Uteroferrin and rose proteins for stimulating hematopoietic cells

Method of using uteroferrin and rose for stimulating proliferation of hematopoietic cells.

FIELD OF THE INVENTION 
This invention relates to uteroferrin and rose proteins and their use to 
stimulate hematopoietic cells. 
DESCRIPTION OF THE BACKGROUND ART 
Uteroferrin is a purple colored, progesterone-induced glycoprotein 
containing two molecules of iron which is secreted by uterine endometrial 
epithelium of pigs (F. W. Bazer and R. M. Roberts, J. Exp. Zool., 228:373, 
1983; R. M. Roberts and F. W. Bazer, Bio Essays, 1:8, 1984). Uteroferrin 
exists as a 35,000 Mr polypeptide having a purple color and as a 
heterodimer (Mr=80,000) with one of three "uteroferrin-associated 
proteins" which have high amino acid sequence homology with serine 
protease inhibitors (M. K. Murray, et al., J. Biol. Chem., 264:4143, 
1989). The heterodimer has a rose color, but the biochemical and 
biological significance of the rose-form of uteroferrin and the 
uteroferrin-associated proteins is not known. Uteroferrin carries high 
mannose carbohydrate with the mannose-6-PO.sub.4 recognition marker for 
lysosomal enzymes (G. A. Baumbach, et al., Proc. Nat. Acad. Sci., U.S.A., 
81:2985, 1984) and has acid phosphatase activity (D. C. Schlosnagle, et 
al, J. Biol. Chem. 249:7574, 1974). During pregnancy, uteroferrin is 
transported from uterine secretions into the fetal-placental circulation 
by specialized placental structures called areolae (R. H. Renegar, et al., 
Biol. Reprod., 27:1247, 1982). The mannose residues on uteroferrin are 
responsible for uteroferrin being targeted to reticuloendothelial cells of 
the fetal liver, the major site of hematopoiesis in fetal pigs (P. T. K. 
Saunders, et al., J. Biol. Chem., 260:3658, 1985). 
Administration of radiolabelled iron to pigs results in endometrial 
secretion of uteroferrin carrying radiolabelled iron and incorporation of 
radiolabelled iron into fetal erythrocytes and cells of liver, spleen and 
bene marrow (C. A. Ducsay, et al., Biol. Reprod., 26:729, 1982; C. A. 
Ducsay, et al., J. Anim. Sci., 59:1303, 1984). Uteroferrin gives up its 
iron to fetal transferrin in allantoic fluid with a half-life of 12 to 24 
hours (W. C. Buhi, et al, J. Biol. Chem., 257:1712, 1982). Further, 
administration of iron dextran to pregnant pigs on days 50, 60 and 70 
(term is at 115 days), the period of maximum secretion of uteroferrin by 
the endometrium, results in a 20% increase in iron stores in neonatal 
piglets (C. A. Ducsay, et al., Biol. Reprod., 26:729, 1982; C. A. Ducsay, 
et al., J. Anim. Sci., 59:1303, 1984). These results suggest a role for 
uteroferrin in transplacental transport of iron. However, after Day 75 of 
gestation, translation of mRNA for uteroferrin decreases rapidly (R. C. M. 
Simmen, et al., Mol. Endocrinol. 2:253, 1988), secretion of uteroferrin by 
endometrial explant cultures declines (S. M. M. Basha, et al., Biol. 
Reprod., 20:431, 1979), and the amount of uteroferrin in allantoic fluid 
decreases dramatically (F. W. Bazer, et al., J. Anim. Sci., 41:1112, 
1975). This suggests that an alternate mechanism for transplacental iron 
transport becomes operative between Days 75 and term when fetal/placental 
demands for iron are increasing (C. A. Ducsay, et al., Biol. Reprod., 
26:729, 1982; C. A. Ducsay, et al., J. Anim. Sci., 59:1303, 1984). 
