Note: Descriptions are shown in the official language in which they were submitted.
1 ~ 9 ~ 7 3
R~X~B~T 00~ ST~ATIN~ F~R-l
The present invention relates to the use of
recombinant technology for production of lymphokines
ordinarily produced in low concentration. More
5 specifically, the invention relates to the cloning and
expression of a DNA sequence encoding human colony
stimulating factor-l (CSF-l).
The ability of certain factors produced in very
low concentration in a variety of tissues to stimulate
the growth and development of bone marrow progenitor
cells into granulocytes and/or macrophages has been
known for nearly 15 years. The presence of such factors
in sera, urine samples, and tissue extracts from a
number of species is demonstrable using an in vitro
15 assay which measures the stimulation of colony formation
by bone marrow cells plated in semi-solid culture
medium. There is no known in vivo assay. Because these
factors induce the formation of such colonies, the
factors collectively have been called Colony Stimulating
20 Factors (CSF).
' 1339~73
More recently, it has been ~hown that there are
at least four subclasses of human CSF proteins which can
be defined according to the types of cells found in the
resultant colonies. One subclass, CSF-l, results in
- 5 colonies contain~ng macrophages predominantly. Other
subclas~es produce colonies which contain both neutro-
philic granulocyte~ and macrophages: which containpredominantly neutrophilic granulocytes; and which contain
neutrophilic and eosinophilic granulocytes and
macrophages.
There are murine factors analogolls to the first
three of the above human CSFs. In addition, a murine
factor called IL-3 induces colonies from murine bone
marrow cells which contain all these cell types plus
megakaryacytes, erythrocytes, and mast cells, in various
combinations. These CSFs have been reviewed by Dexter,
T. M., Nature (1984) 309:746, and Vadas, M. A., et al, J
Immunol (1983) 130:793.
The inve~tion herein is concerned with the
recombinant production of proteins which are members of
the first of these subclasses, CSF-l. This subclass has
been further characterized and delineated by specific
radioimmunoas~ays and radioreceptor assays -- e.g.,
antibodies raised against purified CSF-l are able to
suppress specifically CSF-l activity, without affecting
the bioloqical activities of the other subclasses, and
macrophage cell line J774 contains receptors which bind
CSF-l specifically. A description of these a~says was
published by Das, S. K., et al, Blood (1981) 58:630. ,
Purification methods for various CSF proteins
have been published
Stanley, ~.R., et al, J Biol Chem (1977)
252:4305 reported purification of a CSF protein from
murine L9Z9 cells to a specific activity of about 1 x
.~. ,~
-- 13~987~
108 units/mg, which also stimulated mainly macrophage
prod~ction. Waheed, A., et al, 8100d (1982) 60:238,
described the purification of mou~e L-cell CSF-l to
apparent homogeneity using a rabbit antibody column and
reported the first 25 amino acids of the murine sequence
(Ben-Avram, C.M., et al, Proc Natl Acad Sci (USA) (1985)
882:4~86)
,
Stanley, E.R., et al, J Biol Chem (1977)
25Z:4305-~312 disclosed a purification procedure for
CSF-l from human urine and Das, S.K., et al, Blood
(1981) 58:630: J Biol Chem (1982) 257:13679 obtained a
human urinary CSF-l at a specific activity of 5 x 10
units/mg which produced only macrophage colonies, and
outlined the relationship of glycosylation of the CSF-l
proteins prepared from cultured mouse ~-cells and from
human urine to their activities. Wang, F.F., et al, J
Cell Biochem (1983) 21:263, isolated human urinary CSF-l
to specific activity of 10 U/mg. Waheed, A., et al,
disclosed purification of human urinary CSF-l to a
specific activity of 0.7-2.3 x 10 U/mg on a rabbit
antibody column (EXP Hemat (1984) 12:434).
Wu, M., et al, J Biol Chem (1979) 254:6226
reported the preparation of a CSF protein from cultured
human pancreatic carcinoma (MIAPaCa) cells which
resulted in the growth of murine granulocytic and
macrophagic colonies. The resulting protein had a
specific activity of approximately 7 x 107 units/mg.
Partially purified preparations of various CSFs
have also been reported from human and mouse lung,-cell
conditioned media (Fojo, S.S., et al, BiochemistrY
(1978) 17:3109; Burgess, A.W., et al, J Biol Chem (1977)
Z52:1998); from human T-lymphoblast cells (Lusis, A.J.,
et al, Blood (1981) 57:13; U.S. Patent, 4,~38,032); from
human placental conditioned medium to apparent
13 3 ~ 8 ~ 3
-4-
homogeneity and specific activity of 7 x 107 U/mg (Wu,
M., et al, BiochemistrY (1980) 19:3846).
A significant difficulty in putting CSF
proteins in general, and CSF-l in particular, to any
useful function has been their unavailability in
distinct and characterizable form in sufficient amounts
to make their employment in therapeutic use practical or
even possible. The present invention remedies these
deficiencies by providing purified human and murine
CSF-l in useful amounts through recombinant techniques.
~ A CSF protein of a different subclass, murine
and human GM-CSF has been purified and the cDNAs
cloned. This protein was shown to be distinct from
other CSFs, e.g., CSF-l, by Gough, et al, Nature (1984)
309:763-767. Murine IL-3 has been cloned by Fung, M.
C., et al, Nature (1984) 307:233. See also Yokota, T.,
et al, PNAS (1984) 81:1070-1074; Wong, G.G., et al,
Science (1985) 2?8:810-815; Lee, F., et al,; PNAS (1985)
82:~360-4364: and Cantrell, M.A., et al, PNAS (1985)
82:6250-6254.
Disclosure of the Invention
In one aspect, the present invention relates tO
recombinant CSF-l protein, including the biologically
active proteins containing modifications of primary
amino acid sequence of the native protein. CSF-l
protein in recombinant form can be obtained in quantity,
can be modified advantageously through regulation of the
post-translational processing provided by the host, and
can be intentionally modified at the genetic or protein
level to enhance its desirable properties. For example,
muteins having deletions of substantial portions of the
carboxy terminal one-third of the polypeptide are thus
active. Thus, the availability of CS~'-l in recombinant
1339~73
--5--
form provides both flexibility and certain quantitative
advantages which make possible applications for use of
the protein therapeutically, that are unavailable with
respect to the native protein.
In other aspects, the invention relates to an
isolated DNA sequence encoding recombinant CSF-l, to
recombinant expression systems for this sequence and to
vectors containing them, to recombinant hosts which are
transformed with these vectors, and to cultures
producing the recombinant protein. The invention
further relates to methods for producing the recombinant
protein and to the materials significant in its
production.
In addition, the invention relates to
compositions containing CSF-l which are useful in
pharmceutical and therapeutic applications, and to
methods of use for such compositions.
Brief DescriPtion of the Drawinqs
Figure 1 shows the partial amino acid sequences
of human urinary and murine L-929 cell CSF-l as
determined from purified native proteins.
Figure 2 shows the sequence of certain oligomer
probes for murine CSF-l.
Figure 3 shows the sequence of oligomer probes
used to obtain human genomic CSF-l.
Figure 4 shows the sequenced portion of a 3.9
kb HindIII fragment encoding human CSF-l sequences and
the deduced amino acid sequences for the exon regions.
Figure 5 ~hows the DNA and deduced amino acid
sequences for a cDNA clone encoding CSF-l.
~Figure 6 shows a comparison of the activities
of CSF-l and other colony stimulating factors in
enhanci~g the ability of macrophages to ~ill tumor cells.
6 1~3~87~
Figure 7 shows the results o~ sucrose gradient
fractionation of MIAPaCa mRNA.
Modes for Carryinq Out the Invention
A. ~efinitions
"Colony stimulating factor-l (CSF-l)" refers to
a protein which exhibits the spectrum of activity
understood in the art for CSF-l -- i.e., when applied to
the standard in vitro colony stimulating assay of
Metcalf, D., J Cell PhYsiol (1970) 76:89, it results in
the formation of primarily macrophage colonies. Native
CSF-l is a glycosylated dimer: dimerization may be
necessary for activity. Contemplated within the scope
of the invention and within the definition of CSF-l are
both the dimeric and monomeric forms. The monomeric form
may be converted to the dimer by in vitro provision of
intracellular conditions, and the monomer is per se
useful as an antigen to produce anti-CSF-l antibodies.
Z0 There appears to be so,ne species specificity:
Human CSF-l is operative both on human and on murine
bone marrow cells: murinc CSF-l does not show activity
with human cells. Therefore, ~human~ CSF-l should be
positive in the specific murine radioreceptor assay of
Das, S.K., et al, Blood (1981) 58:630, although there is
not necessarily a complete correlation. The biological
activlty of the protein will generally also be inhibited
by neutralizing antiserum to human urinary CSF-l (Das,
S.K., et al, supra). However, in certain special
circumstances (such a~, for example, where a particular
antibody preparation recognizes a CSF-l epitope not
essential for biological function, and which epitope is
not present in the particular CSF-l mutein being tested)
this criterion may not be met.
-7- ~339~3
Certain other properties of CSF-l have been
recognized more recently, including the ability of this
protein to stimulate the secretion of fieries E
prostaglandins, interleukin-l, and interferon from
mature macrophages (Moore, R., et al, Science (1984)
223:178). The mechanism for these latter activities is
not at present understood, and for purposes of
definition herein, the criterion for fulfillment of the
definition resides in the ability to stimulate the
formation of monocyte/macrophage colonies using bone
marrow cells from the appropriate species as starting
materials, under most circumstances (see above) the
inhibition of this activity by neutralizing antiserum
against purified human urinary CSF-l, and, where
appropriate for species type, a positive response to the
radioreceptor assay. (It is known that the
proliferative effect of CSF-l is restricted to cells of
mononuclear phagocytic lineage (Stanley, E.R., The
Lymphokines (1981), Stewart, W.E., II, et al, ed, Humana
Press, Clifton, NJ), pp. 102-132) and that receptors for
CSF-l are restricted to these cell lines (Byrne, P.V.,
et al, Cell Biol (1981) 91:848)).
As is the case for all proteins, the precise
chemical structure depends on a number of factors. As
ionizable amino and carboxyl groups are present in the
molecule, a particular protein may be obtained as an
acidic or basic salt, or in neutral form. All such
pceparations which retain their activity when -placed in
suitable environmental conditions are included in the
definition. Further, the primary amino acid sequence
may be augmented by derivatization using sugar moieties
(glycosylation) or by other supplementary molecules such
as lipids, phosphate, acetyl groups and the like-, more
commonly by conjugation with saccharides. The primary
1339~73
-8-
amino acid ~tructure may al~o aggregate to form
complexes, most frequently dimers. Indeed, native human
urinary CSF-l i~ isolated as a highly glyco~ylated di~er.
Certain aspects of such augmentation are
accompli~hed through post-translational processing
systems of the producing ho~t: other such modification
may be intcoduced in vitro. In any event, such
modifications are included in the definition so long as
the activity of the protein, as defined above, is not
destroyed. It is expected, of course, that such
modifications may quantitatively or qualitatively affect
the activity, either by enhancing or diminishing the
activity of the protein in the various assays.
Further, individual amino acid residues in the
chain may be modified by oxidation, reduction, or other
derivatization, and the protein may be cleaved to obtain
fragments which retain activity. Such alterations which
do not destroy activity do not remove the protein
sequence from the definition.
Modifications to the primary structure itself
by deletion, addition, or alteration of the amino acids
incorporated into the sequence during translation can be
made without destroying the activity of the protein.
Such substitutions or other alterations result in
proteins having an amino acid sequence which falls
within the definition of proteins ~having an amino acid
sequence substantially equivalent to that of CSF-l".
Indeed, human and murine derived CSF-l proteins have
non-identical but similar primary amino acid sequences
which display a high homology.
