Note: Descriptions are shown in the official language in which they were submitted.
2090121
USES OF COLONY STIMULATING FACTOR-1
The present invention relates to the various therapeutic uses of rtcombinantly
produced human colony stimulating factor-1 (CSF-1).
The ability of curtain factors produced in very low concentration in a variety
of
tissues to stimulate the growth and development of bone marrow progenitor
cells into
macrophages and/or granulocytes 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 ~ v~'~ assay which measures the stimulation of colony
formation by bone marrow cells plated in semi-solid culture medium. There art
no
acceptable in v;vo assays. Because these factors induce the formation of such
colonies,
the factors collectively have been called Colony Stimulating Factors (CSF).
More recently, it has been shown that them arc 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-1, results in colonies containing
predominantly
macrophages. Other subclasses product colonies which contain both neutrophilic
granulocytes and macrophages (GM-CSF); which contain predominantly
neutrophilic
granulocytes (G-CSF); and which contain neutrophilic and eosinophilic
granulocytes,
macrophages, and other myeloid cell types (basophils, erythrocytes, and
megokaryocytes) (11,-3).
GM-CSF is described by Gough, ~ ~., Nature (1984) x:763-767. This
protein is further described in WO 87/02060, published April 9, 1987, as being
useful
to treat cancer patients to regenerate leukocytes after traditional cancer
treatment, and to
reduce the likelihood of viral, bacterial, fungal and parasitic infection in
immunocompromised individuals, such as those having acquired immune deficiency
syndrome (AIDS). Human IL-3 has been cloned by Yank, Y.C., ~ ~., ~j (1986)
~:3.
There art marine factors analogous to the above human CSFs, including a
marine factor called II~3 which induces colonies from marine bone marrow cells
which
contain all these cell types plus megakaryocytes, erythrocytes, and mast
cells, in
various combinations. Marine IL-3 has been cloned by Fung, M.C., ~ ~., ~a~re
(1984) x:233. See also Yokota, T., ~ ~., Proc Natl Acad Sc- (1984)
$,1:1070-1074; Wong, G.G., ~ ~., ,science ( 1985) x$:810-815; Lee, F., ~ al.,
$~
Natl cad Sci (USA) (1985) $x:4360-4364; and Cantrcll, M.A., ~ ~., Proc Natl
Acad
Sci (CISAI (1985) $x:6250-6254.) These CSFs and others have been reviewed by
Dexter, T.M., Nature (1984) ~Q:746, and Vadas, M.A., ~ ~., J. lmmunol. (1983)
r-
P
y'
2090121
1,~Q:793, Clark, S.C., Science (1987) x:1229, and Sachs, L., Science (1987)
x,$:1374.
The cloning and expression of G-CSF is described in U.S. Patent No.
4,810,643 and a method to purify G-CSF from human oral cancer tissue is
described in
U.S. Patent No. 4,833,127.
The invention hetzin is concerned with the recombinant production of proteins
which arc members of the first of these subclasses, CSF-1. This subclass has
been
further characterized and delineated by specific radioimmunoassays and
radiotrceptor
assays to suppress specifically CSF-1 activity, without affecting the
biological activities
of the other subclasses, and macrophage cell line J774 contains receptors
which bind
CSF-1 specifically. A description of these assays was published by Das, S.K.,
~ ~.,
~ (1981) x$:630.
A significant difficulty in putting CSF proteins in general, and CSF-1 in
particular, to any useful function has been their unavailability in distinct
and
charactctizable forth in sufficient amounts to make their employment in
therapeutic use
practical or even possible. The present invention remedies these deficiencies
by
providing purified human and marine CSF-1 in useful amounts through
recombinant
techniques and discloses various therapeutic uses thereof.
Treatment of patients suffering from A)DS with CSF-1, alone or together with
erythropoietin andJor an antiviral agent and/or IL-2 is t~eported in
W087/03204,
published June 4, 1987. U.S. Patent No. 4,482,485, issued November 13, 1984,
starts that CSF isolated from human urine can be used for a supporting role in
the
treatment of cancer. In addition, EP 118,915, published September 19, 1984,
reports
production of CSF for preventing and treating granulocytopenia and
macrophagocytopenia in patients tt;ceiving cancer therapy, for preventing
infections,
and for treating patients with implanted bone marrow.
In a,ddtion, CSF-1 is reported to stimulate nonspecific tumoricid.al activity
(Ralph ~ al., jm,m,~,iQl (1986) x:194-204). Ralph ~ al., dell Immunol (1983)
xø:10-21 reported that CSF-1 has no immediate dirrct role in activation of
macrophages
for tumoricidal and microbiocidal activities against fibrosarcoma 1023,
lymphoma
18-8, and L, amastigotes. Ralph ~t al., Cell Immunol (1987) x:270-279
reports the delayed tumoricidal effect of CSF-1 alone and the added
tumoricidal effect - .
of a combination of CSF-1 and lymphokine on marine sarcoma TUS targets.
European Patent 273778 discloses the synergistic effect of CSF-1 and G-CSF to
stimulate the immune system.
209011
WO 92/03151 PCT/US91/06052
3
In addition, Warren ~ ~., ( 1986) x:2281-2285 discloses that
CSF-1 stimulates monocyoe production of interferon, TNF and colony stimulating
activity. Lee ~ ~., J. of I~y~, (1987) x$:3019-3022 discloses CSF-1-induced
resistance to viral infection in marine macrophages.
In one aspect of the invention, therapeutic treatments based on the ability of
CSF-1 to induce resistance to a number of infectious diseases in mammals,
including
those caused by bacterial, viral or fungal agents, are disclosed. Yet a
further aspect
concerns the ability of CSF-1 to promote the repair of tissue damage for use
in wound
healing. Lastly, the invention provides methods of treating tumor cells in
mammals by
using an effective amount of CSF-1 to that tumor burden. In one aspect of this
indication, the invention relates to methods of enhancing the stimulation of
antibody-dependent targeted cellular cytotoxicity using human effector cells,
such as,
bone marrow derived cells, tissue macrophages, or peripheral blood mononuclear
cells
against tumor cells, all of which are mediated by a bifunctional antibody. In
addition,
the invention relates to pharmaceutical compositions comprising CSF-1 or a
mixture
thereof with a cytokine, lymphokine, or with an excipient for use in the
various
aforementioned indications.
Figure 1 shows a comparison of the activities of CSF-1 and other colony
stimulating factors in enhancing the ability of macrophages to kill tumor
cells.
Figure 2 shows the ability of the heteroconjugate antibody 113F1 F(ab')2-3G8
F(ab')2 at 1 ~tg/ml to mediate lysis of cancer cells by adherent blood
mononuclear cells
(AMC) in the presence and in the absence of CSF-1.
Figure 3 shows the ability of the bispecific antibody 2B 1 to mediate lysis by
AMC preincubated with 14-100 ng/ml of CSF-1, as compared to antibody alone.
Figure 4 shows the results of CSF-1 dose range studies on AMC using 100
ng/ml of 2B 1.
Figure 5 shows a protective effect of CSF-1 against a lethal dose of marine
cytomegalovirus (mCMV).
Figure 6 shows wound closure data in control and CSF-1 treated mice.
"Colony stimulating factor-1 (CSF-1 or M-CSF)" refers to those proteins which
exhibit the spectrum of activity understood in the art for CSF-1, i.e., when
applied to
the standard 'fir ~ colony stimulating assay of Metcalf, D., J. Cell Phvsiol.
(1970)
J~:89, it is capable of stimulating the formation of primarily macrophage
colonies.
Native CSF-1 is a glycosylated dimer; dimerization is reported to be necessary
for
activity as the monomer is not active in the Metcalf colony stimulating assay
(supra) or
various other L vitro bioactivity assays (Ralph, P. ~ g_l., Bl~ (1986) x$:633;
Stanley, E.R., ~~., J Biol Chem (1977) 52:4305). As used herein the number of
WO 92/03151 PCT/US91/06052
~UU01~~
4
CSF-1 units corresponds to the number of colonies in the mouse bone marrow
colony
assay in the titratable range (described in Ralph ~ gl., , ). Contemplated
within the scope of the invention and within the definition of CSF-1 are both
the
dimeric and monomeric forms. The monomeric form may be converted to the
dimeric
foam by in vitro provision of suitable refolding conditions as described in
copending
PCT WO 88/08003, published October 20, 1988, and in U.S. Patent No. 4,929,700,
issued May 29, 1990, and the monomer is per se useful as an antigen to produce
anti-CSF-1 antibodies.
There appears to be some species specificity: Human CSF-1 is operative both
on human and on marine bone marrow cells; marine CSF-1 does not show activity
with human cells. Therefore, "human" CSF-1 should be positive in the specific
marine
radioreceptor assay of Das, 1981, , although there is not necessarily a
complete
correlation. The biological activity of the protein will generally also be
inhibited by
neutralizing antiserum to human urinary CSF-1 (Das, 1981, ). However, in
certain special circumstances (such as, for example, where a particular
antibody
preparation may recognize a CSF-1 epitope not essential for biological
function, and
which epitope is not present in the particular CSF-1 mutein being tested) this
criterion
may not be met
Certain other properties of CSF-1 have been recognized more recently,
including the ability of this protein to stimulate the secretion of series E
prostaglandins,
interleukin-1, and interferon from mature macrophages (Moore, R., ~ ~1.,
Science
(1984) x:178). The mechanism for these latter activities is not presently
understood,
and for purposes of definition herein, the criterion for fulfillment of the
definition
resides in the ability to stimulate the fon~nation of monocyte/macrophage
colonies using
bone marrow cells from the appropriate species as starting materials, and
under most
circumstances (see above), show inhibition of this activity by neutralizing
antiserum
against purified human urinary CSF-1, and, where appropriate for species type,
exhibit
a positive response in the radioreceptor assay. (It is known that the
proliferative effect
of CSF-1 is restricted to cells of mononuclear phagocytic lineage (Stanley,
E.R., ~
1~~ okines (1981 ), Stewart, W.E., II, ~ gj., ed., Humana Press, Clifton, NJ),
pp.
102-132) and that receptors for CSF-1 are found on cells of this lineage
(Byrne, P.V.,
~ ~., Cell Biol (1981) Q~,,:848), placental trophoblasts and some other
cells).
"Effective amount" signifies an amount effective to perform the function
specified, such as to kill tumors or reduce tumor burden or prevent or cure
infectious
diseases.
"Therapeutic treatment" indicates treating a subject after a disease is
contracted,
and includes prophylactic therapy.
2090121
~Mammals" indicates any mammalian species, and includes rabbits, mice, dogs,
cats, primates and humans, preferably humans.
~Immunosuppression" means prevention or diminution of the immune response
due to infections, chemical agents, burns, major trauma, or irradiation.
Individuals
who arc immunocomprrnnised arc also immunosuppr~cssed.
"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 into the host chromosome.
As used herein "ctU", "call line". and "cell culture" art; used
interchangeably
and all such designations include progeny. Thus "transformants" or
"transformed
cells" includes the subject cell and cultures arrived 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 scztened for in the originally
transformed cell are
included. Where distinct designations anc intended, it will be clear from the
context.
CSF-1 apparently occurs in numerous forms all of which are included in the
embodiments of the present invention. Human CSF-1 cDNA clones coding for CSF-1
pre-pro-polypepddes of thr~ec different lengths (256 amino acids; 554 amino
acids; and
438 amino acids) have been isolated from cells expressing the single CSF-1
gene (see
commonly owned U.S. Patent No. 4,847,201 issued July 11, 1989 and U.S. Patent
No. 4,868,119, issued September 19, 1989; Wong. G.G., ~gl,., Science (1987)
x:.1504, Kawasaki, ~ al., ~p~ (1985) ?~Q:291; Leaner ~I al., Embo JJ (1987)
x:2693; Cerretti, D.P. ~ $1., ~ (1988) ~"~:761. The CSF-1 proteins
useful in the therapies disclosed heroin may also be processed by proteolysis
including,
for example, in the long form, at the Lys residue at 238, the Arg residue at
249, and the
Arg residue at 411. It is believed that CSF-1 may occur in nature in one or
more
C-terminally deleted forms. In addition, CSF-1 proteins lacking the first two
or four
amino acids have been isolated in active form from the supernatant of the
human cell
line AGR-0N (equivalent to CEM-ON; ATCC No: CRL-8199; Takahashi, M., ~ al.,
Biochem Bio~hyc Res Comm (1988) x:1401 and U.S. Patent No. 4,675,291 issued
June 23,1987). CSF-1 protein comprising monomers ending at amino acid 145 arc
'
reported to have In v~'ltit biological activity (European Patent (EP)
Publication No.
