U.S. patent application number 09/728342 was filed with the patent office on 2001-04-26 for method for culturing recombinant cells.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Mather, Jennie P..
Application Number | 20010000488 09/728342 |
Document ID | / |
Family ID | 22263548 |
Filed Date | 2001-04-26 |
United States Patent
Application |
20010000488 |
Kind Code |
A1 |
Mather, Jennie P. |
April 26, 2001 |
Method for culturing recombinant cells
Abstract
A method for culturing a recombinant host cell comprising:
determining a polypeptide factor for a polypeptide factor-dependent
host cell; transforming said host cell with nucleic acid encoding
said polypeptide factor; transforming the host cell with nucleic
acid encoding a desired protein; and, culturing the transformed
host cells in a medium lacking the polypeptide factor.
Inventors: |
Mather, Jennie P.;
(Millbrae, CA) |
Correspondence
Address: |
Genentech, Inc.
1 DNA Way
South San Francusco
CA
94080
US
|
Assignee: |
Genentech, Inc.
|
Family ID: |
22263548 |
Appl. No.: |
09/728342 |
Filed: |
December 1, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09728342 |
Dec 1, 2000 |
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08845698 |
Apr 25, 1997 |
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08845698 |
Apr 25, 1997 |
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08633638 |
Apr 17, 1996 |
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08633638 |
Apr 17, 1996 |
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08420619 |
Apr 12, 1995 |
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08420619 |
Apr 12, 1995 |
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08222498 |
Apr 4, 1994 |
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08222498 |
Apr 4, 1994 |
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07097472 |
Sep 11, 1987 |
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Current U.S.
Class: |
435/69.1 ;
435/325; 435/358; 530/303; 530/305 |
Current CPC
Class: |
C07K 14/79 20130101;
C07K 14/59 20130101 |
Class at
Publication: |
435/69.1 ; 435;
435/325; 435/358; 530/303; 530/305 |
International
Class: |
C12P 021/02; C12N
015/17; C12P 021/06; C07K 005/00; C07K 007/00; C07K 016/00; C07K
017/00; A61K 038/28; C12N 005/06; C12N 005/10 |
Claims
Claims
1. A method for culturing a host cell comprising: a. determining a
polypeptide factor for a polypeptide factor-dependent host cell; b.
transforming said host cell with nucleic acid encoding said
polypeptide factor: c. transforming the host cell with nucleic acid
encoding a desired protein; and d. culturing the transformed cells
of step (c) in a medium lacking the polypeptide factor.
2. The method of claim 1 which additionally comprises the step of
recovering the desired protein.
3. The method of claim 1 wherein the medium is serum-free
medium.
4. The method of claim 1 wherein the host cell is a vertebrate host
cell.
5. The method of claim 1 wherein the host cell is a chinese hamster
ovary cell.
6. The method of claim 1 wherein the polypeptide factor is
proinsulin.
7. The method of claim 1 wherein the polypeptide factor is
transferrin.
8. The method of claim 1 wherein the polypeptide factor is
transferrin and insulin.
9. A host cell transformed to express a desired protein and a
polypeptide factor.
10. The host cell of claim 9 which is a vertebrate cell.
11. The host cell of claim 9 which is a chinese hamster ovary
cell.
12. The host cell of claim 11 wherein the polypeptide factor is
proinsulin.
13. The host cell of claim 9 which is a 293 cell.
14. The host cell of claim 9 wherein the polypeptide factor is
proinsulin.
15. The host cell of claim 9 wherein the polypeptide factor is
transferrin.
16. A culture comprising the host cell of claim 9 and a medium
lacking the polypeptide factor expressed by said host cell.
17. The culture of claim 16 wherein the medium is a serum-free
medium.
18. The culture of claim 16 wherein the polypeptide factor lacking
in the medium is insulin.
Description
CROSS-REFERENCE TO RELATED APPLICATION
1. U.S. Ser. No. ______ filed of even date, relates to a method of
preparing heterologous polypeptides in a recombinant host cell and
enhancing the yields of said heterologous polypeptides by
transfecting said host cell with a nucleic acid encoding an
oncogene and culturing those transformed host cells.
FIELD OF THE INVENTION
2. This invention relates to methods for culturing vertebrate host
cells transformed to produce a desired protein. In particular it
relates to the use of recombinant technology to create host cells
which will produce factors necessary for their survival and growth
in culture.
BACKGROUND OF THE INVENTION
3. The last decade has seen an explosive growth in the knowledge of
molecular biology and commercialization of that knowledge. Great
success has been had in the cloning and expression of genes
encoding proteins that were previously available in very small
amounts, such as human growth hormone, tissue plasminogen activator
and various lymphokines, to name just a few. Initially attempts
were made to produce these proteins in bacterial or yeast
expression systems. Many proteins may be preferably produced in
cell culture. The reasons influencing one to use cell culture are:
glycosylation of the desired protein, ease of purification of
secreted products, and correct protein processing with correct
folding and disulfide bond formation.
4. Once the gene encoding the desired protein is expressed in a
mammalian cell line, its production must then be optimized.
Optimization of protein yield in cell culture may be made by
various means. Improvement may be obtained, for example by
optimizing the physicochemical, nutritional, and hormonal
environment of the cell.
5. Mammalian cells in vivo are in a carefully balanced homeostatic
environment. The advantages of obtaining a completely defined
medium for the growth of cells in vitro were recognized very early
in the history of cell culture. (Lewis, M. R. and Lewis, W. H.,
Anat. Rec. 5:277 [1911]). Defined medium typically refers to the
specific nutritional and hormonal chemicals comprising the medium
required for survival or growth. Most cell types have stringent
requirements as to the optimal range of physical parameters for
growth and performance. Physicochemical parameters which may be
controlled in different cell culture systems, for example, are:
temperature, pH, pO.sub.2, and osmolarity. The nutritional
requirements of cells are usually provided in standard media
formulations developed to provide an optimal environment. Nutrients
can be divided into several categories: amino acids and their
derivatives, fatty acids, complex lipids, carbohydrates, sugars,
vitamins and nucleic acid derivatives. Not only the absolute
requirements, but the relative concentrations of nutrients must be
optimized for each individual cell type.
6. Most cell types will not grow and/or secrete proteins optimally
in medium consisting only of nutrients, even when the nutritional
components are optimized. It is for this reason that serum has
remained an essential medium component for the growth of cells in
culture. Various experiments led to the hypothesis that the role of
serum in cell culture was to provide a complex of hormones that
were growth-stimulatory for a given cell type. (Sato, G. H. et al.,
in Biochemical Action of Hormones, Vol. III [G. Litwak, ed.]
Academic Press, N.Y., page 391). A pituitary cell line was grown in
serum-free medium supplemented with hormones, growth factors, and
transferrin. (Hayashi. I. and Sato, G., Nature [Lond] 159:132
[1976]). Subsequently, hormone-supplemented serum-free conditions
were developed for the growth of several cell lines originating
from different tissues (Mather, J. and Sato, G., Exp. Cell Res.
120:191 [1979]; Barnes, D. and Sato, G., Cell 22:69 [1981]). These
studies led to several conclusions concerning the growth of cells
in serum-free medium. Serum can be replaced by a mixture of
hormones, growth factors, and transport proteins. The required
supplements (containing the hormones, growth factors and transport
proteins) to serum-free medium may differ for different cell types.
The supplements, traditionally, have been provided as part of
complex biological mixtures such as serum or organ extracts. The
"hormonal" milieu may be optimized to reduce or eliminate the need
for undefined growth factors, remove inhibitory factors, or provide
critical hormones at desirable levels.
7. Cells frequently require one or more hormones from each of the
following groups: steroids, prostaglandins, growth factors,
pituitary hormones, and peptide hormones. Most cell types require
insulin to survive in serum-free media. (Sato, G. H. et al. in
Growth of Cells in Hormonally Defined Media, [Cold Spring Harbor
Press, N.Y., 1982]). Certain mutant cell lines have been reported
which are insulin-independent. (Mendiaz, E. et al., In Vitro Cell.