For convenience, the mature protein amino acid
~equence of the monomeric portion of a dimeric protein
~hown in Figure 5, deduced from the cDNA clone
illustrated herein, is de~ignated mCSF-l (mature
~ / .. ~
,i
~3~73
g
CSF-l). Figure 5 shows the presence of a 32 ~esidue
putative signal sequence, which is presumably cleaved
upon secretion from mammalian cells; mCSF-l is
represented by amino acids 1-224 shown in that figure.
Specifically included in the definition of human CSF-l
are muteins which monomers and dimers are mCSF-l and
related forms of mCSF-l, designated by their differences
from mCSF-l. CSF-l derived from other species may fit
the definition of "human" CSF-l by virtue of its display
of the requisite pattern of activity as set forth above
with regard to human substrate.
Also for convenience, the amino acid sequence
of mCSF-l will be used as a reference and other
sequences which are substantially equivalent to this in
terms of CSF-l activity will be designated by referring
to the sequence shown in Figure 5. The substitution of
a particular amino acid will be noted by reference to
the amino acid residue which it replaces. Thus, for
example, sergOCSF-l refers to the protein which has
the sequence shown in Figure 5 except that the amino
acid at position 90 is serine rather than cysteine.
Deletions are noted by a V followed by the number of
amino acids deleted from the N-terminal sequence, or by
the number of amino acids remaining when residues are
deleted from the C-terminal sequence, when the number is
followed by a minus sign. Thus, V4cSF-l refers to
CSF-l of Figure 5 wherein the first 4 amino acids from
the N-terminus have been deleted; V130 refers to
CSF-l wherein the last 94 amino acids following amino
acid 130 have been deleted. Illustrated below are for
example asp59CSF-l (which contains an aspartic residue
encoded by the gene (Figure 4) at position 59 rather
than the tyrosine residue encoded by the cDNA and
1339~3
--10--
V158 CSF-l which comprises only amino acids 1-158 of
mCSF-l.
'lOperably linked" refers to juxtaposition such
that the normal function of the components can be
performed. Thus, a coding sequence ~'operably linked" to
control sequences refers to a configuration wherein the
coding sequence can be expressed under the control of
these sequences.
"Control sequences" refers to DNA sequences
necessary for the expression of an operably linked
coding sequence in a particular host organism. The
control sequences which are suitable for procaryotes,
for example, include a promoter, optionally an operator
sequence, a ribosome binding site, and possibly, other
as yet poorly understood, sequences. Eucaryotic cells
are known to utilize promoters, polyadenylation signals,
and enhancers.
"Expression system" refers to DNA sequences
containing a desired coding sequence and control
sequences in operable linkage, so that hosts transformed
with these sequences are capable of producing the
encoded proteins. In order to effect transformation,
the expression system may be included on a vector:
however, the relevant DNA may then also be integrated
z5 into the host chromosome.
As used herein "cell~, "cell linell, and ~cell
culture" are used interchangeably and all such
designations include progeny. Thus "transformants" or
lltransformed cells" includes the primary subject cell
and cultures derived therefrom without regard for the
number of transfers. It is also understood that all
progeny may not be precisely identical in DNA content,
due to deliberate or inadvertent mutations. Mutant
progeny which have the same functionality as screened
1339~73
for in the originally transformed cell, are included.
Where distinct designations are intended, it will be
clear from the context.
B. General De~criPtion
The CSF-l proteins of the invention are capable
both of stimulating monocyte-precursor/macrophage cell
production from progenitor marrow cells, thus enhancing
the effectiveness of the immune system, and of
stimulating such functions of these dif~erentiated cells
as the secretion of lymphokines in the mature
macrophages.
In one application, these proteins are useful
as adjuncts to chemotherapy. It is well understood that
chemotherapeutic treatment results in suppression of the
immune system. Often, although successful in destroying
the tumor cells against which they are directed,
chemotherapeutic treatments result in the death of the
subject due to this side effect of the chemotoxic agents
on the cells of the immune system. Administration of
CSF-l to such patients, because of the ability of CSF-l
to mediate and enhance the growth and differentiation of
bone.marrow-derived precursors into macrophages and
monocytes and to stimulate the function of these mature
cells, res~lts
in a restimulation of the immune system to prevent this
side ef~ect, and thus to prevent the propensity of the
patient to succumb to secondary infection. Other
patients who would be helped by such treatment include
those being treated for leukemia through bone marrow
transplants: they are often in an immunosuppressed state
to prevent rejection. For these patients also, the
immunosuppression could be reversed by administration of
CSF-l.
In general, any subject suf~ering from
immunosuppression whether due to chemotherapy, bone
-12- 13~9873
marrow transplantation, or other, accidental forms of
immunosuppression such as disease (e.g., acquired immune
deficiency syndrome) would benefit from the availability
of CSF-l for pharmacological use. In addition, subjects
could be supplied enhanced amounts of previously
differentiated macrophages to supplement those of the
indigenous system, which macrophages are produced by in
vitro culture of bone marrow or other suitable
preparations treated with CSF-l. These preparations
include those of the patient's own blood monocytes,
which can be so cultured and returned for local or
systemic therapy.
The ability of CSF-l to stimulate production of
lymphokines by macrophages and to enhance their ability
to kill target cells also makes CSF-l directly useful in
treatment of neoplasms and infections.
CSF-l stimulates the production of interferons
by murine-derived macrophage (Fleit, H.B., et al, J Cell
Physiol (1981) 108:347, and human, partially purified,
CSF-l from MIAPaCa cells stimulates the poly IC-induced
production of interferon and TNF from human monocytes as
illustrated below. In addition, CSF-l stimulates the
production of myeloid CSF by human blood monocytes.
Also illustrated below is a demonstration of
the ability of murine CSF-l (from L-cell-conditioned
medium) to stimulate normal C3H/HeN mouse peritoneal
macrophages to kill murine sarcoma TU5 targets. This
activity is most effective when the CSF-l is used as
pretreatment and during the effector phase. The ability
of CSF-l to do so is much greater than that exhibited by
other colony stimulating factors, as shown in Figure 6
hereinbelow. In addition, the ability of murine cells
to attack viruses is enhanced by CSF-l.
:~339~73
Murine CSF-l i8 inconsistently reported to
stimulate murine macrophaqe to be cytostatic to P815
tumor cells (Wing, E.J., et al, J Clin Invest (1982)
69:270) or not to kill other leukemia targets (Ralph, P,
s et al, Cell lmmunol (1983) 76:10). Nogawa, R.T., et al,
Cell Immunol (1980) 53:116, report that CSF-l may
stimulate macrophage to ingest and kill yeast.
Thus, in addition to overcoming immunosup-
pre~sion per ~e, CSF-l can be used to destroy the
invading organisms or malignant cells indirectly by
stimulation of macrophage secretions and activity.
The CSF-l of the invention may be formulated in
conventional ways standard in the art for the
administration of protein substances. Administration by
injection is preferred: formulations include solutions
or suspensions, emulsions, or solid composition for
reconstitution into injectables. Suitable excipients
include, for example, Ringer's solution, Hank's
~olution, water, saline, glycerol, dextrose ~olutions,
and the like. In addition, the CSF-l of the invention
may be preincubated with preparations of cells in order
to stimulate appropriate responses, and either the
entire preparation or the supernatant therefrom
introduced into the subject. As shown hereinbelow, the
materials produced in response to CSF-l stimulation by
various types of blood cells are ef~ective against
desired targets, and the properties of these blood cells
themselves to attack invading viruses or neoplasms may
be enhanced. The subject's own cells may be withdrawn
3~ and used in this way, or, for example, monocytes or
lymphocyte~ from another compatible individual employed
in the incubation.
Although the existence of a pattern of activity
designated CSF-l has been known for some time, the
-14- 133~7~
protein responsible has never been obtained in both
sufficient purity and in sufficient amounts to permit
sequence determination, nor in sufficient purity and
quantity to provide a useful therapeutic function.
Because neither completely pure practical amounts of the
protein nor its encoding DNA have been available, it has
not been possible to optimize modifications to structure
by providing such alternatives as those set forth in l~A
above, nor has it been possible to utilize this protein
in a therapeutic context.
The present invention remedies these defects.
Through a variety of additional purification procedures,
sufficient pure CSF-l has been obtained from human urine
to provide some amino acid sequence, thus permitting the
construction of DNA oligomeric probes. The probes are
useful in obtaining the coding sequence for the entire
protein. One approach, illustrated below, employs
probes designed with respect to the human N-terminal
sequence to probe the human genomic library to obtain
the appropriate coding sequence portion. The human
genomic cloned sequence can be expressed directly using
its own control sequences, or in constructions
appropriate to mammalian systems capable of processing
introns. The genomic sequences are also used as probes
for a human cDNA library obtained from a cell line which
produces CSF-l to obtain cDNA encoding this protein.
The cDNA, when suitably prepared, can be expressed
directly in COS or CV-l cells and can be constructed
into vectors suitable for expression in a wide range of
hosts.
Thus these tools can provide the complete
coding sequence for human CSF-l from which expression
vectors applicable to a variety of host systems can be
constructed and the coding sequence expressed. The
-15~ 9 3 7 3
variety of hosts available along with expression vectors
suitable for such hosts permits a choice among
post-translational processing systems, and of
environmental factors providing conformational
regulation of the protein thus produced.
C. Suitable Hosts, Control Systems and Methods
In general terms, the production of a
recombinant form of CSF-l typically involves the
following:
First a DNA encoding the mature (used here to
include all muteins) protein, the preprotein, or a
fusion of the CSF-l protein to an additional sequence
which does not destroy its activity or to additional
sequence cleavable under controlled conditions (such as
treatment with peptidase) to give an active protein, is
obtained. If the sequence is uninterrupted by introns
it is suitable for expression in any host. If there are
introns, expression is obtainable in mammalian or other
eucaryotic systems capable of processing them. This
sequence should be in excisable and recoverable form.
The excised or recovered coding sequence is then placed
in operable linkage with suitable control sequences in a
replicable expression vector. The vector is used to
transform a suitable host and the transformed host
cultured under favorable conditions to effect the
production of the recombinant CSF-l. Optionally the
CSF-l is isolated from the medium or from the cells;
recovery and purification of the protein may not be
necessary in some instances, where some impurities may
be tolerated. For example, for in vitro cultivation of
cells from which a lymphokine factor will be isolated
for administration to a subject, complete purity is not
required. However, direct use in therapy by
~ 3 ~ 7 ~
-16-
administration to a subject would, of course, require
purificatio~ of the CSF-l produced.
Each of the foregoing steps can be done in a
variety of ways. For example, the desired coding
sequences can be obtained by preparing suitable cDNA
from cellular messenger and manipulating the cDNA to
obtain the complete sequence. Alternatively, genomic
fragments may be obtained and used directly in
appropriate hosts. The constructions for expression
vectors operable in a variety of hosts are made using
appropriate replicons and control sequences, as set
forth below. Suitable restriction sites can, if not
normally available, be added to the ends of the coding
sequence so as to provide an excisable gene to insert
into these vectors.
The control seguences, expression vectors, and
transformation methods are dependent on the type of host
cell used to express the gene, Generally, procaryotic,
yeast, or mammalian cells are presently useful as
hosts. Since native CSF-l is secreted as a glycosylated
dimer, host systems which are capable of proper
post-translational processing are preferred.
Accordingly, although procaryotic hosts are in general
the most efficient and convenient for the production of
recombinant proteins, eucaryotic cells, and, in
particular, mammalian cells are preferred for their
processing capacity. Recombinant CSF-l produced by
bacteria would require in vitro dimerization. In
addition, there is more assurance that the native signal
sequence will be recognized by mammalian cell hosts
making secretion pos~sible, and purification therefore
easier.