261,592 published March 30, 1988). Some biological activity is reported for a
dimeric
WO 92/03151 2 0 9 0121 P~/US91/06052
6
CSF-1 protein composed of monomers ending at amino acid 132 (EP 328,061
published August 16, 1989). The monomeric CSF-1 polypeptide (whether clipped
at
the C-terminus or not) may also refold to form multimers, most frequently
dimers.
Native human urinary CSF-1 has been isolated as a highly glycosylated dimer
of 45-90 kD, depending on the source, method of measurement and identity of
the
reporter. The recombinantly produced unglycosylated CSF-1 reported by Wong, gl
~.,
appears to have a mono~ric molecular weight of approximately 21 kD. On the
other hand, the molecular weight calculated on the basis of the amino acid
sequence
deduced for the "short" 224 amino acid form of CSF (SCSF) by Kawasaki ~ ~.,
() (see also U.S. Patent No. 4,847,201 (gyp and commonly owned PCT
Publication No. W086/04607 published August 14, 1986) is on the order of 26
kD,
while that of the "long" 522 amino acid form (LCSF) is calculated to be on the
order of
55 kD (along, ~ al,1,~~); Ladner ~ ~., (supra); commonly owned EP 272,779,
published June 29, 1988; and U.S. Patent No. 4,868,119 corresponding to PCT
Publication No. W087/06954, published November 19, 1987). When deleted
constructs of these genes are expressed in ~. ~ (where glycosylation does not
occur),
they, of course, give rise to proteins of considerably lower molecular weight.
Other forms of CSF-1 proteins useful in the present invention include the
polymer conjugated CSF-1 described in commonly owned U.S. Patent No.
4,847,325.
The transmembrane region deletion mutants disclosed in EP 249,477 published
December 16, 1987 and the glycosylation site deletion mutants disclosed in EP
272,779, ~unra, are also considered to be useful in the presently disclosed
therapies.
Methods for the production of these various forms of CSF-1 from various
sources are reported in U.S. Patent No. 4,879,227 issued November 7, 1989; WO
86/04587 published August 14, 1986; WO 89/10407 published November 2, 1989;
WO 88/08003; s~,pra and EP 276,551 published August 3, 1988.
It is, of course, well known that bacterially produced mature proteins which
are
immediately preceded by an ATG start codon may or may not include the N-
terminal
methionine, and it is shown in EP 272,779 (that deletion of residues 1 and 2
(both glutamic acid) or residues 1-3 (Glu-Glu-Val) aids in this manner.
Deletions are
noted by a ~ 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. Thus, the N-terminal deletions referred to above
having the
first 2 and the first 3 residues deleted are designated N12 and N13,
respectively.
C-terminal truncations of CSF-1 resulting in proteins of 150, 158, 190 and 221
amino
acids in length for example are referred to as C1150, C~ 158, C~ 190 and
C1221,
respectively. A 221 amino acid CSF-1 molecule derived from LCSF having an
WO 92/03151 ~ ~ ~ ~ PCT/US91/06052
7
N-terminal deletion of 3 amino acids is denoted by LCSF/N13 0221, for example.
Amino acid substitutions are designated by reference to the position of the
amino acid
which is replaced. For example, substitution of the cysteine residue at
position 157 in
Figure 4 of Ladner ~ ~1_., by serine is referred to as CSF-1 Serls~.
In summary, in addition to the N-terminal and C-terminal deletions and
aggregations, individual amino acid residues in the chain may be modified by
oxidation, reduction, deletion or other derivatization, and these proteins may
also be
cleaved andJor polymerized to obtain dit~rie products that retain activity.
Such
alterations which do not destroy activity do not remove the protein sequence
from the
definition, and are specifically included as substantial equivalents. CSF-1
derived from
other species may fit the definition of a protein having activity of "human
CSF-1" by
virtue of its display of the requisite pattern of activity as set forth above
with regard to
human substrate.
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 preparations 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, polyethylene glycols
(PEGS),
and polyoxyethylene glycols (POGs) as shown in U.S. Patent No. 4,847,325.
Certain
aspects of such augmentation are accomplished through post-translational
processing
systems of the producing host; other such modifications may be introduced in
vi . In
any event, such modifications are included in the definition so long as the
activity of the
dimeric 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-1."
2090121
g
Indeed, human- and marine-derived CSF-1 proteins have nonidentical but similar
primary amino acid sequences which display a high homology.
The CSF-1 proteins of the invention arc capable both of stimulating
monocyte-prccursor/macrophage cell production from progenitor marrow cells,
thus
enhancing the efi'ecdveness of the immune system, and of stimulating such
functions of
these differentiated cells as the secretion of lymphokines in the mat~u~e
macrophages.
In one application, these proteins an useful as adjuncts to chemotherapy. It
is
well understood that chemothcrapeutic 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 these toxic agents on the bone marrow cells. Administration of CSF-1
to such
patients, because of the ability of CSF-1 to mediate and enhance the growth
and
differentiation of bone marrow-derived precursors into macrophages and
monocytcs
and to stimulate the function of these tnatun cells, results in a re-
stimulation of the
immune system to prevent this side effect, and thus to prevent the propensity
of the
patient to succumb to secondary infection.
CSF-1 tray also be used to cure leukopenia, a disease involving a deficiency
in
the total number of whist blood cells. Neutropenia reflects a deficiency
affecting
principally the polymorphonuclear leukocytes (neutrophils, granulocytes) and
may be
due to various infections, certain drugs (e.g., cytotoxic drugs) or ionizing
radiations.
Thus, j~ vivo administration of CSF-1 can be used to induce stem cells to
indirzctly
increase the production of polymorphonuclear leukocytes, thereby increasing
the count
of white blood cells.
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-1.
In general, any subject suffering from immunosuppression whether due to
chemotherapy, bone marrow transplantation, or other, forms of
immunosuppression
such as disease (e.g., acquired immune deficiency syndrome) would benefit from
the
availability of CSF-1 for pharmacological use.
Opportunistic infections can be contracted as a result of immunosuppression.
For example, AIDS related opporumistic infections are described in Mills ~i
$1.,1990,
x:51-57 .
Mills ~ a1. show that common opportunistic infections are caused by the
following agents: G~tomegalovirus, pneumocxstis carinii, ~andida , Varicella-
Zoster virus, Epstein-Burr virus, Toxo~ lasma gondii, M"vcobacterium avium,
8
WO 92/03151 2 0 9 01 '~ 1 PCT/US91/06052
9
n, etc. The authors also describe various agents that are used
to treat these infections. It is contemplated that CSF-1 can be combined with
one or
more of those agents to treat these and other opportunistic infections.
Additionally, it is envisioned that CSF-1 is used to treat ~~
infections, such as cryptococcal meningitis. Cryptococcal meningitis is the
most
common form of fungal meningitis in the United States and the thins most
important
agent of neurologic disease after Human Immunodeficiency Virus (HIV) and
Tox~~l_a~ g~,~ji in AIDS patients. Cryptococcosis will develop at some point
during their illness in 5 to 7% of patients with AIDS in the U.S. while
disseminated
cryptococcosis may develop in up to a third of African A117S patients.
The therapeutic treatment for these opportunistic infections can be similar to
that
described below for fungal diseases. For example, CSF-1 can be administered at
similar dose levels and on the same schedule.
In addition, subjects could be supplied enhanced amounts of previously
differentiated macrophages to supplement those of the indigenous system, which
macrophages are produced by ~ ~ culture of bone marrow or from blood
monocytes, or other suitable preparations followed by treatment with CSF-1.
These
preparations include those of the patient's own blood monocytes or bone marrow
derived cells, which can be so cultured and returned for local or systemic
therapy.
The ability of CSF-1 to stimulate production of lymphokines by macrophages
and to enhance their ability to kill target cells also makes CSF-1 directly
useful in
treatment of neoplasms and infections. Moreover, treatment of wounds with CSF-
1
will promote tissue repair.
CSF-1 stimulates the production of interferons by marine-derived macrophages
(Fleit, H.B., ~ ~1., J. Cell Ph, s~ iol. (1981) ~Q$:347), and human, partially
purified,
CSF-1 from MIAPaCa cells stimulates the poly(I):poly(C)- induced production of
interferon and TNF from human monocytes as illustrated in PCT publication
W086/04607, supra. In addition, CSF-1 stimulates the production of myeloid CSF
by
human blood monocytes.
Moreover, for the various uses described herein, CSF-1 can be employed in
conjunction with other efficacious agents, including antibodies; lymphokines;
cytokines; or macrophage activating agents, such as, e.g., IFN-alpha, IFN-
beta,
IFN-gamma, IL-2, TNF; or muramyl dipeptide and analogs thereof to treat
tumors.
Also illustrated below is a demonstration of the ability of CSF-1 (from marine
L-cell-conditioned medium and ~. ~, produced human recombinant CSF-1) to
stimulate normal C3H/HeN mouse peritoneal macrophages to kill marine sarcoma
TUS
targets. This activity is most effective when the CSF-1 is used as
pretreatment and
l0 2090 i ~ i
during the effectar phase. The ability of CSF-1 to do so is much gtzaoer than
that
exh.ibitcd by other colony stimulating factors.
CSF-1 may also be employed to augment the lymphokinc-induced
antibody-dependent cellular cyu~ooxiciry (ADCC) or targeted ADCC by
macrophages or
natural killer ctlls against tumor oclls. This activity is particularly
effective when
CSF-1 is used in combination with IL2, IFN-alpha, IFN-beta or IFN-gamma.
The ability of CSF-1 to erthanct targeted ADCC activity is believed to be
dependent on the land of the antibody, the dose of CSF-1, and the effector to
target
ratio. Targeted cellular cytotoxiciry is thought to rely on cell surface
rrccptors on
cytotoxic cells, such as tnonocytes, macrophages, natural killers, etc. It is
known that
the expression of one of these cell surface receptors, CD16, is enhanced by
culturing
these effector cells in CSF-1, with or without additional lympholdncs, such as
IL-2. If
the cytotoxic cell is positioned up against the target cell, cytotoxiciry is
enhanced A
bifunctional antibody can promote this process by binding to a target cell
through one
of its combining sites, and to the lysis protnoting receptor on the cytotoxic
cell through
its second combining site, thereby joining the two cell types and causing the
cytotoxic
cell to deliver a kill signal. "Bifunctional antibodies" include those
produced by ja vivo
recombination of antibody chains in trioma or hybrid hybridoma txIl lines and
those
produced by 1a ~ chemical conjugation of two antibodies or antibody fragments;
the
latter chemically linked antibodies are referred to as heteroconjugates.
In the instant invention, such bifunctional antibodies include either hybrid
hybridoma derived bispecific or heteroconjugated antibodies that target the
CD16
antigen known as the human Fc receptor III (FcRBI) on leukocytes. 3G8, a
marine
hybridoma secreting an IgGI monoclonal antibody to human FcRIII, is described
in
Unkeless, J.B., ~ ~.. Ann Rev Imm (1988) x:251. The use of this antibody and
monoclonal antibody 52009 to develop a hybrid hybridotna derived bispecific
antibody
2B 1, is described in published PCT application 90/03576.
The resulting bispecific antibody exhibiu binding to the ~16 Fc recxptor III
positive cells, as well as to breast cancer cells that display the positive
proto-oncogene
product erbB-2. 3G8 has also been chemically cross-linked (using chemical
crosslinkers as described by, for example, Karpovsky, ~ al., J. Eu~ ( 1984)
~Q:1686) with 113F1 antibody, a marine monoclonal antibody to a breast cancer
. .
associated antigen (U.S. Patent No. 4,753,894), producing an antibody also
having
bifunctional specificity.
The ~, v~'~ effective dose of CSF-1 in such a targeted cytotoxic assay, is in
the
range of 10-200 ng/ml. However, its jr~ v'v dosage is dependent upon various
factors
including the severity of the cancer, host immune status, body weight, the
ratio of
r_--->
209012
WO 92/03151 ~ PGT/US91/06052
11
effector to target cells, many of which may only be determined on a case-by-
case basis.
