transfection by incubating cells with medium containing 0.05 to 0.1
.mu.M MTX. Amplification of inserted antibody sequences is achieved
by a stepwise increase in MTX concentration up to 5 .mu.M.
[0113] The clone was subjected to gene amplification with MTX
increased stepwise to 0.3 .mu.M, at which point the maximum
productivity (Pmax) of the antibody was increased to about 100
mg/L. To improve cell growth properties, 665.2B9 cells were
transfected with a plasmid expression vector (FIG. 12) containing
the human Bcl-2 gene by electroporation. Bcl-2 gene was excised
from pB4 plasmid purchased from ATCC (pB4, catalog #79804) using
EcoRI sites and inserted into MCS of mammalian expression vector
pZeoSV(+) using the same restriction enzyme. Since zeocin
resistance gene is part of the vector, transfected cells were
placed into medium containing zeocin ranging from 50-300 .mu.g/mL.
Stable clones were selected from media containing 300 mg/ml zeocin
and subcloned in media without zeocin by plating into 96-well
plates at a density of 0.5 cell/100 uL/well. The media without
zeocin was used thereafter.
[0114] Formation of clones in wells was confirmed by visual
observation under a microscope. Cells from the wells with only 1
cluster of cells were expanded. Each 96-well plate produced around
30 clones, from which 14 clones were randomly selected for further
studies. The growth characteristics of these clones were evaluated
by daily cell counting and viability measurements with ViaCount
reagent and Guava PCA. From the 14 clones evaluated in 24-well
plates, one Bcl-2-transfected clone showing improved growth
characteristics (higher cell densities and prolonged cell survival)
was identified and designated as 665.2B9#4 (or clone #4). Comparing
to the parent 665.2B9 clone, clone #4 grew to a higher cell density
(about 1.7-fold) and survived 4 to 6 days longer in T-flasks (not
shown), and as a consequence of better growth, the P.sub.max of
clone #4 was increased to about 170 mg/L as determined by ELISA
titration and Protein A column purification.
[0115] Bcl-2 Expression in 665.2B9#4
[0116] To confirm that the improved growth properties of 665.2B9#4
resulted from transfection of Bcl-2, intracellular level of human
Bcl-2 protein was measured by using Guava Express reagent and Guava
PCA instrument. Briefly, 4.times.10.sup.5 cells placed in 1.5 ml
spin-tubes were centrifuged for 5 minutes at 1500 rpm, washed three
times with 1.times.PBS. Supernatants were carefully aspirated.
Fixation solution (10.times., 60 .mu.L) from Santa Cruz
Biotechnology (SCB), Inc. (cat. #sc-3622) was added to cell pellets
for 15 min and incubated on ice. Fixation solution was removed with
4.times.1 mL PBS at 4.degree. C., each time spinning as
described.
[0117] Permeabilization buffer (0.5 mL) at -20.degree. C. (SCB cat.
#sc-3623) was added dropwise while vortexing, followed by 15 min
incubation on ice. Cells were then spun and washed two times with
0.5 mL FCM wash buffer (SCB cat. #sc-3624). Final cell pellet was
resuspended in 100 .mu.L of FCM wash buffer and stained for Bcl-2
intracellular protein with 10 .mu.L of anti-Bcl-2 mouse monoclonal
antibody conjugated to PE (obtained from SCB). Incubation was
performed at room temperature in dark for one hour. Two washes with
0.5 mL of FCM wash buffer followed. The final cell pellet was
resuspended with 0.4 mL FCM wash buffer and the cells analyzed on
Guava PC. Mean values of the fluorescence intensity (MFI) for each
clone were compared to control staining with non-specific, isotype
mouse IgGI conjugated with PE. The results summarized in Table 2
confirm that clone 665.2B9#4 expresses a higher level of Bcl-2
protein compared to the parental cell line. A zeocin-resistant
clone (#13) that showed a similar growth profile as the parent
665.2B9 was negative for Bcl-2 staining, confirming that Bcl-2
expression is necessary for the improvement of growth.
TABLE-US-00002 TABLE 2 Intracellular level of Bcl-2 determined by
Guava Express. Viability.sup.a Mean FI Cell (%) (AU) 665.2B9 84 42
665.2B9#4 97 110 Clone#13 92 14 Non-specific antibody 12 staining
.sup.aDetermined before the assay to ensure healthy cells were
used. .sup.b665.2B9 cells stained with an isotype-matched mouse
IgG1 antibody, PE- conjugated.
[0118] With Guava Express analysis it was found that the
intensities of fluorescent staining corresponding to Bcl-2 levels
are rising with MTX amplification of clone 665.2B9#4, suggesting
co-amplification of Bcl-2 with the dhfr gene. To compare
intracellular Bcl-2 levels of amplified cells, Western blotting
analysis was performed on cell lysates of clone 665.2B9#4 (Bcl-2
positive) and clone #13 (Bcl-2 negative) using an anti-human Bcl-2
antibody. Densitometric evaluation showed that Bcl-2 signal of
clone 665.2B9#4 growing in 1.0 .mu.M MTX is 2.times. stronger than
the cells in 0.6 .mu.M MTX. A lysate of Clone #13 did not reveal
the presence of Bcl-2 protein (not shown).
Example 6
Generation and Characterization of the SpEEE Cell Line that
Constitutively Expresses a Mutant Bcl-2
[0119] Evidence suggests that a mutant Bcl-2 possessing three point
mutations (T69E, S70E and S87E) exhibits significantly more
anti-apoptotic activity compared to wild type or single point
mutants (Deng et al., PNAS 101: 153-158, 2004). Thus, an expression
vector for this triple mutant (designated as Bcl-2-EEE) was
constructed and used to transfect Sp2/0 cells for increased
survival and productivity, particularly in bioreactors. Clones were
isolated and evaluated for Bcl-2-EEE expression level, growth and
apoptotic properties. The nucleic acid sequence for the Bcl-2-EEE
is depicted as SEQ ID NO. 3; the corresponding amino acid sequence
for the Bcl-2-EEE protein is depicted as SEQ ID NO. 4.
[0120] Molecular Cloning
[0121] A 116 bp synthetic DNA duplex was designed based on the
coding sequence for amino acid residues 64-101 of human Bcl-2. The
codons for residues 69, 70 and 87 were all changed to those for
glutamic acid (E). The entire sequence was extraordinarily GC rich
and had numerous poly G and poly C runs. Conservative changes were
made to several codons to break up the G and C runs and decrease
the overall GC content.
[0122] Two 80-mer oligonucleotides, BCL2-EEE Top and BCL2-EEE
Bottom, were synthesized that, combined, span the 116 bp sequence
and overlap on their 3' ends with 22 bp. The oligonucleotides were
annealed and duplex DNA was generated by primer extension with Taq
DNA polymerase. The duplex was amplified using the PCR primers
BCL2-EEE PCR Left and BCL2-EEE PCR Right. The DNA sequences of
these four oligonucleotides are provided below.
TABLE-US-00003 BCL2-EEE Top (SEQ ID NO: 5)
5'GGACCCGGTCGCCAGAGAAGAACCGCTGCAGACTCCGGCTGCTCCTGG
AGCAGCTGCAGGACCTGCGCTCGAACCGGTGC-3' BCL2-EEE Bottom (SEQ ID NO: 6)
5'CGCCGGCCTGGCGGAGGGTCAGGTGGACCACAGGTGGCACCGGTTCGA
GCGCAGGTCCTGCAGCTGCTCCAGGAGCAGCC-3' BCL2-EEE PCR Left (SEQ ID NO:
10) 5'-TATATGGACCCGGTCGCCAGAGAAG-3' BCL2-EEE PCR Right (SEQ ID NO:
11) 5'-TTAATCGCCGGCCTGGCGGAGGGTC-3 '
[0123] The 126-bp amplimer was cloned into the pGemT PCR cloning
vector (Promega, Madison, Wis.) and the resulting vector
(BCL2-EEE-pGemT) was digested with TthI and NgoMI restriction
endonucleases to obtain the 105-bp fragment, which was ligated with
hBCL2-puc19 vector (ATCC 79804) that had been digested with TthI
and NgoMI to generate hBCL2(EEE)-puc19. The sequence of this
construct was confirmed.