3337~
-17-
C.l. Control Sequences And CorresPondinq Hosts
Procaryotes ~ost ~requently are rèpresented by
various ~trains of E. coli. However, other microbial
strain~ may also be used, such as bacilli, for example
Bacillus subtilis, various species of Pseudomonas, or
other bacterial strains. In such procaryotic systems,
plasmid vectors which contain replication sites and
control sequences derived from a species compatible with
the host are used. For example, E. coli is typically
transformed using derivatives of pBR322, a plasmid
derived from an E. coli species by Bolivar, et al, Gene
(1977) 2:95. pBR322 contains genes for ampicillin and
tetracycline resistance, and thus provides additional
markers which can be either retained or destroyed in
constructing the desired vector. Commonly used
procaryotic control sequences which are defined herein
to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding
site sequences, include such commonly used promoters as
the beta-lactamase (penicillinase) and lactose (lac)
promoter systems (Chang, et al, Nature (1977) 198:1056
and the tryptophan (trp~ promoter system (Goeddel, et al
Nucleic Acids Res (1980) 8:4057 and the lambda derived
P~ promoter and N-gene ribosome binding site
(Shimatake, et al, Nature (1981) 292:128), which has
been made useful as z portable control cassette, as set
forth in U.S. Patent No. 4,711,845, issued ~ -
December 8, 1987 and agsigned to the same assignee.However, any available promoter system compatible with
procaryotes can be used.
In addition to bacteria, eucaryotic microbes,
such as yeast, may also be used as hosts. Laboratory
strain~ of SaccharomYces cerevisiae, Baker's yeast, are
most used although a number of other strains are
1~3~7~
-18-
commonly available. While vectors employing the 2
micron origin of replication are illustrated, Broach, J.
R., Meth Enz (1983) 101:307, other plasmid vectors
suitable for yeast expression are known (see, for
example, Stinchcomb, et al, Nature (1979) 282:39,
Tschempe, et al, Gene (1980) 10:157 and Clarke, L, et
al, Meth Enz (1983) 101:300). Control sequences for
yeast vectors include promoters for the synthesis of
glycolytic enzymes (Hess, et al, J Adv Enzyme Req (1968)
7:149: Holland, et al, Biochemistry (1978) 17:4900).
Additional promoters known in the art include the
promoter for 3-phosphoglycerate kinase (Hitzeman, et al,
J Biol Chem (1980) 255:2073), and those for other
glycolytic enzymes, such as glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase, 3-
phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. Other promoters, which have the additional
advantage of transcription controlled by growth
conditions are the promoter regions for alcohol
dehydrogenase 2, isocytochrome C, acid phosphatase,
degradative enzymes associated with nitrogen metabolism,
and enzymes responsible for maltose and galactose
utilization (Holland, ibid). It is also believed
terminator sequences are desirable at the 3' end of the
coding sequences. Such terminators are found in the 3l
untranslated region following the coding sequences in
yeast-derived genes. Many of the vectors illustrated
contain control sequences derived from the enolase gene
containing plasmid peno46 (Holland, M. J., et al, J Biol
Chem (1981) 2 :1385) or the LEU2 gene obtained from
YEpl3 (Broach, J., et al, Gene (1978) 8:121), however
any vector containing a yeast compatible promoter,
-lg- 1~9,373
origin of replication and other control sequences is
suitable.
It is also, of course, possible to express
genes encoding polypeptides in eucaryotic host cell
cultures derived from multicellular organisms. See, for
example, Tissue Culture, Academic Press, Cruz and
Patterson, editors (1973). Useful host cell lines
include murine myelomas N51, VERO and HeLa cells, and
Chinese hamster ovary (CHO) cells. Expression vectors
for such cells ordinarily include promoters and control
sequences compatible with mammalian cells such as, for
example, the commonly used early and late promoters from
Simian Virus 40 (SV 40) (Fiers, et al, Nature (1978)
273:113), or other viral promoters such as those derived
from polyoma, Adenovirus 2, bovine papilloma virus, or
avian sarcoma viruses, or immunoglobulin promoters and
heat shock promoters, General aspects of mammalian cell
host system transformations have been described by Axel:
U.5. Patent No, 4,399,Z16 issued 16 August 1983. It now
appears, also that "enhancer" regions are important in
optimizing expression: these are, generally, sequences
found upstream of the promoter region. Origins of
replication may be obtained, if needed, from viral
sources. However, integration into the chromosome is a
Z5 common mechanism for DNA replication in eucaryotes.
Plant cells are also now available as hosts, and control
sequences compatible with plant cells such as the
nopaline synthase promoter and polyadenylation signal
sequences (Depicker, A., et al, J ~ol APP1 Gen (1982)
1:561) are available.
.
C.2. Tranformations
Depending on the host cell used, transformation
is done using standard techniques appropriate to such
~3~873
zo
cells. The calcium treat~ent employing calcium
chloride, as described by Cohen, S. N., Proc Natl Acad
Sci (USA) (1972) 69:ZllO is used for procaryotes or
other cells which contain substantial cell wall
barriers. Infection with Aqrobacterium tumefaciens
(Shaw, C. H., et al, Gene (1983) 23:315) is used for
certain plant cells. For mammalian cells without such
cell walls, the calcium phosphate precipitation method
of Graham and van der Eb, Viroloqy (1978) 52:546 is
preferred. Transformations into yeast are carried out
according to the method of Van Solingen, P., et al, J
Bact (1977) 130:946 and Hsiao, C. L., et al, Proc Natl
Acad Sci (USA) (1979) 76:3829.
C.3. Probinq mRNA bY Northern Blot; Probe of cDNA or
Genomic Libraries
RNA is fractionated for Northern blot by
agarose slab gel electrophoresis under fully denaturing
conditions using formaldehyde (Maniatis, T., et al,
Molecular Cloninq (1982) Cold Spring Harbor Press, pp
202-203) or 10 mM methyl mercury (CH3HgOH) (Bailey, J.
M., et al, Anal Biochem (1976) 70:75-85; and Sehgal, P.
B., et al, Nature (1980) 288:95-97) as the denaturant.
For methyl mercury gels, 1.5% gels are prepared by
melting agarose in running buffer (100 mM boric acid, 6
mM sodium borate, 10 mM sodium sulfate, 1 mM EDTA, pH
8.Z), cooling to 60~C and adding 1/100 volume of 1 M
CH3HgOH. The RNA is dissolved in 0.5 x running buffer
and denatured by incubation in 10 mM methyl mercury for
min at room temperature. Glycerol (20%) and
bromophenol blue (0.05%) are added for loading the
samples. Samples are electrophoresed for 500-600
volt-hr with recirculation of the buffer. After
electrophoresis, the gel is washed for 40 min in 10 mM
13~373
2-mercaptoethanol to detoxify the methyl mercury, and
Northern blot~ prepared by transferring the RNA from the
gel to a membrane filter.
cDNA or genomic libraries are screened using
the colony or plaque hybridization procedure. Bacterial
colonies, or the plaques for phage are lifted onto
duplicate nitrocellulose filter papers (S & S type
BA-85). The plaques or colonies are lysed and DNA is
fixed to the filter by sequential treatment for 5 min
with 500 mM NaOH, 1.5 M NaCl. The filters are washed
twice for 5 min each time with 5 x standard saline
citrate (SSC) and are air dried and baked at 80~C for 2
hr.
The gels for Northern blot or the duplicate
filters for cDNA or genomic screening are prehybridized
at 25-42~C for 6-8 hr with 10 ml per filter of DNA
hybridization buffer without probe (0-50% formamide, 5-6
x SSC, pH 7.0, 5x Denhardt's solution (polyvinyl-
pyrrolidine, plus Ficoll and bovine serum albumin: 1 x =
0.02% of each), 20-50 mM sodium phosphate buffer at pH
7.0, 0.2% SDS, 20 ~g/ml poly U (when probing cDNA3,
and 50 ~g/ml denatured salmon sperm DNA). The samples
are then hybridized by incubation at the appropriate
temperature for about 24-36 hours using the
hybridization buffer containing kinased probe (for
oligomers). Longer cDNA or genomic fragment probes were
labeled by nick translation or by primer extension.
The conditions of both prehybridization and
hybridization depend on the stringency desired, and vary,
for example, with probe length. Typical conditions for
relatively long (e.g., more than 30-50 nucleotide)
probes employ a temperature of 42~-55~C and
hybridization buffer containing about 20%-50%
formamide. For the lower stringencies needed for
~L3i~!3873
-22-
oligomeric probes of about 15 nucleotides, lower
temperatures of about 25~-~2~C, and lower formamide
concentrations (0%-20%) are employed. For longer
probes, the filters may be washed, for example, four
times for 30 minutes, each time at 40~-55~C with 2 x
SSC, 0.2% SDS and 50 mM sodium phosphate buffer at pH 7,
then washed twice with 0.2 x SSC and 0.2% SDS, air
dried, and are autoradiographed at -70~C for 2 to 3
days. Washing conditions are somewhat less harsh for
shorter probes.
C.4. Vector Construction
Construction of suitable vectors containing the
desired coding and control sequences employs standard
ligation and restriction techniques which are well
understood in the art. Isolated plasmids, DNA
sequences, or synthesized oligonucleotides are cleaved,
tailored, and religated in the form desired.
Site specific DNA cleavage is performed by
treating with the suitable restriction enzyme (or
enzymes) under conditions which are generally understood
in the art, and the particulars of which are specified
by the manufacturer of these commercially available
restriction enzymes. See, e.g., New England Biolabs,
Product Catalog. In general, about 1 ~g of plasmid or
DNA sequence is cleaved by one unit of enzyme in about
~1 of buffer solution; in the examples herein,
typically, an excess of restriction enzyme is used to
insure complete digestion of the DNA substrate.
Incubation times of about one hour to two hours at about
37~C are workable, although variations can be
tolerated. After each incubation, protein is removed by
extraction with phenol/chloroform, and may be followed
by ether extraction, and the nucleic acid recovered from
1339~73
-23-
aqueous fractions by precipitation with ethanol. If
desired, gize separation of the cleaved fragments may be
performed by polyacrylamide gel or agarose gel
electrophoresis using standard techniques. A general
descri~tion of size separations is found in Methods in
En2YmoloqY (1980) 65:499-560.
Restriction cleaved fragments may be blunt
ended by treating with the large fragment of E. coli DNA
polymerase I (~lenow) in the presence of the four
deoxynucleotide triphosphates (dNTPs) using incubation
times of about 15 to Z5 min at 20 to 25~C in 50 mM Tris
pH 7.6, 50 mM NaCl, 6 mM MgC12, 6 ~M DTT and 5-10 ~M
dNTPs. The Klenow fragment fills in at 5~ sticky ends
but chews back protruding 3' single strands, even though
the four dNTPs are present. If desired, selective
repair can be performed by supplying only one of the, o~
selected, dNTPs within the limitations dictated by the
nature of the sticky ends. After treatment with Klenow,
the mixture is extracted with phenol/chloroform and
ethanol precipitated. Treatment under appropriate
conditions with Sl nuclease results in hydrolysis of any
single-stranded portion.
Synthetic oligonucleotides may be prepared by
the triester method of Matteucci, et al (J Am Chem Soc
(1981) 103:3185-3191) or using automated synthesis
methods. Kinasing of single strands prior to annealing
or for labeling is achieved using an excess, e.g.,
approximately 10 units of polynucleotide kinase to
nmole substrate in the presence of 50 mM Tris, pH 7.6,
lo mM MgC12, 5 mM dithiothreitol, l-Z mM ATP. If
kinasinq is for labeling of probe, the ATP will contain
high specific activity 32~P.
Liqations are performed in 15-30 ~1 volumes
under the following standard conditions and
~, ~
1;~3~73
-24-
temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgC12, 10
mM DTT, ~3 ~g/ml BSA, 10 mM-50 mM NaCl, and either 40
~M ATP, 0.01-0.02 (Wei6s) units T4 DNA ligase at O~C
(for "sticky end" ligation) or 1 mM ATP, 0.3-0.6 (Weiss)
units T4 DNA ligase at 14~C (for "blunt end'l ligation).