CSF-1 should be administered in a dosage which does not cause a systemic toxic
reaction but elicits the potentiating response on the effector cells.
The ja y~ effective dosages of the bifunctional antibody are in the range of 1
ng/ml to 200 ng/ml. jn vivo dosage again depends on a number of factors,
including
the clinical estimate of tumor size, the extent of metastasis and the
biodistribution of the
active drugs and of the cells that an activated. The effective ~ ,~ effector
to target
cell ratio is approximately 10:1 to 80:1. The actual effector to target ratio
in vivo
depends on the accessibility of the tumors to the effector cells and
antibodies.
In addition, the ability of marine cells to attack infectious organisms,
including
viruses such as those from the Herpesvirus genera, for example,
cytomegalovirus;
bacterial agents including those causing Gram-negative sepsis, and fungi is
enhanced
by CSF-1. (Marine CSF-1 is inconsistently reported to stimulate marine
macrophage
to be cytostatic to P815 tumor cells (Wing, E.J., ~~j., J. Clin. Invest.
(1982) x:270)
or not to kill other leukemia targets (Ralph, P, gl ~., Cell Immunol (1983)
7:10).
Nozawa, R.T., ~ ~., Cell Immunol (1980) ,5:116, report that a CSF-1
preparation
may stimulate macrophage ~ vitro to ingest and kill .)
Additionally, it has been discovered that CSF-1 is effective in meating fungal
infections in humans. These fungal infections typically arise in
immunosuppressed
patients (for example, patients that have bone marrow transplants, AIDS,
etc.), but can
also occur in non-immunosuppressed individuals. Fungi from the following
genuses
that can be treated: Candida, ~~;~, ~rhtococcus, Histo lasma, Blastomvces,
Coccidioides, Paracoccidioides, ~, Rhodotorula
oroth,~,~, DermatoRh_, t~,
Pseudallescheria,1'rototheca, Bhino~oridium, or fungi that cause mycetoma or
chromomycosis, etc. (see Principles and Practice of Infectious Diseases, 3rd
Edition,
Mandell ~ ~., Churchell Livingtone [ 1990] pages 1942-2016).
Preferably, CSF-1 is parenterally administered until the fungal infection is
cleared as detemuned by negative culture results. CSF-1 can be administered
subcutaneously, by continuous infusion, by bolus injection, or by constant
infusion.
By "continuous infusion" is meant at least 24 hours of dosing and by "constant
infusion" is meant 24 hours or less of dosing. CSF-1 is preferably
administered by
constant infusion lasting between 1 and 4 hours. The daily CSF-1 dose that is
effective
to treat fungal infections is preferably between 0.01 to 50 mg/M2, more
preferably
between 0.05 to 10 mg/M2, most preferably between 0.5 and 5 mg/M2. Preferably,
CSF-1 is administered for at least 14 days, more preferably for 21-59 days.
Other dose
schedules can also be used if the above treatment causes unwanted effects
(however,
2090 i 21
12
minimal effects have bean observed at doses of up to 30 mg/M2/day by i.v.
bolus) or if
mare efficacious results can be shown.
Preferably, CSF-1 is used to treat ~n _iøg and Asps 'glllus infections as
described above and specifically as shown below. ~tococcus infections such as
cryptococcal meningitis can be ucazed similarly.
Thus, in addition to overcoming itnmunosuppression per se, CSF-1 can be
used to destroy the invading organisms or malignant cells indirectly or
directly by
stimulation of macrophage secretions and activity. This latter activity may be
enhanced
by CSF-1 therapy in combination with an antimicrobial agent such as for
example, one
or more antiviral, antifungal or antibacterial agents.
Examples of antifungal agents include: Amphotericin B, Fluconazole
(Diflucan), 5 fluro-cytosine (Flucytosinc, 5-FC), Ketoconazole, Miconazolc,
Intraconazole, etc. (sec also Mandell ~ g1. above at pages 361-370 or Mills ~t
~. at
pages 54-55). As an example of the clinical spectrum of one of the above
antifungal
agents, Amphotericin typically inhibits the following fungi and protozoa:
Wiper 'grllus
~misat~, Paracoccidioides brasiliensis, Coccidioides ' mi ' , ø~tococcus
ncofarmans, Fiistovlasma caT~m Mucor ~ Rhodotorula spp., ~~Qrothrix
n k'i, Blastomvces ~crmatitidis, ndida spp., L,~ishmania spp., Nae-,~na spp.,
and ~.canthamo~ba spp. The antifungal agents can be administered before,
during, or
after the CSF-1 is given. Doses of these agents are known to those of ordinary
skill in
the art and include daily dose ranges such as 0.4 to 0.6 mg/kg/day for
Amphotericin B,
200 mg/day to 400 mg/day for Fluconazole, about 150 mg/kg/day for 5-Fluro-
cytosine,
and 200 mg/day to 400 mg/day for Ketoconazole. These ranges are merely
exemplary
and arc not to be construed as limiting in any way. Also, it is contemplated
that CSF-1
can be combined with other agents that arc, or will be, effective at treating
fungal
infections. The antifungal agents can be conjugated to polymers to adjust
their j~ of vo
half life and toxicity, see published PCT application 90/015628.
As stated above CSF-1 can be combinod with ottzcr anamicrobial agents, such
as antibacterials. Examples of antibiotics that can be combined with CSF-1
include
those selected from the following categories: ~i-lactam rings (penicillins),
amino sugars
in glycosidic linkage (aminoglycosidcs), macrocyclic lactone rings
(tnacrnlides),
polycyclic derivatives of napthacenecarboxamide (tetracyclines), nitrobenzene
derivatives of dichloroacetic acid, peptides (bacitracin, gramicidin, and
polymyxin),
large rings with a conjugated double bond system (polycnes), sulfa drugs
derived from
sulfanilamide (sulfonamides), S-vitro-2-furanyl groups (nitrofurans),
quinolone
CA 02090121 2004-04-26
13
carboxylic acids (i.e., nalidixic acid), and many others. The groups of
antibiotics
mentioned above are examples of preferred antibiotics, examples of antibiotics
within
those graups are: peptide antibiotics, such as amphomycin, bacitracin,
bleomycin,
eactinomycin, capreomycias, eolistin, dactinomycin, enduracidin, gramicidin A,
gramicidin J(S), mikamycins, polymyxins, stendomycin, thiopeptin,
thiostrepton,
tynxidines, viomycin, virginiamycins, and actinomycin (see Encyclopedia of
Chemical Technology, 3rd edition, Kirk-Othmer Editors, Volute 2, at page 991
(1978) ; aminoglycosides,
such as streptomycin, neomycin, paromomycin, gentamycin, ribostamycin,
IO tobramycin, amikacin, and lividomycin (see Kirk-Othmer, Voluax 2 at pages 8-
19); (3-
lactams, such as benrylpenicillin, methicillin, oxacillin,
hetacillin,:piperacillin,
amoxiciliin, and carbenicillin (see Kirk-Othmer, Volume 2 at page 871);
chloramphenicol (see Kirk-Othmer, Volume 2 at page 920); lincosaminides, such
as
clindamycin, lincomycin celesticetin, desalicetin (see Kirk-Othmer, Volume 2
at page
930); macrolides, such as erythromycin A-E, Iankamycin, Ieucomycins , and
picromycin (see Kirk-Othmer, Volume 2 at page 937); nucleosides, such as 5-
azacytidine, amicedn,.puromycin, and septacidin (see Kirk-Othmer, Volume 2 at
page
962); oligosaccharides, such as curamycin, and everninomicin B (see Kirk-
Othmer,
Volume 2 at page 986); phenaaines, such as, myxin, lomofungin, iodinin, etc.
(see
Kirk-Othmer, Volume 3 at page I); polyenes, such as amphotericins, candicidin,
nystatin, etc. (see Kirk-Othmer, Volume 3 at page 22 ); polyethers (see Kirk-
Othmer,
Volume 3 at page 47); tetracyclines, such as chlortetracycline,
oxytetracycline,
demecIocycIine, methacycline, doxycycline, and minocycline (see Kirk-Othmer,
Volume 3 at page b5); sulfonamides, such as sulfathiazole, sulfadiazine,
sulfapyrazine,
and sulfanilamide (see Kirk-Othmer, volume 2, page 795); nitrofurans, such as
nitrofurazone, furazolidone, nitrofurantoin, furium, nitrovin, and. nifuroxime
(see Kirk-
Othmer, volume 2, page 790); quinolone carboxylic acids, such as nalidixic
acid,
piromidic acid, pipemidic acid, and oxolinic acid (see Kirk-Othmer, volume 2,
page
782).
CSF-1 can also be combined with antiviral agents, such as amantadine,
rimantadine arildone, ribavirin, acyclovir, 9-[(1,3-dihydroxy-2-
propoxy)methyl]guanine (DHPG), vidarabine (ARA-A), ganciclovir, enviroxime,
foscarnet, interferons (alpha-, beta- and gamma-), ampligen, podophyllotoxin,
2;3-
didioxycytodine (DDC), iododeoxyuridine (>DU), trifluorothymidine (TFZ'),
dideoxyinosine (ddI), dideoxycytodine (ddC), zidovudine and specific antiviral
immune globulins (see Sanford, J.P. Guide to Antimicrobial Th~,ranv_, West
2 49.0121
WO 92/03151 PCT/US91/06052
14
Bethesda:Antimicrobial Therapy, Inc., 1989, pp.88-93; and Harrison's
Princj~les of
j~"gl Medicine, l lth Edition, Braunwald, E. et al. Eds. New York:McGraw-Hill
Book Co., 1987, pp 668-672). ,'~
Finally, the CSF-1 may be used to promote the repair of tissues for wound
S healing when applied either locally or systemically. CSF-1 may recruit
macrophages
(Wang, J.M., ~ ~., Z j~ (1988) x,:575), as well as induce them to provide
connective tissue growth factors such as platelet-derived growth factor
(PDGF), and
active factors including tumor necrosis factor (TNF), as the stimulus for cell
proliferation. Wound macrophages are reported to release substances that
stimulate
fibroplasia, collagen synthesis, and angiogenesis ~ vivo (Hunt T.K., ~ ~,.,
Surgery
( 1984) Qø:48).
The CSF-1 of the invention may be formulated in conventional ways standard
in the art for the administration of protein substances. Administration by
injection is
one preferred route; and such formulations include solutions or suspensions,
emulsions, or solid composition for reconstitution into injectables or gel
formulations.
Suitable excipients include, for example, Ringer's solution, Hank's solution,
water,
saline, glycerol, dextrose or mannitol solutions, and the like. While liquid
solutions of
CSF-1 may be used directly on or under wound dressings, reconstituted
compositions
are useful for salves, gel formulations, foams and the like for wound healing.
Reconstituted gel formulations provide a controlled delivery system for CSF-1
at a
wound site. Controlled release refers to drug release sufficient to maintain a
therapeutic
level over an extended period of time, such as up to 24 hours or more,
preferably in the
range of 24-72 hours. Increased contact time of growth factors may be
necessary to
achieve a significant increase in the rate of wound healing.
In addition, the CSF-1 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-1 stimulation by various types of blood
cells are
effective against desired targets, and the properties of these blood cells
themselves to
attack invading organisms or neoplasms may be enhanced. The subject's own
cells
may be withdrawn and used in this way, or, for example, monocytes or
lymphocytes
from another compatible individual employed in the incubation.
As discussed previously and particularly with regard to the subject matter
disclosed in U.S. Patent No. 4,847,201 (, the complete coding sequences for a
number of human CSF-1 proteins are now available, and expression vectors
applicable
to a variety of host systems have been constructed and the coding sequence
expressed.
In addition to those expression systems provided in U.S. Patent No. 4,847,201,
2090121
expression systems employing insect cells utilising the control systems
provided by
baculovirus vectors have bean described (Miller, D.W., ~ ~., in Genetic Engine
(1986) Setlow, J.K., ~ ~., eds., Plcnum Publishing, Vol. 8, pp. 277-279, U.S.
Patent No. 4,745,051, issued May 17, 1988, and. published PCT application
89/01038. ~~t ~v-based expression can'bc in m .
