Note: Descriptions are shown in the official language in which they were submitted.
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ENHANCEMENT OF HEMATOPOIETIC CELLS
Cross-Reference to Related Applications
This application is related to and claims the benefit of provisional
application
serial number 60/035,875, filed January 21, 1997.
Background of the Invention
During fetal life, the generation of all blood cells occurs initially in blood
islands
and then in the liver and spleen. This function is gradually taken over by the
bone
marrow and increasingly the flat bones, so that by puberty this process occurs
mostly in
the sternum, vertebrate, iliac bones, and ribs. The red marrow that is found
in these bones
consists of a sponge-like reticular framework located between long trabeculae.
The
spaces in this framework are filled with fat cells, which mature and exit via
the dense
network of vascular sinuses to become part of the circulatory system.
All blood cells originate from a common stem cell that becomes committed to
differentiate along particular lineages (i.e., erythroid, megakaryocytic,
granulocytic,
monocytic) and lymphocytic). The proliferation and maturation of precursor
cells in the
bone marrow are stimulated by certain cytokines. Many of these cytokines are
also called
"colony-stimulating factors" because they are assayed by their ability to
stimulate the
growth and development of various leukocyte colonies from marrow cells. While
it is
known that different cytokines promote the proliferation and maturation of
different
lineages of bone marrow precursor cells, little is known about the nature of
the self-
renewing pluripotent stem cell or the mechanisms that regulate its commitment
to specific
lineages.
Increasingly these cytokines are being studied, and in some cases used, for
potential clinical applications related to their hematopoietic modulating
properties.
Because of their potential, the discovery of any substance with similar
properties may
also have the potential for clinical applications. A need exists to discover
such
substances.
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2
Summary of the Invention
The invention relates to a method for enhancing hematopoiesis by contacting
hematopoietic pluripotent stem cells or progenitor cells with a composition
containing
prolactin. Preferably the prolactin used is recombinant prolactin. Stimulation
of
hematopoesis can serve to replace hematopoietic cells as they become ablated
because of
a therapeutic drug or treatment. The enhancement can also function to recruit
new or
additional cell lineages to a depleted or poorly-functional repertoire of
cells.
The invention further relates to a method for treating an animal to improve
hematopoiesis or prevent hematopoietic-suppression by administering a
pharmaceutically
acceptable composition containing prolactin.
The invention further relates to a composition comprising a cytokine that can
enhance hematopoiesis and prolactin.
The invention further relates to a composition comprising a therapeutic that
can
cause hematopoietic-suppression and a prolactin.
Detailed Description of the Drawint=s
Figure 1 A shows a graph demonstrating that recombinant human prolactin
promotes the growth of hematopoietic progenitor cells in long term bone marrow
culture
as verified by improved cumulative celluiarity.
Figure 1 B graphically illustrates that recombinant human prolactin promotes
the
growth of hematopoietic progenitor cells in long term bone marrow cultures as
measured
by colony forming unit-culture assay.
Figure 1 C shows a graph demonstrating that recombinant human prolactin
promotes the growth of hematopoietic progenitor cells in long term bone marrow
cultures
as measured by burst forming unit-erythroid assay.
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Figure ? graphically illustrates that azidothymidine (AZT) significantly
lowers
hematopoietic progenitor content in the bone marrow cells and that prolactin
counteracts
the effect as measured by hematocrit.
Figure 3A shows a graph illustrating that prolactin counteracts the
myelosuppressive effects of AZT lowering the hematopoietic progenitor content
in the
bone marrow cells as verified by improved cumulative cellularity.
Figure 3B graphically illustrates that prolactin counteracts the
myelosuppressive
effects of AZT lowering the hematopoietic progenitor content in the bone
marrow cells as
measured by colony forming unit-culture assay.
Figure 3C shows a graph demonstrating that prolactin counteracts the
myelosuppressive effects of AZT lowering the hematopoietic progenitor content
in the
bone marrow cells as measured by burst forming unit-erythroid assay
Figure 4 graphically illustrates that prolactin prevents the myelosuppressive
effects of AZT in pretreated mice as measured by hematocrit.