Uteroferrin from pig uterus is a tartrate-resistant acid phosphatase with 
many properties in common with the Type 5 acid phosphatase in human 
placenta (C. M. Ketcham, et al., J. Biol. Chem., 260:5768, 1986), 
chondrocytes of humans with osteoclastic bone tumors and spleens of humans 
with hairy cell leukemia, Gaucher's disease and Hodgkin's disease. In 
addition, uteroferrin has characteristics similar to those for purple acid 
phosphatases from bovine, rat, mouse, and pig spleen, as well as bovine 
milk, bovine uterine secretions, equine uterine secretions, and rat bone 
(C. M. Ketcham, et al., J. Biol. Chem. 260:5768, 1985). 
In the medical community there has long been a recognition of various 
disorders involving the hematopoietic system. These disorders include the 
anemias; myeloproliferative diseases; primary bone marrow dysfunctions, 
especially those involving pancytopenia; and the leukemias. 
Depending on the nature of the hematopoietic disorder, various therapies 
may be used. Unfortunately, for many of these disorders, no adequate 
therapeutic approach is available and treatment consists primarily of 
basic management of the patient. Alternatively, where therapeutic agents 
are available, there are often significant toxic side effects associated 
with their use. 
In order to circumvent the toxic side effects often associated with 
traditional chemotherapy, in recent years considerable research has 
focused on the discovery and use of "natural" hematopoietic growth 
factors, such as erythropoietin, or bone marrow transplants, as 
alternative forms of therapy. Although these modalities appear promising, 
they also have their limitations. For example, the use of bone marrow 
transplants has been severely limited due to the extreme difficulty in 
obtaining bone marrow which is histocompatible with the recipient. As a 
result, there continues to be considerable need for other agents capable 
of stimulating cells of the hematopoietic system. 
SUMMARY OF THE INVENTION 
The present invention arose out of the discovery that uteroferrin and rose 
could be used as hematopoietic growth factors to stimulate hematopoietic 
cells. This stimulation appears to act at a more primitive level of stem 
cell development than has previously been observed with other 
hematopoietic growth factors. Thus, the present invention relates to the 
therapeutic use of uteroferrin and rose as hematopoietic growth factors. 
Various other aspects and attendant advantages of the present invention 
will be more fully appreciated from an understanding of the following 
detailed description in combination with the accompanying example.

DETAILED DESCRIPTION OF THE INVENTION 
Uteroferrin and rose may be obtained by a variety of different methods. 
These substances may be obtained from uterine flushings of pigs (Baumbach, 
et al., Proc. Natl. Acad. Sci., U.S.A., 81:2985, 1984) or allantoic fluid 
(Baumbach, et al., J. Biol. Chem., 261:12869, 1986). Human uteroferrin, 
also referred to as human placental Type V acid phosphatase, can be 
purified as described by C. M. Ketcham, et al., (J. Biol. Chem., 260:5768, 
1986). Uteroferrin has also been produced by recombinant techniques 
(Simmen, et al., Molecular Endocrinology, 2:253, 1988; C. M. Ketcham, et 
al., J. Biol. Chem., 264:557, 1989). 
The term "hematopoietic growth factor" is intended to include uteroferrin 
and multichain variants of uteroferrin, such as rose, and their functional 
derivatives. The term "functional derivatives" pertains to polypeptides 
that may contain or lack one or more amino acids that may not be present 
in uteroferrin or rose, wherein these polypeptides are functionally 
similar to uteroferrin or rose. Such polypeptides are termed "functional 
derivatives", if they demonstrate hematopoietic activity which is 
substantially similar to uteroferrin or rose. 
The term "therapeutically effective" means that the amount of hematopoietic 
growth factor used is of sufficient quantity to stimulate the 
proliferation of hematopoietic cells. The dosage ranges for the 
administration of the hematopoietic growth factor of the invention are 
those large enough to produce the desired effect in which the 
hematopoietic cells show some degree of stimulation. The dosage should not 
be so large as to cause adverse side effects, such as unwanted 
cross-reactions, anaphylactic reactions, and the like. Generally, the 
dosage will vary with the age, condition, sex and extent of the disease in 
the patient and can be determined by one of skill in the art. The dosage 
can be adjusted by the individual physician in the event of any 
contraindications. Dosage can vary from about 10 .mu.g/kg/dose to about 
500 .mu.g/kg/dose, preferably about 20 .mu.g/kg/dose to about 200 
.mu.g/kg/dose, most preferably from about 50 to about 100 .mu.g/kg/dose in 
one or more dose administrations daily, for one or several days. 