[0124] A 948-bp insert fragment was excised from hBCL2(EEE)-puc19
with EcoRI and ligated with pZeoSV2+ vector (Invitrogen, Carlsbad,
Calif.) that was digested with EcoRI and treated with alkaline
phosphatase. Insertion in the proper orientation was confirmed by
digestion with BamHI with a correct clone having 650 bp and 3812 bp
fragments (reverse orientation has 344 bp and 4118 bp). The
resulting construct was designated hBCL2(EEE)-pZeoSV2+.
[0125] Cell Culture
[0126] Sp2/0-Ag14 cells (ATCC CRL#1581), and SpEEE cells were
maintained as suspension cultures in Hybridoma Serum-Free Media
(H-SFM) supplemented with 10% fetal bovine serum (FBS), 4 mM
L-glutamine and 100 units/ml penicillin-streptomycin (10% H-SFM;
Invitrogen Life Technologies, Carlsbad, Calif.). Cell culture
flasks, plates, vials and tubes were purchased from Corning
(Lowell, Mass.). All cells were passaged in T-25 flasks at
37.degree. C. and 5% CO.sub.2. Viable cell concentration and
percent viability were determined at each passage using Guava
ViaCount and Guava PCA instrumentation (Guava Technologies, Inc.,
Hayward, Calif.)
[0127] Transfection of Sp2/0 cells with hBCL2(EEE)-pZeoSV2+
[0128] Sp2/0 cells (5.6.times.10.sup.6) were then transfected by
electroporation with 60 .mu.g of hBcl-2 (EEE)-pZeoSV2+ via
electroporation (450 volts, 25 .mu.F) using a Gene Pulser
electroporation apparatus (BioRad, Hercules, Calif.). Cells were
resuspended in 60 ml of 10% H-SFM and plated onto six 96-well
tissue culture plates. After 48 hours, 10% H-SFM containing zeocin,
at a final concentration of 1.6 mg/ml, was added to each well.
Zeocin-resistant clones were expanded for evaluation of Bcl-2-EEE
expression.
[0129] Cells from 40 wells were expanded to 24-well plates and
analyzed by Western blot with anti-hBcl-2 and anti-beta actin. All
but 5 of the 40 showed medium to high levels of Bcl-2-EEE
expression (not shown) An Sp2/0 derived hMN 14 cell line (Clone
664.B4) that was previously transfected with wild type Bcl-2 was
used as a positive control. As was demonstrated by Deng et al., the
Bcl-2-EEE migrates slightly slower than wild type Bcl-2 in
SDS-PAGE.
[0130] A transgene encoding a constitutively active Bcl-2 mutant
was stably transfected into Sp2/0-Ag14 myeloma cells. Previously,
the triple-mutant Bcl-2 (T69E, S70E and S87E) was shown to potently
enhance survival of multiple cell lines in response to stress (Deng
et al., 2004). Over-expression of the anti-apoptotic protein may
lead to improved growth characteristics and enhance performance of
a host cell line used for the production of antibodies and other
proteins. Forty random transgenic clones were evaluated for Bcl-2
expression by anti-Bcl-2 immunoblotting (FIG. 1). Three of the
strongest positive clones (#7, 25, and 87) were subcloned by
limiting dilution and further analyzed for Bcl-2 expression using
Guava Express (FIG. 2). Clones #7-12, 7-16, 87-2 and 87-10 were
expanded for further analysis. Subsequently, some initially slower
growing subclones were similarly analyzed and one clone, 87-29,
gave a signal that was 20% higher than any other clone and was
expanded for further analysis.
[0131] The level of Bcl-2-EEE expression in the new clones (shown
for #87-29 and #7-16 in FIG. 3A) is about 20-fold higher than the
endogenous level of Bcl-2 found in cell lines, such as Raji and
Daudi. The parent Sp2/0 cells do not express Bcl-2. These
observations were confirmed by anti-Bcl-2 immunoblot analysis with
an anti-Bcl-2 mAb that recognizes mouse, rat, and human Bcl-2
(FIGS. 3B & 3C). We estimate that if there is any Bcl-2
expressed in Sp2/0 cells, it is at a level that is more than 2
orders of magnitude less than the Bcl-2-EEE in clone #87-29.
[0132] Growth curves were generated to compare the growth
properties of five high-expressing Bcl-2-EEE subclones to Sp2/0
cells (FIG. 4). Four of the five subclones possess improved growth
properties, achieving higher cell density and increased viability
compared to Sp2/0. The two subclones (#87-29 and #7-16) showing
superior survival were evaluated for growth and survival in
low-serum or serum-free media.
[0133] Cultures that were carried in 10% FBS were used to seed
media supplemented with 10% FBS, 1% FBS or 0% FBS. In 10% FBS,
subclone #87-29 grew to a high density and had more than 4 days
increased survival compared to Sp2/0 cells (FIGS. 5A & 5B). In
1% FBS, all cells grew to approximately 35-40% of the density
achieved in 10% FBS, with both subclones having a distinct survival
advantage over Sp2/0 cells (FIGS. 5C & 5D). When transferred
directly into serum-free media, the Sp2/0 cells only reached
6.times.10.sup.5 cells/ml, while #87-29 grew to a two-fold higher
density (FIG. 5E). In addition, #87-29 cells survived 4-6 days
longer than Sp2/0 cells when cultured in serum-free medium (FIG.
5F). Because of its superior growth properties, #87-29 was selected
for further development and referred to as SpEEE.
[0134] The methotrexate (MTX) sensitivity was determined for 87-29
(not shown). The data suggests that a minimum MTX concentration of
0.04 .mu.M is sufficient for initial selection of MTX-resistant
clones. Therefore, the same selection and amplification protocols
used for Sp2/0 cells can be employed with the SP-EEE cells.
[0135] Bcl-2 is a pro-survival/anti-apoptotic protein. It has been
demonstrated by several groups that a Bcl-2 deletion mutant missing
the flexible loop domain (FLD) has an enhanced ability to inhibit
apoptotosis (Figueroa et al., 2001, Biotechnology and
Bioengineering, 73, 211-222; Chang et al., 1997, EMBO J.,16,
968-977). More recently, it was demonstrated that mutation of 1 to
3 S/T residues in the FLD of Bcl-2 to glutamic acid, which mimics
phosphorylation, significantly enhances its anti-apoptotic ability
(Deng et al. 2004, PNAS, 101, 153-158). The triple mutant (T69E,
S70E and S87E) provided the most significant survival enhancement.
Here, a similar Bcl-2 triple mutant construct (Bcl-2-EEE), was used
to stably transfect Sp2/0 cells.
[0136] All the aforementioned experiments demonstrate that
expression of Bcl-2-EEE reduces apoptosis rates in Sp2/0 cells.
This effect was largely dose dependent, in that clones with higher
expression levels survived longer than those with lower levels. The
best clone, 87-29 (SpEEE), grows to a 15-20% higher cell density
and survives an additional 4-6 days compared to untransfected Sp2/0
cells.
[0137] The Bcl-2-EEE level in clone SpEEE is approximately 20-fold
higher than normal levels in Daudi or Raji cells. No Bcl-2
expression was detected in untransfected Sp2/0 cells.
hMN-14-expressing Sp2/0 cells were transfected with a similar
construct for expression of wild type Bcl-2 and a clone with
exceptional growth properties and enhanced productivity was
isolated. When this clone (664.B4) was amplified further with MTX,
the Bcl-2 levels increased significantly. Ultimately, the amplified
(3 .mu.M MTX) cell line was sub-cloned and the Bcl-2 level of one
clone (664.B4.1C1) was two-fold higher than 664.B4. This particular
subclone has superior productivity and growth properties. The
Bcl-2-EEE level in SpEEE is approximately two-fold higher than the
level of Bcl-2 in the amplified 664.B4.1C1. SpEEE cells have a
growth rate that is comparable to that of Sp2/0 cells and can
apparently continue to grow for one additional day and reach a
maximal density that is 15-20% higher than Sp2/0. A similar
property was found for the E6/E7 expressing Sp-E26 cell line. The
Bcl-2-EEE expressing SpEEE clone, which provides an additional 4-6
days survival over the parental Sp2/0 cells, is superior to the
Sp-E26 clone, which only survives one additional day.
[0138] The SpEEE cell line as represented by the 87-29 clone is
useful as an apoptosis-resistant host for expressing a recombinant
protein upon transfection with a suitable vector containing the
gene for that recombinant protein. In order for this cell line to
be useful it must maintain its Bcl-2-EEE expression and survival
advantage following transfection and amplification and during
extended culture. It is unlikely that the stably transfected
Bcl-2-EEE gene will be lost during subsequent transfection and
therefore the survival properties should not diminish. It is
possible that MTX amplification could even improve the survival of
producing clones via increasing expression of Bcl-2 proteins.