Figure 5 shows a graph demonstrating that prolactin can reverse the
myelosuppressive effects of AZT as measured by hematocrit.
Figure 6A graphically illustrates that proiactin increases platelet content.
Figure 6B shows a graph demonstrating that prolactin increases white blood
cell
count.
Figure 7A and 7B graphically demonstrates through differential analysis that
the
lymphocyte and neutrophil percentage in blood was significantly increased,
suggesting
prolactin improved the peripheral lymphocyte and neutrophil development.
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Figure 8 shows a graph illustrating that prolactin influenced B-cell
progenitor
cells by improving responsiveness to keyhole limpet hemocyanin (KLH) as
measured by
increased production of KLH-specific IbG and IgM.
Figure 9 graphically illustrates that prolactin increases natural killer
function, as
assessed by cytotoxicity.
Detailed Description of the Invention
Definitions
As used herein, "prolactin" refers to a polypeptide obtained from tissue
cultures or
by recombinant techniques and other techniques known to those of skill in the
art,
exhibiting the spectrum of activities characterizing this protein. The word
includes not
only human prolactin (hPRL), but also other mammalian prolactin such as, e.g.,
mouse)
rat, rabbit, primate, pig (ovine) and cow (bovine) prolactin. The recombinant
PRL
(r-PRL) includes any active fragment or active prolactin sequence.
The term "recombinant prolactin", designated as r-PRL, preferably human
prolactin, refers to prolactin having comparable biological activity to native
prolactin
prepared by recombinant DNA techniques known by those of skill in the art.
The term "hematopoiesis" or "hemopoiesis" refers to the conventional meaning
of
the word which encompasses the formation and development of various types of
cells
including pluripotent stem cells, myeloid progenitor cells and lymphoid
progenitor cells
as well as blood products derived therefrom such as platelets.
The phrase "pharmaceutically acceptable composition" refers to any formulation
or preparation that when administered to an animal will be tolerated by said
animal.
Administration includes oral administration and injection including
subcutaneous,
intraperitoneal, intravenous, intradermal, intramuscular, etc.
The term "hematopoietic-suppression" include myelosuppression or lymphoid-
suppression as caused by such treatment as AZT, irradiation, cytoreductive
treatment,
chemotherapy, cytolytic therapy, immunocytolytic, or combinations thereof.
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It is intended that the term "cytokine" or "cytokines" as used herein means
any
cytokine or growth factor or colony-stimulating factor that can stimulate the
expansion
and differentiation of stem cells or progenitor cells. Cytokines include
interleukin- I ,
interleukin -2, interleukin 3, interleukin-4, interleukin-6, interleukin-7)
interleukin-9,
interleukin-1 1, interleukin -15, c-Kit ligand, granulocyte-monocyte colony-
stimulating
factor, monocyte-colony-stimulating factor, granulocyte-colony-stimulating
factor, Flt3
ligand) Mpl ligand, erythropoietin (Epo), thrombopoietin, (Tpo), growth
hormone, (GH),
insulin-growth factor, (IGF), transforming-growth factor-f3, (TGF-f3), and
mixtures
thereof.
Example 1: Effect of nrolactin on hematopoietic progenitor content in vivo.
BALBIc and C57BL/6 (B6) mice (8-12 weeks of age) were injected with IONg of
recombinant human prolactin (r-hPRL, Genzyme Corporation) that was resuspended
in
0.2 mL Hanks' Balanced Salt Solution (HBSS) Mediatech, Inc. Herndon, VA. The
animals received i.p. injections every other day for 10 days (a total of five
injections).
Mice were weighed weekly. Blood was collected from the mice via the lateral
tail
vein, using EDTA as an anticoagulant.
Complete blood counts were performed using an HC 820 Hematology Analyzer
(Danam Electronics, Inc., Dallas TX) or by Metpath (Rockville, MD). The
cellularity of
bone marrow and spleen were assayed using a Coulter Counter (Coulter
Electronics,
Hialeah, FL).