Studies indicate that the hematopoietic growth factor uteroferrin is 
phenotypically conserved. Thus, according to the method of the invention, 
uteroferrin from a first species can be used in a second species to 
stimulate the hematopoietic cells of the second species, although the 
activity of uteroferrin of a given species is usually optimal when used to 
treat a member of that same species. 
The hematopoietic growth factor of the invention can be administered 
parenterally or by gradual perfusion over time. The hematopoietic growth 
factor of the invention can be administered intravenously, 
intraperitoneally, intramuscularly, subcutaneously, intracavity, or 
transdermally. 
Preparations for parenteral administration include sterile aqueous or 
non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous 
solvents are propylene glycol, polyethylene glycol, vegetable oils such as 
olive oil, and injectable organic esters such as ethyl oleate. Aqueous 
carriers include water, alcoholic/aqueous solutions, emulsions or 
suspensions, including saline and buffered media. Parenteral vehicles 
include sodium chloride solution, Ringer's dextrose, dextrose, and sodium 
chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include 
fluid and nutrient replenishers, electrolyte replenishers (such as those 
based on Ringer's dextrose), and the like. Preservatives and other 
additives may also be present such as, for example, antimicrobials, 
anti-oxidants, chelating agents, and inert gases and the like. In order to 
form a pharmaceutically acceptable composition suitable for effective 
administration, such compositions will contain an effective amount of the 
hematopoietic growth factor, or its functional derivatives, together with 
a suitable amount of a carrier vehicle. 
Additional pharmaceutical methods may be employed to control the duration 
of action. Controlled release preparations may be achieved by the use of 
polymers to complex or adsorb the hematopoietic growth factor or its 
functional derivatives. The controlled delivery may be exercised by 
selecting appropriate macromolecules (for example, polyesters, polyamino 
acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, 
carboxymethylcellulose, and protamine sulfate) and the concentration of 
macromolecules as well as the methods of incorporation in order to control 
release. Another possible method to control the duration of action by 
controlled release preparations is to incorporate the hematopoietic growth 
factor into particles of a polymeric material such as polyesters, 
polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate 
copolymers. Alternatively, instead of incorporating the hematopoietic 
growth factor into these polymeric particles, it is possible to entrap the 
hematopoietic growth factor in microcapsules prepared, for example, by 
coacervation techniques or by interfacial polymerization, for example, 
hydroxymethylcellulose or gelatin-microcapsules and poly 
(methylmethacrylate) microcapsules, respectively, or in colloidal drug 
delivery systems, for example, liposomes, albumin microspheres, 
microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such 
teachings are disclosed in Remington's Pharmaceutical Sciences (16th Ed., 
A. Oslo, ed., Mack, Easton, Penn., 1980 ). 
The method of the invention can also be used ex vivo to stimulate 
hematopoietic cell proliferation. For example, where a patient is 
receiving an allogeneic bone marrow graft from a donor, the donor cells 
can be treated with the hematopoietic growth factors of the invention and, 
if desired, co-cultured for a period of time, before infusion into the 
recipient. Such treatment can also be utilized, for example, where it is 
desirable for the patient to undergo an autologous marrow graft wherein 
the patient's marrow is treated chemotherapeutically and/or 
radiotherapeutically before re-infusion into the same patient. Ex vivo 
treatment using the hematopoietic growth factors of the invention is 
particularly relevant, for example, where the abnormal bone marrow must be 
eliminated and replaced with normal marrow. Other diseases where ex vivo 
therapy would be useful are acute leukemia, and other hematologic 
malignancies, where the complete destruction of the leukemic cell 
population and, unavoidably, normal marrow cells by intensive 
chemoradiotherapy is required. Techniques for ex vivo treatment of bone 
marrow cells are well known, or readily discernable, to those of skill in 
the art without undue experimentation. 