Indeed, this was the case with the hMN-14 664.B4 cell line, which
was transfected with wild type Bcl-2. Following amplification and
sub-cloning, the Bcl-2 level increased several fold and cell
survival improved significantly.
[0139] The final SpEEE clone (#87-29) has a growth rate that is
comparable to the parental Sp2/0 cells. However, the SpEEE 87-29
cells continue to grow for one additional day, reach a maximal
density that is 15-20% greater and display an additional 4-6 days
survival compared to Sp2/0. Further, the SpEEE cell line was
considerably more tolerant to serum deprivation compared to Sp2/0
cells.
Example 7
SpEEE Based Cell Line Stability
[0140] The SpEEE-based cell lines were developed to enhance the
growth and survival of the resulting transfectants. In order for
this cell line to be useful it must maintain its Bcl-2-EEE
expression and survival advantage following transfection and
amplification and during extended culture without the selection
agent, zeocin. To determine the stability of the Bcl-2 gene,
several cell lines at different stages of development were analyzed
by Guava Express for intracellular Bcl-2 expression.
[0141] Cells were counted using Guava ViaCount reagent and Guava
PCA instrumentation (Guava Technologies, Inc). Approximately
1.times.10.sup.6 cells were pelleted, and washed 2 times with PBS.
FCM Fixation buffer (Santa Cruz Biotechnology, Inc., Santa Cruz,
Calif.) was diluted 1:10 in PBS and 600 .mu.l added to the cell
pellet and incubated on ice for 15 minutes. Cells were washed 2
times with PBS and 0.5 ml of FCM Permeabilization buffer (Santa
Cruz Biotechnology, Inc.) was added drop-wise to the cell pellet,
which was incubated on ice for 15 minutes. Cells were washed twice
and resuspended in 100 .mu.l of FCM wash buffer (Santa Cruz
Biotechnology, Inc.). Cells were then stained with 10 .mu.l of
phycoerythrin (PE)-conjugated mouse anti-human Bcl-2 antibody
(Santa Cruz Biotechnology, Inc.), and incubated in the dark for 1
hour. Cells were washed twice and resuspended in 600 .mu.l of FCM
wash buffer. Stained cells were then analyzed with Guava Express
software and Guava PCA instrumentation (Guava Technologies,
Inc.).
[0142] Whole cell lysates from a known amount of cells were
resolved by SDS-PAGE using 4-10% polyacrylamide gels. Proteins were
electrophoretically transferred to a polyvinylidene fluoride (PVDF)
membrane. The membrane was blocked with a solution containing 5%
milk, 0.05% Tween in PBS. After blocking, the membrane was
incubated in a solution containing either mouse anti-human Bcl-2 or
mouse anti-rat, mouse and human Bcl-2 (Santa Cruz Biotechnology,
Inc.). After washing with PBS containing 0.05% Tween (PBS-T), the
membrane was incubated for 1 hour in peroxidase-conjugated
anti-mouse IgG diluted 1:500 in 1% PBS-T (1% BSA in PBS containing
0.025% Tween 20) and washed again with PBS-T. The membrane was
developed using LumiGLO Peroxidase Chemiluminescent Substrate Kit
(KPL Protein Research Products, Gaithersburg, Md.) and visualized
using Kodak Image Station 4000R (Eastman Kodak Company, Rochester,
N.Y.).
[0143] As shown in FIG. 10, Sp2/0 and A-IgG, which is an
IgG-expressing Sp2/0 cell line, were negative for Bcl-2. Production
of SpEEE by transformation with Bcl-2-EEE is described above. Two
derivative cell lines, SpESF and SpESF-X are described in the
following Examples. Three of the Bcl-2-EEE transfected cell lines,
SpESF-X2, SpESF-X10 and SpEEE, which have been continuously
cultured in media containing zeocin, were positive for Bcl-2-EEE
expression. Three different Fab-expressing SpESF cell lines that
were grown in the absence of zeocin for over 50 passages were found
to express Bcl-2-EEE at levels even higher than those found for the
parental cell lines, which suggests that the Bcl-2-EEE transgene
may be co-amplified with the recombinant protein.
Example 8
Improved Production of Recombinant Proteins with the SpEEE Cell
Line
[0144] There are two paths that can be taken when developing a cell
line with enhanced survival for production of recombinant proteins.
One method, which has been accomplished quite successfully,
involves stable transfection of an already producing cell line with
a pro-survival gene, such as Bcl-2. However, this method requires
additional transfection, selection and cloning steps, thereby
lengthening the cell line development process by at least two
months and possibly much more. Further, screening for the "best"
clone is rather involved, since a number of parameters need to be
determined for each clone, including growth/survival, Bcl-2
expression level and productivity. Thus, only a small number of
clones can be evaluated. It is quite possible that clones with the
highest productivity may not have superior survival and vice versa.
An alternative strategy, employed here, is to develop a parental
cell line with superior growth and survival properties, which is
subsequently transfected with the expression vector for production
of the desired protein.
[0145] Compared to Sp2/0 cells, the SpEEE cells continue to grow
for one additional day, reach a maximal density that is 15-20%
higher, and survive an additional 4-6 days in culture. The cells
retain their enhanced growth and survival properties when
subsequently transfected with genes for the production of
recombinant proteins, such as IgG, antibody fragments and fusion
proteins, growth factors, such as G-CSF, GM-CSF, EPO
(erythropoietin), EGF (epidermal growth factor), VEGF (vascular
endothelial growth factor), cytokines, such as an interleukin
family member (IL-1-IL-31), or interferon family members (such as
alpha, beta or gamma interferon), oligonucleotides, peptides,
hormones, enzymes, or vaccines (e.g., Hepatitis A, B or C, as well
as others described above).
[0146] A DNA vector, such as pdHL2, containing one or more
expression cassettes for recombinant protein(s), such as an IgG, is
used to transfect SpEEE cells by standard methods, such as
electroporation. The transfectants are plated in 96-well plates and
clones are analyzed for protein production by established
techniques such as ELISA or Biacore. Productive clones are
subjected to increasing concentrations of MTX in the culture media
over several months to amplify the genetic copy number. Since the
Bcl-2-EEE-expressing clones grow to about 20% higher cell density
and survive at least an additional 4 days as compared to clones
generated in Bcl-2 negative Sp2/0 cells, the former will produce at
least 20% more recombinant protein in standard flask or roller
bottle culture. An even greater increase is realized in suspension,
perfusion or fed-batch bioreactor cultures.
Example 9
Improved Ab-Production of Bcl-2 Transfected Clones Cultivated in a
Bioreactor
[0147] Both 665.2B9#4 and the parent clone 665.2B9 of Example 5
were weaned into serum-free media. The cells were adapted to a
customized formulation of hybridoma serum-free medium (HSFM)
(Immunomedics PN 10070) containing 3 .mu.M MTX by continuous
subculture in T-flasks for several months. The adapted cells were
scaled up from T-flasks to roller bottles for banking. A master
cell bank (MCB) for each cell line was created with
1.times.10.sup.7 viable cells in each 1-mL vial using an FBS-free
cryopreservation solution composed of 45% conditioned medium
(medium that is collected as supernatant after centrifugation of a
culture in the exponential growth phase), 10% DMSO and 45% HSFM.
The MCB cell lines were designated 665.2B9.1E4 (without Bcl-2 gene)
and 665.B4.1C1 (with Bcl-2 gene), respectively. The growth
properties and antibody production of these two clones were
compared under batch culture conditions.
[0148] Experiments were conducted in 3-L bench-scale bioreactors
using the above cells expanded from the MCB. The 3-L bioreactor
system is the scale-down model of a 2500-L cGMP bioreactor system.
Therefore, the evaluation results would support the suitability of
these cell lines for large-scale commercial manufacturing.
[0149] The same growth HSFM as that used in creating the MCB
(Immunomedics PN 10070) was used to maintain the cell line and
prepare the inoculum. Basal HSFM, a customized formulation based on
the growth HSFM with customized modifications (Immunomedics PN
10194), was used in the 3-L fed-batch bioreactor process. Both
media contain insulin and transferrin as the only trace proteins.