Spleen cells and bone marrow cells (BMC) obtained from one tibia were washed
and resuspended in Iscove's modified Dulbecco's medium (IMDM) with 10% fetal
bovine serum (FBS), 1 °lo L-glutamine, and antibiotics.
Long term bone marrow cultures (LTBMC) were maintained in LTBMC serum-
free medium purchased from Stem Cell Technologies (British Columbia, Canada),
which
contained all nutrients and cytokines for BMC growth in vitro.
BMC from the mouse femur were cultured at an initial concentration of 1X106
cells/mL in 24-well plates. Every third day, the cultures had half of their
volume
exchanged with fresh medium. When the cell concentration was more than
2x106/mL,
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the culture was diluted and separated into two wells. Every 10 days the
cultures were
evaluated for their cellularity and by colony assays for CFU-c and BFU-e.
The colony forming unit-culture (CFU-c) assay was performed as described by
Murphy, et. al. ( 1992) Blood 80:1443-1447. Briefly, for CFU-c assay, the
cells were
plated in 0.3% bactoagar (Difco Laboratories, Detroit, MI) in 35-mm Lux petri
dishes
(Miles Laboratories, Inc., Naperville, IL) at a concentration of 1 x 106
spleen cells or
2x 105 BMC per plate. Colony formation was stimulated with predetermined
optimal
doses of growth-promoting cytokines such as recombinant murine granulocyte
macrophage stimulating factor (GM-CSF) at 10 ng/mL (Amgen Corp., Thousand
Oaks,
CA) and recombinant murine interleukin-3 (IL-3), 10 ng/mL) supplied by the
Biological
Response Modifiers Program Repository (Frederick, MD). Plates were incubated
at 37oC
for 7 days in 100% humidity, 5% C02 atmosphere and then colonies were counted.
A
colony was defined as a clustered growth of more than 50 cells.
The burst forming unit-erythroid (BFU-e) assay was performed as described by
Stephenson JR, et.ai ( 1971 ) Proc Natl Acad USA, 68:1542. For the burst BFU-a
assay, a
S-mL volume of the suspension included: 1.5 mL cells, 1.5 mL FBS, 0.5 mL 10%
BSA,
0.5 mL of 1.0 mM 2-mercaptoethanol, 0.5 mL Epo, and 0.5 mL recombinant murine
IL-3
(rmIL-3). The final concentration of erythropoietin (Epo) Stem Cell
Technologies,
British Columbia, Canada) was 2 U/mL, rmIL-3 was 20 ng/mL and the cells were
at
1 x 1 OS/mL. BFU-a were scored after 12 days of incubation. A BFU-a was
defined as a
group containing 50 or more benzidine-positive cells. All assays had at least
three mice
per group and were performed at least three times, with a representative
experiment being
shown.
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Table 1
Effects
of r-hPRL
on hematopoietic
progenitor
content
in vivo
Strain Organ TreatmentNo. of cellsCFU-c BFU-a
(X 106)
BALB/c Spleen HBSS 86.9+/-9.8 3.7+/-0.2 2.1+/-0.1
BALB/c Spleen r-hPRL 87.8+/-9.2 5.8+/-0.6**7.6+/-0.4**
BALB/c BMC HBSS 21.9+/-3.5 34.8+/-4.8 22.3+/-2.1
BALB/c BMC r-hPRL 22.8+/-3.2 54.9+/-5.5**55.2+/-5.9**
C57BL/6 Spleen HBSS 95.4+/-9.8 5.4+/-0.4 3.1+/-0.3
C57BL/6 Spleen r-hPRL 97.2+/-8.9 8.6+/-0.7**9.6+/-1.0~'*
C57BL/6 BMC HBSS 27.8+/-3.3 42.3+I-3.7 25.6+/-3.3
C57BL/6 BMC r-hPRL 28.2+/-2.8 65.7+/-7.6**61.2+/-6.7**
** <0.01
The soft agar colony assays indicated that prolactin could effect the
hematopoietic
progenitor cell content of BMC and spleen. The results shown in Table 1 above
demonstrated
that the administration of r-hPRL resulted in significant (p<0.01 ) increases
in splenic and BMC
colony-forming units-culture (CFU-c) and burst-forming unit-erythroid (BFU-e)
in both strains
of mice. However, no significant effects were detected on BMC and splenic
cellularities (Table
1 shown above) or on peripheral blood differential counts in the normal
recipients. even when
100Ng dose of r-hPRL were administered (data not shown). In addition. no
significant increases
in body weight were noted in the recipients receiving lOpg injections of r-
hPRL (data not
shown). Therefore r-hPRL appears to exert significant hematopoietic growth-
promoting effects
after in vivo administration.