The ex vivo method of the invention can also be modified by the addition of 
an iron source, or compounds of similar biologic activity such as 
transferrin or FeCl.sub.3, to the proliferating cell culture, in 
combination with uteroferrin or rose. Co-culturing with transferrin 
appears to enable much lower concentrations of hematopoietic growth factor 
to be utilized than when the hematopoietic growth factor is administered 
alone. When transferrin is administered in combination with the 
hematopoietic growth factor a dosage of transferrin of from about 100 to 
about 700 .mu.g/ml, preferably from about 400 to about 600 .mu.g/ml, most 
preferably from about 500 to about 600 .mu.g/ml, is utilized. 
The invention also relates to a method for preparing a medicament or 
pharmaceutical composition comprising the hematopoietic growth factor of 
the invention, the medicament being used for therapy to stimulate the 
proliferation of hematopoietic cells. 
The above disclosure generally describes the present invention. A more 
complete understanding can be obtained by reference to the following 
specific examples which are provided herein for purposes of illustration 
only, and are not intended to limit the scope of the invention. 
EXAMPLE 
COMATIVE STIMULATORY EFFECT OF UTEROFERRIN AND ROSE ON VARIOUS 
HEMATOPOIETIC CELL TYPES 
Uteroferrin, the rose-colored heterodimer, and an unidentified basic 
protein with a molecular weight slightly greater than that of "rose" were 
individually purified (FIG. 1) from uterine secretions of pseudopregnant 
pigs (G. M. Baumbach, et al., J. Biol. Chem., 261:12869, 1986) as 
previously described (Baumbach, et al., Proc. Nat. Acad. Sci., U.S.A., 
81:2985, 1984; Murray, et al., J. Biol. Chem., 264:4143, 1989). These 
purified components were tested for colony stimulating factor (CSF) 
activity with respect to GM cells (Iscove, et al., Blood, 37:1, 1971), E 
cells (Ogawa, et al., Blood, 48:407, 1976) and GEMM cells (Fauser, et al., 
Blood, 53:1023, 1979). 
These assays detect colony forming units (CFU) of granulocytes (CFU-GM), 
erythrocytes (CFU-E), or the mixed lineage primitive 
granulocyte/macrophage/erythrocyte/megakaryocyte colonies (CFU-GEMM). 
Light density nonadherent mononuclear cells were isolated from human and 
pig bone marrow and fetal bone marrow from pigs (Days 65 and 101 of 
gestation). Additionally, tissue samples were obtained from neonatal 
piglet liver (Days 65 and 101 of gestation) and embryonic yolk sac cells 
(Day 21 of gestation). Cells were cultured in Minimal Essential 
Medium-.alpha. (MEM.alpha.) with 0.9% methyl cellulose, 20-30% fetal 
bovine serum and 0.6% I-glutamine for 7 days (CFU-E) and for 14 days 
(CFU-GM and CFU-GEMM) in a humidified incubator containing 5% CO.sub.2 in 
air. The positive control cultures contained recombinant human GM-CSF 
(rhGM-CSF, 50 units/ml or 1 ng) to stimulate CFU-GM, recombinant human 
erythropoietin (rhEPO, 2 units/ml or 20 ng) to stimulate CFU-E or 
rhGM-CSF+rhEPO to stimulate CFU-GEMM. 
Uterine secretions were obtained from a pseudopregnant pig and fractionated 
into basic and acidic proteins (S. M. M. Basha, et al., Biol. Reprod., 
20:431, 1979). The acidic and basic proteins were then fractionated 
further using Sephadex S-300 gel filtration chromatography. None of the 
acidic proteins had detectable hematopoietic growth factor activity. Only 
uteroferrin and "rose", in basic proteins from uterine secretions from 
pigs, had hematopoietic growth factor. Uteroferrin and rose were purified 
further (FIG. 1) and tested at 10.sup.-9, 10.sup.-7, 10.sup.-5, 10.sup.-3, 
and 10.sup.-1 mg/ml for hematopoietic growth factor activity. A basic 
protein fraction eluting from the Sephadex S-300 gel filtration prior to 
"rose" was used as the negative control protein(s) in this study since 
those proteins had been subjected to identical chromatographic procedures 
as uteroferrin and rose. These negative control proteins are noted as 
"yellow" herein, due to their yellow coloration. 