Intermolecular "sticky end" ligations are usually
performed at 33-100 ~g/ml total DNA concentrations
(5-100 nM total end concentration). Intermolecular
blunt end ligations (usually employing a 10-30 fold
molar excess of linkers) are performed at 1 ~M total
ends concentration.
In vector construction employing "vector
fragments'l, the vector fragment is commonly treated with
bacterial alkaline phosphatase (BAP) in order to remove
the 5' phosphate and prevent religation of the vector.
BAP digestions are conducted at pH 8 in approximately
150 mM Tris, in the presence of Na and Mg using
about 1 unit of BAP per ~g of vector at 60~ for about
one hour. In order to recover the nucleic acid
fragments, the preparation is extracted with
phenol/chloroform and ethanol precipitated.
Alternatively, religation can be prevented in vectors
which have been double digested by additional
restriction enzyme digestion of the unwanted fragments.
C.5. Modification of DNA Sequences
For portions of vectors derived from cDNA or
genomic DNA which require sequence modifications, site
specific primer directed mutagenesis is used. This
technique is now standard in the art, and is conducted
using a primer synthetic oligonucleotide complementary
to a single stranded phage DNA to be mutagenized except
for limited mismatching, representing the desired
mutation. Briefly, the synthetic oligonucleotide is
1~39873
-25-
used as a primer to direct synthesis of a strand
complementary to the phage, and the resulting
double-stranded DNA is transformed into a
phage-supporting host bacterium. Cultures of the
transformed bacteria are plated in top agar, permitting
plaque formation from single cells which harbor the
phage.
Theo~etically, 50% of the new plaques will
contain the phage having, as a single strand, the
mutated form; 50% will have the original sequence. The
plaques are hybridized with kinased synthetic primer at
a temperature which permits hybridization of an exact
match, but at which the mismatches with the original
strand are sufficient to prevent hybridization. Plaques
which hybridize with the probe are then picked,
cultured, and the DNA recovered. Details of site
specific mutation procedures are described below in
seecific examples.
C.6. Verification of Construction
In the constructions set forth below, correct
ligations for plasmid construction are confirmed by
first transforming E. coli strain MM294, or other
suitable host with the ligation mixture. Successful
transformants are selected by ampicillin, tetracycline
or other antibiotic resistance or using other markers
depending on the mode of plasmid construction, as is
understood in the art. Plasmids from the transformants
are then prepared according to the method of Clewell, D.
B., et al, Proc Natl Acad Sci (USA) (1969) 62:1159,
optionally following chloramphenicol amplification
(Clewell, D. B., J Bacteriol (1972) 110:667). The
isolated DNA is analyzed by restriction and/or sequenced
by the dideoxy method of Sanger, F., et al, Proc Natl
-26- 1~39873
Acad Sci (USA) (1977) 74:5463 as further described by
Messing, et al, Nucleic Acids Res (1981) 9:309, or by
the method of Maxam, et al, Methods in EnzYmoloqY (ls80)
65:499.
C.7. Hosts ExemPlified
Host strains used in cloning and expression
herein are as follows:
~ or cloning and sequencing, and for expression
lo of construction under control of most bacterial
promoters, E. coli strain MM294 obtained from E. coli
Genetic Stock Center GCSC #6135, was used as the host.
For expression under control of the PLNRBS promoter,
E. coli strain K12 MC1000 lambda lysogen, N7N53cI857
SusP80, ATCC 39531 is used.
For M13 phage recombinants, E. coli strains
susceptible to phage infection, such as E. coli K12
strain DG98 are employed. The DG98 strain has been
deposited with ATCC 13 July 1984 and has accession
number 39768.
Mammalian expression has been accomplished in
COS-7 and CV-l cells.
D. Preferred Embodiments
The recombinant CSF-l of the invention can be
considered a set of muteins which have similar but not
necessarily identical primary amino acid sequences, all
of which exhibit, or are specifically cleavable to a
mutein which exhibits, the activity pattern
characteristic of CSF-l - i.e. they are capable of
stimulating bone marrow cells to differentiate into
monocytes, preponderantly, and, within the limitations
set forth in the Definitions section above, are
immunoreactive with antibodies raised against native
133g87~
CSF-l and with the receptors associated with CSF-l
activity. Certain e~bodiments of these muteins are,
however, preferred.
The primary sequence shown in Figure 5 for
mCSF-l has the required activity, and it is, of course,
among the preferred embodiments. Also preferred are
muteins wherein certain portions of the sequence have
been altered by either deletion of, or conservative
substitution of, one or more amino acids in mCSF-l. By
a "conservative" amino acid substitution is meant one
which does not change the activity characteristics of
the protein, and in general is characterized by chemical
similarity of the side chains of the two residues
interchanged. For example, acidic residues are
conservatively replaced by other acidic residues, basic
by basic, hydrophobic by hydrophobic, bulky by bulky,
and so forth. The degree of similarity required
depends, of course, on the criticality of the amino acid
for which substitution is made, and its nature. Thus,
in general, preferred substitutions for cysteine
residues are serine and alanine; for aspartic acid
residues, glutamic acid; for lysine or arginine
residues, histidine, leucine, isoleucine, or valine; for
tryptophan residues, phenylalanine or tyrosine; and so
forth.
Regions of the CSF-l protein which are most
tolerant of alteration include those regions of known
low homology between human and mouse species (residues
15-20 and 75-84); regions which confer susceptibility to
proteolytic cleavage (residues 51 and 5Z and residues
191-193); cysteine residues not participating in
disulfide linkages, or which are not absolutely
essential for activity (residues 158-224).
1~3~73
-2B-
Therefore, particularly preferred are those
CSF-l muteins characterized by the deletion or
conservative substitution of one or more amino acids
and/or one or more sequences of amino acids between
positions 158 and 224 inclusive of mCSF-l. Also
preferred are those characterized by- the deletion or
conservatiYe substitution of one or more of the amino
acids at positions 51 and 52 and/or positions 191, 192
and 193 of mCSF-l. Since they tepresent regions of
apparently low ho~ology, another preferred set of
embodiments is that characterized by the deletion or
conservative substitution of one or more of the amino
acids at positions 15-20 and/or positions 75-84 of
mCSF-l. Also preferred are those muteins characterized
by the deletion or conservati~e substitution of the
cysteine residue at any position not essential for
disulfide bond formation. Also preferced are those
proteins characterized by the deletion or substitution
of the tyrosine residue at position 59 of mCSF-l;
particularly substitution by an aspartic acid residue.
E. Cloninq and ExPression of Human CSF-l
The following illustrates the methods used in
obtaining the coding sequence for human CSF-l, for
disposing this seguence in expression vectors, and for
obtaining expression of the desired protein.
E.l. Purification of Native Human CSF-l and Probe Desiqn
Human urinary CSF-l was partially purified by
standard methods as described by Das, S. K., et al,
Blood (1981) 58:630, followed by an affinity
purification step using a rat monoclonal antibody to
murine CSF-l, designated YYG106, attached to a Sepharose
B column (Stanley, E.R., Methods Enzymol (1985)
* Trade Mark
~ :~339873
-29-
116:~64). The ~inal step in purification was reverse
phase HPLC in a 0.1% T~A/30% acetonitrile - 0.1
TFA/60~ acetonitrile buffe~ system.
For MlAPaCa CSF-l, which was produced
serum-free by induction with phorbol myristic acetate,
the cell superatant was sub3ected to calcium phosphate
gel chromatography (according to Das (supra)), followed
by af~inity chromatography using lentil lectin (in place
of the ConA affinity step of Das), and then to the
immunoa~finity step employing the YYG106 monoclonal
antibody conjuqated to Sepharose~ B and to the reverse
phase HPLC, both as above described.
The urinary and MIAPaCa proteins, having been
puri~ied to homogeneity, were subjected to amino acid
sequencing using Edman degradation on an automated
sequencer. Sufficient N-terminal sequence of human CSF
was determined to permit construction of probes shown in
Figure 3.
E.2. PreParation of the Human Genomic Sequence
A human genomic sequence encoding CSF-l was
obtained from the Maniatis human genomic library in
phage Charon 4 using probes designed to encode the
~-terminal sequence of hu~an protein. The library was
constructed using partial HaeIII/AluI digestion of the
human genome, ligation to EcoRI linkers, and insertion
of the fragments into EcoRI diqested Charon 4 phage. A
Charon 4A phage containing the CSF-l sequence as judged
by hybridization to probe as described below, and
designated pHCSF-l, wa~ deposited with the American Type
Culture Collection (ATCC) on 2 April, 1985 and has
accession no. 40177. Upon later study of this phage, it
was ~ound that rearrangements and/or deletions had
occurred and the correct sequences were not
* trademark
1339~73
-30-
maintained. Therefore, an alternative colony obtained
from the genomic library in identical fashion, and
propagated to confirm stability through replication, was
designated pHCSF-a and was deposited with ATCC on 21 May
1985, and given accession number 40185. pHCSF-la
contained an 18 kb insert and was capa-ble of generating
restriction enzyme digests which also hybridized to
probe, and was used for sequence determination and
additional probe construction as outlined below.
If the CSF-l encoding sequence is present in
its entirety its presence can be demonstrated by
expression in COS-7 cells, as described by Gluzman, Y.,
Cell (1981) 23:175. The test fragment is cloned into a
plasmid derived from pBR322 which has been modified to
contain the SV40 origin of replication (pGRI Ringold,
G., J Mol Appl Genet (1982) 1:165-175). The resulting
high copy number vectors are transformed into COS-7
cells and expression of the CSF-l gene assayed after 24,
48, and 72 hours by the radioreceptor assay method
described by Das (supra). Expression is under control
of the native CSF-l control sequences. The HindIII
digests of the approximately 18 kb insert of pHCSF-la
tested in this manner failed to express, thus indicating
that HindIII digests into the gene. This was confirmed
by subsequent mapping.
However, for initial sequencing, a 3.9 kb
HindIII fragment was obtained from the pHCSF-la phage
and cloned into M13 cloning vectors.
The HindIII fragment has been partially
sequenced, and the results are shown in Figure 4, along
with a deduced peptide sequence. It contains the
correct codons for the portion of the human CSF-l
protein for which the amino acid sequence had been
determined, as set forth in Figure 1. The presence of
1~9~73
-31-
an intron of approximately 1400 bp was deduced from the
available amino acid sequence. In addition, based on
the genomic sequence encoding amino acids 24-34 (see
overlined portion of Figures 4 and 5), a 32-mer probe
for the cDNA library was constructed and employed as
described below.
In more detail, to obtain the genomic clone,
pHCSF-la, the Maniatis library was probed using two
mixtures of oligomers shown in Figure 3. EK14 and EK15
were selected, although the other oligomers shown are
useful as well. A "full length" probe for the
N-terminal sequence, EK14 was used as a mixture of
sixteen 35-mers. A shorter oligomer EK15, was employed
as a mixture of sixty-four 18-mers. Phage hybridizing
to both kinased probes were picked and cultured by
infection of E. coli DG98 or other competent strain.
Specific conditions for probing with EK14 and
EK15 are as follows: for EK14, the buffer contained 15%
formamide, 6 x SSC, pH 7.0, 5x Denhardt's, 20 mM sodium
phosphate, 0.2% SDS and 50 ~g/ml denatured salmon
sperm DNA. Prehybridization and hybridization were
conducted at 42~C and the filters were washed in 2 x SSC
at 52~C. For EK15, similar conditions were used for
hybridization and prehybridization except for the
formamide concentration, which was 0%; washing was at a
slightly lower temperature, 42~C.