Example II. Effect of prolactin on hematopoietic proeenitor content in vitro.
In order to verify direct effects of r-hPRL on hematopoietic growth, a short-
term colony
culture system (CFU-c and BFU-e) and a long term suspension culture system
(LTBMC) were
evaluated. The methods for these soft agar colony assays were described in
Example 1, above.
The results of these experiments demonstrated that r-hPRL promoted CFU-c and
BFU-a
formation in a dose dependent manner (Table 2 shown below). The optimal dose
for murine
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CFU-c and BFU-a formation was 50 ng of PRL, while the optimum for the human
system was
100ng. It was also noted that r-hPRL promoted the growth of hematopoietic
progenitor cells in
LTBMC, showing that the cumulative suspension culture cellularity increased
during 50 days of
culture (Figure 1 a). The hematopoietic progenitor cell content (CFU-c and BFU-
e) in long-term
BM culture also increased after r-hPRL treatment (Figure 1 b and Figure 1 c).
Table 2
Effects of
r-hPRL on
growth of
hematopoietic
progenitor
content
in vitro
Strain Source Treatment CFU-c BFU-a
BALB/c BMC HBSS 48.3+/-4.7 42.1+/-3.7
BALB/c BMC 12 ng r-hPRL 46.4+/-4.5 41.3+/-3.2
BALB/c BMC 25 ng r-hPRL 54.3+/-3.2 58.3+I-4.1
**
BALB/c BMC 50 ng r-hPRL 58.9+/-4.5**64.3+/-5.8**
BALB/c BMC 100 ng r-hPRL 55.2+/-4.3 40.1+/-4.8
Human Cord BloodHBSS 35.2+/-3.2 25.6+/-3.1
Human Cord Blood1 ng r-hPRL 34.6+/-3.7 33.2+/-2.3**
Human Cord Blood10 ng r-hPRL 40.2+/-2.8 37.2+/-3.5**
Human Cord Blood100 ng r-hPRL 46.2+/-3.7**37.8+/-4.1
**
Human Cord Blood1000 ng r-hPRL34.2+/-4.6 26.1+/-2.5
I * * <0.01
Example III. Effects of ~rolactin on hematopoietic~ro~enitor content in mice
administered AZT
as a means of inducing mvelosuppression.
Because one of the dose-limiting toxicities of AZT is anemia resulting from
its
myelotoxic properties, studies were done to determine whether concurrent
treatment of mice with
r-hPRL and AZT results in an improvement in the hematologic parameters.
B6 mice were placed on AZT (2.5 mg/mL in drinking water) for several weeks.
Upon
analysis, these mice exhibited significantly lower (p<0.01 ) BMC hematopoietic
progenitor
content as measured with CFU-c and BFU-a (Table 3 shown below) as well as
significantly
lower hematocrit (HCT) than normal mice (Figure 2). These effects became more
pronounced
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the longer the mice were placed on AZT) with most hematologic values
approaching nearly half
the control values.