At peak activity (10.sup.-3 mg/ml) colony forming responses to uteroferrin 
and rose were greater (P&lt;0.01) than for the negative control proteins when 
added to cultures of hematopoietic stem cells from human bone marrow 
(FIGS. 2A, B, and C), neonatal piglet bone marrow (FIGS. 3A, B, and C) and 
neonatal piglet liver (FIGS. 4A, B, and C). Uteroferrin and rose were not 
as stimulatory to human bone marrow cells as human rhGM-CSF, rhEPO, or 
rhGM-CSF+rhEPO. However, both uteroferrin and rose were more stimulatory 
than recombinant human hematopoietic growth factors when nonadherent 
hematopoietic stem cells were from neonatal piglet bone marrow and liver; 
a result consistent, since it would be expected that a factor would be 
optimal when used within the same species from which the factor was 
derived. In general, uteroferrin has greater CFU-GM activity, while rose 
has greater CFU-E and CFU-GEMM activity regardless of the source of stem 
cells. It is especially significant that uteroferrin and rose alone can 
stimulate CFU-GEMM whereas the positive control cultures required a 
combination of rhGM-CSF and rhEPO to obtain similar biological activity. 
In another study, uteroferrin and rose were found to possess hematopoietic 
growth factor activity when added to cultures of pig hematopoietic stem 
cells from fetal spleen, fetal bone marrow, and fetal liver from Days 65 
and 101 of gestation (Table 1). In each case, uteroferrin and rose had 
more potent CFU activities than rhGM-CSF, rhEPO and rhGM-CSF+rhEPO. 
TABLE 1 
__________________________________________________________________________ 
CELL DAY 65 DAY 101 
SOURCE 
TREATMENT CFU-GM 
CFU-E 
CUF-GEMM 
CFU-GM 
CFU-E 
CUF-GEMM 
__________________________________________________________________________ 
FETAL UTEROFERRIN 32 .+-. 3 
22 .+-. 3 
17 .+-. 2 
26 .+-. 2 
10 .+-. 1 
10 .+-. 1 
PIG ROSE PROTEIN 26 .+-. 1 
15 .+-. 2 
21 .+-. 3 
20 .+-. 2 
10 .+-. 2 
13 .+-. 1 
SPLEEN 
YELLOW PROTEIN 
6 .+-. 2 
8 .+-. 1 
3 .+-. 1 
2 .+-. 1 
0 .+-. 0 
0 .+-. 0 
POSITIVE CONTROL 
13.sup.a 
.sup. 9.sup.b 
.sup. 7.sup.c 
14 9 6 
NEGATIVE CONTROL.sup.d 
0 0 1 0 0 0 
FETAL UTEROFERRIN 28 .+-. 5 
22 .+-. 3 
11 .+-. 1 
29 .+-. 1 
21 .+-. 5 
14 .+-. 1 
PIG ROSE PROTEIN 17 .+-. 3 
5 .+-. 1 
7 .+-. 3 
31 .+-. 2 
27 .+-. 1 
18 .+-. 2 
BONE YELLOW PROTEIN 
1 .+-. 1 
1 .+-. 1 
0 1 .+-. 1 
1 .+-. 1 
1 .+-. 1 
MARROW 
POSITIVE CONTROL 
9 6 4 16 14 6 
NEGATIVE CONTROL 
0 0 0 0 0 0 
FETAL UTEROFERRIN 22 .+-. 3 
8 .+-. 2 
16 .+-. 1 
25 .+-. 2 
8 .+-. 1 
22 .+-. 2 
PIG ROSE PROTEIN 15 .+-. 3 
8 .+-. 2 
16 .+-. 3 
29 .+-. 2 
10 .+-. 1 
15 .+-. 1 
LIVER YELLOW PROTEIN 
0 .+-. 0 
3 .+-. 1 
1 .+-. 1 
1 .+-. 1 
0 0 
POSITIVE CONTROL 
9 5 3 21 5 12 
NEGATIVE CONTROL 
0 1 0 0 0 0 
__________________________________________________________________________ 
.sup.a positive control for CFUGM is rhGMCSF (0.1 mg/ml) 
.sup.b positive control for CFUE rhEPO (2 ng/ml) 
.sup.c positive control for CFUGEMM is rhGMCSF + rhEPO 
.sup.d negative control for CFUGM CFUE, and CFUGEMM is the absence of 
rhGMCSF and rhEPO 
Another study was done to investigate the possible role of uteroferrin as 
an iron donor in stimulating hematopoietic cell stimulation. Various 
hematopoietic cell types from various tissues were purified as described 
above and cultured in the presence of different concentrations of 
uteroferrin in the absence or presence of a constant amount of 
transferrin. Here, the effect of hematopoietic growth factor was measured 
using a method which combines CFU-GEMM and BFU-E (burst forming units) in 
a single system along with the CFU-GM method (Ash, et al., Blood, 58:309, 
1981). The media used in this technique was modified by using Iscove's 
Modified Dulbecco's Medium with 2ME (2.times.10.sup.-5 M). 