Additional 0.1% Pluronic F68 was incorporated into the formulation
to protect cells from shear caused by agitation and aeration. This
media also contained 3 .mu.M MTX.
[0150] The fed-batch experiments were conducted in 3 L Bellco
spinner-flask bioreactor systems (Bellco glasses, Vineland, N.J.)
with 2 L of working volume. The bioreactor temperature, pH and
dissolved oxygen (DO) were monitored and controlled by single loop
controllers. The reactor temperature was controlled at 37.degree.
C. by a heating blanket. The culture pH was controlled at 7.3 by
the addition of CO.sub.2 or 6% Na.sub.2CO.sub.3. Aeration was
performed through a cylindrical sintered sparger at 10 ml/min. DO
was controlled above 40% of air saturation by intermittent sparging
of O.sub.2 into the medium. A constant agitation rate of 50 about
60 rpm was used throughout the cultivation.
[0151] A frozen vial from MCB was thawed and recovered in T-flasks
in approximately 1 to 2 weeks. The cells were then expanded from
T-flasks to roller bottles prior to inoculation into the
bioreactors. Cells were cultured at 37.degree. C. in a 5% CO.sub.2
atmosphere and maintained in the exponential growth phase
throughout the expansion process.
[0152] Prior to the inoculation, 1.2 liters of Basal HSFM was
pump-transferred into the bioreactor aseptically. The medium was
air saturated to calibrate the dissolved oxygen (DO) probe. A
medium sample was also taken to calibrate the pH probe. Once pH
probes and DO probes were calibrated, both controllers were set to
AUTO modes. Once the system reached set points of pH (7.3) and
temperature (37.degree. C.), calculated amount of inoculum from
roller bottle was pump transferred into the bioreactor. The
post-inoculation viable cell density (VCD) was around
2.times.10.sup.5 vial cells/ml.
[0153] The feeding strategy is as follows. During the cultivation,
concentrated nutrient solutions were fed into the bioreactor to
provide the cells with necessary and non-excessive nutrients.
Concentrated nutrient solutions were delivered to the culture via
continuous feeding and pulse feeding. The continuous feeding
solutions were pump transferred into the reactor continuously using
peristaltic pumps (Watson-Marlow 101U/R). The pulse feeding
solutions were pulse fed once a day into the culture.
[0154] During the cultivation, bioreactor samples were taken
periodically for off-line analysis. The viable cell density (VCD)
and the cell viability were measured by microscopic counting using
a hemocytometer after staining with 0.4% trypan blue dye. The
glucose, lactate, glutamine, ammonia concentrations were measured
using a Nova Bioprofile 200. The antibody concentration was
determined by HPLC using a protein A affinity chromatography column
(Applied Biosystems, P/N 2-1001-00).
[0155] The specific antibody productivity was calculated by
dividing the cumulative antibody produced by the time integral of
the total viable cell in the culture:
Q [ MAb ] = ( [ Mab ] t 1 V t 1 - [ Mab ] t 0 V t 0 .intg. 0 t 1
VCD V t , in which ##EQU00001## .intg. 0 t 1 VCD V t is
approximated by the ##EQU00001.2## Trapezium Rule : ( VCD t 0 V t 0
+ VCD t 1 V t 1 ) ( t 1 - t 0 ) 2 ##EQU00001.3##
[0156] As compared to 665.2B9.1E4 cells, 665.B4.1C1 cells exhibited
much better growth (not shown). The antibody yields of two cell
lines were also compared. The final yield of 665.2B9.1E4 cells was
0.42 g/L in one culture process and 0.55 g/L in a second culture
process. For comparison, 665.B4.1C1 cells delivered a higher final
yield of 1.5 g/L in both processes.
[0157] The daily specific antibody productivities (per cell basis)
were calculated and the 665.2B9.1E4 cells had an average daily
Q.sub.[MAb] of approximately 15 pg/cell/day throughout the course
of cultivation for both processes. The 665.B4.1C1 cells showed a
daily Q.sub.[MAb] between about 20 to 25 pg/cell/day until day 9.
Thereafter the productivity declined.
[0158] Compared with the 665.2B9.1E4 cell line, the 665.B4.1C1 cell
line exhibited a higher specific antibody productivity of about 25
pg/cell/day as compared to 15 pg/cell/day. Combining with its
better growth, the 665.B4.1C1 cell line tripled the final antibody
yield to 1.5 g/L as compared to 0.55 g/L achieved by the
665.2B9.1E4 cell line. These results demonstrate that transfection
of Bcl-2 or its analogs, such as Bcl-2-EEE, into cell lines grown
in serum-free media in a bioreactor modeled for large-scale
commercial preparation of a recombinant protein, in this case an
antibody for clinical use, show the same increase in protein
production observed under batch cultivation.
Example 10
Development of SpESF Serum-Free Pre-Adapted Cell Line
[0159] Since the SpEEE cell line showed enhanced growth and
survival properties as well as superior tolerance to serum
deprivation, it was decided to explore the feasibility of
developing an SpEEE cell line derivative that is pre-adapted to
growth in serum-free media and to use this line for transfection,
cloning and amplification. The following describes the development
of the SpESF (SpEEE serum-free) cell line. Feasibility for
production of cloned proteins, such as antibodies or fragments, was
demonstrated by transfection with the C-AD2-Fab-h679-pdHL2
expression vector.
[0160] Adaptation to Growth in Serum Free Media and Subcloning
[0161] The enhanced survivability of SpEEE cells in serum-free
media led to the successful development of the entirely
serum-independent host cell line, SpESF. SpEEE cells were adapted
to serum-free media over an 8-week period via step-wise reduction
of serum from the media. Once the cell line was adapted to growth
in serum-free media, a limiting dilution was performed to determine
if the cells were capable of surviving at such low densities, as
would be necessary for future transfections and subcloning. Seven
subclones resulted from the limiting dilution and the growth
properties of 4 of the 7 subclones were compared to those of the
parental SpEEE cell line. FIG. 6 shows that subclone #3 survived
for one additional day and gave an area under the curve (AUC) 38%
greater than the parental Sp-EEE or other subclones. In addition,
subclones #3 and #1 reached higher maximal cell density (3.2 to 3.3
million/mL) than the other clones (not shown). Since subclone #3
appeared to be better adapted to undergo successful transfection,
it was selected for further development and designated as the SpESF
cell line.
[0162] After several months of continuous culture and full
adaptation of the SpESF cell line to growth in serum-free media,
growth curves were compared to Sp2/0 and SpEEE in media
supplemented with 10% FBS. FIG. 7 shows that the SpESF cell line
grown in serum-free media is superior to Sp2/0 cells in 10% FBS,
with the former surviving for an additional 3 days. Further, SpESF
is equivalent or better than the SpEEE cell line (in 10% FBS) in
terms of both maximum cell density and longevity.
[0163] Transfection of SpESF Cells with h679-AD2
[0164] Based on the above data SpESF cells (subclone #3) were
transfected by electroporation with 30 .mu.g of h679-AD2-pdHL2.
After 48 hours cells were selected with 0.1 .mu.M MTX. As a
control, SpEEE cells in 10% FBS were also transfected with
h679-AD2-pdHL2 by electroporation under the same conditions. After
10 days plates were ready for screening via ELISA using BSA-IMP-260
coated plates. For both transfections approximately 130 of 400
wells contained positive clones. Positive SpESF cells from wells
with the 40 highest OD readings were transferred to 24-well plates
and the MTX was increased to 0.2 .mu.M MTX. After the cells in the
24-well plates reached terminal, further screening by BIACORE
analysis using an HSG sensorchip was performed. Four of the
screened clones had a productivity of >50 mg/L. The highest
producing clone (h679-AD2-SF #T6) had an initial productivity of 82
mg/L. These initial productivity results were very similar to those
obtained from a previous transfection of this construct using SpEEE
cells in 10% FBS.
[0165] Amplification With MTX
[0166] The h679-AD2-SF# T6 clone was selected for MTX
amplification. After 2 weeks the MTX concentration was increased
from 0.2 .mu.M to 0.4 .mu.M. After only 2 MTX increases, some
amplification in productivity can already be observed (Table
3).