Table 3
Effects
of r-hPRL
on hematopoietic
progenitor
content
in mice
placed
on AZT
Treatment CFU-c/Femur BFU-e/Femur
At day
14
BMC HBSS 5,525+!-435 3,045+/-324
B MC AZT 3,634+/-37 8 1, 806+/-213
BMC AZT+lpg r-hPRL 5,145+/-467** 3,485+/-378**
BMC AZT+lOpg r-hPRL 5,645+/-546** 3,765+/-423**
BMC AZT+100pg r-hPRL 5,485+/-579** 4,556+/-476**
At day
28
BMC HBSS 5,055+/-543 4,759+/-435
BMC AZT 3,120+/-321 2,506+/-198
BMC AZT+Ipg r-hPRL 3,856+/-410 3>569+/-371**
BMC AZT+l0pg r-hPRL 5,436+/-432** 5,236+/-543**
BMC AZT+100pg r-hPRL 5,215+/-576** 6,045+/-701**
** <0.01
Groups of mice concurrently received 1, 10, or 1001r g r-hPRL ip every other
day for 20
days. Celluiarity and colony assays (CFU-c and BFU-e) were determined after l4
or 28 days.
The results, shown in Table 3 above, demonstrate that hematopoietic and
hematologic parameters
improved significantly (p<0.01 ) with concurrent administration of prolactin.
The CFU-clFemur
or BFU-e/Femur were calculated as: colony number/2x i OS x cellularity of
femur. The
hematopoietic progenitor cell content (CFU-c and BFU-e) fully recovered to
normal or even
higher. The HCT value also increased in response to r-hPRL treatment (Figure
2), increasing
from 29.5+/-1.3% to 40.3+l-3.2% with mice administered AZT and examined at day
14 after
concurrent r-hPRL treatment. Similar results were obtained after 28 days.
Additionally, the mice
exhibited no apparent pathologic effects from repeated r-hPRL injections. The
mice appeared to
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be in good health throughout the study. They maintained a constant weight and
mice sacrificed
at the end of the study showed no gross pathologic abnormality.
Experiments were also done to determine whether r-hPRL could directly
counteract the
myelosuppressive effects of AZT. The CFU-c and BFU-a colony assays were done
in the
presence of AZT and varying doses of r-hPRL. Prolactin could, in a dose-
dependent manner,
counteract the AZT-induced suppression of CFU-c and BFU-a formation in murine
and human
colony cultures (Table 4 shown below). In addition, in the long-term BM
culture system, (Figure
3 ) performed as described previously, r-hPRL reversed the AZT-growth-
inhibition as
demonstrated by improved cumulative cellularity (Figure 3a) and greater
hematopoietic
progenitor cell content (CFU-c, Figure 3b and BFU-e, Figure 3c).
Table 4
Effects of
r-hPRL on
growth of
hematopoietic
progenitor
cells with
AZT
Treatment CFU-c BFU-a
Murine
BMC AZT 30.2+/-1.9 25.0+/-3.1
BMC AZT+6.25ng r-hPRL 36.0+/-2.8* 28.9+/-3.3
BMC AZT+12.5ng r-hPRL 34.2+/-3.3 29.2+/-2.6
BMC AZT+25ng r-hPRL 37.3+/-3.3 35.5+/-3. i
* **
BMC AZT+50ng r-hPRL 42.5+/-4.9**37.6+/-4.1 **
BMC AZT+lOUng r-hPRL 47.3+/-4.3**33.3+/-3.1'~
BMC AZT+200ng r-hPRL 45.2+/-3.8**29.5+/-3.6
BMC AZT+400ng r-hPRL 37.2+/-3.9 28.8+/-2.4
Human
Cord Blood AZT 28.1+/-2.1 17.3+/-1.8
Cord Blood AZT+ l ng r-hPRL 34.9+/-3.3 23.5+/-2.1
*
Cord Blood AZT+l0ng r-hPRL 38.6+/-4.2**25.1+!-3.1**
Cord Blood AZT+100ng r-hPRL 35.5+/-2.7**32.4+/-3.5**
Cord Blood AZT+1000ng r-hPRL 29.6+/-3.1 21.1+/-2.3
* <0.05 ,
* * <0.01
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Example IV. Early administration of~rolactin prevents AZT-induced
mvelosu~~ression in mice.