Interleukin-3(IL-3) was added (100 U) to the controls and rhEPO was used 
at a concentration of 1 U(10 ng). 
TABLE 2 
______________________________________ 
EFFECT OF TRANSFERRIN AND UTEROFERRIN 
INTERACTION ON HUMAN HEMATOPOIETIC 
STEM CELLS 
TREATMENT.sup.a 
DOSE CFU-GM BFU-E.sup.b 
CFU-GEMM 
______________________________________ 
UTEROFERRIN 
10.sup.-1 
26 .+-. 2 51 .+-. 3 
12 .+-. 1 
10.sup.-3 
49 .+-. 6 142 .+-. 27 
23 .+-. 2 
10.sup.-5 
38 .+-. 9 124 .+-. 22 
19 .+-. 2 
10.sup.-7 
26 .+-. 4 76 .+-. 9 
14 .+-. 1 
10.sup.-9 
20 .+-. 2 41 .+-. 8 
6 .+-. 1 
.sup. 10.sup.-11 
8 .+-. 1 12 .+-. 2 
1 .+-. 1 
UTERO- 10.sup.-1 
6 .+-. 1 16 .+-. 1 
3 .+-. 1 
FERRIN + 10.sup.-3 
12 .+-. 2 36 .+-. 6 
6 .+-. 1 
TRANSFERRIN 
10.sup.-5 
18 .+-. 6 87 .+-. 11 
7 .+-. 1 
(600 .mu.g/ml) 
10.sup.-7 
37 .+-. 4 
126 .+-. 24 
16 .+-. 3 
10.sup.-9 
28 .+-. 5 107 .+-. 16 
11 .+-. 2 
.sup. 10.sup.-11 
6 .+-. 1 43 .+-. 7 
4 .+-. 1 
______________________________________ 
.sup.a None of the cell types showed colony growth when tested with 
transferrin alone (600 .mu.g/ml). 
.sup.b burst forming units 
As shown in Table 2, the presence of transferrin enabled comparable levels 
of cell stimulation to be achieved at significantly lower levels of 
uteroferrin. 
Results of this study are the first to indicate that progesterone-induced 
uterine secretory proteins, uteroferrin and rose, effect differentiation 
of primitive nonadherent hematopoietic stem cells and that the effect is 
not species specific. 
In order to investigate the stimulatory effect of uteroferrin from other 
species, human uteroferrin was purified and tested with various human 
hematopoietic cell types (Table 3). In this study, positive and negative 
controls were as previously described. 
TABLE 3 
______________________________________ 
EFFECT OF HUMAN UTEROFERRIN ON VARIOUS 
HUMAN BONE MARROW CELLS 
TREATMENT CFU-GM BFU-E CFU-GEMM 
______________________________________ 
UTEROFERRIN 42 127 22 
POSITIVE CONTROL 
88 197 35 
NEGATIVE CONTROL 
2 0 0 
______________________________________ 
These results show that uteroferrin is capable of inducing significant 
proliferation of the bone marrow cells tested and, unlike the positive 
control, could act alone to stimulate GEMM cells. 
The invention now being fully described, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
without departing from the spirit or scope of the invention.