These systems arc also successful in producing CSF-1. Mammalian expression has
been accomplished in COS-7, GHO, mouse, and CV-1 cells, and also can be
accomplished in COS-A2, hamster, and marine cells.
The 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. Thus, the availability of this information provides CSF-1 proteins
in
sufficient quantity for application in the various therapies discussed herein.
Among the vectors disclosed in the aforementioned patent publications, the
plasmids pLCSF221 A (which contains the gene encoding asp59LCSF/N13C1'221 )
and pcCSF-17 (which contains the gene encoding SCSF) aro preferred
forprocaryotic
and eukaryotic expression, respectively, of human CSF-1. The plasmid pCSF221A
(hereinafter ~. ~ (221 )) transformed into ~. ~1_i strain DG 116, was
deposited with
the ATCC on April 14, 1987 under the Accession No. 67390. The plasmid pcCSF-17
in ~. ~ MM294 was deposited with the ATCC on June 14, 1985 under the Accession
No. 53149.
Also preferred arc CSF-1 proteins which comprise the amino acid sequences
containing the first 3-150 or 4-150 amino acids of SCSF and LCSF and the C-
terminal
deletions, such as LCSF/~221.
The activity of CSF-1 was determined in the following examples using partially
purifxd MIAPaCa CSF-1, marine L cell CSF-1, CV-1-produced recombinant material
or ~, ~-produced human CSF-1. CSF-1 was shown to enhance the production of
interferon (IFN) and tumor necrosis factor ~ by induced human monocytes by up
to 10 to 30-fold. CSF-1 also was demonstrated to stimulate macrophage
antitumor
toxicity la ~, to inhibit tumor growth j~ viva, to protect mice 5~otn lethal
bacterial
infection, to promote the repair of tissue damage jn viva, to inhibit the
growth of
cytotnegalovirus ~ viva, and to inhibit the growth of yeast ~ viva.
The following examples are illustrative, not limiting, of the therapeutic
uses.
clairned herein.
B;
WO 92/03151 2 0 9 01 ~ i pL'1'/US91/06052
16
.~ J ~ ~. l ...
MIAPaCa CSF-1 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
each. One flask was treated with 1000 U/ml CSF-1 purified as above. After 3
days,
the cells wem harvested, and washed, and resuspended at a cell concentration
of 5 x
105/ml and plated in 24-well plates at 0.5 ml/well. The wells were treated
with 10
~tg/ml lipopolysaccharide (LPS) and 20 ng/ml PMA for 48 hr and the
supernatants were
harvested for TIVF assay. Cells treated with CSF showed TNF secretions
approximately 9-fold higher than the untreated cells ( 1500 U/ml, compared to
162
U/ml).
Stimulation of Interferon Production by Human Monocvtes
In an analogous experiment to determine the effect of CSF-1 on interferon
production, peripheral blood-adherent cells were incubated for 3 days in the
presence
and absence of 1000 U/ml CSF-1, as described above, harvested, resuspended at
5 x
105/ml, and plated in a 24-well plate, as described above. The cells were
induced for
interferon production by addition of varying amounts of poly(I):poly(C). The
supernatants were assayed for interferon production by their cytopathic effect
on
VSV-infected GM 2504 cells. The CSF-1-stimulated cells showed production of
100
U/ml when induced with 50 itg/ml poly(I):poly(C), whereas comparably induced
untreated cells produced less than 3 U/ml.
Stimulation of Myeloid CSF Production by Human Monorvt~c
Monocytes were incubated ~ CSF-1 for 3 days and then induced for production
of myeloid CSF as in Table 1. The three representative experiments shown used
blood
from different donors.
WO 92/03151 2 0 9 0121 p~/US91/06052
17
Exp. 1 Exp. Exp. 3
2
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 ~tg/ml 0 70072 4020 20020 10312
LPS
37757
0.1 ~.g/ml - - 61750 993101 112082
128060
LPS + 2
ng/ml PMA
l~tg/mILPS 28342 983252 36092 1400180 53747
1080122
2 ng/ml
PMA
2 ng/ml - 37017 2976 18315 38052
PMA
716+76
Therefore, CSF-1 stimulates myeloid CSF or colony stimulating activity
production.
Stimulation of Tumor Cell Killing by Marine Macro~ge,~
Comrarison to other Colon~r Stimulating Factors
To assay macrophage stimulation, marine CSF-1 obtained from
L-cell-conditioned medium, was used as a model for the recombinantly produced
CSF-1 from pcCSF-17 in an assay which showed stimulation of the ability of
marine
macrophages to kill sarcoma targets. In this assay, 2 hr adherent C3H/HeN
mouse
peritoneal macrophages were incubated for 1 day jp vitro with and without CSF-
1 and
then mixed at a 20:1 ratio with 3H-thymidine-labeled mouse sarcoma TUS cells
along
with 10% (v/v) ConA-induced (10 ~tg/ml) spleen lymphokine (LK), which contains
gamma interferon. The LK preparation can be replaced by purified gamma
interferon in
this assay. The release of labeled thymidine over the following 48 hours was
used as a
measure of tumor cell killing. The effect of adding CSF-1 as marine L-cell
conditioned medium containing 1200 U/ml CSF-1 is shown in Table 2.
-2090 ~ 21
WO 92/03151 PCT/US91/06052
18
Purified marine CSF-1 and rhCSF-1 from CV-l and ~. ~ (221) have also
been effective in this assay.
Increase Due
Treatment Kjj1 to CSF-1
DAY 1 DAY 13 % %
__ _. 13 _-
-- LK 39 --
-- CSF-1+LK 49 26
CSF-1 LK 51 31
CSF-1 CSF-1+LK 60 54
__ __ 3 __
-- LK 35 --
-- CSF-1+LK 47 34
CSF-1 -- 7 _-
CSF-1 LK 49 40
CS F-1 CS F-1 +LK 69 97
Increase in the ability to kill the target cells was noted whether CSF-1 was
added during the preliminary 1 day of growth or during the period of
induction;
however, the most dramatic effects were observed when CSF-1 was present during
both of these periods.
The possibility of contaminating bacterial LPS as the cause of stimulation of
monocytes and macrophages was excluded: The LPS content of the applied CSF-1
was
low (<0.3 ng/3000 U CSF-1, by Limulus amoebocyte lysate (LAL) assay); activity
was removed by application to an anti-CSF-1 column; polymyxin B was used to
neutralize LPS; the macrophages from C3H/HeJ mice respond to CSF-1 but not to
LPS.
Effect of Other N~yeloid _SFs
CSF-GM was prepared from 6 mouse lungs obtained 5 hours after intravenous
(i.v.) administration of 5 ~tg LPS. The lungs were chopped and incubated for 3
days in
serum free medium, and the supernatant was depleted of CSF-1 using a YYG106
immunoaffmity column as described in WO 86/04587, , (CSF-1 content reduced
from 270 U/ml to 78 U/ml). CSF-G was prepared from similarly treated LD 1
(melanoma cell line) serum free 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-1 for 1 day,
and
then incubated for 48 hours either with additional medium or with LK, and
assayed for
TUS killing as described above.
,.':
WO 92/03151 PCT/US91/06052
19
The results are shown in Figure 1. While CSF-1 showed marked enhancement
of toxicity to TUS, neither CSF-G nor CSF-GM had any effect.
CSF-1 purified from MIAPaCa cell line (approximately 40% purity, specific
activity approximately 2 x 10~ U/mg), marine L-cell conditioned medium
(specific
activity approximately 2.3 x 105 U/mg), and recombinant human (rh) from CV-1
(>95% purity, specific activity approximately 4 x 10~ were found to stimulate
mouse
macrophage ADCC to tumor targets in combination with IL-2 or alpha-, beta- or
gamma-IFN.
In the ADCC assay, female C3H/HeN or C3H/FieJ mice were injected
intraperitoneally (i.p.) with 1.5 ml proteose peptone (Difco Laboratories,
Detroit, MI).
After 3 days, the peritoneal exudate cells at 3 x 105 big cells/0.5 ml alpha-
MEM
medium plus 10% heat-inactivated fetal calf serum were adhered in replicate 1
ml wells
in parallel sets. After 2 hours, the wells were washed thoroughly 3 times with
PBS,
and CSF-1 or lymphokine visas added and incubated for 2 days at 37°C.
The cell
population was >95% macrophage by morphology and cell numbers recovered in
parallel wells at day 2 were similar for the different treatments. On day 2,
heat-inactivated antiserum (anti-Thy-1, rabbit anti-mouse brain, Accurate
Chemicals,
Westbury, NY) was added to one of the parallel sets at various dilutions. The
target,
Rl.l, a T-lymphoma cell line, was added to macrophage wells and to parallel
wells
without macrophages ~ CSF-1, lymphokine, or antiserum.
Since high concentrations (1 p.g/ml) of bacterial LPS stimulate macrophage
ADCC, the marine and human CSF-1 preparations were tested using the LAL assay
and had less than 0.2 ng/ml LPS.
The macrophages were then tested at 3:1 effectoraarget ratio for ADCC by
introducing 105 Rl.l targets plus or minus antiserum and counting live target
cells at 9,
24, 48 and 96 hr. Growth of Rl.l with control macrophages plus antibody was
the
same as that with control or cytokine-treated macrophages in the absence of
antibody or
that of Rl.l alone ~ antibody ~ cytokines. The results are shown in Table 3.
209012
WO 92/03151 PCT/US91/06052
5
Macrophage Treatment Percent ADCC* Kill with Time
9 hours 24 hours 48 hours
Medium 0 0 0
M-CSF 0 . 0 0
IFN-gamma 44~5 63~3 72~3
M-CSF
10 (1000U) + IFN-gamma 88~1 >96 98~1
M-CSF
(100U) + IFN-gamma 83~6 93~3 92~5
* %ADCC kill = 100 (y-x)/y where y = target cell number minus
15 antiserum and x = target cell number plus 1:20,000 dilution antiserum.
IFN-gamma was used at 5 U/ml.
IFN-alpha and IFN-beta at 50 U/ml had about the same ADCC-stimulation
effect as 5 U/ml IFN-gamma. IFN-alpha and IFN-beta at 5 U/ml had essentially
no
effect on ADCC, but in the presence of CSF-1 stimulated tumor killing to
levels seen
20 using 50 U/ml of either IFN alone. Similar effects were seen with rhIL-2:
treatment of
macrophages for two days with 5 U/ml IL-2 alone significantly boosted
macrophage
ADCC. CSF-1 moderately enhanced this strong IL-2 induced activity. However,
when IL-2 was used at lower, ineffective concentrations of 1 U/ml or 0.2 U/ml,
the
addition of CSF-1 showed a strong enhancing effect on tumoricidal activity.
Other lymphokines were tested as primary stimulators of ADCC. Incubation of
macrophages for two days with rhTNF at 1, 10 or 100 U/ml alone or with 1000
U/ml
CSF-1 did not significantly induce ADCC activity. rhlL-1 alpha or beta at 0.2
to 50
U/ml and marine rIL-4 at 1 to 100 U/ml also did not stimulate ADCC alone or
with
CSF-1. Attempts were made to find other cofactors which could substitute for
CSF-1.
Marine rGM-CSF and rIL-3 tested at 10, 100 or 1000 U/ml did not boost ADCC
alone
or with IFN-gamma in the standard two-day pretreatment of macrophages, in
contrast
to CSF-1. These cytokines, after incubation 2 days in medium, had no effect on
growth of Rl.l targets in the absence of macrophages.
In Vitro Targeted ADCC Assay,
The effects of CSF-1 on cultured human peripheral blood monocytes were
studied to deterniine whether pretreatment with CSF-1 could enhance targeted
ADCC.
Human effector cells were isolated from donor buffy coats obtained from
Stanford University Blood Bank (Palo Alto, CA). Mononuclear cells were
separated
2~~~1~~
21
by Ficoll-Hypaque differential centrifugation. To isolate adherent mononuclear
cells
(A.'viC), total mononuclear cells were plated into 24-well tissue cultunc
plates and
allowed to adhere for 30 minutes at 37'C, 5% C4z. Nonadherent cells were
washed
off with warm Hank's balanced salt solution with 50 ~tgftnl gcntamicin. The
viabilities
of all effector cell proparations were >9596 by trypan blue dye exclusion.