The approximately 18 kb DNA insert isolated
from the positively hybridizing phage pHCSF-la was
treated with HindIII and the fragments were subjected to
electrophoresis on agarose gel according to the method
of Southern. The gels were replicated onto nitro-
cellulose filters and the filters were probed again with
EK14 and EK15. Both probes hybridized to a 3.9 kb
fragment.
~3~73
-32-
The positive fragment was excised from the gel,
eluted, and subcloned into HindIII-treated M13mpl9 for
dideoxy sequencing. A partial ~equence is shown in
Figure 4. The underlining corresponds precisely to the
previously determined N-terminal sequence of human
CSF-l: the residues with dot subscripts are homologous
to the murine sequence.
In Figure 4, the 1.4 kb intron region between
the codons for amino acids 22 and 23, as deduced from
the human sequence determined from the purified protein,
is shown untranslated. The sequence upstream of the
N-terminal residues contains the putative leader; the
translation of the portion of this leader immediately
adjacent to the mature protein, which was tentatively
verified by the preliminary results of sequencing of the
cDNA clone (see below) is shown. The upstream portions
are, however, not shown translated; these portions are
confirmed by comparison to the cDNA to comprise an
intron.
Further sequencing to obtain about 13 kb of the
entire 18 kb gene shows that the gene contains 9 exons
separated by 8 introns. The regions of the mature
protein cDNA correspond exactly to the genomic exon
codons except for codon 59, as further described below.
An additional M13 subclone was obtained by
digestion of the HindIII 3.9 kb fragment with PstI to
generate a 1 kb PstI/PstI fragment which includes the
known N-terminal sequence and about 1 kb of additional
upstream sequence.
E.3. cDNA Encodinq Human CSF-l
The human derived pancreatic carcinoma cell
line MIAPaCa-2 was used as a source of mRNA to validate
probes and for the formation of a cDNA library
3987~
containing an intronless form of the human CSF-l coding
sequence. The MIAPaCa cell line produces CSF-l at a
level approximately 10 fold below that of the murine
L-929 cells.
Negative control mRNA was prepared from MIAPaCa
cells maintained in serum-free medium, i.e. under
conditions wherein they do not produce CSF-l. Cells
producing CSF-l were obtained by reinducing CSF-l
production after removal of the serum.
Cells were grown to confluence in roller
bottles using Dulbecco's Modified Eagles' Medium (DMEM)
containing 10% fetal calf serum, and produce CSF-l at
2000-6000 units/ml. The cell cultures were washed, and
reincubated serum-free to suppress CSF-l formation. For
negative controls, no detectable CSF-l was produced
after a day or two. Reinduced cells were obtained by
addition of phorbol myristic acetate (100 ng/ml) to
obtain production after several days of 1000-2000
units/ml.
The mRNA was isolated by lysis of the cell in
isotonic buffer with 0.5% NP-40 in the presence of
ribonucleoside vanadyl complex (Berger, S.L., et al,
Biochemistry (1979) 18:5143) followed by phenol
chloroform extraction, ethanol precipitation, and oligo
dT chromatography, and an enriched mRNA preparation
obtained. In more detail, cells are washed twice in PBS
(phosphate buffered saline) and are resuspended in IHB
(140 mM NaCl, 10 mM Tris, 1.5 mM MgC12, pH 8)
containing 10 mM vanadyl adenosine complex (Berger, S.
L., et al, supra).
A non-ionic detergent of the ethylene oxide
polymer type (NP-40) is added to 0.5% to lyse the
cellular, but not nuclear membranes. Nuclei are removed
by centrifugation at 1,000 x g for 10 min. The
1~9~73
-34-
post-nuclear supernatant is added to two volumes of TE
(10 m~ Tris, 1 mM ethylenediaminetetraacetic acid
(EDTA), pH 7.5) saturated phenol chloroform (1:1~ and
adjusted to 0.5% sodium dodecyl sulfate (SDS) and 10 mM
EDTA. The supernatant is re-extracted 4 times and phase
separated by centrifugation at 2,000 x g for 10 min.
The RNA is precipitated by adjusting the sample to 0.25
M NaCl, adding 2 volumes of 100% ethanol and storing at
-Z0~C. The RNA is pelleted at 5,000 x g for 30 min, is
washed with 70% and 100% ethanol, and is then dried.
Polyadenylated (poly A ) messenger RNA (mRNA) is
obtained from the total cytoplasmic RNA by
chromatography on oligo dT cellulose (Aviv, J., et al,
Proc Natl Acad Sci (1972) 69:1408-1412). The RNA is
dissolved in ETS (10 mM Tris, 1 mM EDTA, 0.5% SDS, pH
7.5) at a concentration of 2 mg/ml. This solution is
heated to 65~C for 5 min, then quickly chilled to 4~C.
After bringing the RNA solution to room temperature, it
is adjusted to 0.4 M NaCl and is slowly passed through
an oligo dT cellulose column previously equilibrated
with binding buffer (500 mM NaCl, 10 mM Tris, 1 mM EDTA,
pH 7.5 0.05% SDS). The flow-through is passed over the
column twice more. The column is then washed with 10
volumes of binding buffer. Poly A mRNA is eluted
with aliquots of ETS, extracted once with TE-saturated
phenol chloroform and is precipitated by the addition of
NaCl to 0.2 M and 2 volumes of 100% ethanol. The RNA is
reprecipitated twice, is washed once in 70% and then in
100% ethanol prior to drying.
Total mRNA was subjected to 5-Z0% by weight
sucrofie gradient centrifugation in 10 mM Tris HCl, pH
7.4, 1 mM EDTA, and 0.5% SDS using a Beckman SW40 rotor
at 20 ~C and 27,000 rpm for 17 hr. The mRNA fractions
were then recovered from the gradient by ethanol
133~87~
-35-
precipitation, and in3ected into XenoPus oocytes in the
standard translation assay. The oocyte products of the
RNA fractions were assayed i~ ~e bone marrow
proliferation assay as described
by Moore, R.N., et al, J Immunol (1983) 131:2374,
and of Prystowsky, M.B., et al, Am J Pathol (1984)
114:149) and the fractions themselves were assayed by
dot blot hybridization to a 32-mer probe corresponding
to the DNA in the second exon of the genomic sequence
(exon II probe). (The Gverlining in Figures 4 and 5
shows the exon II probe.) These results are summarized
in Figure 7.
The broken line in figure 7A shows the response
in the bone marrow proliferation assay of the
supernatants from the XenoPus oocytes: Figure 7B shows
the d~t-blo~ ~sults. T.~e ,~gst strongly hybridizina
fraclron, l~, corresponds to a size slightly larger than
the 18S marker, while the most active fractions 8 and 9
correspond to 14-16S. Fractions 8, 9 and 11 were used to
20 form an enriched cDNA library as described below.
(The mRNA was also fractionated on a denaturing
formaldehyde gel, transferred to nitrocellulose, and
probed with exon II probe. Several distinct species
ranging in size from 1.5 kb to ~.5 kb were found, even
under ~tringent hybridization conditions. To eliminate
the possibility of multiple genes encoding CSF-l,
digests of genomic DNA with various restriction enzymes
were sub3ected to Southern blot and probed using
pcCSF-17 DNA. The re~triction pattern was consistent
with the presence of only one gene encoding CSF-l.)
The enriched mRNA pool was prepared by
combining the mRNA from the gradient fractiongS and 9
having the highest bone marrow proliferative activity, although
their ability to hybridize to probe is relatively low
. ~3~7~
(14S-165) with the fraction(1) hybridizing most intensely
to probe ~slightly larger than 18S). H~gher molecular weight
fractions which also hybridized to exon II probe were not
included because corresponding mRNA from uninduced MIAPaCa
cells also hybridized to exon II probe.
cDNA libraries were prepared from total or
enriched human ~NA in two w~ys. One ~ethod ~ses
~gtlO phage vector~ and i~ de~cribed by Huynh, T.V.,
et al, in DNA Cloninq Techniques: A Practical APproach
IRL Press, Oxford 1984, D. GloYer, Ed.
~ preferred method uses oligo dT pri~ing of the
poly A tails and AMV reverse transcriptase employing the
method of Okayama, H., et al, Mol Cell Biol (1983)
3:280-289. This ~
method result~ in a higher proportion of full length
clones than does poly dG tailing and effecti~ely uses as
host vecto~ portions of two vectors therein described,
-- and readily obtainable from the authors, pcDVl and pLl.
The resulting ~ectors contain the insert between vector
fragment~ containing proximal BamHI and XhoI restriction
sites; the vector contains the pBR322 origin of
replication, and Amp resistance gene and SV40 control
elements which result in the ability of the vector to
effect expre~sion of the inserted sequences in C05-7
cells.
A 300,000 clone library obtained from above
enriched MIAPaCa ~RNA by the Okayama and Berg method was
then probed under conditions of high sttingency, using
the exon II probe. Ten colonies hybridizing to the
probe were picked and colony purified. These clones
were assayed for the presence of CSF-l encoding
sequences by transient expression in COS-7 cells. The
cloning vector, which contains the SV40 p~omoter was
used per se in the transformation of COS-7 cells.
1~3~8~3
Plasmid DNA was purified from the 10 positive
clones using a CsCl gradient, and the COS-7 cells
transfected using a modification (Wang, A.M., et al,
Science (1985) 228:149) of the calcium phosphate
coprecipitation technique. After incubation for three
days, CSF-l production was assayed by subjecting the
culture supernatants to the radioreceptor assay
performed substantially as disclosed by Das, S.K., et
al, Blood (1981) 58:630, and to a colony stimulation
(bone marrow proliferation) assay performed
substantially as disclosed by Prystowsky, M.B., et al,
Am J Pathol (1984) 114: 149. Nine of the ten clones
picked failed to show transient CSF-l production in
COS-7 cells. One clone, which did show expression, was
cultured, the plasmid DNA isolated, and the insert was
sequenced. The DNA sequence, along with the deduced
amino acid sequence, are shown in Figure 5. The full
length cDNA is 1.64 kb and encodes a mature CSF-l
protein of 224 amino acids. The clone was designated
CSF-17 with Cetus depository number CMCC 2347 and was
deposited with the American Type Culture Collection on
14 June 1985, as accession no. 53149. The plasmid
bearing the CSF-l encoding DNA was designated pcCSF-17.
E.4. Transient Expression of CSF-l
The expression of plasmid DNA from CSF-17
(pcCSF-17) in COS-7 cells was confirmed and quantitated
using the bone marrow proliferation assay, the colony
stimulation assay and the radioreceptor assay. It will
be recalled that the specificity of the bone marrow
proliferation assay for CSF-l resides only in the
ability of CSF-l antiserum to diminish activity; that
for the colony stimulation assay, in the nature of the
~L3.~9~73
-38-
colonies obtained. Both assays showed CSF-l production
to be of the order of several thousand units per ml.
Bone Marrow Proiiferation
For the bone marrow stimulation assay, which
measures biological activity of the protein, bone marrow
cells from Balb/C mice were treated with serial
dilutions of the 72 hour supernatants and proliferation
of the cells was measured by uptake of labeled
thymidine, essentially as described by Moore, R.N., et
al, J Immunol (1983) 131:2374: Prystowsky, M.B., et al,
Am J Pathol (1984) 114:149. The medium from induced
MIAPaCa cells was used as control. Specificity for
CSF-l was confirmed by the ability of rabbit antisera
raised against human urinary CSF-l to suppress thymidine
uptake. The results for COS-7 cell supernatants
transfected with pcCSF-17 (CSF-17 supernatant) at a 1:16
dilution are shown in Table 1.
Table 1
3H-thymidine incorporation
(cpm)
no normal antihuman
add'ns serum CSF-l serum
medium 861 786 2682
MIAPaCa supernate12255 16498 3302
CSF-17 supernate16685 21996 2324
(The antihuman CSF-l serum was prepared as
described by Das, et al, supra.)