TABLE-US-00004 TABLE 3 MTX Concentration Productivity 0.1 .mu.M MTX
82 mg/L 0.2 .mu.M MTX 93 mg/L 0.4 .mu.M MTX 103 mg/L
[0167] Conclusions
[0168] The data presented above for SpESF indicate that
transfection, cloning by limiting dilution and MTX amplification
can all be accomplished under serum-free conditions in less than a
month. This was demonstrated with the transfection of the
h679-Fab-AD2-pdHL2 expression vector, resulting in the initial very
high production of 82 mg/L, which could be amplified to 103 mg/L in
two weeks. Further amplification is expected with a longer time of
MTX exposure. The initial productivity of the best clone (T6) of 82
mg/L surpasses the initial productivity of the best h679-AD2-pdHL2
clone (5D8) from the original transfection of the parent SpEEE cell
line carried out in 10% FBS, which was around 50 mg/L. SpESF cells
have also been transfected with EPO-DDD2-pdHL2 for production of
erythropoietin.
[0169] As shown in Table 4, which compares the key parameters of
SpESF with those of the existing PER.C6 cell line (Jones et al in
Biotechnol. Prog. 2003, 19: 163-168), Sp/ESP is superior to PER.C6
in many categories.
TABLE-US-00005 TABLE 4 Sp/ESP PER.C6 Parental Cell Mouse myeloma
Human embryonic line retina + E1 Anti-apoptotic Bcl-2-EEE None gene
Transfection Method Electroporation Lipofectamine Efficiency
130/400 ? Growth Suspension Adherent Medium SFM 10% FBS Screening
Growth Suspension Adherent Medium SFM 10% FBS Selected clones
Growth Suspension Suspension Medium SFM SFM Adaption time None 4
weeks Doubling time ~12 h 30-33 h Cell culture Vessel T-25 Roller
bottle Medium SFM SFM Maximal 3.3 .times. 10.sup.6/mL 5 .times.
10.sup.6/mL density Productivity 103 mg/L of Fab* 300-500 mg/L of
IgG *Equivalent to 300 mg/L of IgG
Example 11
Use of SpESF for Protein Production
[0170] The approximately 11-Kb plasmid vector, pdHL2, used for
high-level expression of humanized mAb in myeloma cell lines has
been described previously (Gillies et al., 1989, J Immunol Methods
125:191-202; Qu et al., 2005, Methods 36:84-95). The vector
contains expression cassettes for IgG heavy and light chains under
transcriptional control of the MT1 promotor and the dhfr gene,
which encodes dihydrofolate reductase conferring resistance to
methotrexate (MTX) for selection of transfected clones and gene
amplification for improved protein expression.
[0171] A-IgG-pdHL2 and X, Y, or Z-Fab-pdHL2 (where A, X, Y and Z
represent four different humanized antibodies) were transfected
into SpESF cells via electroporation. Plasmid DNA (20-30 .mu.l) was
linearized with SalI, added to SpESF cells and pulsed twice at 450
volts, 25 .mu.F using a Gene Pulser (BioRad Laboratories, Inc.,
Hercules, Calif.). Cells were resuspended in 80 ml of 0% H-SFM and
plated onto eight 96-well tissue culture plates. After 48 hours,
selection media containing MTX was added to each well. Screening of
MTX-resistant clones was performed 1-2 weeks later via sandwich
ELISA to select high-level antibody-expressing clones. Selected
clones were then transferred to 24-well tissue culture plates for
further testing and expansion.
[0172] For transfected cells expressing antibodies, antibody
expression was determined by sandwich ELISA using mouse anti-human
IgG kappa chain (SouthernBiotech, Birmingham, Ala.) coated on
plates (Nalge Nunc, Rochester, N.Y.). Media supernatant fluid was
diluted in 1% PBS-T and incubated in the ELISA plate for 1 hour at
room temperature. The wells were then washed 3 times with PBS-T.
Horseradish peroxidase (HRP)-conjugated goat anti-human IgG
(Fab').sub.2 specific second antibody (Jackson ImmunoResearch
Laboratories Inc., West Grove, Pa.) was added to the wells for 1
hour. The plate was washed 3 times and substrate solution
containing 4 mM ortho-phenylenediamine (OPD, Sigma, St. Louis, Mo.)
and 0.012% H.sub.2O.sub.2 in PBS was added to the wells and allowed
to develop in the dark for approximately 15 minutes. The reaction
was stopped with the addition of 4N H.sub.2SO.sub.4 and plates were
read at OD.sub.490 using an EnVision plate reader (Perkin Elmer,
Waltham, Mass.). An exemplary result for antibody production is
shown in Table 5.
[0173] To date, more than 20 recombinant proteins have been
produced in SpESF transfectants with the transfection, selection,
amplification, and expression steps all carried out in serum-free
medium. Moreover, following the same amplification protocols that
have been used successfully for the parental Sp2/0 cells, we have
shown similar amplification of recombinant protein expression upon
increasing concentration of MTX. Table 5 summarizes the
productivity of an IgG- and a Fab-expressing cell line derived from
SpESF at each step of MTX amplification.
[0174] Typical productivity for an IgG upon complete amplification
is 150-200 mg/ml in commercially available serum-free media in
roller bottles grown in batch cultures, which is expected to
increase with media optimization and fed-batch cultures.
TABLE-US-00006 TABLE 5 Amplification progress of 2 different
constructs, A-IgG and Z-Fab. Productivity was evaluated by sandwich
ELISA. A-IgG Productivity Z-Fab Productivity MTX (.mu.M) (mg/L) MTX
(.mu.M) (mg/L) 0.1 50 0.2 89 0.4 102 0.8 84 0.7 128 1.5 97 1 133
3.0 127 2 177 4.5 141 3 189
Example 12
Transfection of SpESF With C-DDD2-Fab-hMN-14
[0175] Linearized C-DDD2-Fab-hMN-14-pdHL2 DNA (40 .mu.g) was used
to transfect 2.4.times.10.sup.6 SpESF cells by electroporation
using standard conditions. Cells were plated into sixteen 96-well
plates and selected with 0.15 82 M MTX. Approximately 1000 positive
clones were identified, 32 of which were high-level producers. Some
of the high producers were amplified with increasing MTX, resulting
more than a two-fold increase in productivity. Following
amplification, high producing cell lines were subcloned by limiting
dilution at 0.3 cells/well in three 96-well plates resulting in
sixty-two viable subclones representing survival efficiency of
>70%.
Example 13
Development of SpESF-X Cells
[0176] For further enhancement, the SpESF cells were subjected to
iterative rounds of stressful growth conditions in the hope that
even more robust cell lines could be obtained. SpESF cells were
allowed to overgrow until the viability reached 50-75%. At this
point the cells were allowed to recover in fresh media, followed by
another typical passage in fresh media. This cycle was repeated
over 4 months and then subcloning by limiting dilution was
performed, which resulted in 14 subclones, designated SpESF-X1
through 14. FIG. 8 summarizes the growth properties and viability
of the 14 SpESF-X subclones. AUCs were calculated for each
subclone. Based on these data, 5 subclones were compared to the
parental SpESF-X (before subcloning), Sp2/0, and SpEEE cell lines
(FIG. 9). SpESF-X2 survived for a longer period of time than the
other subclones and had the highest AUC. By comparison, SpESF-X6
reached a higher density than any other subclone, but had the
lowest AUC. To further select the best X clone as the host cells,
transfection may need to be performed with each subclone to
determine if or how the improved growth characteristics of each
clone will actually translate into increased protein expression.
Additional examples of recombinant proteins expressed in SpESF or
SpESF-X are listed in Table 6.