& Dev. Biol. 22[2]:66 [1986]; Serrero, G., In Vitro Cell. &
Biol. 21[9]:537 [1985]). In addition to the hormones, cells may
require transport proteins such as transferrin (plasma iron
transport protein), ceruloplasmin (a copper transport protein), and
high density lipoprotein (a lipid carrier) to be added to cell
media. The set of optimal hormones or transport proteins will vary
for each cell type. Most of these hormones or transport proteins
have been added exogenously or, in a rare case, a mutant cell line
has been found which does not require a particular factor.
8. Recently, cellular proliferation has been studied to elaborate
the events necessary to lead from quiescent growth arrest to the
cellular commitment to proliferate. Various factors have been found
to be involved in that transformation. These transformed cells have
been found to produce peptide growth factors in culture. (Kaplan,
P. L. et al., PNAS 79:485-489 [1982]). The secretion from a cell of
a factor to which that same cell can respond has been referred to
as an "autocrine" system. Numerous factors have been described as
autocrine: bombesin, interleukin-2 (Duprez, V. et al. PNAS 82:6932
[1985]); insulin, (Serrero, G. In Vitro Cellular & Dev. Biol.
21[9]:537 [1985]); transforming growth factor alpha (TGF-.alpha.),
platelet-derived growth factor (PDGF); transforming growth
factor-beta (TGF-.beta.), (Sporn, M. B. & Roberts, A. B.,
Nature 313:745 [1985]); sarcoma growth factor (SGF), (Anzano, M. A.
et al., PNAS 80:6264 [1983]); and, hemopoietic growth factor,
granulocyte-macrophage colony stimulating factor (GM-CSF), (Lang,
R. A. et al., Cell 43:531 [1985)).
9. It is an object of the present invention to provide a defined
medium for particular recombinant host cells. Another object of
this invention is to eliminate problems associated with the supply
of necessary polypeptide factors for the maintenance and growth of
recombinant host cells. For example, certain polypeptide factors,
such as insulin, are unstable in some culture conditions. It is an
object of the invention to provide a local environment for the host
cell that is optimal for growth or survival. More particularly, it
is an object of the invention to eliminate the need for preliminary
testing, for example of purity, of polypeptide factors necessary
for the host cells in cell culture. Yet another object of this
invention is to lower the risk of contamination of a cell culture
by eliminating the need of adding exogenous factors. Another object
is to produce a more robust host cell line by providing autocrine
production of polypeptide factors necessary for the survival and
growth of recombinant host cells in culture. A further object is to
produce recombinant host cells that are less sensitive to medium
conditions. Still another object is to provide a localized
environment for cell growth or survival. Yet another object is to
improve the efficiency of cell culture through autocrine production
of necessary polypeptide factors. And yet another advantage is to
the lower the cost of the defined medium.
SUMMARY OF THE INVENTION
10. The objects of this invention are accomplished by a novel
method for culturing a recombinant host cell comprising: selecting
a polypeptide-dependent host cell that requires a polypeptide
factor for its survival or growth; transforming the host cell with
a nucleic acid encoding the particular polypeptide factor;
transforming a host cell with nucleic acid encoding a desired
protein; and, culturing the transformed host cells in a medium
lacking the particular polypeptide factor. The cells made in accord
with this invention can survive or grow in a medium lacking the
polypeptide factor. The recombinant host cell itself is satisfying
its need for the polypeptide factor. It was not appreciated until
the instant invention that a host cell could be made using
recombinant means to supply the polypeptide factor(s) necessary for
its own survival or growth in culture. Surprisingly, supply of the
necessary polypeptide factor did not limit the host cell's
capability to produce the desired protein in usable quantities.
This invention provides significant economic savings in the culture
of recombinant cells. This savings in the context of large scale
production of a desired protein is on the order of tens of millions
of dollars. Accordingly, in one aspect the invention is directed to
a method for culturing a host cell in a medium lacking necessary
polypeptide factor(s) for survival or growth. In another aspect the
invention is directed to a host cell transformed to express a
polypeptide factor necessary for its own growth or survival. Yet
another aspect of the invention is the culture comprising
polypeptide factor-transformed host cells in a medium lacking the
polypeptide factor(s) necessary for the host cells' growth and
maintenance.
BRIEF DESCRIPTION OF THE DRAWINGS
11. FIG. 1. Construction of a human preproinsulin expression
vector, pSVEHIGDHFR, used to establish an insulin-independent cell
line for production of a desired protein.
12. FIG. 2. Construction of a human preproinsulin expression
vector, pSVEHIGNeo, used to establish an insulin-independent cell
line for production of a desired protein.
13. FIG. 3. Construction of pCVSVD22/preUK54 an intermediate
plasmid for construction of pSVEHIGNeo.
14. FIG. 4. Construction of an ornithine decarboxylase (ODC)
expression vector used for amplification of the ODC gene and the
cotransfected preproinsulin gene.
15. FIG. 5. (a) Growth of two insulin-independent cell lines and
control cell line in presence of 5% whole FBS.
16. (b) Growth of two insulin-independent cell lines and the
control cell line in 1% charcoal/dextran extracted FBS (treated to
remove insulin from the medium).
17. FIG. 6. (a) Growth of control cells (CHO/DHFR.sup.-, no
preproinsulin) in serum-free medium in the presence of 0 to 10
.mu.g/ml exogenous insulin.
18. (b) Typical growth pattern of clones 7 and 12 subjected to
varying insulin concentration under serum-free conditions.
19. FIG. 7. In serum-free culture in the absence of insulin the
DFMO pool (100 .mu.M) and the unamplified clone 13 C2B-proinsulin
line (Cl.13) which was selected for insulin independence
demonstrated titers that were vastly elevated over C2B (control)
under identical conditions. The C2B/clone 13 cell ultimately
achieved tPA titers equivalent to the C2B control with 20 .mu.g/ml
insulin (C2B+ insulin).
20. FIG. 8. Diagram of an expression vector, pRKTF, encoding
transferrin.
21. FIG. 9. Construction of the Expression vector pRK5 into which
the cDNA encoding transferrin was inserted.
DETAILED DESCRIPTION
22. As used herein, npolypeptide factor," refers to any protein
necessary for the survival or growth of a host cell in culture. The
polypeptide factor may be a hormone, growth factor, peptide
hormone, autocrine factor, transport protein,
oncogene/proto-oncogene and the like. Examples of polypeptide
factors that are hormones are, for example, insulin, proinsulin,
follicle stimulating hormone (FSH), calcitonin, leutinizing hormone
(LH), glucagon, parathyroid hormone (PTH), thyroid stimulating
hormone (TSH), thyroid releasing hormone (TRH), thyroxine
(T.sub.3), growth hormone. Additional examples of polypeptide
factors are the transport proteins, such as, transferrin, serum
albumin, ceruloplasm, low density lipoprotein (LDL) and high
density lipoprotein (HDL). Other examples of polypeptide factors,
often described as autocrine because, in some instances, the cell
they are secreted from can respond to the secreted factor, are
interleukin-2, insulin, insulin-like growth factor I and II,
transforming growth factor alpha (TGF-.alpha.), platelet-derived
growth factor (PDGF), bombesin, erythropoietin, transforming growth
factor-beta (TGF-.beta.), sarcoma growth factor (SGF), epidermal
growth factor (EGF), fibroblast growth factor (FGF), thrombin,
nerve growth factor, hemopoietic growth factor and
granulocyte-macrophage colony stimulating factor (GM-CSF). Yet
other examples of polypeptide factors are peptides resulting from
the expression of certain oncogenes/proto-oncogenes. The proteins
encoded by these proto-oncogenes which come within the polypeptide
factors of this invention are growth factors, transducing proteins
and membrane receptors. Examples of a growth factor is PDGF (.beta.
subunit) encoded by the sis oncogene. Examples of peripheral
membrane proteins are the truncated cell surface receptor for EGF
encoded by erb-B, the cell surface receptor for M-CSF/CSF-1 encoded
by fms and the receptors encoded by neu and ros. An example of a
transducing protein is tyrosine kinase at the inner surface of the
plasma-membrane encoded by abl. While these polypeptide factors
encoded by oncogenes/proto-oncogenes are typically not added to a
culture medium, they may be substituted for another polypeptide
factor which is necessary. The growth factors of this invention are
non-enzymatic and thus do not include such proteins as
dihydrofolate reductase (DHFR), ornithine decarboxylase (ODC),
thymidine kinase or phosphotransferase.