Adherent
mononuclear cells were stained with FTTC-anti L,euM3 (Becton Dickinson,
Mountain
View, CA) and analyzed on an EPICS V cell sorter to be >85% LeuM3 positive.
The antibody heteroconjugatc 113F1 F(ab')2-3G8 F(ab')2, a chemically linked
antibody recognizing both a beast cancer associated antigen and the human
FcRIII
(CD 16), and the hybrid hybridoma derived bispecific antibody 2B 1, which has
anti-CD 16 and anti-erbB-2 activities, were used in the following experiments.
Both of
these antibodies mediate good specific lysis of SK-Br-3 tumor target cells
using human
total mononuclear cells as effectors.
Onc day before an ADCC assay, target cells in T75 flasks (50% confluent) were
labeled with 62.5 p.Ci of 3H thymidine (New England Nuclear, 6.7 ~tCi/mmole)
in 25
ml medium. After 30 hours, the cells wen trypsinized off the flasks and washed
3
times. Forty thousand labeled target cells were used per well in the assays.
Medium used throughout the assays for diluting effector cells, antibodies and
CSF-1 was AIM.V serum free medium (Gibco) with 8 mM glutamine. Final total
volume was 1 ml/well. For CSF-1'meatment, medium with or without CSF-1 was
added to effector cells and incubated for 2-3 days. Antibodies and labeled
target cells
were then added. Tritium release in the supernatant was measured after 3 days
with
Cytoscint (ICN) as the scintillation fluid.
Each sample was tested in 4 replicates in each experiment. The well to well
variation of the replicates was usually less than ~ 20% of the mean value. The
mean
tritium release of the replicates was used to calculate the percent specific
lysis, using the
formula: (mean sample release - spontaneous release) (maximum release -
spontaneous
release). To measure spontaneous please, labeled target cells were incubated
in
medium alone, and the supernatant was counted after 3 days. Spontaneous
release of
tritium from the target cells (in the absence of effector cells) averaged less
than 10% in
all experiments. Neither antibodies nor CSF-1 increased spontaneous lysis when
incubated with the target cells alone. To measure maximum r~clease, labeled
target cells
were Iysed in a final concentration of 0.5°k SDS.
The ability of the heteroconjugatc 113F1 F(ab')2-3G8 F(ab')2 at 1 ~tg/ml to
mediate lysis with AMC from two donors is shown in Figure 2. When AMC plus the
hetcroconjugatc were tested against SK-Br-3 at an E:T ratio of 20:1, the
average
WO 92/03151 2 0 9 0121 PCT/US91/06052 --
22
antibody dependent lysis observed was 28%. As shown in Figure 2, when the
effector
cells were preincubated with 14 ng/ml of CSF-1, heteroconjugate mediated lysis
was
enhanced by approximately 110%.
Adherent cells from a total of 11 donors were tested with the F(ab')2 fragment
of bispecific antibody 2B 1. To avoid the possible involvements of FcRI and
FcRII on
human AMC, 2B 1 F(ab')2 was used in the study instead of whole 2B 1. (An
experiment was also conducted to show that the F(ab')2 fragments of the 2
parental
antibodies of 2B 1; 52009 and 3G8, did not ~diate any specific lysis whereas
2B 1
F(ab')Z caused almost complete lysis of the target cells.) The effects of CSF-
1 at
10-100 ng/ml were studied to observe whether CSF-1 was effective in augmenting
lysis mediated by 2B1 F(ab')2. AMC preincubated with 14 to 100 ng/ml of CSF-1
gave higher specific lysis than AMC preincubation without CSF-1 (Figure 3).
The total
amount of target cell lysis obtained varied from donor to donor, although
increases in
specific lyric activity with CSF-1 treatment were reproducible observed in all
monocyte
preparations. As higher levels of CSF-1 may be necessary to stimulate the AMC
of
minimally responsive or unresponsive donors, CSF-1 dose response studies were
conducted using 100 ng/ml of 2B 1 F(ab')2 and the results are shown in Figure
4.
Preincubating AMC with increasing concentrations of CSF-1, prior to
introduction of
2B 1 F(ab')2, gave higher specific lysis starting at 10 ng/ml and did not yet
plateau at
100 ng/ml in all donors.
In Vivo Test of CSF-1 for Anti-Tumor Efficacy
A. Meth A Sarcoma Model
Recombinantly produced CSF-1 (C 158) from CV-1 cell line (LAL assay:2 ng
LPS/ml, 8 ng LPS/mg CSF, 2 x 10~ U/mg) was injected i.p. at 50 ~tg/dose twice
a day
for five days into a 20 g mouse (3 mice per group) implanted subcutaneously
with a
Meth A sarcoma tumor 7 days earlier. For 6 days after the beginning of the CSF-
1
treatment, the 3 untreated and 3 treated mice were evaluated for body weights
and
tumor volumes. There was no evidence of toxicity as measured by change in body
weight. On day 7, one mouse from each group was sacrificed for comparative
histopathological analysis (no gross signs). The four remaining mice were
evaluated
for the usual 14-day period in the Meth A model. The results are provided in
Table 4
below:
WO 92/03151 2 0 9 0121 PCT/LJS91/06052
23~
Treatment Percent
Day CSF-1 Buffered Saline O TV Treated
O TV Control
Mean Change in Tumor Volume (~ TV)
3 2.0 2.2 91
6 2.6 6.8 38
7 4.1 8.0 51
8 5.7 11.0 52
14 13.9 29.4 47
O TV = Ratio of the mean tumor volume at the day indicated to the mean
tumor volume at day 0 within a single group of mice.
The results show that there was evidence for CSF-1-mediated efficacy,
particularly at the day 6 tumor volume measurements. The differences between
the
CSF-1 and control groups were greatest during a period starting several days
after the
commencement of treatment and several days thereafter, after which the tumor
returned
to its usual rate of growth. These data suggest that multiple daily dosing
(continuous
infusions to improve efficacy at this dose level, for longer periods of time)
or a higher
dose level and altered schedule to include drug holiday may enhance efficacy.
Similar results were seen using LCSF C~ 190, and LCSF/C1221 from ~
Eli. The protocol was performed using a group of 5 mice; 50 ~tg/dose of each
product
were used and the administration (twice daily for 5 days) was similar except
for the ~
0150 and 0190 material wherein the schedule was increased to 10 days and
administration was 7-8 hours apart. Table 5 provides results as a percentage
of ATV
Treated divided by OTV Control for each CSF-1-derived material.
WO 92/03151 j ~ 1 PCT/US91/06052
24
CV-1 N~3
Day (C~ 158) C~ 150 C~ 190 C~221 C~221
3 21 0 0 36 14
4 23 0 0 38 46
5 46 5 -- 44 48
6 59 7 57 50 40
7 61 15 70 61 40
8 64 7 81 40 39
13/14 56 30 28 20 26
B . B 16 Metastases Model
CSF-1 was tested in the B16 experimental metastasis model to assess its effect
on the prevention of pulmonary metastases.
1 x 105 tumor cells, suspended in 0.2 ml of Ca+2 and Mg+z-free HBSS, were
inoculated into unanesthetized mice in the lateral tail vein. Twenty-one days
after tumor
cell inoculation, the mice were sacrificed and necropsied. During necropsy,
the lungs
and brain were removed, rinsed in water, and weighed. The lungs were then
fixed in
Bouin's solution, and the number of surface tumor nodules per pair of lungs
was
determined with the aid of a dissecting scope.
Recombinant human CSF-1 (N~3C 221), was used for all experiments.
CSF-1 was freshly obtained prior to each experiment from frozen stocks and
diluted
immediately prior to injection in USP 0.9% saline. CSF-1 was delivered
intravenously
on a once a day (QD) x 10 day schedule. The dosing levels used are given in
the
following table. As a negative control consisting of a non-specific and non-
therapeutic
protein, either USP human serum albumin (HSA) or boiled CSF-1 was used. CSF-1
was boiled for 30 minutes to inactivate the CSF-1 activity.
The efficacy data shown in Table 6 demonstrates that CSF-1 given QD x 10,
intravenously, starting 3 days before intravenous inoculation of 1 x 105 tumor
cells
produces a significant reduction in the median number of pulinonary
metastases. In
contrast, if the CSF-1 therapy was initiated one day post tumor cell
inoculation, no
significant decrease in the median number of pulmonary metastases was
observed. No
oven toxicity, as measured by lethality, was observed at this dose level (2.5-
5.0
m~g)~
20901~.~
WO 92/03151 PCT/US91/06052
25
Table 6
Day of Median Number
Initiation of Pulmonary
Group Dose of Therapy Metastases (Range)
1. Saline. -- +1 , 55 (5, 29, 48, 52,
52
~ 58, 58, 74, 80, 91
)
2. M-CSF 2.5 mg/kg +1 38 (11, 11, 13, 2~8,
32, 44, 50, 64, 67,
90)
3. M-CSF 5 mg/kg +1 50 (22, 32, 48, 48,
48, 52, 57, 62, 65,
76)
4. M-CSF 2.5 mg/kg -3 7 g (0, 0, 2, 5, 6,
8,9,11,12,19)
Difference is significant between this group and control at p = 0.002
(Mann-Whitney).
In a second experiment, CSF-1 was administered i.v. QD x 5. B 16-W 10 tumor
cells were harvested by a brief one min trypsinization, centrifuged, and then
prepared
as a single cell suspension in Ca- and Mg-free HBSS. On day 0, 8 x 104 cells
were
injected per mouse, in a total volume of 0.2 ml in the lateral tail vein. CSF-
1 (~.
N13C~221) therapy (0.25 to 5.0 mg/kg/day) was administered QD x 5, i.v.,
beginning on day -3. On day 14 the mice were sacrificed, the lungs removed,
rinsed in
water, and then fixed on Bouins fixative. Surface tumor nodules were counted
with the
aid of a dissecting scope. As shown below in Table 7, CSF-1 was able to
significantly
decrease the median number of pulmonary metastases when administered i.v. at
either
1.0, 2.5 or 5.0 mg/kg/day, QD x 5.
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WO 92/03151 ' PCT/US91/06052
26
Table 7
Group Dose Number of Lung Metastases p-VALUE
(mg/kg/day) Median (individual values)
HSA 5.0 199.5 (94,142,176,181,187, --
212,22'1,223,236,250)
CSF-1 5.0 27.0 (2,4,11,18,25,29,31
33,39,98) 0.000
CSF-1 2.5 132.0 (12,33,43,67,105,159,
161,178,206,239) 0.034
CSF-1 1.0 85.5 (19,61,62,64,64,107,
114,160,201,234) 0.013
CSF-1 0.5 173.5 (23,114,127,159,171,
176,191,194,200,236) 0.173
80,000 tumor cells i.v. on day 0
Treatment: HSA or M-CSF QDxS i.v. beginning day 3
10 BDF-1 mice/group, sacrificed on day 14
The following experiment shows that CSF-1 may be administered by the
subcutaneous (s.c.) or intraperitoneal (i.p.) route which are equally
effective as CSF-1
when administered i.v.
Tumor cells were prepared as taught above. On day 0, 7.5 x 104 cells were
injected per mouse (5-10 female BDF-1 mice/group), in a total volume of 0.2 ml
in the
lateral vein. CSF-1 at 5 mg/kg/day, QD x 5 beginning on day -3 was
administered by
the three different routes. On day 18 the mice were sacrificed and the lungs
were
prepared as taught above. The results are shown below in Table 8.
r
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27
Table 8
Groups Route Number of Lung Metastases
Median Individual values
1.HSA i.v. 26.5 0,1,19,20,26,27,35,
43,59,76
2.M-CSF i.v. 1.0 0,0,1,10,30
3.HSA i.p. 22.0 16,17,22,33,34
4.M-CSF i.p. 1.0 0,1,1,3,4b
5.M-CSF s.c. 2.0 0,1,2,2,9b
6.HSA i.v. 85.5 2,8,23,30,50,61,64,
68,73,84,87,91,91,
93,98,112,147,150,
150,150
7.M-CSF i.v. 6.0 0,1,2,5,5,7,7,13,15,
18b
8.M-CSF i.v. 2.0 0,0,0,1,1,3,4,4,6,33b
9.M-CSF s.c. 2.5' 0,0,0,2,2,3,3,4,4,72b
Gzoups a separate experiment from Groups
6-9 1-5
wen
run
in
p-value less than
is 0.05
compared
to the
proper
HSA control
The following experinxnt compares the efficacy of CSF-1 administered by
continuous infusion performed subcutancously using Alzet osmotic pumps and
subcutaneou~~bolus dosing. CSF 1 was administered either as a s.c. bolus or as
a s.c.
continuous infusion iz; 0.9~ NaCI. Continuous infusions were performed using
s.c.
implanted Abet pumps, models 1003D and 2001, which dclivend CSF-1 for either 3
days or 14 days, respectively. The 1003D model pump has a mean pumping rate of
1
Etl/hour and a mean fill volume of 87 Nl. The 2001 model pump has a mean
pumping
rate of 0.417 Etl/hour and a taean fill volume of 20? ~tl. For pump
implantation; BDF-1
female (18-20 g) were anesthetized with Meoofane~and a small dorsal incision
was made
in the skin. Pumps were implanted under the skin with the flow moderator
pointing
away from the incision. The incision was closed with a wound clip. All therapy
was
initiated on day -3.