Studies were undertaken to determine whether prolactin, administered before
myelosuppression, such as induced by AZT, would protect and improve
hematopoietic
parameters. B6 mice were injected with 10-lrg of r-hPRL ip every other day for
14 days and then
administered AZT in their drinking water (2.5 mg/mL in drinking water), for
another 14 days
without r-hPRL injections. Cellularity and progenitor cell content (CFU-c and
BFU-e) were
determined at various time points. Significant protection of myelosuppression
induced by AZT
was observed. The HCT value was significantly (p<0.01 ) enhanced at day 29 to
day 34 (Figure
4). Hematopoietic progenitor cells (CFU-c and BFU-e) were also significantly
(p<0.01 )
increased in r-hPRL pretreated mice during AZT administration (Table 5 shown
below). These
results suggest that r-hPRL may protect the progenitor cells in vivo and
increase their ability to
resist myelosuppression.
Table 5
Early administration
of r-hPRL
prevents
AZT-induced
myelosuppression
Treatment CFU-c/Femur BFU-e/Femur
BMC HBSS 4,365+/-523 2,217+/-312
BMC HBSS, AZT 3,829+/-341 1,287+/-121
BMC r-hPRL) AZT 4,839+/-367** 2,418+/-212**
** <0.01
Example V. Later administration of proiactin reverses AZT-induced
myelosuppression in mice.
Studies were undertaken to determine whether the administration of r-hPRL
after
myelosuppression, such as induced by AZT, would improve hematopoietic and
hematologic
parameters. B6 mice were administered AZT in drinking water (2.5 mg/mL in
drinking water)
for 14 days. After this two week period, the mice were evaluated to confirm
they were
myelosuppressed. The animals subsequently received l Op g r-hPRL ip
administered every other
day for another 20 days (total of 10 injections). The animals were evaluated
for cellularity and
progenitor cell content at various time points. Significant improvement of BMC
hematopoietic
cell content was noted in r-hPRL treated mice (Table 6 shown below) at day 29
(7 r-hPRL
injections). The CFU-c/Femur or BFU-e/Femur were calculated as: colony
numberJ2x 105 x
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92
cellularity of femur. V clues are representative of three experiments
containing three to four mice
per group. HCT values also recovered to nearly normal levels by day 24 ( 10
days after r-hPRL
treatment) and were significantly higher than HBSS control (Figure 5). These
findings suggest
that r-hPRL can reverse myelosuppression induced by AZT.
Table 6
Later administration
of r-hPRL
reverses
AZT-induced
myelosuppression
in mice
Treatment CFU-clFemur BFU-e/Femur
BMC 0, HBSS 4,065+/-452 2,709+/-312
BMC AZT, HBSS 3,218+/-278 2,231+l-214
BMC AZT, r-hPRL 4,787+/-513** 3,189+/-324**
* ~ <0.01
Example VI. Prolactin accelerates hematopoietic reconstitution after lethal
irradiation followed
by hone marrow transplantation.
Recipient BALB/c mice (8-12 weeks of age) were exposed to a 137Cs irradiation
source
in order to lethally irradiate the animals for cellular reconstitution
studies. The mice received
850 cGy total body irradiation. These mice then received 1x106 syngeneic BMC
intravenously
(iv). This procedure is referred to as syngeneic bone marrow transplantation
(SBMT). There
were five mice per group and each experiment was performed 3-4 times.
At day 1 after syngeneic bone marrow transplantation (SBMT) the mice started
their
treatment of either lOpg r-hPRL or Hanks Balanced Salt Solution (HBSS) as
control. r-hPRL
was resuspended in 0.2 mL HBSS and injected i.p. every other day until the
mice were assayed or
received a total of 10 injections over 20 days. Mice were weighed weekly.
Blood was collected from mice via the lateral tail vein, using EDTA as
anticoagulant.