23. "Desired protein" refers to a protein which is desired to be
expressed in a host cell, but which the host cell either normally
does not produce itself or produces in small amounts, and which is
not normally necessary for the cells' continued existence. The
desired protein includes a protein having as few as about five
amino acids to much larger proteins such as factor VIII. Such a
protein includes any molecule having the pre- or prepro- amino acid
sequence as well as amino acid or glycosylation variants (including
natural alleles) capable of exhibiting a biological activity in
common with the desired protein. Examples of such proteins are:
growth hormone, insulin, factor VIII, tissue plasminogen activator,
tumor necrosis factor alpha and beta, lymphotoxin, enkephalinase,
human serum albumin, oullerian inhibiting substance, relaxin,
tissue factor protein, inhibin, erythropoietin, interferon alpha,
beta and gamma, superoxide dismutase, decay accelerating factor,
viral antigen such as, for example, a portion of the AIDS envelope,
and interleukin. The desired protein could also be a polypeptide
factor.
24. The term "cell culture" or "culture" refers to populations of
vertebrate cells grown from a single cell such that the population
grows or survives for one or more generations. The growth or
survival of vertebrate cells in culture, sometimes referred to as
tissue culture, has become a routine procedure. See for example
Mammalian Cell Culture. The Use of Serum-Free Hormone-Supplemented
Media, Ed. Mather, J. P. (Plenum Press, N.Y., 1984).
25. The term "host cell" refers to those vertebrate cells capable
of growth in culture and expressing a desired protein and a
polypeptide factor(s). Suitable host cells include for example:
monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);
human embryonic kidney line (293, Graham, F. L. et al., J. Gen
Virol. 36: 59 [1977]); baby hamster kidney cells (BHK, ATCC CCL
10); chinese hamster ovary-cells-DHFR (CHO, Erlaub and Chasin, PNAS
(USA) 77:4216 [1980].backslash.0; mouse Sertoli cells (TM4, Mather,
J. P., Biol. Reprod. 23:243-251 [1980]); monkey kidney cells (CV1
ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC
CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);
canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells
(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75);
human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT
060562, ATCC CCL51); and, TR1 cells (Mather, J. P. et al., Annals
N.Y. Acad. Sci. 383:44-68 [1982]). While the preferred host cells
of this invention are vertebrate cells, other eukaryotic cells may
be used, such as for example, insect cells.
26. The host cells may be transformed with nucleic acid encoding
the polypeptide factor either before, after or simultaneously with
nucleic acid encoding the desired protein. It is preferred to
introduce the nucleic acid encoding the polypeptide factor before
thus providing a "polypeptide factor-independent host cell" capable
of being transformed with the nucleic acid encoding a desired
protein.
27. "Polypeptide factor-dependent host cell" refers to a host cell
requiring one or more polypeptide factors in the culture medium for
growth or survival. The polypeptide factor(s) for a particular host
cell is determined using general methods known to the ordinarily
skilled artisan as described below. Elimination of the polypeptide
factor from the medium may result in death of the cell or in
inhibited growth. Which result depends upon the particular host
cell, the polypeptide factor, culture conditions and other factors
such as cell density.
28. The term "medium" refers to the aqueous environment in which
the vertebrate cells are grown in culture. The medium comprises the
physicochemical, nutritional, and hormonal environment.
Traditionally the medium has been formulated by the addition of
nutritional and growth factors necessary for growth or survival.
"Serum-free medium" refers to a medium lacking serum. The hormones,
growth factors, transport proteins, peptide hormones and the like
typically found in serum which are necessary for the survival or
growth of particular cells in culture are typically added as a
supplement to serum-free medium. A "defined medium" refers to a
medium comprising nutritional and hormonal requirements necessary
for the survival and growth of the cells in culture such that the
components of the medium are known. A defined medium provided by
the method of the instant invention establishes a local environment
for a particular host cell that may differ from the general
environment of the medium.
29. Determining the particular polypeptide factor(s) and in turn
providing a defined medium required by a recombinant host cell can
be accomplished by the ordinarily skilled artisan in cell culture.
Cell lines are routinely carried in a serum-supplemented medium.
Most established cell lines have been grown in serum-supplemented
medium for a period of years. It can be assumed that to a greater
or lesser extent the serum-supplement is providing these cells with
the hormones required for growth and survival in vivo and/or the
cells have adapted to the absence of, or reduced levels of, some
hormones required.
30. There are several approaches to defining the polypeptide factor
requirements for a given cell line. The method of choice will
depend on the cell line. Several possibilities are known to the
ordinarily skilled artisan of which the following are exemplary.
The initial step is to obtain conditions where the cells will
survive and/or grow slowly for 3-6 days. In most cell types this
is, in part, a function of inoculum density. For a cell which will
attach and survive in serum-free media, it is only necessary to
select the proper inoculum density and begin testing hormones for
growth-promoting effects. Once the optimal hormone supplement is
found, the inoculum density required for survival will decrease. In
some cases the plating efficiency in hormones will be similar to
that in serum, although this is not true for all cell types. This
may be due to added requirements for attachment factors or growth
factors needed only initially or at higher concentrations than
those needed when cells are plated at high densities. Many cells,
both transformed and normal, are capable of producing substances
which are required for their attachment or growth.
31. However, some cell lines will not survive even 24 hours or will
not attach to the dish in serum-free medium. For these cells
several initial approaches are possible: pre-coat the dish with
serum; plate cells in serum-containing medium for 12-24 hours and
then change to serum-free; reduce serum concentrations to the point
where the cells will survive but not grow; and use various
attachment factors.
32. The various polypeptide factors can then be tested under these
minimal conditions. When optimal conditions for growth are found,
the serum (or pre-incubation step) can then be omitted and/or
replaced with purified attachment and/or polypeptide factors.
33. Cells in serum-free medium generally require insulin and
transferrin in a serum-free medium for for optimal growth. These
two factors should be tested first. Most cell lines require one or
more of the growth factors. These include epidermal growth factor
(EGF), fibroblast growth factor (FGF), insulin like growth factors
I and II (IGFI, IGFII), nerve growth factor (NGF), etc. Other
classes of factors which may be necessary include: prostaglandins;
steroids; transport and binding proteins (e.g., ceruloplasmin, high
and low density lipoprotein [HDL, LDL], albumin); hormones; and
fatty acids.
34. Polypeptide factor testing is best done in a stepwise fashion
testing new polypeptide factors in the presence of those found to
be growth stimulatory. This is essential in some cases as
polypeptide factor effects are seldom simply additive.
Alternatively, some polypeptide factors can stimulate growth singly
but their effects when added together cancel or are inhibitory.
35. A complete replacement of serum by polypeptide factor would
ideally allow for a doubling time and plating efficiency equal to
(or in some cases greater than) that seen for that cell type in
serum and the ability to carry the cell line through successive
subcultures in the polypeptide factor-supplemented serum-free
medium. It would be expected that the dose of each polypeptide
factor added should fall within the physiologic range for that
factor. It should be noted, however, that this is not always the
case. In some cases a higher level is required (e.g., insulin at
5-10 .mu.g/ml) and in others, a lower range (e.g., TF 0.50-50
.mu.g/ml). Finally, a more highly purified preparation of added
polypeptide factors may elicit a different response than a less
pure form. Additionally, the optimal amount of a given polypeptide
factor added to the media may vary in different media, for cells
grown on different substrates, or in the presence of other
polypeptide factors.
36. For undefined media it is sufficient to grow cells in
conditions in which the polypeptide factor is known to be absent or
inactive (e.g., depleted serum) (Nishikawa et al. Proc. Natl. Acad.