~'~.-.:
WO 92/03151 2 0 9 0 l 21 PCT/US91/06052
28
On day 0, to 7.5 x 104 B 16-W 10 tumor cells (prepared as previously
described)
were injected per mouse, in a total volume of 0.2 ml in the lateral tail vein
using a
27-gauge needle. On day 18, the mice were sacrificed, the lungs removed,
rinsed in
water, and then fixed in Bouins fixative.
As shown in Table 9, CSF-1 administered by s.c. continuous infusion at doses
as low as 0.2$ mg/kg/day was highly effective at decreasing the median number
of
pulmonary metastases.
These studies suggest that CSF-1 when administered by s.c. continuous
infusion is at least 10-fold more potent than the same dose given over the
same period
of time by once a day s.c. boluses.
Daily Lung Metastases p-Value
Group Dose Route* Schedule Median Individual value vs HSA vs s.c. bolus
1$ mg/kg
1. HSA 1.0 s.c. QDxl4d-3 58.5 21,27,54,58.59,66,71,
> 100, 100 -- --
2. HSA 1.0 14d pump d-3 56.0 2,40,40.42,44,68,69,
87,>100, 100 -- 0.820
3. CSF-1 1.0 s.c. QDxl4 d-3 37.0 4,25,26,28,33.41,42.
7s,>loo.loo o.3os --
4. CSF-1 1.0 14d pump d-3 9.s 2,s.6.7,9,10,21,21.24,36 0.002 0.005
5. CSF-1 0.5 s.c. QDxl4d-3 66.5 28,32,36,45,65,68,81,
>loo.loo.loo o.g49 --
6. CSF-1 0.5 14d pump d-3 19.0 2,11.15,16,19,20,25,
34.35 0.002 0.000
3$ 7. CSF-1 0.25 l4dpump d-3 14.0 0,5.8,10,11.17,20,25.
28,29 0.005 --
8. CSF-1 s.0 i.v. QDxSd-3 12.0 0.0,2.10,11.13.14.15
52.91 0.015 --
* Mice were implanted with Alzet osmotic pumps subcutaneously.
In Vitro Test of CSF-1 Alone and with IFN-Yfor Anti-Tumor Fffi~aw
The present example tested a number of cytokines, besides CSF-1 and IFN-y,
4$ for enhancement of tumor cell cytotoxicity.
Mononuclear cells (MNC) were separated from either heparinized venous blood
or buffy coats of normal healthy volunteers by density gradient centrifugation
on
lymphocyte separation medium (LS~VI-..Or~anon Teknika Corp., Durham, N.C.).
MNC
29
TM
were then washed twice with PBS and layered on 49.2% isotonic Percoll
tPharmacia)
and centrifuged for 25 minuoes at 1500 x g. The monocyte sand at the interface
(_>80%
pug monocytes by morphological analysis of cyto-centrifuge przparations) was
harvested and further purified by plastic adherence at 37'C. Adherence was
done in 96
well plates at a density of 12 x 105 cells per well. After one hour, non-
adherent cells
wcrt removed by vigorous washing, leaving approximately 1 x 105 cells per
well.
Purified monocytes were cultured for 3 days in 0.1 % FCS containing either
CSF-1 (E,~ N~3C~221), II,-1, II,-3, IL-4, GM-CSF (all from Genryme Corp.),
1L-2 (fetus Core.), or medium alone (1' induction). After 3 days, monocytes
went
washed and then incubated for an additional 2 days with 2' inducers. 1'
induction with
or without CSF-1 was also carried out in flasks in several experiments.
Monocytes
were adhered directly in tissue culture flasks, non-adherent cells were
rtmoved, and 1'
inducers wcrz added. After 1' induction, monocytes were harvested by
trypsinization
and gentle scraping; viable cell counts were done by trypan blue exclusion,
and 1 x 105
ctlls per well were plated in 96 well plates for the remaining 2 days of the
protocol.
The WEHI 164 tumoricidal assay (Colotta ~ ~1., I. Immunol. (1984) X2:936)
was used to test the cytotoxicity of the cytokines. Briefly, WEHI 164 target
cells in
active log phase were either pre-treated for 3 hours with actinomycin D (act
D) at 1
~tg/ml, washed, and labeled for 1 hour with 200 ~tCi of SlCr, or treated
simultaneously
with act D and StCr for 1 hour at 37'C in 5% C4Z. After removing 100 ~t1 of
culture
supernatant from the monocytes, labeled target cells were added in a 100 ~1
volume to
the effector cells to achieve an effector to target ratio of 10:1, unless
otherwise noted
The cells, in a volume of 200 l,tl, were allowed to incubate for 6 hours at
37'C in 5%
CO2. The plates were then centrifuged for 5 minutes at 1200 rpm in a table-top
swinging bucket centrifuge. 100 Etl of supernatant was removed from each well
and
counted in a gamma counter.
P815 target cells we~nc treated similarly, except the actinomycin D pre-
ucarment
was omitted, and the target cells and effector culls were co-incubated for 18
hours:
Percent induced cytotoxicity was calculated using the formula:
axnerimental cnm -~onta_~eous cod X 1o0
maximum cpm - spontaneous cpm
where: experimental cpm = efhector cells + target cells + induocrs
spontaneous cpm - effcctor cells + target cells + rrxdia
maximum cpm - target cells alone lysed with 1 % SDS
The results art shown in Tables 10 and 11:
WO 92/03151 PGT/US91/06052 ~
209012.
Table 10
Percent otoxici~~v Induced by CSF-1
5 Induction Protocol
1 Media CSF-1 CSF-1
2 CSF-1 CSF-1 Media
10
Experiment 1 19% 34% 35%
Experiment 2 12% 49% 37%
Experiment 3 4% 8% 5%
15 Experiment 4 7% 19% 9%
Although the level of activation
induced by CSF-1 was variable,
16 out of 40
such donors showed between 10%
and 49% enhanced tumoricidal
activity upon
stimulation by CSF-1 alone.
20 Table 11, CSF-1 was used inducer prior to addition of
as a 1 a variety of 2
inducers.
WO 92/03151
PCT/US91/06052
Table 11
2' Stimulation Percent of otoxic itv
1' Stimulation M-CSF at 1000 U/ml
Medium Control 0 2
M-CSF 1000 U/ml 1 11
LPS 1 ~tg/ml 4 26
IFN-y 1 U/ml 0 7
IFN-y 100 U/ml 2 13
LPS 1 ~tg/ml +
IFN-y ( 1 U/ml) 11 29
LPS 1 ~tg/ml +
IFN-y ( 100 U/ml) 7 49
LPS 1 ~g/ml +
PMA 2 ng/ml 22 53
LPS 10 ~g/ml +
PMA 2 ng/ml 24 45
IL-2 50 U/ml 2 5
1L-2 500 U/ml 2 7
Other factors tested as 2' inducers and found to have no effect with or
without
CSF-1 included IL-1, IL-3, IL-4 and GM-CSF at up to 500 U/ml.
In Vitro Stimulation of Murin . Anr;~al Actiwtv
Adherent marine thioglycolate-elicited macrophages were incubated with CSF-1
for 3 days and infected with VSV overnight (Lee, M.T., ~ X1.,1. Immunol.
(1987)
1_x$:3019-3022). Table 12 shows crystal violet staining as measured by
absorbance at
550 nm of cells remaining adherent.
WO 92/03151 2 0 9 012 ~ PCT/US91/06052
32
Absorbance
Treatment (Mean) (S.D.)
10
Mcdium/No virus 0.346~0.02
Medium + virus 0.170+0.02
CSF-1, 1000 U/ml + virus 0.264~0.02
CSF-1, 2000 U/ml + virus 0.356~0.04
CSF-1 treated cells, therefore, showed protection of the macrophage against
VSV.
In Vivo Treatment of CMV Infection with CSF-1
Outbred CD-1 mice were treated with the CSF-1 (C 158) produced from the
CV-1 cell line at doses of 400 ~g/kg, i.p., once a day for 5 days, starting 2
days before
infection with a sub-lethal dose of cytomegalovirus (CMV). Mice were
sacrificed on
the third day after infection and the extent of viral replication in target
organs such as
the spleen was evaluated by plaque assay. The results showed that mice treated
with
CSF-1 have significantly lowered (57.8% reduction in) spleen viral titer
compared to
the saline-treated control mice, indicating that CMV infection is less severe
in
CSF-1-treated mice.
In a second experiment, 20 g Balb/C mice (5 per group) were infected with a
sublethal dose of mCMV (2x104 pfu/mouse, i.p.) 4 hours after the last CSF-1
dosing.
CSF-1 was administered, i.p., once a day for 4 days at 4 dose levels (3.6,
0.9, 0.23
and 0.06 mg/kg/day). In this subacute infection model, mice were assessed for
severity of infection by assaying viral titers (plague forming units on mouse
embryo
cells) in blood and organs (spleen, liver and kidney) at 7 days after
infection.
CSF-1-pretreated mice showed 75-97% reduction in viral titers in spleens,
kidneys and
livers as compared to saline treated control (P<0.01). The CSF-1 effect is
dose
dependent; mean spleen viral titer reduction of 97.8, 95.3, 80.9 and 63.1 % at
CSF-1
dose levels of 3.6, 0.9, 0.23 and 0.06 mg/kg/day, respectively were observed
as
shown in Table 13:
WO 92/03151 2 0 9 0121 PGT/US91/0605Z
33
Mean Vital Titer (% Decrease)a
CSF-1 Dose SPLEEN LIVER IQ1DNEY
(mg/kg/day) x 104 pfu/g x 102 pfu/g x 102 pfu/g
3.6 16.02.1(97.8)b 17.52.5(92.1)b 2.52.5(95.7)b
0.9 32.96.4(95.3)b 35.010.0(84.3)b 17.52.5(69.6)b
0.23 135.025.0(80.9) 97.512.5(56.2) 7.57.5(87.0)
0.06 260.025.0(63.1) 90.05.0(59.6)b 2.50(56.5)
HSA/saline 705.041.7 222.547.5 57.512.5
a = Data represent mean organ titer from five animals assayed individually.
Value in parenthesis refers to percent decrease in organ titer as compared
to HSA saline control.
b = p<0.01
c = p<0.05
Separately, CSF-1 producedin ~. ~ (N13C1221) has been tested in a lethal
mCMV infection model (this is in contrast to the above experiment using
sublethal
doses of CMV, in which organ titers were monitored): When 50 ~tg/ 0.05 ml CSF-
1
was administered i.p. to 13.5-14.5 g Balb/C mice at day -1 or day -1, 0, 1, 2
and 3
(single dose given per mouse) before viral challenge (4 x 105 pfu/mouse, 0.2
ml i.p.),
after 7 days there was a significant increase in survival, shown in Table 14,
as
compared to saline-treated control.
T 1 14
Group Percent Survival
Controls 3/10
CSF-1
~Y-1 8/10
CSF-1
days -1,0,1,2,3 7/10
For prophylactic effect in the acute infection model, 13-14 g Balb/C mice were
infected with a lethal dose of mCMV (5 x 104 to 2 x 105 pfu/mouse, i.p.) 4
hours after
the last CSF-1 dosing (using the dose levels between 0.9 mg/kg/day up to 7.2
WO 92/03151 PCT/US91/06052
2090121
34
mg/kg/day) and survival monitored for 14 days. In this acute infection model,
CSF-1
significantly enhanced survival in mice challenged with mCMV at 50,000
pfu/mouse as
shown in Figure 5, although lower doses of CSF-1 (0.9 mg/kg and 3.6 mg/kg)
appeared to be less effective in mice challenged with mCMV at 100,000
pfu/mouse.