TABLE-US-00007 TABLE 6 Recombinant proteins expressed in SpESF or
SpESF-X Construct Antigen Structure Host Cell Line
C-DDD2-Fab-hMN-14 CEACAM5 (Fab).sub.2 SpESF hA20-Fab-DDD2 CD20
(Fab).sub.2 SpESF h679-Fab-AD2 HSG Fab SpESF and SpESF-X hL243-IgG
HLA-DR IgG SpESF hL243-IgG-AD2 HLA-DR IgG SpESF and SpESF-X
hA19-Fab-DDD2 CD19 (Fab).sub.2 SpESF-X hR1-IgG-AD2 IGF1R IgG SpESF
and SpESF-X hPAM4-Fab-DDD2 Muc1 (Fab).sub.2 SpESF hL243-Fab-DDD1
HLA-DR (Fab).sub.2 SpESF-X hL243-Fab-DDD2 HLA-DR (Fab).sub.2
SpESF-X CDDD2-Fab-hRS7 EGP-1 (Fab).sub.2 SpESF-X hMN-15-Fab-DDD2
CEACAM6 (Fab).sub.2 SpESF Epo-DDD2 N/A N/A SpESF hR1-Fab-DDD2 IGF1R
(Fab).sub.2 SpESF hA20-IgG-AD2 CD20 IgG SpESF hLL2-IgG-AD2 CD22 IgG
SpESF hLL1-Fab-DDD2 CD74 (Fab).sub.2 SpESF
[0177] Conclusions
[0178] Bcl-2 is a pro-survival/anti-apoptotic protein. It has been
demonstrated by several groups that a Bcl-2 deletion mutant missing
the flexible loop domain (FLD) has an enhanced ability to inhibit
apoptosis (Figueroa et al., 2001; Chang et al., 1997). More
recently, it was demonstrated that mutation of 1 to 3
serine/threonine residues in the FLD of Bcl-2 to glutamic acid,
which mimics hyper-phosphorylation, significantly enhances its
anti-apoptotic ability (Deng et al., 2004). Phosphorylation at
these or other residues in the positive regulatory domain (aa
69-87) of the FLD appears to block binding of p53 to the negative
regulatory domain (aa 32-68) and functions to maintain Bcl-2's
survival function (Deng et al., 2006, Mol Cell Biol
26(12):4421-4434).
[0179] We have generated three murine myeloma host cell lines,
which carry the BCL-2 triple mutant (T69E, S70E and S87E), for the
expression of recombinant proteins. The transfection efficiency of
the hBCL2(EEE)-pZeoSV2+ vector was very high for SP2/0 cells. A
total of 40 wells were chosen randomly from the plates with the
highest zeocin concentration and analyzed by anti-Bcl-2 immunoblot.
A wide range of expression levels was observed and the cells with
the highest Bcl-2-EEE were immediately sub-cloned by limiting
dilution.
[0180] Over-expression of Bcl-2-EEE appears to inhibit apoptosis in
SP2/0 cells. This effect was largely dose-dependent, since clones
with higher expression levels had a tendency to survive longer than
those with lower levels. Bcl-2 expression was not detected in
untransfected SP2/0 cells. The highest Bcl-2-EEE-expressing clone
grew to a 15-20% higher cell density and survived an additional 4-6
days in batch culture compared to SP2/0 cells.
[0181] Serum-deprivation experiments demonstrated that SpEEE clone
#87-29 (SpESF) possessed enhanced survival function, presumably due
to its resistance to apoptosis. This property allowed the facile
adaptation to growth in serum-free media and eliminated the
requirement of serum over the entire cell line development process,
including transfection, gene amplification, subcloning, and
cryopreservation. This and other qualities make these attractive
host cell lines for recombinant protein expression. Expression of
Bcl-2-EEE, which is stable in the absence of zeocin selection, has
resulted in the generation of a robust cell line that reaches high
cell-density and sustains high cell viability for an extended
period of time. The absence of serum reduces many potential risks
associated with the use of animal products, such as the
introduction of adventitious agents (Merten, 1999, Dev Biol Stand
99:167-180). And finally, the development of stable recombinant
protein-expressing cell lines is expedited because no additional
serum-weaning step is required. Both SpESF and SpESF-X have been
shown to be suitable host cells for generating mAb-production cell
lines from transfection to expression, all in serum-free medium,
reaching 4.times.10.sup.6 cells/ml and yielding 150 to 200 mg/mL in
batch cultures.
[0182] The PERC.6 and NSO-PFCF are two promising serum-free cell
line platforms for monoclonal antibody production; however, they
both require supplementation with 10% FBS during transfection to
help the cells recover (Jones, et al., 2003; Hartman, et al., 2006,
Biotechnol Bioeng 96:294-306). In addition, several serum-free cell
lines exist that have been used for large-scale, transient
transfection of recombinant proteins (Rosser, et al., 2005; Pham,
et al., 2003, Biotechnol Bioeng 84:332-342; Derouazi, et al., 2004,
Biotech Bioeng. 87(4):537-545). As far as the inventors are aware,
this is the first successful application of a serum-free cell line
for the stable production of recombinant proteins.
Example 14
Production of Human Growth Hormone in SpESF-X Cells
[0183] A cDNA encoding human growth hormone (e.g, GenBank Accession
No. NM 000515) is cloned into a mammalian expression vector and
transfected into SpESF-X cells by electroporation as disclosed in
Example 12. Cells are plated into 96-well plates and selected with
0.15 .mu.M MTX. Production of hGH is confirmed by immunoassay using
antibody against hGH. High-level producing clones of hGH are
selected and subcloned by limiting dilution. Several subcloned cell
lines are maintained in cell culture. The SpESF-X cells are stably
transfected with the hGH expression vector and produce hGH at a
level of over 150 mg protein/mL of growth medium.
Sequence CWU 1
1
1119780DNAArtificial SequenceDescription of Artificial Sequence
Synthetic nucleotide construct 1ttccataggc tccgcccccc tgacgagcat
cacaaaaatc gacgctcaag tcagaggtgg 60cgaaacccga caggactata aagataccag
gcgtttcccc ctggaagctc cctcgtgcgc 120tctcctgttc cgaccctgcc
gcttaccgga tacctgtccg cctttctccc ttcgggaagc 180gtggcgcttt
ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
240aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
atccggtaac 300tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
cactggcagc agccactggt 360aacaggatta gcagagcgag gtatgtaggc
ggtgctacag agttcttgaa gtggtggcct 420aactacggct acactagaag
gacagtattt ggtatctgcg ctctgctgaa gccagttacc 480ttcggaaaaa
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
540ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
agatcctttg 600atcttttcta cggggtctga cgctcagtgg aacgaaaact
cacgttaagg gattttggtc 660atgagattat caaaaaggat cttcacctag
atccttttaa attaaaaatg aagttttaaa 720tcaatctaaa gtatatatga
gtaaacttgg tctgacagtt accaatgctt aatcagtgag 780gcacctatct
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
840tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
gataccgcga 900gacccacgct caccggctcc agatttatca gcaataaacc
agccagccgg aagggccgag 960cgcagaagtg gtcctgcaac tttatccgcc
tccatccagt ctattaattg ttgccgggaa 1020gctagagtaa gtagttcgcc
agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc 1080atcgtggtgt
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
1140aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