Sci. USA 72:483-487 [1975]; Kato et al. Exptl. Cell Res. 130:73-81
[1980]; McAuslan et al. Exptl. Cell Res. 128:95-101 [1980]; and
Ross et al. Cell 14:203-210 [1978]) The growth of cells in the
presence or absence of the polypeptide factor can then be measured
to determine whether the factor is required for growth stimulation
or survival. The polypeptide factor tested should be of sufficient
purity to be able to conclude with reasonable certainty that it is,
in fact, the known peptide which is responsible for the growth
stimulation.
37. "Control region" refers to specific sequences at the 5' and 3'
ends of eukaryotic genes which may be involved in the control of
either transcription or translation. Virtually all eukaryotic genes
have an AT-rich region located approximately 25 to 30 bases
upstream from the promoter, the site where transcription is
initiated. Another sequence found 70 to 80 bases upstream from the
start of transcription of many genes is a CXCAAT region where X may
be any nucleotide. At the 3' end of most eukaryotic genes is an
AATAAA sequence which may be the signal for addition of the poly A
tail to the 3' end of the transcribed mRNA.
38. Preferred promoters controlling transcription from vectors in
mammalian host cells may be obtained from various sources, for
example, the genomes of viruses such as: polyoma, Simian Virus 40
(SV40), adenovirus, retroviruses, hepatitis-B virus and most
preferably cytomegalovirus, or from heterologous mammalian
promoters, e.g. beta actin promoter. The early and late promoters
of the SV40 virus are conveniently obtained as an SV40 restriction
fragment which also contains the SV40 viral origin of replication.
Fiers et al., Nature, 273: 113 (1978). The immediate early promoter
of the human cytomegalovirus is conveniently obtained as a HindIII
E restriction fragment. Greenaway, P. J. et al., Gene 18: 355-360
(1982). Of course, promoters from the host cell or related species
also are useful herein.
39. Transcription of a DNA encoding a polypeptide factor or desired
protein by higher eukaryotes is increased by inserting an enhancer
sequence into the vector. Enhancers are cis-acting elements of DNA,
usually about from 10-300 bp, that act on a promoter to increase
its transcription. Enhancers are relatively orientation and
position-independent having been found 5' (Laimins, L. et al., PNAS
78: 993 [1981]) and 3' (Lusky, M. L., et al., Mol. Cell Bio. 3:
1108 [1983]) to the transcription unit, within an intron (Banerji,
J. L. et al., Cell 33: 729 [1983]) as well as within the coding
sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293
[1984]). Many enhancer sequences are now known from mammalian genes
(globin, elastase, albumin, .alpha.-fetoprotein and insulin).
Typically, however, one will use an enhancer from a eukaryotic cell
virus. Examples include the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.
40. Expression vectors used in eukaryotic host cells, including
vertebrate host cells, will also contain sequences necessary for
the termination of transcription which may affect mRNA expression.
These regions are transcribed as polyadenylated segments in the
untranslated portion of the mRNA encoding the polypeptide factor or
the desired protein. The 3' untranslated regions also include
transcription termination sites.
41. Expression vectors for expression of the desired protein or the
polypeptide factor may contain a selection gene, also termed a
selectable marker. Examples of suitable selectable markers for
mammalian cells are dihydrofolate reductase (DHFR), thymidine
kinase or phosphotransferase. When such selectable markers are
successfully transferred into a mammalian host cell, the
transformed mammalian host cell can survive if placed under
selective pressure. There are two widely used distinct categories
of selective regimes. The first category is based on a cell's
metabolism and the use of a mutant cell line which lacks the
ability to grow independent of a supplemented media. Two examples
are: CHO DHFR.sup.- cells and mouse LTK.sup.- cells. These cells
lack the ability to grow without the addition of such nutrients as
thymidine or hypoxanthine. Because these cells lack certain genes
necessary for a complete nucleotide synthesis pathway, they cannot
survive unless the missing nucleotides are provided in a
supplemented media. An alternative to supplementing the media is to
introduce an intact DHFR or TK gene into cells lacking the
respective genes, thus altering their growth requirements.
Individual cells which were not transformed with the DHFR or TK
gene will not be capable of survival in non supplemented media.
42. The second category is dominant selection which refers to a
selection scheme used in any cell type and does not require the use
of a mutant cell line. These schemes typically use a drug to arrest
growth of a host cell. Those cells which have a novel gene would
express a protein conveying drug resistance and would survive the
selection. Examples of such dominant selection use the drugs
neomycin, Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327
(1982), mycophenolic acid, Mulligan, R. C. and Berg, P. Science
209: 1422 (1980) or hygromycin, Sugden, B. et al., Mol. Cell. Biol.
5: 410-413 (1985). The three examples given above employ bacterial
genes under eukaryotic control to convey resistance to the
appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic
acid) or hygromycin, respectively.
43. "Amplification" refers to the increase or replication of an
isolated region within a cell's chromosomal DNA. Amplification is
achieved using a selection agent e.g. methotrexate (MTX) which
inactivates DHFR. Amplification or the making of successive copies
of the DHFR gene results in greater amounts of DHFR being produced
in the face of greater amounts of MTX. Amplification pressure is
applied notwithstanding the presence of endogenous DHFR, by adding
ever greater amounts of MTX to the media. Amplification of a
desired gene can be achieved by cotransfecting a mammalian host
cell with a plasmid having a DNA encoding a desired protein and the
DHFR or amplification gene permitting cointegration. One ensures
that the cell requires more DHFR, which requirement is met by
replication of the selection gene, by selecting only for cells that
can grow in the presence of ever-greater MTX concentration. So long
as the gene encoding a desired heterologous protein has
cointegrated with the selection gene replication of this gene gives
rise to replication of the gene encoding the desired protein. The
result is that increased copies of the gene, i.e. an amplified
gene, encoding the desired heterologous protein express more of the
desired heterologous protein.
44. "Transformation" means introducing DNA into an organism so that
the DNA is replicable, either as an extrachromosomal element or by
chromosomal integration. Unless indicated otherwise, the method
used herein for transformation of the host cells is the method of
Graham, F. and van der Eb, A., Virology 52: 456-457 (1973).
However, other methods for introducing DNA into cells such as by
nuclear injection, protoplast fusion, electroporation or liposomes
may also be used. If prokaryotic cells or cells which contain
substantial cell wall constructions are used, the preferred method
of transfection is calcium treatment using calcium chloride as
described by Cohen, F. N. et al., Proc. Natl. Acad. Sci. (USA), 69:
2110 (1972).
45. Construction of suitable vectors containing the desired coding
and control sequences employ standard recombinant DNA techniques.
Isolated plasmids or DNA fragments are cleaved, tailored, and
religated in the form desired to form the plasmids required.
46. For analysis to confirm correct sequences in plasmids
constructed, the ligation mixtures are used to transform E. coli
K12 strain 294 (ATCC 31446) and successful transformants selected
by ampicillin or tetracycline resistance where appropriate.
Plasmids from the transformants are prepared, analyzed by
restriction and/or sequenced by the method of Messing et al.,
Nucleic Acids Res. 9: 309 (1981) or by the method of Maxam et al.,
Methods in Enzymology 65: 499 (1980).
47. "Transfection" refers to the taking up of an expression vector
by a host cell whether or not any coding sequences are in fact
expressed. Numerous methods of transfection are known to the
ordinarily skilled artisan, for example, CaPO.sub.4 and
electroporation. Successful transfection is generally recognized
when any indication of the operation of this vector occurs within
the host cell.
48. In order to facilitate understanding of the following examples
certain frequently occurring methods and/or terms will be
described.
49. "Plasmids" are designated by a lower case p preceded and/or
followed by capital letters and/or numbers. The starting plasmids
herein are either commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids
in accord with published procedures. In addition, equivalent
plasmids to those described are known in the art and will be
apparent to the ordinarily skilled artisan.