These data demonstrate that CSF-1 can be used as an immunoprophylaxis for
viral infection in clinical medicine. CSF-1 may be used alone or in
combination with
another lympholcine or antiviral agent in the treatment of viral infections in
general, and
in particular, may be beneficial in immunosuppressive viral infections such as
acquired
immune deficiency syndrome (AIDS).
The preferred dosage range administered i.p. is about 0.05-8.0 mg/kg CSF-1
per mouse per day.
In Vivo ProR]lylactic Treatment of Bacterial Infection with CSF-1
Outbred CD-1 mice were individually administered CSF-1, produced from the
CV-1 cell line (short clone 0158), one dose (10 ~.g/dose), administered i.p. 1
day
before challenge with a lethal dose (LD1~ = 6 x 10~ cfu) of a clinical isolate
of E.
(SM18), a bacterium responsible for causing Gram-negative sepsis upon
introduction
into a host. The mice were then monitored for survival for 7 days post-
infection. The
data show that pretreatment with CSF-1 enhanced the survival of mice
challenged with
lethal doses of ~. ~.
This experiment was also conducted using the LCSF N13C~221 bacterially
produced CSF-1. Each lot of CSF-1 was tested in 4-fold dilutions for a total
dose
range of 1 x 10~ to 1.7 x 10g units/kg body weight. Minimum protective dose
was
defined as single daily dose (administered once a day for 5 days, i.p.) before
i.p.
infection with E_. Eli, (6 x 10~ cfu/mouse) which produced statistically
significant (p
value less than 0.05 Fisher's Exact test) enhancement of the survival of
treated mice as
compared to saline or boiled CSF-1 control mice. The results are shown in
Table 15.
Table 15
Percent Survivals
Saline -- 0
CSF-1 2.97 mg/kg 100
CSF-1 0.74 mg/kg 90
CSF-1 0.18 mg/kg 10
Boiledb CSF-1 2.97 mg/kg 0
Boiled CSF-1 0.74 mg/kg 20
Boiled CSF-1 0.18 mg/kg 0
Data represent percent of animals surviving at day 7 after infection.
Heat inactivated controls.
WO 92/03151 2 D 9 0121 p~'/US91/06052
Experiments were conducted to~~tudy the effect of dose scheduling on the
induction of CSF-1 effects on host resistance. Groups of mice (10) were
administered
CSF-1 at 0.9 mg/kg/dose per day for either 1, 2, 3, 4 or 5 days. Mice were
then
challenged with ~. ~ (6 x 10~ efu/mouse) i.p. 4 hrs after the last CSF-1
injection.
5 To induce a protective effect the data shown in Table 16 indicate that
multiple
doses of CSF-1 starting between 52 and 100 hours before bacterial infection
are
effective.
10 CSF-1a Time Befor,eb Percent Survivah pd
Dose Schedule Infection Day 7 Value
QD x 1 4 10 ~ --
15 QDx2 28 ~ 10
QD x 3 52 60 <0.05
QD x 4 76 90 <0.01
QD x 5 100 100 <0.01
Saline 76 0 --
20
a = Groups of 10 mice were treated with CSF-1, i.p. (at 0.9
mg/kg/dose/day, for one, two, three, four or five days (i.e. QD X 1 to
QD X 5) before ~. ~ infection at four hours after the last CSF-1 dose.
b = Duration in hours between the first dose of CSF-1 and the time of
infection with ~. ~.
Data represent percent of animal surviving at 7 days after infection.
d = By Fisher's Exact test, as compared with the saline control group.
Single bolus injection (0.2 to 9.0 mg/kg) at either 4, 18, 28, 52 or 76 hrs
before
infection was not effective at inducing enhanced host resistance.
The data show that pretreatment with CSF-1 significantly enhances survival of
mice challenged with lethal doses of ~. ~. The effect is dependent, however,
on the
dose of CSF-1, the timing, and the schedule of administration. At the higher
doses of
approximately 0.7 to 3.0 mg/kg/day, nearly complete protection was seen. At
the
lower dose of 0.2 mg/kg, there was also protection but the effect was smaller.
L~uko~nic Infection Model
CSF-1 induced protection in ~, ~ infection in mice pretreated with 50 mg/kg
cyclophosphamide (CY). The LDSO of CY for mice is at about 400 mg/kg, the
lower
CY dose used represents 1/8 of the LDSO. This dose, when injected i.p. 3 days
earlier,
WO 92/03151 2 O ~ ~ ~' PCT/US91/06052
36
induced a decrease in total white blood cells and neutrophils, and rendered
mice more
susceptible to ~ ~ infection (e.g., infection with 3 x 10~ cfu/mouse killed
100% of
CY treated mice but only 20% of mice not given this dose of CY). When CSF-1
was
given to CY treated mice (CSF-1 at 0.89 mg/kg, once/day for 4 days, i.p.),
there was
100% survival as compared to 309~o in mice given saline instead.
In Vivo Effect of CSF-1 on Candida Albicans
CSF-1 was administered to outbred CD1 mice (27-28g, females) as daily i.p.
doses for 3 to 4 days before challenge with a lethal dose of ~. albicans (1.5
x 10g yeast
cells/mouse, i.p.). This challenge dose resulted in a median survival time
(MST) of 3.0
days in non-treated and saline treated mice. CSF-1 treated mice showed MST of
15
and 13 days at doses of 1.9 and 0.1 mg/kg, respectively. This 4-fold
enhancement in
survival time is significant at p=0.01 (Log Range Tests). To minimize any
possible
interference from endotoxin, highly purified CSF-1 was used which was
virtually free
of endotoxin (<0.05 ng/mg protein). It was also shown that the prophylactic
effect was
abrogated by heat-inactivation of CSF-1 test material. This protection was
associated
with increased numbers of peripheral blood circulating monocytes and
neutrophils and
a 2- to 3-fold increase in peritoneal macrophages. Therefore, CSF-1 enhanced
host
resistance against ~. i an infection and that this effect is probably mediated
by
activation of macrophages and neutrophils.
CSF-1 was also tested in an additional model in which ~. lbicans was
delivered systemically (i.v.). CSF-1 at a dose of 1.9 mg/kg/day QD x 4 was
administered either i.p. or i.v. 2 x 105 cfu/mouse were injected i.v. 4 hours
after the
last dose of CSF-1. Either of these administrations resulted in a significant
enhancement of survival when compared to saline injected control mice.
Survival Time of Rats With Fungal Infections _A__ft_e_r C_'SF-1 Treatment
Using CSF-1 in conjunction with the antifungal agent fluconazole, we
demonstrated that dailing bolus subcutaneous (SQ) injection of CSF-1 given
therapeutically improved the median survival time of infected animals in a rat
Candidiasis model from 5 days (in animals receiving fluconazole alone) to
great than
30 days (in animals receiving a combination of CSF-1 and fluconazole).
Male Fisher-344 rats weighing 200-250 grams were obtained from Charles
River Laboratories. Rats were housed at 5 per cage and received water and
laboratory
chow ad libitum. Serological testing for adventitious viruses and mycoplasma
is
routinely negative in these animals.
2090121
37
Candida albicans strain ATCC No. 18804 from the American Type CuIturt
CoL-~tion (Bethesda, Ice) was grown overnight at 3TC in trypticase soy broth
and
then centrifuged. The blastospor~es wen resusRended and aliquoted in 9596
fetal
bovine serum and 596 dimethyl sulfoxide and frozen at -80'C. On the day of
each
experiment two aliquots wen pooled, and then diluted in phosphate buffered
saline
(PBS) to 4 x 106 blastosports per mL for injxtion. Each experiaxntal inoculum
was
plated on Yeast Extract Peptone agar to confirm the viable organism count.
Fisher-34.4 rats were infected with 2 x I06 Candida albicarts blastospores
(0.5
mLs) by i.v. injection into a lateral tail vein. In the CSF-1 efficacy
studies, a single 0.3
mg/Kg dose of fluconazole (Diflucan, Pfizer, New York, NY) was administered
subcutaneous (SQ) 2 hours later. Immediately following the fluconazole
treatment, a
0.5 mL SQ dose of CSF-1 was given at a different site. CSF-1 treatment was
continued for an additional 9 days for a total of 10 doses. Animals were
assessed daily
for mortality for 30 days.
The dose of Candida albicans needed to produce 100% mortality in untreated F-
344 rats was determined to be 2 x 106 blastospores (Table 17). This dose
resulted in
mortalities in 3 to 6 days, with a median survival time of 4 days.
Table 18 pnscnts the t~csults of four experiments in which fluconazoIe was
combined with the therapeutic SQ administration of CSF-1 after Candida
albicarrs
infection. Doses of CSF-1 at 0.3 and 1.0 mg/Kg/day appear to be the most
efficacious,
resulting in a median survival time of >30 days, at least 6 times the median
survival
time of control animals (5 days).
Table l7
Morality Table of Fishy 344 Rats Infected with Carradidn Albicarss
No. of C. albicer~s Median Mean Survival Time of
Blastos~ores Injezced No. of Mimals Percent Overall Survival Lethally
lrrfected
(s 10~s) Tested Mortality Tune (Days) Mima>: (pays)
1.25 5 0 >30
2.5 - 5 20 >30
S 16 3 8 >30 5.0
16 88 4 4.4
50 100 4 4.4
40 5 100 4 ~ 4.4
100 8 100 2 2.8
330 8 100 1 1.5
1000 4 100 1 1.0
r. c.~ li .,,.
WO 92/03151 _ _ PGT/US91/06052 ~-
2n~0121
38
Therapeutic Use of CSF-1 with Fluconazole in Candida Albicans Infected Rats
w~.r
&~rrirwartohl &~rvlwl
CtF-1 Dow Tolal4 Msdri % &uvhral
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Eighteen patients with invasive fungal infection which was refractory to
standard therapy were treated with CSF-1. Those patients who were referred to
a bone
marrow transplant (BMT) center were eligible for study, but patients with a
malignancy in relapse were ineligible. No exclusions were made for diagnosis,
age,
graft versus host disease (GVHD) status or patient's overall condition. The
clinical
characteristics of the 18 patients are shown in Table 19.
The diagnosis of invasive fungal infection required demonstration of organisms
in biopsy specimens, cultures of closed body fluids (i.e., cerebral spinal
fluid) or
radiologic evidence for invasive disease in the presence of positive blood
cultures.
Demonstration of fungi solely in bronchoscopic lavage fluid, in
gastrointestinal biopsy
specimens, or in single blood cultures were considered insufficient proof of
invasive
disease. The infecting fungal organism, the organs involved, and diagnostic
modalities
used to follow the infections are shown in Table 19.
All patients were examined and had routine blood cultures taken daily, as well
as having blood chemistries and complete blood cell counts performed daily.
Generally, bone marrow aspirates and biopsies were performed prior to therapy
to rule
out malignancy. The fungal infections were re-evaluated using the initial
diagnostic
procedures on a periodic basis and at autopsy.
The study was designed as a Phase I dose escalation trial in which patients
were
enrolled consecutively. All patients werq n~ai~ted on the best available anti-
fungal
WO 92/03151
PGT/US91 /06052
39
therapy at the maximally tolerated dose. CSF-1 (specific activity
approximately 3-10 x
10~ U/mg) was given at a dose between 0.05 to 2.0 mg/M2. CSF-1 was
adrriinistered
in 100 ml of normal saline with 0.25% albumin by central venous catheter as a
single
two-hour intravenou9 infusion daily for seven days. If after seven days there
was no
evidence for a clinical response, the dose was doubled and administezzd for an
additional seven days. Three dose escalations were allowed in a single
patient. If anti-
fungal efficacy was noted, CSF-1 was continued until resolution of the fungal
infection
could be documented. Patients treated with 2 mg/M2 were maintained at that
dose.
CSF-1 was well tolerated at all dose levels. There were no specific symptoms
or changes in blood chemistries that could be ascribed to it. There were no
significant
adverse effects on the severity of preexisting GVHD during the CSF-1 infusion
course.