cggtcctccg 1200atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
tggttatggc agcactgcat 1260aattctctta ctgtcatgcc atccgtaaga
tgcttttctg tgactggtga gtactcaacc 1320aagtcattct gagaatagtg
tatgcggcga ccgagttgct cttgcccggc gtcaacacgg 1380gataataccg
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
1440gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
acccactcgt 1500gcacccaact gatcttcagc atcttttact ttcaccagcg
tttctgggtg agcaaaaaca 1560ggaaggcaaa atgccgcaaa aaagggaata
agggcgacac ggaaatgttg aatactcata 1620ctcttccttt ttcaatatta
ttgaagcatt tatcagggtt attgtctcat gagcggatac 1680atatttgaat
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
1740ggccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
aataggcgta 1800tcacgaggcc ctttcgtctt caagaattcc gatccagaca
tgataagata cattgatgag 1860tttggacaaa ccacaactag aatgcagtga
aaaaaatgct ttatttgtga aatttgtgat 1920gctattgctt tatttgtaac
cattataagc tgcaataaac aagttaacaa caacaattgc 1980attcatttta
tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
2040ctctacaaat gtggtatggc tgattatgat ctaaagccag caaaagtccc
atggtcttat 2100aaaaatgcat agctttagga ggggagcaga gaacttgaaa
gcatcttcct gttagtcttt 2160cttctcgtag acttcaaact tatacttgat
gcctttttcc tcctggacct cagagaggac 2220gcctgggtat tctgggagaa
gtttatattt ccccaaatca atttctggga aaaacgtgtc 2280actttcaaat
tcctgcatga tccttgtcac aaagagtctg aggtggcctg gttgattcat
2340ggcttcctgg taaacagaac tgcctccgac tatccaaacc atgtctactt
tacttgccaa 2400ttccggttgt tcaataagtc ttaaggcatc atccaaactt
ttggcaagaa aatgagctcc 2460tcgtggtggt tctttgagtt ctctactgag
aactatatta attctgtcct ttaaaggtcg 2520attcttctca ggaatggaga
accaggtttt cctacccata atcaccagat tctgtttacc 2580ttccactgaa
gaggttgtgg tcattctttg gaagtacttg aactcgttcc tgagcggagg
2640ccagggtcgg tctccgttct tgccaatccc catattttgg gacacggcga
cgatgcagtt 2700caatggtcga accatgaggg caccaagcta gctttttgca
aaagcctagg cctccaaaaa 2760agcctcctca ctacttctgg aatagctcag
aggccgaggc ggcctcggcc tctgcataaa 2820taaaaaaaat tagtcagcca
tggggcggag aatgggcgga actgggcgga gttaggggcg 2880ggatgggcgg
agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca
2940tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac
taattgagat 3000gcatgctttg catacttctg cctgctgggg agcctgggga
ctttccacac cctaactgac 3060acacattcca cagtcgacta gaatatggat
agtgggtgtt tatgactctg gataagcctg 3120aacaattgat gattaatgcc
cctgagctct gttcttagta acatgtgaac atttacttgt 3180gtcagtgtag
tagatttcac atgacatctt ataataaacc tgtaaatgaa agtaatttgc
3240attactagcc cagcccagcc catactaaga gttatattat gtctgtctca
cagcctgctg 3300ctgaccaata ttgaaaagaa tagaccttcg actggcagga
agcaggtcat gtggcaaggc 3360tatttgggga agggaaaata aaaccactag
gtaaacttgt agctgtggtt tgaagaagtg 3420gttttgaaac actctgtcca
gccccaccaa accgaaagtc caggctgagc aaaacaccac 3480ctgggtaatt
tgcatttcta aaataagttg aggattcagc cgaaactgga gaggtcctct
3540tttaacttat tgagttcaac cttttaattt tagcttgagt agttctagtt
tccccaaact 3600taagtttatc gacttctaaa atgtatttag aatttcgacc
aattctcatg tttgacagct 3660tatcatcgct gcactccgcc cgaaaagtgc
gctcggctct gccaaggacg cggggcgcgt 3720gactatgcgt gggctggagc
aaccgcctgc tgggtgcaaa ccctttgcgc ccggactcgt 3780ccaacgacta
taaagagggc aggctgtcct ctaagcgtca ccacgacttc aacgtcctga
3840gtaccttctc ctcacttact ccgtagctcc agcttcacca gatccctcga
ctctagaggc 3900cttaagggcc ttactgagca cacaggacct caccatggga
tggagctgta tcatcctctt 3960cttggtagca acagctacag gtaaggggct
cacagtagca ggcttgaggt ctggacatat 4020atatgggtga caatgacatc
cactttgcct ttctctccac aggtgtccac tccgacatcc 4080agctgaccca
gagcccaagc agcctgagcg ccagcgtggg tgacagagtg accatcacct
4140gtaaggccag tcaggatgtg ggtacttctg tagcctggta ccagcagaag
ccaggtaagg 4200ctccaaagct gctgatctac tggacatcca cccggcacac
tggtgtgcca agcagattca 4260gcggtagcgg tagcggtacc gacttcacct
tcaccatcag cagcctccag ccagaggaca 4320tcgccaccta ctactgccag
caatatagcc tctatcggtc gttcggccaa gggaccaagg 4380tggaaatcaa
acgtgagtag aatttaaact ttgcttcctc agttggatcc cgcaattcta
4440aactctgagg gggtcggatg acgtggccat tctttgccta aagcattgag
tttactgcaa 4500ggtcagaaaa gcatgcaaag ccctcagaat ggctgcaaag
agctccaaca aaacaattta 4560gaactttatt aaggaatagg gggaagctag
gaagaaactc aaaacatcaa gattttaaat 4620acgcttcttg gtctccttgc
tataattatc tgggataagc atgctgtttt ctgtctgtcc 4680ctaacatgcc
ctgtgattat ccgcaaacaa cacacccaag ggcagaactt tgttacttaa
4740acaccatcct gtttgcttct ttcctcagga actgtggctg caccatctgt
cttcatcttc 4800ccgccatctg atgagcagtt gaaatctgga actgcctctg
ttgtgtgcct gctgaataac 4860ttctatccca gagaggccaa agtacagtgg
aaggtggata acgccctcca atcgggtaac 4920tcccaggaga gtgtcacaga
gcaggacagc aaggacagca cctacagcct cagcagcacc 4980ctgacgctga
gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat
5040cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta
gagggagaag 5100tgcccccacc tgctcctcag ttccagcctg accccctccc
atcctttggc ctctgaccct 5160ttttccacag gggacctacc cctattgcgg
tcctccagct catctttcac ctcacccccc 5220tcctcctcct tggctttaat
tatgctaatg ttggaggaga atgaataaat aaagtgaatc 5280tttgcacctg
tggtttctct ctttcctcat ttaataatta ttatctgttg ttttaccaac
5340tactcaattt ctcttataag ggactaaata tgtagtcatc ctaaggcgca
taaccattta 5400taaaaatcat ccttcattct attttaccct atcatcctct
gcaagacagt cctccctcaa 5460acccacaagc cttctgtcct cacagtcccc
tgggccatgg taggagagac ttgcttcctt 5520gttttcccct cctcagcaag
ccctcatagt cctttttaag ggtgacaggt cttacagtca 5580tatatccttt
gattcaattc cctgagaatc aaccaaagca aatttttcaa aagaagaaac
5640ctgctataaa gagaatcatt cattgcaaca tgatataaaa taacaacaca
ataaaagcaa 5700ttaaataaac aaacaatagg gaaatgttta agttcatcat
ggtacttaga cttaatggaa 5760tgtcatgcct tatttacatt tttaaacagg
tactgaggga ctcctgtctg ccaagggccg 5820tattgagtac tttccacaac
ctaatttaat ccacactata ctgtgagatt aaaaacattc 5880attaaaatgt
tgcaaaggtt ctataaagct gagagacaaa tatattctat aactcagcaa
5940ttcccacttc taggggttcg actggcagga agcaggtcat gtggcaaggc
tatttgggga 6000agggaaaata aaaccactag gtaaacttgt agctgtggtt
tgaagaagtg gttttgaaac 6060actctgtcca gccccaccaa accgaaagtc
caggctgagc aaaacaccac ctgggtaatt 6120tgcatttcta aaataagttg
aggattcagc cgaaactgga gaggtcctct tttaacttat 6180tgagttcaac
cttttaattt tagcttgagt agttctagtt tccccaaact taagtttatc
6240gacttctaaa atgtatttag aatttcgacc aattctcatg tttgacagct
tatcatcgct 6300gcactccgcc cgaaaagtgc gctcggctct gccaaggacg
cggggcgcgt gactatgcgt 6360gggctggagc aaccgcctgc tgggtgcaaa
ccctttgcgc ccggactcgt ccaacgacta 6420taaagagggc aggctgtcct
ctaagcgtca ccacgacttc aacgtcctga gtaccttctc 6480ctcacttact
ccgtagctcc agcttcacca gatccctcga gcacacagga cctcaccatg
6540ggatggagct gtatcatcct cttcttggta gcaacagcta caggtaaggg
gctcacagta 6600gcaggcttga ggtctggaca tatatatggg tgacaatgac
atccactttg cctttctctc 6660cacaggtgtc cactcccagg tccaactgca
ggagagcggt ggaggtgttg tgcaacctgg 6720ccggtccctg cgcctgtcct
gctccgcatc tggcttcgat ttcaccacat attggatgag 6780ttgggtgcga
caggcacctg gaaaaggtct tgagtggatt ggagaaattc atccagatag
6840cagtacgatt aactatgcgc cgtcgctaaa agatagattt acaatatcgc
gagacaacgc 6900caagaacaca ttgttcctgc aaatggacag cctgagaccc
gaagacaccg gggtctattt 6960ttgtgcaagc ctttacttcg gcttcccctg