50. "Digestion" of DNA refers to catalytic cleavage of the DNA with
a restriction enzyme that acts only at certain sequences in the
DNA. The various restriction enzymes used herein are commercially
available and their reaction conditions, cofactors and other
requirements were used as would be known to the ordinarily skilled
artisan. For analytical purposes, typically 1 .mu.g of plasmid or
DNA fragment is used with about 2 units of enzyme in about 20 .mu.l
of buffer solution. For the purpose of isolating DNA fragments for
plasmid construction, typically 5 to 50 .mu.g of DNA are digested
with 20 to 250 units of enzyme in a larger volume. Appropriate
buffers and substrate amounts for particular restriction enzymes
are specified by the manufacturer. Incubation times of about 1 hour
at 37.degree. C. are ordinarily used, but may vary in accordance
with the supplier's instructions. After digestion the reaction is
electrophoresed directly on a polyacrylamide gel to isolate the
desired fragment.
51. Size separation of the cleaved fragments is performed using 5
to 8 percent polyacrylamide gel described by Goeddel, D. et al.,
Nucleic Acids Res., 8: 4057 (1980).
52. "Dephosphorylation" refers to the removal of the terminal 5'
phosphates by treatment with bacterial alkaline phosphatase (BAP).
Alternatively, calf alkaline phosphatase in BRL cove restriction
buffer could be used. This procedure prevents the two restriction
cleaved ends of a DNA fragment from "circularizing" or forming a
closed loop that would impede insertion of another DNA fragment at
the restriction site. Procedures and reagents for dephosphorylation
are conventional. Maniatis, T. et al., Molecular Cloning pp.
133-134 (1982). Reactions using BAP are carried out in 50 mM Tris
at 68.degree. C. to suppress the activity of any exonucleases which
may be present in the enzyme preparations. Reactions were run for 1
hour. Following the reaction the DNA fragment is gel purified.
53. "Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with ATP in the
presence of a nucleotide kinase. A synthetic oligonucleotide will
ligate to a fragment that has not been dephosphorylated.
54. "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Maniatis,
T. et al., Id., p. 146). Unless otherwise provided, ligation may be
accomplished using known buffers and conditions with 10 units of T4
DNA ligase ("ligase") per 0.5 .mu.g of approximately equimolar
amounts of the DNA fragments to be ligated.
55. "Filling" or "blunting" refers to the procedures by which the
single stranded end in the cohesive terminus of a restriction
enzyme-cleaved nucleic acid is converted to a double strand. This
eliminates the cohesive terminus and forms a blunt end. This
process is a versatile tool for converting a restriction cut end
that may be cohesive with the ends created by only one or a few
other restriction enzymes into a terminus compatible with any
blunt-cutting restriction endonuclease or other filled cohesive
terminus. Typically, blunting is accomplished by incubating 2-15
.mu.g of the target DNA in 10 mM MgCl.sub.2, 1 mM dithiothreitol,
50 mM NaCl, 10 mM Tris (pH 7.5) buffer at about 37.degree. C. in
the presence of 8 units of the Klenow fragment of DNA polymerase I
and 250 .mu.M of each of the four deoxynucleoside triphosphates.
The incubation generally is terminated after 30 min. phenol and
chloroform extraction and ethanol precipitation.
56. Host cells are transformed with vector(s) expressing the
polypeptide factor and the desired protein and cultured in a
conventional manner. Various cell culture systems are known to the
ordinarily skilled artisan. For example, plate systems grow cells
attached to a surface. Solid support matrices, such as steel,
glass, organic polymer or ceramic material, contained in a culture
chamber may be used. Another system consisting of a suspension of
microcarrier beads with attached anchorage-dependent cells, or of
cells grown within or trapped in suspended bead matrices may also
be used. Yet another system is suspension culture which provides
ease of monitoring conditions and scale-up potential. The choice of
culture system would be made by one of ordinary skill after
considering several variables, such as: the particular host cell
and whether that cell is anchorage-dependent; manipulations to be
performed; various cell properties such as, for example, lactic
acid production; whether secretion is density-dependent; the
desired protein to be produced by the host cell; and, the volume in
which the culture is to be maintained.
57. The following examples merely illustrate the best mode now
known for practicing the invention, but should not be construed to
limit the invention. All literature citations herein are expressly
incorporated by reference.
EXAMPLE 1
Construction of Human Proinsulin Expression Vector
58. The cDNA clone of the insulin gene, pHI3, provided the coding
sequence of the human preproinsulin gene for construction of
plasmids to direct the expression of preproinsulin in transfected
mammalian cells. The vector pSVEHIGDHFR containing the SV40
promoter, the cDNA encoding human preproinsulin, the hepatitis B
virus surface antigen polyadenylation site and the cDNA encoding
mouse dihydrofolate reductase was constructed.
59. FIG. 1 shows the steps for construction of the preproinsulin
expression vector used to establish an insulin-independent host
cell line. The three parts of the construction of pSVEHIGDHFR are
detailed below:
60. a) pSVEHIGDHFR
61. 1) The cDNA encoding human preproinsulin was obtained in a 440
bp fragment from pHI3 by a (NcoI-XhoII) digest. pHI3 is described
in Sures, I. et al., Science 208:57 (1980). The 440 bp fragment
containing the cDNA encoding preproinsulin was isolated.
62. 2) A 63 bp XbaI-NcoI fragment was isolated from the 5' end of
the insulin receptor plasmid (pCVSVE-HIRc-2, European Publication
No. 0192392, published Aug. 27, 1986). This fragment functioned as
a linker-adapter to fuse the 5' end of the cDNA encoding
preproinsulin to the SV40 early promoter.
63. 3) The vector, pCVSVD22/preUK54, providing the plasmid backbone
which is ligated to the 63 bp linker and preproinsulin gene coding
sequences was prepared as described below. pCVSVD22/preUK54, the
plasmid backbone, is the product of a three fragment ligation as
diagramed in FIG. 2.
64. i) The SV40 early promoter is obtained by digesting plasmid
pCVSVE-HBV (European Patent Application Publication No. 0117060,
published Aug. 29, 1984) with PvuI and XbaI.
65. ii) The fragment containing the preurokinase cDNA was obtained
from plasmid p preUK54 trp207-I (European Patent Application
Publication No. 0092182, published Oct. 26, 1983). The plasmid was
digested with ClaI. The ClaI ends are made blunt by a filling
reaction. The Klenow fragment of DNA polymerase I plus all 4
deoxyribonucleotide triphosphates are added to fill in the ClaI
protruding single stranded ends. After the fill-in, plasmid DNA is
digested with the second enzyme, XbaI. The XbaI-ClaI (filled)
preUK54 cDNA fragment was then isolated.
66. iii) The vector fragment containing the bacterial origin of
replication, the DHFR cDNA, eukaryotic expression unit, and the 3'
untranslated region of hepatitis virus surface antigen was derived
from pEHED22 (U.S. Pat. No. 4,624,918, issued Nov. 25, 1986). The
plasmid was first cut with BamHI. The protruding BamHI ends were
then blunted by a filling reaction with Klenow DNA polymerase I as
in the procedure detailed for ClaI blunting described above.
Following the BamHI digestion and fill-in, the DNA was cut with
XbaI and the large 4.3 Kb fragment isolated.
67. These three fragments were mixed together and ligated in a
three fragment, concerted ligation. The recombinant
pCVSVD22/preUK54 was recovered. Ligation of a filled ClaI site to a
filled BamHI site results in an intact BamHI site at this
junction.
68. To construct pSVEHIGDHFR, pDVSVD22/preUK54 was digested with
XbaI and BamHI and the vector fragment isolated.
69. The final three part ligation to yield pSVEHIGDHFR used: a) the
440 bp NcoI-XhoII fragment containing the cDNA for preproinsulin;
b) a 63 bp XbaI-NcoI fragment from pCVSVE-HIRc-2 to link the cDNA
to the SV40 early promoter; and, c) the XbaI-BamHI vector fragment
from pCVSVD22/preUK54 containing the SV40-DHFR transcription unit,
the ampicillin resistance marker origin of replication in E. coli,
the hepatitis surface antigen 3' end with the polyadenylation and
transcription termination site. The three fragments were ligated
together in a concerted three-way ligation and transformed into E.
coli. Transformants were analyzed and the desired recombinant
identified.