-39- ~ 3
The MIAPaCa supernatant (at the 1:16
dilution used aboYe) contained 125 U/ml CSF activity
corresponding to Z000 U/ml in the undiluted supernatant,
where 1 unit of colony stimulating activity is defined
as the amount of CSF needed to produce one colony from
bone marrow cells/ml in the assay of Stanley,
E.R., et al, J Lab Clin Med (1972) 79:657.)
These data show that the bone marrow
stimulating activity is associated with CSF-l, since
thymidine uptake is inhibited by anti-CS~-l serum.
Regression of results in this bone marrow proliferation
assay obtained at four dilutions ranging from 1:8 to
1:64 gave an estimated activity for CSF-l in CSF-17
supernatants of 2358 U/ml, which was diminished to 424
U/ml in the presence of antiserum, but showed an
apparent increase to 3693 U/ml in the presence of
non-immune serum. This was comparable to the levels
shown in the radioreceptor assay below.
Colony Stimulation
Direct assay of the CSF-17 supernatants for
colony stimulation (Stanley, E.R., et al, J Lab Clin Med
(supra) showed 4287 U/ml, which was substantially
unaffected by the presence of non-immune serum but
reduced to 0 U/ml in the presence of rabbit antihuman
CSF-l. This compares to 2562 U/ml in the MIAPaCa
supernatants. Eighty-five percent of the pcCSF-17
transformed COS-7 supernatant induced colonies had
mononuclear morphology: MIAPaCa supernatant induced
colonies showed a 94% macrophage-6% granulocyte ratio.
Radioreceptor Assay
The radioreceptor assay measures competition
between I-labeled CSF-l and the test compound for
_40_ 13~7~
specific receptors on J774.2 mouse macrophage cells.
MIAPaCa supernatant, assayed for colony stimulating
activity as above, was used as a standard (2000 U/ml).
The CSF-l concentration of the pcCSF-17 transformed
COS-7 supernatant was found to be 2470 U/ml based on a
1:10 dilution and 3239 U/ml based on a 1:5 dilution.
Thus, comparable values for CSF-l
concentration in the media of COS-7 cells transformed
with pcCSF-17 were found in all assays.
E.5. Stable Expression of CSF-l
The COS-7 system provides recombinant CSF-l
by permitting replication of and expression from the
vector sequences. It is a transient expression system.
The human CSF-l sequence can also be stably
expressed in procaryotic or eucaryotic systems. In
general, procaryotic hosts offer ease of production,
while eucaryotes permit the use of the native signal
sequence and carry out desired post-translational
processing. This may be especially important in the
case of CSF-l since the native protein is a dimer.
Bacteria produce CSF-l as a monomer, which would then be
subjected to dimerizing conditions after extraction.
Procaryotic Expression
For procaryotic expression, the cDNA clone,
or the genomic sequence with introns excised by, for
example, site-specific mutagenesis, is altered to place
an ATG start codon immediately upstream of the glutamic
acid at the N-terminus, and a HindIII site immediately
upstream of the ATG in order to provide a convenient
site for insertion into the standard host expression
vectors below. This can be done directly using
insertion site-specific mutagenesis with a synthetic
3 ~3~873
oliqomer containing a new sequence complementary to the
desired AAGCTTATG, flanked by nucleotide sequences
complementary to the native leader and N-terminal coding
sequences.
For cDNA obtained using the method of
Okayama and Berg, the DNA fragment containing the entire
coding sequence is excised from pcCSF-17 by digestion
with ~hoI (at sites retained from the host cloning
vector), isolated by agarose gel electrophoresis, and
recovered by electroelution. To carry out the
mutagenesis, the host bacteriophage M13mpl8 DNA is also
treated with SalI and ligated with the purified fragment
under standard conditions and transfected into frozen
competent E. coli K12 strain DG98. The cells are plated
on media containing 5 x 10 M isopropyl
thiogalactoside (IPTG) obtained from Sigma Chem. (St.
Louis, MO) and 40 ~g/ml X-gal. Non-complementing
white plaques are picked into fresh media.
Mini-cultures are screened for recombinant single strand
phage DNA of the expected size, and the structure of
the desired recombinant phage, is confirmed using
restriction analysis.
A 34-mer complementary to the N-terminal and
leader encoding portions of the CSF-l sequence, but
containing the complement to the desired AAGCTTATG
sequence is synthesized and purified according to the
procedures set forth in ~C.4. A portion of this 34-mer
preparation is radiolabeled according to a modification
of the technique of Maxam and Gilbert (Maxam, A., et al,
Methods in Enzymoloqy (1980) 68:521, Academic Press) as
set forth in C.4 above.
To perform the mutagenesis the above
prepared recombinant bacteriophage is prepared in E.
coli K12 strain DG98 and the single strand phage DNA
13~37~
-42-
purified. One pmole of single strand phage DNA and 10
pmoles of the above synthetic nucleotide primer (not
kina6ed) are annealed by heating for 1 min at 67~C, and
then 30 min at 37~C in 15~1 20 ~M Tris-Cl, pH 8, 20 mM
MgC12, 100 mM NaCl, 20 mM 2-mercaptoethanol. The
annealed DNA is incubated with DNA polymerase I (Klenow)
and 500 ~M dNTPs for 30 min, 0~C and then brought to
37~C. Aliquots (0.05 or 0.25 pmole) are removed after 5
min, 20 min, and 45 min, transformed into E. coli K12
strain DG98 and plated.
After growth, the plates are chilled at 4~C
and plaques lifted with PalI membranes obtained from
Biodyne or S~S filters (1-2 min in the first filter,
more than 10 min for the second filter. The filters are
denatured in 2.5 M NaCl, 0.5 M NaOH (5 min). The
denaturing medium is neutralized with 3 M sodium acetate
to pH 5.5, or with 1 M Tris-Cl, pH 7.5 containing 1 M
NaCl, the filters baked at 80~C in vacuo for 1 hr, and
then prehybridized at high stringency. The filters are
then probed with the kinased synthetic 34-mer prepared
above at high stringency, washed, and autoradiographed
overnight at -70~C.
The RF form of the desired mutated phage is
treated with EcoRI, blunted with Klenow, and then
digested with HindIII to excise the gene as a
HindIII/blunt fragment. (In a strictly analogous
manner, the CSF-l encoding sequence from pMCSF may be
obtained and modified.)
This fragment containing the human (or
murine) CSF-l encoding sequence is then ligated with
HindIII/BamHI (blunt) digested pPLOP or pTRP3 (see
below) to place the coding sequence containing the ATG
start codon immediately downstream from the PL or trp
promoter respectively. These resulting plasmids are
133~873
-43-
transformed into E. coli MC1000 lambda lysogen or MM294,
and the cells grown under non-inducing conditions and
then induced by means appropriate to the promoter. The
cells are harvested by centrifugation, sonicated and the
liberated CSF-l solubilized. The presence of human (or
murine) CSF-l is confirmed by subjecting the sonicate to
the colony stimulating assay set forth above.
Eucaryotic Expression
The Okayama-Berg plasmid pcCSF-17,
containing the cDNA encoding human CSF-l under control
of the SV40 promoter can also be used to effect stable
expression in monkey CV-l cells, the parent cell line
from which the COS-7 line was derived. The host monkey
CV-l cells were grown to confluence and then
cotransformed using 10 ~g pcCSF-17 and various amounts
(1, 2, 5 and 10 ~g) of pRSV-NEO2 (Gorman, C., et al,
Science (1983) 221:551-553) per 500,000 cells. The
transformants were grown in DMEM with 10% EBS medium
containing 100 ~g/ml of G418 antibiotic, to which the
pRSV-NEO2 plasmid confers resistance. The CV-l cell
line showed a G418 transformation frequency of 10
colonies per 10 cells per ~g DNA.
The CV-l cells were cotransformed as
described above and selected in G418-containing medium.
Resistant clones were tested for stability of the
G418-resistant phenotype by growth in G418-free medium
and then returned to G418-containing medium. The
ability of these cultures to survive when returned to
antibiotic-containing medium suggests that the pRSV-NEO2
DNA was integrated permanently into the cell genome.
Since cells stably transformed with a marker plasmid
have about 50% probability of having integrated the DNA
of a cotransfecting plasmid, about half of the~e cells
i~39~73
-44-
will also contain pcCSF-17 DNA in their chromosomal
DNA.
Several clones of the G418-resi6tant pools
of CV-l cells which were demonstrated to be stably
transformed as above were picked and grown in duplicate
flasks to near confluence. One flask of each duplicate
was infected with SV-40 virus at a multiplicity of
infection of 5, and the medium was harvested 6 days
after infection for assay for CSF-l using a
radioimmunoassay. The immunoassay is based on
competition of I-labeled MIAPaCa CSF-l for ~Rabbit
52" polyclonal antiserum raised against purified human
urinary CSF-l.
One of the selected CV-l clones showed 2335
U/ml production of CSF-l, according to this assay,
whereas cells not infected with SV-40 showed less than
20 U/ml. Controls using COS-7 cells transformed with 10
~g pcCSF-17 showed 2400 U/ml CSF-l production without
SV-40 infection.
The CSF-l producing CV-l cell line contains
the pcCSF-17 DNA stably integrated into its genome, and
thus can be used for stable production of CSF-l upon
infection with SV-40. Infection is presumed to "rescue"
the pcCSF-17 DNA from the genome, and provide the SV-40
T-antigen necessary for replication of the rescued DNA.
Without SV-40 infection, the integrated pcCSF-17 DNA is
not effectively expressed.
Optimization of the expression of the CSF-l
encoding sequence by the CV-l (CSF-17) cell line showed
6500-8000 U/ml when measured by the radioimmunoassay six
days after SV-40 infection using a multiplicity of
infection of at least 1, and a 10% FBS medium. Studies
on expression levels at a multiplicity of 10 showed
comparable production, but production was reduced upon
~45~ ~ 3 ~ 9 8 ~ 3
removal of the ~BS from the medium on the second day
after infection.
In the alternative, appropriate control
systems and host vectors permitting expression in
eucaryotic hosts may be used to receive the CSF-l
encoding inserts.
E.6. ActivitY of CSF-l
Additional definition of the activity of
CSF-l was provided using partially purified MIAPaCa
CSF-l or murine L cell CSF-l as models for the
CV-l-produced recombinant material. CSF-l was shown to
enhance the production of interferon and tumor necrosis
factor (TNF) by induced human monocytes by up to
10-fold. CSF-l also was demonstrated to stimulate
macrophage antitumor toxicity.
Stimulation of TNF Production bY Human MonocYtes
MIAPaCa CSF-l was purified from the
supernatant by calcium phosphate gel filtration and
lentil lectin chromatography. For assay of lymphokine
production, peripheral blood-adherent cells were
incubated in duplicate flasks containing 10 cells
e~ach. One flask was treated with 1000 U/ml CSF-l
purified as above. After 3 days, the cells were
harvested, and washed, and resuspended at a cell
concentration of 5 x 10 /ml and plated in 2~-well
plates at 0.5 ml/well. The wells were treated with 10
~g/ml LPS and 20 ng/ml PMA for 48 hr and the
supernatants were harvested for TNF assay. Cells
treated with CSF showed TN~ secretions approximately
(-J 1;~39~73
-46-
nine-~old higher than the untreated cells (1500 U/ml,
compared to 16~ U/ml).
Stimulation of Interferon Production by Human MonocYtes
In an analogous experiment to determine the
effect of CSF-l on interferon production, peripheral
blood-adherent cells were incubated for 3 days in the
presence and absence of 1000 U/ml CSF-l, as described
above, harvested, resuspended at 5 x 105/ml, and
plated in a 25-well plate, as described above. The
cells were induced for interferon production by addition
of varying amounts of poly IC. The supernatants were
assayed for interferon production by their cytopathic
effect on VSV-infected GM 2504 cells. The
CSF-l-stimulated cells showed production of 100 U/ml
when induced with 50 ~g/ml poly IC, as described by
McCormick, F., et al, Mol Cell Biol (1984) 4:166,
whereas comparably induced untreated cells produced less
than 3 U/ml.