Complete blood counts were performed using an HC 820 Hematology Analyzer
(Danam
Electronics Inc., Dallas, TX) or by Metpath (Rockville, MD). The cellularity
of bone marrow,
spleen, and thymus were assayed using a Coulter Counter (Coulter Electronics,
Hialeah, FL).
Spleen cells and bone marrow cells were evaluated for their cellularity and
for their progenitor
content as determined by their ability to form colonies.
Briefly, the assays for in vitro hematopoiesis were performed as described
previously.
Briefly, spleen cells and bone marrow cells (BMC) were obtained from one tibia
and washed and
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resuspended in Iscove's modified Dulbecco's medium (IMDM) with 10% fetal
bovine serum
(FBS), 1 % L-glutamine, and antibiotics. The colony forming assays (CFU-c and
BFU-e) were
performed as described above in Example 1.
The results shown in Table 7(shown below) demonstrate the administration of r-
hPRL
significantly increased (p<0.01 ) both the BMC and splenic progenitor cell
content as determined
by CFU-c and BFU-a at 14 days or 21 days after SBMT. The total progenitor
number per femur
or spleen {femur cellularity/2x 105 BMC X CFU-c or BFU-e/plate for femur or
spleen) were
increased in mice receiving r-hPRL. The total BMC hematopoietic progenitor
(CFU-c) number
was enhanced 6.2 fold at day 14 and 11.7 fold at day 21. Splenic CFU-c were
enhanced 5.4 fold
at day 14 and 10.8 fold at day 21 in mice receiving r-hPRL.
Table 7
Effects
of r-hPRL
on splenic
and BM
cellularity
and hematopoietic
progenitor
content
After Organ TreatmentCell No. CFU-c/Plate BFU-e/Plate
BMT ( 106)
Day 14 Spleen HBSS 23.4+/-2.4 26.8+/-5.4 12.2+/-3.2
Spleen r-hPRL 45.5+!-3.8**63.2+/-9.6**48.8+/-7.6**
BMC HBSS 7.6+/-0.4 21.1+1-4.2 9.4+/-2.1
BMC r-hPRL 11.2+/-1.2**90.2+/-15.6**27.2+I-4.5**
Day 21 Spleen HBSS 43.5+/-3.4 30.3+/-7.3 12.8+/-3.2
Spleen r-hPRL 53.2+/-3.6*144.3+/-21.6**31.2+/-4.9**
BMC HBSS 12.7+/-1.3 25.2+/-4.5 8.7+/-1.4
BMC r-hPRL 16.4+/-l.l*127.2+/-24.6**41.3+/-5.7**
* <0.05. <0.01
* *
Since these results suggested that r-hPRL could promote hematopoietic
reconstitution
after SBMT, peripheral blood was examined for subsequent changes in mature
cell populations.
As shown in Figure 6a, administration of lOlrg r-hPRL significantly increased
platelet content at
days 15,18,21 (p<0.01 ) and day 28 (p<0.05). At this concentration, prolactin
also caused
significantly (p<0.01 at days 7,15) 18) and p<0.05 at day 21 ) increased white
blood cell counts
(Figure 6b). Differential analysis (Figure 7 ) showed that the lymphocyte and
neutrophil
percentage in blood was significantly (p<0.01 ) increased at day 7 to day 21,
suggesting that r-
hPRL improved the peripheral lymphocyte and neutrophil development.
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The effect of prolactin administration on granulocytes was also evaluated in
these mice by
flow cytometry. The mice receiving r-hPRL every other day until day 14 or 21
were put into four
groups while the control animals receiving HBSS are put into four other
groups. Single cell
suspensions from a tibia (BM) were taken from two of the groups of the r-hPRL
treated mice and
from two of the groups of the HBSS treated mice. Single cell suspensions from
the spleen were
taken from the two remaining groups from the r-hPRL and HBSS treated groups.