70. b) pSVEHIGNeo
71. FIG. 3 shows the steps for construction of the preproinsulin
expression vector pSVEHIGNeo.
72. This vector was constructed via a two fragment construction.
The first fragment was a HindIII fragment from pSVEHIGDHFR
described above. Included in the fragment was the cDNA encoding
preproinsulin and the SV40 early promoter that initiates
transcription of the DNA encoding DHFR. The plasmid backbone
comprising the second fragment was obtained by digestion at the
unique HindIII site just downstream of the SV40 promoter of
pSVENEOBa16 (European Publication No. 0160457, published Nov. 6,
1985). The linearized plasmid was then treated with calf alkaline
phosphatase to prevent recircularization. The HindIII fragment from
pSVEHIGDHFR was inserted at the unique HindIII site of pSVENeoBa16
such that the SV40 promoter originally transcribing the mouse
SV40-DHFR transcription unit is upstream of the preproinsulin gene.
After ligation the plasmid is transformed into E. coli 294
cells.
73. Recombinant cells are identified by restriction analysis to
insure proper orientation of the fragment containing the
preproinsulin cDNA. In the proper orientation the SV40 promoter
which originally transcribed the bacterial Neo gene is now upstream
and initiates transcription of the preproinsulin cDNA.
74. c) pEO
75. A vector containing the ornithine decarboxylase (ODC) cDNA
under control of the SV40 promoter, having a hepatitis B
polyadenylation sequence and an ampicillin gene for selection in E.
coli, was constructed. The endogenous ODC gene can be amplified in
mammalian cells by selection with the ODC inhibitor, alpha
difluoromethylornithine (DFMO). (McConlogue, L. & Coffino, P.,
J. Biol. Chem. 258,8384-8388 [1983]; McConlogue, L. & Coffino,
P., J. Biol. Chem. 8:12083-12086 [1983]).
76. FIG. 4 shows the steps for construction of pEO via a two
fragment ligation.
77. 1. A 1688 bp ODC fragment containing the entire coding region
of ODC was obtained from a plasmid containing ODC cDNA cloned into
pBR322 (McConlogue, L. et al. Proc. Natl. Acad. Sci. USA 81:540-544
[1984]; Gupta, M & Coffino, P. J. Biol. Chem. 260:2941-2944
[1985]). The plasmid was cut with SalI and PvuII. The ends were
blunted by filling in with Klenow, and the 1688 pair ODC fragment
was isolated on a gel.
78. 2. A 3593 bp fragment containing the SV40 early promoter, the
hepatitis polyadenylation sequence, and the AMP gene for selection
in E. coli was isolated from plasmid pSVPADHFR. (European Patent
Application Publication No. 0,093,619, referred to therein as
pETPER which was modified by the addition of 192 bp fragment at the
SV40 promoter 5' to the DNA encoding tPA. The additional 192 bp
fragment included an extra HindIII site.) The plasmid was cut with
HDIII and cII and the ends were filled in with Klenow DNA
polymerase and the 3593 fragment was isolated on a gel.
79. These two fragments were then ligated together in a two-part
ligation to form pEO. (See FIG. 4). The orientation and
configuration of the fragments in the final plasmid was checked by
restriction analysis.
EXAMPLE 2
Selection of Insulin-Independent Cells
80. Determination of the requirement for particular polypeptide
factor, in this case proinsulin, for a polypeptide factor-dependent
host cell, in this case CHO cells, was done by supplementing
insulin-free medium with proinsulin. It was known that most cells
require insulin to survive in serum-free media. (Sato, G. H. et al.
supra). Surprisingly, proinsulin was shown to be a replacement for
insulin in the case of the CHO host cell in culture. Thus
CHO/DHFR.sup.- cells were transfected with the preproinsulin vector
to provide proinsulin in an autocrine fashion.
81. CHO/DHFR.sup.- cells were transformed with the pSVEHIGD plasmid
by calcium phosphate precipitation (Simonsen, C. C. & Levinson,
A. D., PNAS 80:2495-2499 [1983]) and were selected for
insulin-independent growth by passaging the cells at low density
into serum-free (350 m Osm), insulin-free F-12/DME (Gibco) medium
(SFIF). F-12/DME comprises: high glucose; 1.times.GHT (0.01
g/1-glycine, 0.015 g/1-hypoxanthine, and 0.005 g/l thymidine); 10
mM HEPES; 1.0 mg/L transferrin; trace elements (McKeehan, W. L. et
al. PNAS 72:2023 [1976]; Johnson Mathew Chemicals); 1 uM linoleic
acid; 1.times.10.sup.-10 M T3 and 1.times.10.sup.-8M
hydrocortisone, estradiol and progesterone. After two weeks in this
medium, surviving cells were rescued with medium containing 5%
dialyzed, charcoal-dextran DEAE extracted, heat-treated FBS
(ChX-FBS). The CHO DHFR- cells will grow in whole serum but not
CHX-FBS unless supplemented with insulin. The ChX-FBS is, however,
capable of providing other necessary factors as can be seen by
comparing the growth rate in the presence of ChX-FBS +insulin
compared to insulin alone. Thus, the addition of ChX-FBS alone
would lead to an increased replication rate ("rescue") of those
cells which were providing their own proinsulin. Processing of the
serum using charcoal extraction was necessary to remove active
insulin. Thus, the sole source of insulin was the transformed host
cell. Insulin-independent cells were cloned on the basis of colony
morphology and size. Clones were subsequently screened for
insulin-independent growth in 1% ChX-FBS. Under insulin-free
conditions the parent line is severely limited in its ability to
replicate (1-2 divisions/week) while the transformed clones
exhibited a 30-40 fold increase in cell number in the same time
period.
82. Two clones which demonstrated the capacity to survive and grow
when carried under insulin-free conditions over extended periods of
time were labelled DP 7 and DP 12, respectively. These
insulin-independent cells were further selected in SFIF in spinners
and on plates. Those cells placed in spinners (500 ml) were
inoculated at 1.times.10.sup.5 cells/ml in SFIF. Plated cells (100
mm plates) were at a seeding density of 2.times.10.sup.5 cells/60
mm plate. After nearly two weeks of selection for
insulin-independence, surviving cells were rescued from both the
plates and the spinners, with medium containing 5% dialyzed,
extracted FBS. Cells from the spinner cultures were removed at that
time to plates. Cells were cloned by limiting dilution using serial
dilutions. The cells from these colonies were then serially diluted
to 1 cell/well. All cloning was done in the presence of F-12/DME,
high glucose, 5% charcoal extracted FBS medium. Approximately one
month later, cells which grew out of the initial cloning were again
serially diluted to one cell/well. The clones which survived and
grew were then taken to 100 mm plates. These cells were carried in
SFIF plus 500 nM aethotrexate and subcultured weekly.
83. Clones DP 7 and 12 demonstrated the capacity to survive and
grow. As shown in FIG. 5 the insulin-independent cells were able to
survive and grow in an insulin-free milieu while the control cells
were not. The insulin-independence of the cells of this invention
is shown in FIG. 6. As the concentration of insulin in the medium
is reduced growth of the insulin-independent cell line is
maintained while the number of cells/plate for the control cells
declined with decreasing concentration of insulin in the
medium.
EXAMPLE 3
tPA Production by an Insulin-Independent Cell Line
84. Expression of t-PA in the culture medium was assayed
quantitatively in a radioimmunoassay. Purified tPA and purified
iodinated tracer tPA were diluted serially to include concentration
of 12.5 to 400 ng/ml in phosphate buffered saline, pH 7.3, 0.5
percent bovine serum albumin, 0.01 percent Tween 80, and 0.02
percent sodium azide. Appropriate dilutions of medium samples to be
assayed were added to the radioactively labelled tracer proteins.