gtttgcttat tggggccaag ggaccccggt 7020caccgtctcc tcaggtgagt
ccttacaacc tctctcttct attcagctta aatagatttt 7080actgcatttg
ttggggggga aatgtgtgta tctgaatttc aggtcatgaa ggactaggga
7140caccttggga gtcagaaagg gtcattggga gccccaagct ttctggggca
ggccaggcct 7200gaccttggct ttggggcagg gagggggcta aggtgaggca
ggtggcgcca gccaggtgca 7260cacccaatgc ccatgagccc agacactgga
cgctgaacct cgcggacagt taagaaccca 7320ggggcctctg cgccctgggc
ccagctctgt cccacaccgc ggtcacatgg caccacctct 7380cttgcagcct
ccaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc
7440tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga
accggtgacg 7500gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca
ccttcccggc tgtcctacag 7560tcctcaggac tctactccct cagcagcgtg
gtgaccgtgc cctccagcag cttgggcacc 7620cagacctaca tctgcaacgt
gaatcacaag cccagcaaca ccaaggtgga caagagagtt 7680ggtgagaggc
cagcacaggg agggagggtg tctgctggaa gccaggctca gcgctcctgc
7740ctggacgcat cccggctatg cagccccagt ccagggcagc aaggcaggcc
ccgtctgcct 7800cttcacccgg agcctctgcc cgccccactc atgctcaggg
agagggtctt ctggcttttt 7860cccaggctct gggcaggcac aggctaggtg
cccctaaccc aggccctgca cacaaagggg 7920caggtgctgg gctcagacct
gccaagagcc atatccggga ggaccctgcc cctgacctaa 7980gcccacccca
aaggccaaac tctccactcc ctcagctcgg acaccttctc tcctcccaga
8040ttccagtaac tcccaatctt ctctctgcag agcccaaatc ttgtgacaaa
actcacacat 8100gcccaccgtg cccaggtaag ccagcccagg cctcgccctc
cagctcaagg cgggacaggt 8160gccctagagt agcctgcatc cagggacagg
ccccagccgg gtgctgacac gtccacctcc 8220atctcttcct cagcacctga
actcctgggg ggaccgtcag tcttcctctt ccccccaaaa 8280cccaaggaca
ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg
8340agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga
ggtgcataat 8400gccaagacaa agccgcggga ggagcagtac aacagcacgt
accgggtggt cagcgtcctc 8460accgtcctgc accaggactg gctgaatggc
aaggagtaca agtgcaaggt ctccaacaaa 8520gccctcccag cccccatcga
gaaaaccatc tccaaagcca aaggtgggac ccgtggggtg 8580cgagggccac
atggacagag gccggctcgg cccaccctct gccctgagag tgaccgctgt
8640accaacctct gtcctacagg gcagccccga gaaccacagg tgtacaccct
gcccccatcc 8700cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc
tggtcaaagg cttctatccc 8760agcgacatcg ccgtggagtg ggagagcaat
gggcagccgg agaacaacta caagaccacg 8820cctcccgtgc tggactccga
cggctccttc ttcctctata gcaagctcac cgtggacaag 8880agcaggtggc
agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac
8940cactacacgc agaagagcct ctccctgtct ccgggtaaat gagtgcgacg
gccggcaagc 9000ccccgctccc cgggctctcg cggtcgcacg aggatgcttg
gcacgtaccc cgtctacata 9060cttcccaggc acccagcatg gaaataaagc
acccaccact gccctgggcc cctgcgagac 9120tgtgatggtt ctttccacgg
gtcaggccga gtctgaggcc tgagtggcat gagggaggca 9180gagcgggtcc
cactgtcccc acactggccc aggctgtgca ggtgtgcctg ggccgcctag
9240ggtggggctc agccaggggc tgccctcggc agggtggggg atttgccagc
gtggccctcc 9300ctccagcagc agctgcctcg cgcgtttcgg tgatgacggt
gaaaacctct gacacatgca 9360gctcccggag acggtcacag cttgtctgta
agcggatgcc gggagcagac aagcccgtca 9420gggcgcgtca gcgggtgttg
gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga 9480tagcggagtg
tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
9540catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
caggcgctct 9600tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
ggctgcggcg agcggtatca 9660gctcactcaa aggcggtaat acggttatcc
acagaatcag gggataacgc aggaaagaac 9720atgtgagcaa aaggccagca
aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 97802420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic nucleotide
sequence 2caatggtcga accatgaggg caccaagcta gctttttgca aaagcctagg
cctccaaaaa 60agcctcctca ctacttctgg aatagctcag aggccgaggc ggcctcggcc
tctgcataaa 120taaaaaaaat tagtcagcca tggggcggag aatgggcgga
actgggcgga gttaggggcg 180ggatgggcgg agttaggggc gggactatgg
ttgctgacta attgagatgc atgctttgca 240tacttctgcc tgctggggag
cctggggact ttccacacct ggttgctgac taattgagat 300gcatgctttg
catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac
360acacattcca cagtcgacta gaatatggat agtgggtgtt tatgactctg
gataagcctg 4203720DNAArtificial SequenceDescription of Artificial
Sequence Synthetic nucleotide sequence 3atggcgcacg ctgggagaac
ggggtacgat aaccgggaga tagtgatgaa gtacatccat 60tataagctgt cgcagagggg
ctacgagtgg gatgcgggag atgtgggcgc cgcgcccccg 120ggggccgccc
ccgcaccggg catcttctcc tcccagcccg ggcacacgcc ccatccagcc
180gcatcccgcg acccggtcgc cagagaagaa ccgctgcaga ctccggctgc
tcctggagca 240gctgcaggac ctgcgctcga accggtgcca cctgtggtcc
acctgaccct ccgccaggcc 300ggcgacgact tctcccgccg ctaccgccgc
gacttcgccg agatgtccag ccagctgcac 360ctgacgccct tcaccgcgcg
gggacgcttt gccacggtgg tggaggagct cttcagggac 420ggggtgaact
gggggaggat tgtggccttc tttgagttcg gtggggtcat gtgtgtggag
480agcgtcaacc gggagatgtc gcccctggtg gacaacatcg ccctgtggat
gactgagtac 540ctgaaccggc acctgcacac ctggatccag gataacggag
gctgggatgc ctttgtggaa 600ctgtacggcc ccagcatgcg gcctctgttt
gatttctcct ggctgtctct gaagactctg 660ctcagtttgg ccctggtggg
agcttgcatc accctgggtg cctatctggg ccacaagtga 7204239PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein 4Met
Ala His Ala Gly Arg Thr Gly Tyr Asp Asn Arg Glu Ile Val Met 1 5 10
15Lys Tyr Ile His Tyr Lys Leu Ser Gln Arg Gly Tyr Glu Trp Asp Ala
20 25 30Gly Asp Val Gly Ala Ala Pro Pro Gly Ala Ala Pro Ala Pro Gly
Ile 35 40 45Phe Ser Ser Gln Pro Gly His Thr Pro His Pro Ala Ala Ser
Arg Asp 50 55 60Pro Val Ala Arg Glu Glu Pro Leu Gln Thr Pro Ala Ala
Pro Gly Ala 65 70 75 80Ala Ala Gly Pro Ala Leu Glu Pro Val Pro Pro
Val Val His Leu Thr 85 90 95Leu Arg Gln Ala Gly Asp Asp Phe Ser Arg
Arg Tyr Arg Arg Asp Phe 100 105 110Ala Glu Met Ser Ser Gln Leu His
Leu Thr Pro Phe Thr Ala Arg Gly 115 120 125Arg Phe Ala Thr Val Val
Glu Glu Leu Phe Arg Asp Gly Val Asn Trp 130 135 140Gly Arg Ile Val
Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu145 150 155 160Ser
Val Asn Arg Glu Met Ser Pro Leu Val Asp Asn Ile Ala Leu Trp 165 170
175Met Thr Glu Tyr Leu Asn Arg His Leu His Thr Trp Ile Gln Asp Asn
180 185 190Gly Gly Trp Asp Ala Phe Val Glu Leu Tyr Gly Pro Ser Met
Arg Pro 195 200 205Leu Phe Asp Phe Ser Trp Leu Ser Leu Lys Thr Leu
Leu Ser Leu Ala 210 215 220Leu Val Gly Ala Cys Ile Thr Leu Gly Ala
Tyr Leu Gly His Lys225 230 235580DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 5ggacccggtc
gccagagaag aaccgctgca gactccggct gctcctggag cagctgcagg 60acctgcgctc
gaaccggtgc 80680DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 6cgccggcctg gcggagggtc
aggtggacca caggtggcac cggttcgagc gcaggtcctg 60cagctgctcc aggagcagcc
8074PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Asp Glu Val Asp 1824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8atgtttcagg acccacagga gcga 24924DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 9ttatggtttc tgagaacaga tggg
241025DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10tatatggacc cggtcgccag agaag 251125DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11ttaatcgccg gcctggcgga gggtc 25
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