Stimulation of Myeloid CSF Production by Human MonocYtes
Monocytes were incubated ~ CSF-l for 3 days
and then induced foc production of myeloid CSF as in
Table 2. The three representative experiments shown,
used blood from different donors.
-47- 133~7~
Table 2
Myeloid CSF (U/ml)
Exp. 1 Exp. 2 Exp. 3
Induction -CSF +CSF -CSF +CSF CSF ~CSF
medium 0 0 0 0 0 0
0.1 ~g/ml - - 0 0 0 80+17
LPS
1 ~g/ml LPS 0700+72 40+20 200+20 103+12 377+57
o.l ~g/ml - - 617+50 993+101 1120+82 1280+60
LPS + 2 ng/ml
, PMA
1 ~g/ml LPS283+42 983+252 360+92 1400+180 537+47 1080+122
~ 2 ng/ml PMA
2 ng/ml PMA- 370+17 297+6 183+15 380+52 716+76
Stimulation of Tumor Cell Killinq bY Murine MacroPhaqe:
Comparison to other Colony Stimulatinq Factors
To assay macrophage stimulation, murine CSF-l
obtained from L-cell-conditioned medium, was used as a
model for the recombinantly produced CSF-l from pcCSF-17
in an assay which showed stimulation of the ability of
murine macrophages to kill sarcoma targets. In this
assay, normal 2 hr adherent C3H/HeN mouse peritoneal
macrophages were incubated for 1 day in vitro with and
without CSF-l and then mixed at a 20:1 ratio with
H-thymidine-labeled mouse sarcoma TU5 cells along
with 10~ v/v conA-induced (10 ~g/ml) spleen lymphokine
(LK), which contains gamma interferon. The release of
labeled thymidine over the following 48 hr was used as a
measure of tumor cell killing. The effect of adding
-48- 13338~3
CSF-l as murine L-cell-conditioned medium containing
1200 U/ml CSF-l is shown in the following table.
Treatment Kill Increase Due
DAY DAY to CSF-l
0~1 1~3 % %
__ __ 13
-- LK 39
-- CSF-l+LK 49 26
CSF-l LK 51 31
CSF-l CSF-l+LK 60 54
15 ~~ LK 35
-- CSF-l+LK 47 34
CSF-l -- 7
CSF-l LK 49 40
CSF-l CSF-l+LK 69 97
Increase in the ability to kill the target
z5 cells was noted whether CSF-l was added during the
preliminary 1 day of growth or during the period of
induction: however, the most dramatic effects were
observed when CSF-l was present during both of these
periods.
~he possibility of contaminating bacterial
lipopolysaccharide (LPS) as the cause of stimulation of
monocytes and macrophages was excluded: The LPS content
of the applied CSF-l was low (<0.3 ng/3000 U CSF-l. by
Limulus amoebocyte lysate assay); activity was removed
13398~3
-49-
by application to an anti-CSF-l column; polymyxin B was
used to neutralize LPS; the macrophages from C3H/HeJ
mice respond to CSF-l but not to LPS.
CSF-GM was prepared from 6 mouse lungs obtained
hours after IV administration of 5 ~g LPS. The
lungs were chopped and incubated for 3 days in serum
free medium, and the supernatant was depleted of CSF-l
using a YYG106 affinity column (CSF-l content reduced
from 270 U/ml to 78 U/ml). CSF-G was prepared from
similarly treated LDI serum fee medium. Both CSF-GM and
CSF-G contents were assayed at 2000 U/ml by colony
stimulating assay.
The peritoneal macrophages were incubated with
40% of either of the foregoing media or with L-cell
medium assayed at 2000 U/ml CSF-l for 1 day, and then
incubated for 48 hours either with additional medium or
with LK, and assayed for TU5 killing as described
above.
The results are shown in Figure 6. While CSF-l
showed marked enhancement of toxicity to TU5, neither
CSF-G nor CSF-GM had any effect.
Stimulation of Murine Antiviral Activity
Adherent murine thioglycolate-elicited macro-
phages were incubated with CSF-l for 3 days and infected
with VSV overnight. Polymyxin B was added to test
samples to block the LPS induction of interferon. The
following table shows crystal violet staining of cells
remaining adherent.
~0
-50- 13 3 9 ~7 3
Table 3
Treatment Cry~tal Violet
-Polymyxin B +Polymyxin B
(mean)(S.D.)
Medium/No VSV .158+.019
Medium + VSV .0583+.02 .049+.009
CSF-1625 U/ml ~ VSV .139+.018 .177+.04
1250 ~ VSV .167+.06 .205+.07
2500 + VSV .160+.06 .219+.04
5000 + VSV .150+.03 .Z02+.06
CSF-l treated cells, therefore, showed protection of the
macrophage against VSV.
E.7 Formulation of CSF-l
The recombinantly produced human CSF-l may be
formulated for administration using standard
pharmaceutical procedures. Ordinarily CSF-l will be
prepared in injectable form, and may be used either as
the sole active ingredient, or in combination with other
proteins or other compounds having complementary or
similar activity. Such other compounds may include
alternate antitumor agents such as adriamycin, or
lymphokines, such as IL-l, -2, and -3, alpha-, beta-,
and gamma-interferons and tumor necrosis factor. The
effect of the CSF-l active ingredient may be augmented
or improved by the presence of such additional
components. As described above, the CSF-l may interact
in beneficial ways with appropriate blood cells, and the
compositions of the invention therefore include
incubation mixtures of such cells with CSF-l, optionally
in the presence of additional lymphokines. Either the
supernatant fractions of such incubation mixtures, or
13398~3
--51--
the entire mixture containing the cells as well may be
used.
F. Murine CSF-l
An intronless DNA sequence encoding murine
CSF-l is prepared using a murine fibroblast cell line
which produces large amounts of CSF-l. The L-929 line,
obtainable from ATCC, is used as a source for mRNA in
order to produce a cDNA library. Using oligomeric
probes constructed on the basis of the known murine
N-terminal and CNBr-cleaved internal peptide sequence,
this cDNA library is probed to retrieve the entire
coding sequence for the murine form of the protein.
Murine CSF-l is believed to be approximately 80%
homologous to the human material because of the homology
of the N-terminal sequences, the ability of both human
and murine CSF-l preparations to stimulate macrophage
colonies from bone marrow cells, and limited
cross-reactivity with respect to radioreceptor and
radioimmunoassays (Das, S. K., et al, Blood (1981)
58:630).
F.l. Protein Purification
Murine CSF-l was purified by standard methods
similar to those that are disclosed by Stanley, E. R. et
al, J Immunol Meth (1981) 42: 253-284 and by Wang, F.
F. et al, J Cell Biochem (1983) 21:263-275.
or SDS gel electrophoresis as reviewed by Hunkapiller,
M. W., et al, Science (1984) 226:304.
Amino acids 1-39 of the murine sequence were
obtained, taking advantage of cyanogen bromide cleavage
at position 10 to extend the degradation procedure. An
internal cleavage fragment from the mouse protein was
also obtained and sequenced.
~ 339~73
Overall composition data for the mouse protein
were also obtained as shown below. These data ~how
correct relative mole % for those amino acids showing
good recoveries; however the numbeEs are not absolute,
as histidine and cysteine were not recovered in good
yield.
Amino Acid mole % residues/125
Asp 2~.1 25.1
10 Glu 20.0 25.0
His
Ser 6.0 7.5
Thr 5.9 7.4
Gly 5.4 6.8
15 Ala 6.8 8.5
Arg 3.0 3.8
Pro 6.7 8.4
Val 5.3 6.6
Met 1.1 1.4
20 Ile 3.9 4.9
Leu 8.5 10.6
Phe 6.0 7.5
Lys 3.5 4.4
Tyr 4.1 5.1
The conversion to residues/125 was based on an
approximation of sequence length from molecular weight.
F.2. Preparation of Murine CSF-l cDNA
The amino acid sequence 5-13 of the murine
CSF-l and the internal sequence were used as a basis for
probe construction.
Three sets of oligomers corresponding to the
murine sequence were prepared. One sequence was
~33~73
prepared to encode "region A" - i.e., amino acids 9-13;
another was prepared to "region B" - i.e., amino acids
5-9, as shown in Figure 2; a third to encode positions
0-6 of an internal sequence, "region C". Because of
codon redundancy, each of these classes of oligomers is
highly degenerate.
Thus, 15-mers constructed on the basis of
region A number 48; 14-mers constructed on the basis of
region B (deleting the last nucleotide of the codon for
histidine) also number 48; 20-mers constructed on the
basis of region C number 32. Alternatively stated, a
15-mer constructed so as to encode region A may have a
mismatch in four of the fifteen positions; a particular
14-mer constructed with respect to region B may have a
mismatch in six positions; a particular 20-mer
constructed with respect to region C may have a mismatch
in five positions.
As described below, by suitable protocol
design, an enriched messenger RNA fraction may be found
for the production of the desired enriched murine cDNA
library, and the precisely correct oligomers for use as
probes also ascertained.
Total messenger RNA is extracted and purified
from murine L-929 cells. Murine L-929 cells are
cultured for 8 days on DME medium and then harvested by
centrifugation. Total cytoplasmic ribonucleic acid
(RNA) was isolated from the cells by the same protocol
as set forth above for MIAPaCa mRNA.
The mRNA is fractionated on gels for Northern
blot as described in paragraph C.3. The 15-mer
sequences corresponding to region A are divided into
four groups of twelve each. Each of these groups was
used to hybridize under low stringency both to control
and to murine L-929 mRNA slabs and the resulting
133!38~3
-54-
patterns viewed by radioautography. Under the low
stringency conditions employed, hybridization occurs to
fractions not containing the proper sequence, as well as
those that do. Also, because the control cell line is
different from that of the L-929 line in ways other than
failure to produce CSF-l, hybridization occurs in a
number of size locations not related to CSF-l in the
L-929 cell gels which are not present in the controls.
Comparable sets of control and L-929 gels are
probed with segregants of the 48 14-mers representing
region B and segregants of the 32 20-mers representing
region C. Only the bands of messenger RNA which
hybridize exclusively in the L-929 slabs for either
regions A or B, and C probes are then further
considered.
The RNA band which continues to bind to one of
the A region 15-mer mixture or one of the region B
14-mer mixture and one of the region C 20-mer mixture
under conditions of increasingly higher stringency is
selected.
When the correct mRNA band is found, each of
the groups of region A 15-mers is used to probe at
various stringency conditions. The group binding at
highest stringency presumably contains the correct
15-mer exactly to complement the mRNA produced. The
correct 15-mer is ascertained by further splitting the
preparation until a single oligomer is found which binds
at the highest stringency. A similar approach is used
to ascertain the correct 14-mer or 20-mer which binds to
region B or C. These specific oligomers are then
available as probes in a murine cDNA library which is
prepared from the enriched mRNA fraction.
The mRNA fraction identified as containing the
coding sequence for CSF-l is then obtained on a
-55- ~339~73
preparative scale. In this preparation, the poly A
mRNA wa~ f~actionated on a sucrose gradie~t in 10 mM
Tris-HCl, pH 7.4, 1 ~M EDTA, and 0.5% SDS. After
centrifugation in a ~eckman SW40 rotor at 30,000 rpm for
17 hr, mRNA fractions are recovered from the gradient by
ethanol precipitation. RNA fractions recovered from the
gradient were each injected into XenoPus oocytes in a
standard translation assay and the products assayed fo~
CSF-l using radioimmunoassay with antibodies raised
against murine CSF-l. Fractions for which positive
results were obtained were pooled and used to construct
the cDNA library. These same fractions hybridize to the
oligomeric probes.