The antigens were allowed to incubate overnight at room temperature
in the presence of a 1:10,000 dilution of the IgG fraction of a
rabbit anti-tPA antiserum. Antibody-antigen complex was
precipitated by absorption to goat anti-rabbit IgG Immunobeads
(Biorad) for two hours at room temperature. The beads were cleared
by the addition of saline diluent followed by centrifugation for
ten minutes at 2000.times.g at 4.degree. C. Supernatants were
discarded and the radioactivity in the precipitates was monitored.
Concentrations were assigned by comparison with the reference
standard. It has been shown that various polypeptide factors affect
protein secretion as well as affecting survival or growth of the
host cell. Polypeptide factors such as follicle stimulating hormone
(FSH), epidermal growth factor (EGF), insulin and transferrin have
been shown to effect protein secretion from cultured cells. (Rich,
K. A. et al. Endocrinology 113(6):2284 [1983]). Thus, a transformed
host cell (C2B) producing a desired protein, tissue plasminogen
activator, was made insulin-independent to assess
production/secretion of the desired protein.
85. In order to determine whether endogenously produced proinsulin
would be sufficient to support the secretion of a desired protein
(e.g. tPA) in an insulin-independent fashion, a transfection was
performed in a manner similar to that described in example 2, but
using a host cell previously transformed to express a desired
protein, in this case tPA. The vector, pSVEHIGNeo, described in
example 1 was transfected into the CHO cell line containing
amplified tPA and DHFR (referred to as C2B) (European Publication
No. 0093619). Transfection was by the calcium-phosphate
coprecipitation method. (Simonsen, C. C. & Levinson, A. D.,
PNAS 80:2495-2499 [1983]; Wigler, M. et al., PNAS [USA]
76:1242-1255 [1979]). Transfected cells expressing the Neo gene
were selected by growth in medium containing G418.
86. The C2B preproinsulin transfected cells were selected for
insulin independence in serum-free, insulin-free (SFIF) spinners
and plates. The serum-free medium was standard 350 mOsm
insulin-free F-12/DME medium described above: glucose; 2.times.GHT;
10 mM Hepes; 10 Mg/L transferrin; 1.times.trace elements; 1 .mu.M
linoleic; 1.times.10.sup.-10M T.sub.3 and 1.times.10.sup.-8M
hydrocortisone, estradiol and progesterone.
87. After nearly two weeks of selection for insulin-independence,
surviving cells were rescued from both the plates and the spinners
with medium containing 5% dialyzed, extracted FBS, and 23 clones
were derived by limiting dilution. These clones were screened for
tPA production under serum-free conditions in the absence of
insulin and in the presence of varying insulin concentrations
(including the optimal concentration of 20 .mu.g/ml insulin). Clone
13 was picked as the most promising for further work.
88. An alternative method for the creation of an
insulin-independent cell to the transfection/selection described in
Example 2 and immediately above is by amplification and in turn
increased expression of proinsulin. Thus, C2B cells producing tPA
were cotransfected with the pSVEHIGNeo vector described in Example
1(b) and the pEO vector of example 1(c). This would permit
amplification using DFMO after selection. A similar
cotransfection-coamplification methodology is described by
Simonsen, C. C. and Levinson, A. D., supra.
89. The C2B cells cotransfected with the preproinsulin-Neo vector
and the ODC vector, pEO, were first selected in medium containing
G418. G418 resistant cells were then grown in increasing
concentrations, 25, 100, 300 and 500 .mu.M DFMO to amplify the
transfected ODC gene and coamplify the preproinsulin gene. After
this amplification procedure methotrexate was added to the medium
with DFMO to maintain selective pressure on the amplified tPA, the
desired protein. The C2B preproinsulin transfected cells were
tested for insulin-independence in serum-free, insulin-free (SFIF)
spinners and plates. The serum-free medium was standard 350 mOsm
insulin-free F-12/DME medium described above: glucose; 2.times.GHT;
10 mM Hepes; 1.0 Mg/L transferrin; 1.times.trace elements; 1 .mu.M
linoleic; 1.times.10.sup.-10M T.sub.3 and 1.times.10.sup.-8M
hydrocortisone, estradiol and progesterone.
90. FIG. 5 shows the production of r-tPA by the CHO
insulin-independent cells transfected with the preproinsulin gene
and selected and the alternative method comprising transfection
with pSVEHIGNeo and amplification. C2B (control) cells, C2B/clone
13 insulin-independent cells and the 100 .mu.M DFMO amplified pool
were rinsed three times in SFIF medium and resuspended in SFIF
medium. Clone 13 and the 100 .mu.M DFMO insulin-independent cell
lines produced tPA in the absence of insulin at titers equivalent
to those achieved by the C2B control cell line in the presence of
optimal concentrations of insulin.
EXAMPLE 4
Construction of Transferrin Expression Vector
91. a) Isolation of Human Transferrin cDNA
92. Messenger RNA (mRNA) was prepared from the liver of an adult
male accident victim by guanidine thiocyanate
homogenization/lithium chloride precipitation (Cathala, G. et al.
DNA 2:329 [1983]).
93. Double-stranded complementary DNA (ds-cDNA) was synthesized
using the above mRNA as a template and employing a commercially
available kit utilizing oligo(dT)-priming (Amersham Corporation)
according to the manufacturer's instructions (which are based on
Okayama, H., and Berg, P., Mol. Cell. Biol. 2:161 [1982] and
Gubler, U. and Hoffman, B. J., Gene 25:263 [1983]).
94. DNA oligonucleotide linkers were ligated to both ends of the
blunt-ended ds-cDNA as shown:
1 ds-cDNA - - - - ----------- G G T C G A C G A G C T C G A G - - -
- ----------- + C C A G C T G C T C G A G C T C T T A A +E,uns
+E,uns +E,uns +E,uns SalI +E,uns SstI +E,uns +E,uns EcoRI +E,uns
XhoI
95. yielding ds-cDNA terminating in EoRI restriction sites.
96. The ds-cDNA was fractionated by polyacrylamide gel
electrophoresis and the ds-cDNA migrating above 2000 base pairs was
recovered from the gel by electroelution. The size-fractionated
ds-cDNA was ligated into the bacteriophage lambda vector gt10
(Hyunh, T. V. et al. in DNA Cloning Techniques, A Practical
Approach, D. Glover (ed.) [IRL Press, Oxford, 1985]) that had been
cut with EcoRI and packaged using a commercial bacteriophage lambda
packaging extract (Stratagene).
97. The packaged bacteriophage were plated on E. coli strain C600
hfI.sup.- (Hyunh, T. V. et al. Construction and Screening cDNA
Libraries in .lambda. gt10 and .lambda. gt11 in DNA Cloning ed.
Glover, D. M., [IRL Press Oxford, Washington, D.C.], [1985])., and
bacteriophage DNA was transferred to replicate nitrocellulose
filters (Maniatis, T. et al. Molecular Cloning: A Laboratory
Manual, [Cold Spring Harbour Laboratory, 1982]).
98. b) Identification of Recombinant Clones Containing the
Transferrin cDNA
99. Six of the nitrocellulose filters were probed with the
synthetic oligonucleotide shown below. Its sequence was designed to
hybridize to the sequence of the human transferrin cDNA from
nucleotide #110 to #175 as reported by Yang et al. Proc. Natl.
Acad. Sci. [USA] 81:2752-2756 (1984).
5' GTG TGC AGT GTC GGA GCA TGA GGC CAC TAA CTG CCA GAG TTT CCG CGA
CCA TAT GAA AAC CGT CA 3'
100. The oligonucleotide was radiolabelled by the addition of a
radioactive phosphate group to the 5' end of the oligonucleotide in
a standard kinase reaction (Maniatis, T. et al., supra at 125). The
hybridization was carried out as described by Maniatis, (Ibid pg.
326) using 30% formamide in the hybridization buffer. Positively
hybridizing plaques were identified using autoradiography
(Maniatis, Ibid pg. 326) and six individual phage plugs were picked
for purification (Maniatis, Ibid pg 64).