Eleven patients had invasive candidiasis, four had aspergillosis, two were
diagnosed with yeast sp., and one was diagnosed with Rhodatorula (Table 19).
Three
patients received CSF-1 prior to BMT for progressive fungal disease that had
not
responded to amphotericin B. One of these patients had under undergone two
sinus
cavity resections for aspergillosis. However, CT scan demonstrated a residual
sinus
mass, and aspiration cultures continued to demonstrate aspergillus. When
cavitating
mufti-lobar pulmonary nodules developed and the patient became progressively
azotemic on 1 mg/kg of amphotericin, CSF-1 was started and the amphotericin
dose
was reduced to 0.5 mg/kg. After 35-days of therapy, the CT scan showed
clearing of
the sinus cavities and cultures for aspergillus became negative. All but the
two largest
cavitary pulmonary nodules resolved. Both pulmonary nodules were subsequently
resected and shown to be free of fungal elements. The patient remains off
antifungal
therapy awaiting an unrelated donor BMT. The second patient treated prior to
BMT
received two grams of amphotericin for an i al i ns (involving the liver and
spleen) without a clinical orradiologic response. After 35 doses of CSF-1,
marked
radiologic evidence of improvement was noted, and the liver was rebiopsied
with no
evidence of residual fungal infection. The third patient had progressive
hepatic
candidiasis. She had clinical and radiologic resolution of the fungal
infection after
receiving 28 doses of CSF-1.
Thirteen of the eighteen patients showed resolution or clinical improvement in
their fungal infections (Table 19). Several patients had complete resolution
of their
fungal infections and were discharged from the hospital. Two patients were
well
enough to receive a bone marrow transplant after their infections cleared. Of
the
remaining five patients, one patient was not evaluable, another patient may
not have had
an invasive fungal infection and expired due to pancreatitis, and the
remaining three
patients showed no response, but received CSF-1 treatment for 8 days or less.
WO 92/03151 ~ ~ ~ ~ ~ ~ PCT/US91/06052
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42
In Yivo Stimu~tion of ite BI ~~lj.,Count
Outbred CD-1 mice were administered purified recombinant human CSF-1, at
2 mg/kg per dose, throe times a day for five consecutive days. Total white
blood cell
count increased to 12,000-13,000/ltl in CSF-1-treated mice from 8,700/N1 in
saline-trraocd control mice. In addition, neutrophil count increased to
6,821/~Cl in
CSF-1-treated mice as compared w 1,078/~tl in saline-treated control mice.
This effect is dependent on the dose of CSFtl and the schedule of
administration. The incncase in peripheral blood neutrophils was de=tcctable 2-
4 hours
after a single doss of CSF 1 was administered i.p. These =tsults~ indicate
that CSF-I
administration may be useful in clinical ar veterinary medicine as a stimulus
of
granulocyte production and an enhancer of white blood count.
CSF-1 in Wognd_,~'e,~ing
CSF-1 is assayed for wound healing using animal models and protocols such
as the Goretcx miniature wound healing model of Goodson and Hunt (fig. Res.,
1982, 3:394) in which implanted Gorctex tubes fill up,with invading
macrophages,
fibroblasts and ocher connective tissue cells, and collagen and fibrin
deposition.
Healing is assessed by examining tube contents microscopically. A second modtl
is
the excisional wound healing model of Eisenger, ~ ~,., (19$8; EN~g;$ SA1.
x:1937)
in which wounds are observed visually and punch biopsies arc taken to monitor
healing, density-of cells, and number of epidermal cell layers arising from
hair follicles.
A third model is a scrosal model such as the heat-injured testicular serosa of
Fotev; gl
,~j., (1987, ,Z Pathos,, ,l,~,j,:209),. in which healing is assessed in fixed
sections by
degree of mesotheIial resurfacing of the injured site.
Generally, CSF-I is applied to the site of the wound by soaking a nonadhesive
surgical dressing in 104 to 108 U/ml of CSF 1 in saline as described in the
cxcisional
wound healing model nef~nertce using epidermal cell derived factor (EGF) for
topical
wounds. Alternatively, similar amounts of CSF 1 are introduced into Goret~ac
tubes at
the time of implantation as described in Goodson and Hunt, r , or CSF 1 tray
also
lx incorporated into a slow-rtlease mattvt and applied at the site of the
wound (in
Gort;tcx tubes, in or under dressings, in a slow release gelatin or.collagen-
based matrix
or by injection in the peritoneal cavity) by systemic treatment 1-3 times a
day (i.v., i.p.,
or s.c.) at a dose of 10 to 10,000 p.g/kg/day. .
In a full thiclrness dermal excisional model, experimental groups of five
female
BDF-1 mice, weighing 18 to 20 grams, were anesthetized by methoxyflurane
inhalation
(Metofane, Pitman-Moore, Inc., Washington Crossing, NJ). Wounds were made
WO 92/03151 ~ PCT/US91/06052
43
using a clean surgical technique. Hair on the dorsum and sides was clipped,
and the
skin swabbed with 70% ethanol and dried. A strip of transparent tape was
applied over
the back, approximately midway between the sacrum and scapulae. The skin was
elevated parallel to the length of the mouse, and full thickness excisional
wounds were
created using a 6 mm diameter punch. The tape was removed, exposing an 8 to 10
mm
wide strip of intact skin between the left and right circular bilateral
wounds. Triple
antibiotic ointment (polymyxin B- bacitracin-neomycin) was applied to the
fresh wound
using a cotton tipped swab stick.
Wounds were treasured with hand held calipers in their anterior-posterior and
transverse dimensions on days 0, 1, 2, 3, 4, 5, 7, 9 and 10 (with day 0
representing
the day of wounding). Although the wounds went circular initially, they tended
to heal
in a elliptical shape. For this reason, approximate wound area was calculated
using the
formula for the area of an ellipse A = pi(BxC)/4, where A = area (mm2), B =
wound
diameter (mm) of the anterior-posterior axis, and C = wound diameter (mm) of
the
transverse axis. Percent wound closure was calculated for each wound by
dividing the
area at any given time point by initial wound area on day 0.
~. Eli produced long clone CSF-1 N13C1221 (specific activity >6.0 x 10~
units/mg) was diluted in 0.9% sodium chloride, USP, and delivered
intravenously via
the lateral tail vein in a final injection volume of 100 ~tl. Doses of CSF-1
ranging from
0.5 mg/kg/day ( 10 ~,g/day) to 10.0 mg/kg/day (200 ~g/day) were administered
daily
for a total of 7 days, with the first dose occurring approximately 4 hours
after
wounding. Human serum albumin (HSA) diluted in 0.9% NaCI was chosen as a
non-specific protein control, and was administered to control animals at a
dose of 5.0
mg/kg/day.
Statistical analysis was done by individual Student t test comparisons between
treatment groups on each day. For a.ll comparisons, statistical significance
was noted
when p<.05.
Little or no hemorrhage occurred in fresh wounds. A thin fibrinous covering
was evident over wounds within 12-24 hours, progressing to a scab within 1-2
days.
The scabs contracted as they dried, gradually becoming less adherent to the
underlying
granulation tissue, but did not distort the wound area measurements. Mean.
wound area
was calculated from measurements of the right and left wounds of 5 mice for
each
group and day. Initial wound area did not differ among any of the treatment
groups.
As shown in Figure 6, wound closure was more rapid in CSF-1 treated mice
than in HSA treated controls, where closure was defined as the percent
reduction in
initial wound area on a given day. The values provided in Figure 6 represent
the means
of 5 mice at each time point. CSF-1 treated group differed from the controls
at all time
WO 92/03151 ~ ~ ~ ~ ~ ~ ~ PCT/US91/06052
44
points (p<.05). The "square" represents the control; the "+" represents CSF-1
(10.0
mg/kg/day); the "triangle" represents CSF-1 (5.0 mg/kg/day); and the "x"
represents
CSF-1 (0.5 mg/kg/day). The greatest enhancement in wound closure was observed
in
mice receiving 10.0 mg/kg/day. Intermediate (5.0 mg/kg/day) and low (0.5
mg/kg/day) doses of CSF-1 also significantly increased rates of wound closure,
while
at these two doses the response was nearly equivalent.
The enhanced rate of wound closure seen in CSF-1 treated wounds appeared to
result from a more rapid initial phase which occurred within the first few
days. During
this period, closure was enhanced approximately 40% by CSF-1 as shown in Table
18.
Thereafter, the rate of closure in all groups was nearly identical and began
to decline
steadily until the wounds were completely healed Wounds of CSF-1 treated mice
reached 50% closure 1 to 2 days sooner than HSA treated controls (Figure 6),
yet the
period of time required for wounds to reach 100% closure was approximately 10
days
for all groups.
Table 20
Control ~CSF-1
0 Days Wotmd Standard Wound Wound Standard Wound F~nced~ P value
Post-Wound Area Deviation Closure Area Deviation Closure Closure Relative to
(mm2) (mm2) (~o) (mm2) (mm2) (9'0) (~o) Control
0 35.2 75 0.0 34.4 8.1 0.0 -- 0.828
1 25.2 6.9 28.4 18.4 3.6 46.6 64.0 0.017
2 19.4 4.8 44.7 11.6 2.3 66.1 47.9 0.000
3 17.4 5.2 50.5 9.0 2.4 73.9 46.4 0.000
4 11.2 3.7 68.1 7.8 2.6 77.2 13.4 0.037
5 9.9 3.1 71.8 5.1 1.9 85.2 18.7 0.001
7 5.0 3.1 85.7 2.6 1.0 92.4 7.9 0.045
10 0.0 0.0 100.0 0.0 0.0 100.0 0.0 ---
' CSF-1 was administered to mice intravenously at a dose of 5.0 mg/kg/day for
a total of 7
days, beginning 4 hours after wounding.
Enhanced closure calculated as: g'o closure (treated) - 4'o closure (control)
4'o closure (control)
Gross observations made at time points from day 3 to day 10 suggested that
changes in the vascular content of the skin surrounding the wound space
occurred
during the course of healing in control and CSF-1 treated wounds. As early as
3 days
post-wounding, increased vasculature (larger and more tortuous vessels) was
evident
by the naked eye in regions of skin surrounding the wound sites in control
mice. By
WO 92/03151 PGT/US91/06052
day 7, the vascular content of skin surrounding control wounds had decreased
while
the.skin surrounding the wounds in CSF-1 treated mice showed significantly
greater
vascularization than did the control mice, both in areas around the wound and
in vessels
not adjacent to the site of wounding. This response appeamd to include both a
greater
5 number of vessels and more extensive branching.
CSF-1 may also be used in combination with other growth factors to promote
wound healing such as epidermal growth factor (EGF), fibroblast growth factor
(basic
and acidic FGF), platelet derived growth factor (PDGF) or transforming growth
factors
(TGF alpha and beta), IL-1, IL~2, platelet derived wound healing factor
(PDWI~) and
10 other substances such as somatomedin C and vitamin C.
The recombinantly produced human CSF-1 may be formulated for
administration using standard pharmaceutical procedures. Depending on ultimate
indication, CSF-1 will be prepared in an injectable or a topical form, and may
be used
15 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, e.g., chemotherapeutic agents such as adriamycin,
or
lymphokines, such as IL-1, -2, -3, -4, and -6, alpha-, beta-, and gamma-
interferons,
CSF-GM and CSF-G, and tumor necrosis factor. The effect of the CSF-1 active
20 ingredient may be augmented or improved by the presence of such additional
components. As described above, the CSF-1 may interact in beneficial ways with
appropriate blood cells, and the compositions of the invention therefore
include
incubation mixtures of such cells with CSF-1, optionally in the presence of
additional
lymphokines or cytokines. Either the supernatant fractions of such incubation
25 mixtures, or the entire mixture containing the cells as well, may be used.
Staggered
timing may be preferred for some combinations, such as CSF-1 followed one to
two
days later by gamma interferon.
The CSF-1 described herein is generally administered therapeutically in
amounts of between 0.01-10 mg/kg per day, whether single bolus administration
or
30 fractionated over 24 hr, for all indications, e.g., treatment of infectious
disease, wound
healing, restoration of myelopoiesis and immunity, and cancer.
The scope of the invention is not to be conswed as limited by the illustrative
embodiments set forth herein, but is to be determined in accordance with the
appended
claims.