Other methods of preparing cDNA libraries are,
of course, well known in the art. One, now classical,
method uses oligo dT primer, reverse transcriptase,
tailing of the double stranded cDNA with poly dG, and
annealing into a ~uitable vector, ~uch as pBR322 or a
derivative thereof, which has been cleaved a~ the
desired restriction site and tailed with poly dC. A
detailed description ~f this alterna~ method is found,
for example, in US Patent No. 4,518,584 issued May 21, 1985
and assigned to the same assignee.
Z5 In the method used here, ~he enriched mRNA (5
~g) is denatured by treat~.ent with 10 mM methyl
me~cury at 22~C for 5 min and detoxified by the addition
of 1~0 mM 2-mercaptoethanol (Payvar, F., et al, J Biol
Chem (1979) 254:7636-7642). Plasmid pcDVl is cleaved
with Kpnl, tailed with dTTP, and annealed to the
denatured mRNA. This oligo dT primed mRNA is treated
with reverse transcriptase, and the newly synthesized
DNA ~trand tailed with dCTP. Finally, the unwanted
portion of the pcDVl vector is removed by cleavage with
~339~73
HindIII. Separately, pLl is cleaved with PstI, tailed
with dGTP, cleaved with HindIII, and then mixed with the
poly T tailed mRNA/cDNA complex extended by the pcDVl
vector fragment, ligated with E. coli ligase and the
mixture treated with DNA polymerase I (Klenow) E. coli
ligase, and RNase H. The resulting vectors are
transformed into E. coli K12 MM294 to Amp .
The resulting cDNA library is then screened
using the oligomer probes identified as complementary to
the mRNA coding sequence as described above. Colonies
hybridizing to probes fcom regions A or B and C are
picked and grown; plasmid DNA isolated, and plasmids
containing inserts of sufficient size to encode the
entire sequence of CSF-l isolated. The sequence of the
insert of each of these plasmids is determined, and a
plasmid preparation containing the entire coding
sequence including regions A and B at the upstream
portion is designated pcMCSF.
F.3 Expression of Murine CSF-l DNA
In a manner similar to that set forth above for
the human cDNA, the murine cDNA is tested for transient
expression in COS cells, and used for expression in
stably transformed CV-l. In addition, the appropriate
HindIII/ATG encoding sequences are inserted upstream of
the mature protein by mutagenesis and the coding
sequences inserted into pPLOP or pTRP3 for procaryotic
expression.
G. Host Vectors
pPLOP is a host expression vector having the
PL promoter and N gene ribosome binding site adjacent
a HindIII restriction cleavage site, thus permitting
convenient insertion of a coding sequence having an ATG
_~7_ ~33~873
start codon preceded by a HindIII site. The backbone of
this vector is a temperature-sensitive high copy number
pla~mid derived from pCS3. pPLOP was deposited at ATCC
on 18 December 1984, and has accession number 39947.
pTRP3 i~ a host expression vector containing a
trp promoter immediately upstream of a HindIII
re~triction site, thus permitting insertion of a coding
sequence in a manner analogous to that above for pPLOP.
The backbone vector for pTRP3 is pBR322. pTRP3 was
deposited with ATCC on 18 December 1984, and has
accession number 39946.
Construction of PPLOP
Oriqin of RePlication
pCS3 provides an origin of replication which
confers high copy number of the pPLOP host vector at
high temperatures. pCS3 was deposited 3 June 1982 and
assigned ATCC number 39142.
pCS3 is derived from pEW27 and pOP9. pEW27 is
described by E.M. Wong, Proc Natl Acad Sci (USA) (1982)
79:3570. It contains mutations near its origin of
replication which pro~ide for temperature regulation of
2s copy number. As a result of these mutations replication
occurs in high copy number at high temperatures, but at
low copy number at lower temperatures.
pOP9 is a high copy number plasmid at all
temperatures which was constructed by inserting ,into
pBR322 the EcoRI/PvulI origin containing fragment from
Col El type plasmid pOP6 (Gelfand, D., et al, Proc Natl
Acad Sci (USA) (1978) 75:5869). Before insertion, this
fragment was modified as follows: 50 ~g of pOP6 was
dige~ted to completion with 20 units each BamHI and
:;
l339873
-58-
SstI. In order to eliminate the SstI 3' protruding ends
and "fill in~ the BamHI 5' ends, the digested pOP6 DNA
was treated with E. coli DNA polymerase I (Klenow in a
two-stage reaction first at 20~C for elimination of the
3' SstI protruding end and then at 9~C for repair at the
5' end. The blunt ended fragment was digested and 0.02
pmole used to transform competent DG75 (O~Farrell, P.,
et al, J Bacterioloqy (1978) 134:645-654).
Transformants were selected on L plates containing 50
~/ml ampicillin and screened for a 3.3 kb deletion,
loss of an SstI site, and presence of a newly formed
BamHI site.
One candidate, designated pOP7, was chosen and
the BamHI site deleted by digesting 25 ~g of pOP7 with
20 units BamHI, repairing with E. coli DNA polymerase I
fragment (Klenow), and religating with T4 DNA ligase.
Competent DG75 was treated with 0.1 ~g of the DNA and
transformants selected on L plates containing 50 ~g/ml
ampicillin. Candidates were screened for the loss of
the BamHI restriction site. pOP8 was selected. To
obtain pOP9 the AvaI(repaired)/EcoRI Tet fragment
from pBR322 was prepared and isolated and ligated to the
isolated PvuII(partial)/EcoRI 3560 bp fragment from pOP8.
Ligation of 1.42 kb EcoRI/AvaI(repair) Tet
(fragment A) and 3.56 kb EcoRI/PvuII Amp (fragment B)
used 0.5 ~g of fragment B and 4.5 ~g of fragment A
in a two-state reaction in order to favor intermolecular
ligation of the EcoRI ends.
Competent DG75 was transformed with 5 ~1 of
the ligation mixture, and transformants were selected on
ampicillin (50 ~g/ml) containing plates. pOP9,
isolated from Amp Tet transformants showed high
copy number, colicin resistance, single restriction
sites for EcoRI, BamHI, PvuII, HindIII, 2 restriction
1339~73
sg
sites for HincII, and the appropriate size and HaeIII
digestion pattern.
To obtain pCS3, 50 ~g pEW27 DNA was digested
to completion with PvuII and the EcoRI. Similarly, 50
~g of pOP9 was digested to completion with PvuII and
EcoRI and the 3.3 kb fragment was isolated.
0.36 ~g (0.327 pmoles) pEW27 fragment and
0.35 ~g (0.16 pmoles) pOP9 fragment were ligated and
used to transform E. coli MM294. AmpR TetR
transformants were selected. Successful colonies were
initially screened at 30~C and 41~C on beta-lactamase
assay plate and then for plasmid DNA levels following
growth at 30~C and 41~C. A successful candidate,
designated pCS3, was confirmed by sequencing.
Preparation of the PL_RBs Insert
The DNA sequence containing PL phage promoter
and the ribosome binding site for the N-gene (NRBS)
was obtained from pEC5, and ultimately from a derivative
of pKC30 described by Shimatake and Rosenberg, Nature
(1981) 292:128. pKC30 contains a 2.34 kb fragment from
lambda phage cloned into the HindIII/BamHI vector
fragment from pBR322. The PL promoter and NRBS
occupy a segment in pKC30 between a BglII and HpaI
site. The derivative of pKC30 has the BglII site
converted to an EcoRI site.
The BglII site immediately preceding the PL
promoter was converted into an EcoRI site as follows:
pKC30 was digested with BglII, repaired with Klenow and
dNTPs and ligated with T4 ligase to an EcoRI linker
(available from New England Biolabs) and transformed
into E. coli K12 strain MM294 lambda . Plasmids were
isolated from AmpR TetR transformants and the
desired sequence confirmed by restriction analysis and
133!~873
-60-
sequencing. The resulting plasmid, pFC3, was
double-digested with PvuI and HpaI to obtain an
approximately 540 bp fragment isolated and treated with
Klenow and dATP, followed by Sl nuclease, to generate a
blunt ended fragment with the 3' terminal sequence
-AGGAGAA where the -AGGAGA portion is the NRBS. This
fragment was restricted with EcoRI to give a 347 base
pair DNA fragment with 5'-EcoRI (sticky) and HinfI
(partial repair, Sl blunt)-3' termini.
To complete pFC5, p~I-Zi5 was used to create a
HindIII site 3~ of the NR~S. p~I-Z15 was deposited 13
January 1984, ATCC No. 39578, and was prepared by fusing
a sequence containing ATG plus 140 bp of ~-IFN fused to
lac Z into pBR322. In p~I-Z15, the EcoRI site of pBR322
is retained, and the insert contains a HindIII site
immediately preceding the ATG start codon of ~-IFN.
p~I-Z15 was restricted with HindIII, repaired with
Klenow and dNTPs, and then digested with EcoRI. The
resulting EcoRI/HindIII (repaired) vector fragment was
ligated with the EcoRI/HinfI (repaired) fragment above,
and the ligation mixture used to transform
MC1000-39531. Transformants containing the successful
construction were identified by ability to grow on
lactose minimal plates at 34~ but not at 30~.
(Transformations were plated on X-gal-Amp plates at 30~
and 34~ and minimal-lactose plates at 30~ and 34~.
Transformants with the proper construction are blue on
X-gal-Amp plates at both temperatures, but on minimal
lactose plates, grow only at 34~.) The successful
construct was designated pFC5.
Completion of pPLOP
pCS3 was then modified to provide the PL and
NRBS control sequences. pCS3 was digested with
1339~3
HindIII, and then digested with EcoRI. The vector
fragment was ligated with an isolated EcoRI/HindIII from
pFC5 containing the PLNRBS and transformed into E.
coli MM294. The correct construction of isolated
plasmid DNA was confirmed by restriction analysis and
sequencing and the plasmid designated pPLOP.
Preparation of PTRp3
To construct the host vector containing the trp
control sequences behind a HindIII site, the trp
promoter/operator/ribosome binding site sequence,
lacking the attenuator region, was obtained from pVH153,
obtained from C. Yanofsky, Stanford University. Trp
sequences are available in a variety of such plasmids
known in the art. pVH153 was treated with HhaI (which
cuts leaving an exposed 3' sticky end just 5~ of the trp
promoter) blunt ended with Klenow, and partially
digested with TaqI. The 99 bp fragment corresponding to
restriction at the TaqI site, 6 nucleotides preceding
the ATG start codon of trp leader were isolated, and the
ligated to EcoRI (repair)/ClaI digested, pBR322 to
provide pTRP3.
On 2 April 1985, Applicants have deposited with
the American Type Culture Collection, Rockville, MD, USA
(ATCC) the phage pHCSF-l in E. coli DG98, accession no.
40177. On 21 May 1985, pHCSF-la, designated CMCC 2312
in the Cetus collection and pHCSF-l ~ Charon 4A for
deposit, was deposited with ATCC and has accession no.
40185. On 14 June 1985, CSF-17 in E coli MM294,
designated CMCC 2347, was deposited with ATCC and has
accession no. 53149. These deposits were made under the
provisions of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the
1~3~7~
-62-
Purposes of Patent Procedure and the Regulations
thereunder (Budapest Treaty). This assures maintenance
of a viable culture for 30 years from date of deposit.
The deposits will be made available by ATCC under the
terms of the Budapest Treaty, and subject to an
agreement between Applicants and ATCC which assures
permanent and unrestricted availability upon issuance of
the pertinent US patent. The Assignee herein agrees
that if the culture on deposit die or be lost or
destroyed when cultivated under suitable conditions, it
will be promptly replaced upon notification with a
viable specimen of the same culture. Availability of
the deposits is not to be construed as a license to
practice the invention in contravention of the rights
granted under the authority of any government in
accordance with its patent laws.
The scope of the invention is not to be
construed as limited by the illustrative embodiments set
forth herein, but is to be determined in accordance with
the appended claims.