101. The phage from each plug were replated at low density and
after a 16 hour growth phase bacteriophage DNA was again
transferred to nitrocellulose filters. These filters were screened
as described above using the same oligonucleotide probe. A single
isolated plaque was picked from each of the six plates. These phage
were used to infect a culture of a susceptible strain of E. coli,
c600 hFI.sup.- (Hyunh, T. V. et al., supra).
102. Phage DNA was prepared from each of the six clones using a
standard small scale phage preparation (Maniatis, Ibid pg.
373).
103. 40 .mu.g of DNA from each clone was digested with the
restriction enzyme, SstI (Goff, S. P. and Rambach, A., Gene 3:347
[1978]). These digests were run out on 1% low melting point agarose
gels (Struhl, K., Biotechniques 3:452 [1985]). Three of the clones
showed inserts of approximately the correct size of 2.3 Kb (Yang et
al., supra). The insert bands were cut out of the gels and
subcloned (Struhl, supra) into the M13 based vector mp19
(Yanish-Perron et al., Gene 33:103-119 [1985] and Norrander, J. et
al., Gene 26:101 [1983]). Recombinant phage clones (white plaques)
were picked and the ends sequenced.
104. One of the clones showed perfect coding region identity to the
published transferrin sequence (Yang et al., supra). The insert
from this clone was subcloned (Struhl, supra) into pUC19
(Yanish-Perron, supra) in the LstI site. Recombinant clones were
identified as white colonies on plates containing transferrin-gal
(Yanish-Perron, supra). Plasmid DNA was purified from a single
clone in which the transferrin coding region was oriented in the
direction opposite the lacZ promoter region.
105. The transferrin coding region was excised from the pUC vector
as a 2.3 Kb EcoRI-XbaI fragment from an XbaI-EcoRI partial digest.
This unique fragment was purified from a 1% low melting point gel
and subcloned (Struhl, supra) into an EcoRI-XbaI digested pRK5
vector. Construction of this vector is described below and in FIG.
9. This created pRKTFN.
106. c) Construction of pRK5
107. The starting plasmid was pCIS2.8c28D (described in copending
U.S. patent application Ser. Nos. 07/071,674 and 06/907,297). The
base numbers in paragraphs 1 through 6 refer to pCIS2.8c28D with
base one of the first T of the EcoRI site preceding the CMV
promoter. The cytomegalovirus early promoter and intron and the
SV40 origin and polyA signal were placed on separate plasmids.
108. 1. The cytomegalovirus early promoter was cloned as an EcoRI
fragment from pCIS2.8c28D (9999-1201) into the EcoRI site of pUC118
(Yanish-Perron et al. Gene 33:103 [1985]). Twelve colonies were
picked and screened for the orientation in which single stranded
DNA made from pUC118 would allow for sequencing from the EcoRI site
at 1201 to the EcoRI site at 9999. This clone was named
pCMVE/P.
109. 2. Single stranded DNA was made from pCMVE/P in order to
insert an SP6 (Green, M. R. et al., Cell 32:681-694 [1983])
promoter by site-directed mutagenesis. A synthetic 110 mer which
contained the SP6 promoter (See Nucleic Acids Res. 12:7041 [1984]
FIG. 1; sequences from -69 to +5 of SP6 promoter were used along
with 18 bp fragments on either end of the oligomer corresponding to
the CMVE/P sequences. Mutagenesis was done by standard techniques
and screened using a labeled 110 mer at high and low stringency.
Six potential clones were picked and sequenced. A positive was
identified and labelled pCMVE/PSP6.
110. 3. The SP6 promoter was checked and shown to be active, for
example, by adding SP6 RNA polymerase and checking for RNA of the
appropriate size.
111. 4. A Cla-NotI-Sma adapter was made to be inserted from the
ClaI site (912) to the SmaI site of pUC118 in pCMVE/P (step 1) and
pCMVE/PSP6 (step 2). This adapter was ligated into the ClaI-SmaI
site of pUC118 and screened for the correct clones. The linker was
sequenced in both and clones were labelled pCMVE/PSP6-L and
pCMVE/P-L.
112. 5. pCMVE/PSP6-L was cut with SmaI (at linker/pUC118 junction)
and HindIII (in pUC118). A HpaI (5573) to HindIII (6136) fragment
from pSVORAA.DELTA.RI 11, described below, was inserted into
SmaI-HindIII of pCMVE/PSP6-L. This ligation was screened and a
clone was isolated and named pCMVE/PSP6-L-SVORAA.DELTA.RI.
113. a) The SV40 origin and polyA signal was isolated as XmnI
(5475)-HindIII (6136) fragment from pCIS2.8c28D and cloned into the
HindIII to SmaI sites of pUC119. This was named pSVORAA.
114. b) The EcoRI site at 5716 was removed by partial digest with
EcoRI and filling in with Klenow. The colonies obtained from
self-ligation after fill-in were screened and the correct clone was
isolated and named pSVORAA.DELTA.RI 11. The deleted EcoRI site was
checked by sequencing and shown to be correct.
115. c) The HpaI (5573) to HindIII (6136) fragment of
pSVORAA.DELTA.RI 11 was isolated and inserted into pCMVE/PSP6-L
(see 4 above).
116. 6. pCMVE/PSP6-L-SVOrAA.DELTA.RI (step 5) was cut with EcoRI at
9999, blunted and self-ligated. A clone without an EcoRI site was
identified and named pRK.
117. 7. pRK was cut with SmaI and BamHI. This was filled in with
Klenow and religated. The colonies were screened. A positive was
identified and named pRK.DELTA.Bam/Sma 3.
118. 8. The HindIII site was converted to a HpaI site using a
converter. (A converter is a piece of DNA used to change one
restriction site to another. In this case one end would be
complimentary to a HindIII sticky end and at the other end have a
recognition site for HpaI.) A positive was identified and named
pRK.DELTA.Bam/Sma, HIII-HpaI 1.
119. 9. pRK.DELTA.Bam/Sma, HIII-HpaI 1 was cut with PstI and NotI
and a RI-HIII linker and HIII-RI linker were ligated in. Clones for
each linker were found. However, it was also determined that too
many of the HpaI converters had gone in (two or more converters
generate a PvuII site). Therefore, these clones had to be cut with
HpaI and self-ligated.
120. 10. RI-HIII clone 3 and HIII-RI clone 5 were cut with HpaI,
diluted, and self-ligated. Positives were identified. The RI-HIII
clone was named pRK5.
EXAMPLE 5
Selection of Transferrin-Independent Cells
121. DP7 insulin-independent cells were transfected with pRKTFN
described in example 4 above. Transfection was by the calcium
phosphate coprecipitation method of Simonsen and Levinson, supra.
Transfected cells are selected for hygromycin-resistance. The
hygromycin-resistant cell pool is cloned and several colonies are
picked. Cloning decreases the possibility of cross-feeding
non-producer cells in the subsequent selection step. Cell lines
making transferrin are selected by growing the above clones in a
serum-free (350 m Osm) transferrin-free F-12/DME medium. F-12/DME
is as described above, except that no iron is added. However, under
these conditions iron is introduced as a contaminant of other
medium components (e.g. water, NaCl, etc.). This small amount of
iron is insufficient to support optimal cell growth in the absence
of transferrin, but can support cell growth in the presence of
transferrin (Mather, J. P. and Sato, G. H., Exptl. Cell Res.
120:191-200 [1979]; Perez-Infante, U. and Mather, J. P., Exptl.
Cell Res. 142:325-332 [1982]) presumably due to increased
efficiency of iron-uptake via the transferrin-receptor system.
Cells which survive for 1-2 weeks in this
serum-free/transferrin-iron-free medium are then rescued with
F-12/DME medium containing 5% extracted FBS. Clones are
subsequently tested for transferrin independence by comparing the
growth of the clones and the untransfected parent line in the
low-iron medium with and without added human transferrin. Clones
with the capacity to survive and grow when carried under
transferrin-iron-free conditions are selected further in spinners
and plates.
122. The selected transferrin-independent clones are subsequently
tested for insulin-independence by comparing the growth of those
clones and the untransfected lines in serum-free, insulin-free,
transferrin-free and low iron medium with and without insulin and
transferrin.
* * * * *