Both BM and
spleens cells were analyzed by double-color cytometric analysis as described
previously (Murphy
et.al.1992. J. Immunol 148: 3799-3805). Briefly, the cells were stained with
rat FITC-labeled
anti-SE6 (natural killer (NK) cell) antibody and rat PE-labeled 8C5
(granulocyte) antibody
obtained from Becton Dickinson (Mountain View, CA). The cells were fixed in
100%
paraformaldehyde and analyzed on a EPCIS flow cytometer. FITC or PE-labeled
normal rat
immunoglobuiin (NRIg) was used as a control and each group had 3-4 mice per
group. Using
this method, it was noted that 8C5+ (granulocyte marker) cell content was
increased in both BM
and spleen. Because the cellularity of both BM and spleen also increased (BM
group vs. BM+r-
hPRL group: 7.8x 106 vs. 12.6x 106 for BM; 54.Sx 106 vs. 78.Sx 106 for
spleen)) the absolute
number of 8C5+ granulocytes in BM and spleen increased 2.24 fold and 2.85 fold
at day 14.
This cell population increased 2.03 fold and 1.92 fold at day 21.
Collectively, the results from these experiments demonstrate that treatment
with r-hPRL
promoted BMC engraftment resulting in improved development of hematopoietic
progenitor
cells which gave enhanced numbers of mature cell populations.
Example VII. Treatment with prolactin after BMT improves B-cell lineage
development.
B-cell progenitor content and B-cell mitogen responses as a B-cell function
assay were
evaluated in mice after SBMT. Six mouse groups received r-hPRL treatment (
lOpg/injection,
every other day until day 14 or day 21 ) while 6 groups of control mice,
groups received HBSS.
The B-cell progenitor content was determined by flow cytometry using the dual-
labeling method
described above and FI1'C-labeled anti-B220 and PE-labeled sIgM, both obtained
from Becton
Dickinson. B-cell progenitors would stain positive for the B220 marker and
negative for surface
IgM (sIgM). The B-cell progenitor (B220+ sIgM cell) content increased after r-
hPRL treatment
in BM and lymph node (LN ) cells, but not in the spleen at days 14 and 21. At
day 14 after
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CA 02277482 1999-07-09
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treatment, the absolute number of B-cell progenitors in BM and LN increased
2.56 and 3.78 fold
respectively (cellularity x positive cells), suggesting that r-hPRL
accelerated the B-cell lineage
engraftment and development. It was also noted that the mature B-cell
{B220+/sIgM+ cells)
content increased in both spleen and LN at day 14 and day 21 after SBMT.
In addition to increased number of B-cells, the functionality of splenic B
cells was
evaluated. This was done by assessing their proliferative response to the B
cell mitogen LPS,
using a standard tritiated thymidine proliferation assay. As shown in Table 8
below, the
splenocytes from mice receiving r-hPRL demonstrated an enhanced proliferative
response to the
B-cell mitogcn, with the CPM of the r-hPRL treatment group significantly
higher than the control
(p<0.01 for any dose of LPS) and the stimulating index (SI) enhanced at each
dose of LPS. The
BMC-transplanted mice were further evaluated for B cell function by immunizing
the animals
with KLH and measuring their IgG and IgM response over time. At day 21 (e.g.
two weeks after
KLH immunization), the spleens were harvested and the cell suspension used to
evaluate KLH-
specific responses. Both KLH-specific IgG and IgM were increased in the r-hPRL
treated mice
(see Figure 8).
Table 8
Effects
of r-hPRL
on splenic
B-cell
mito~en
responsiveness
after SBMT
LPC Concentration L)
l /m
Treatment Cell Opg l~tg l0~tg 2S~tg
Yield
( 106)
BMT 54.5+/- 5,110+/- 4,755+/-2057,474+/-2,1209.381+/-3,478
7.4 112
SI=0.9 SI=1.S SI=1.8
BMT+r-hPRL 78.5+/- 4,788+/- 7,960+/- 15>577+/- 18,895+/-
5.6** 321 412* 1,102* 4,321*
S I=1.7 S I=3.3 S I=4.0
*P<0.05,
** <0.01
a, roliferative
res once.
c m
b. Stimulatin
index (SI)=c
m + LPS/c
m - LPS
These findings verify that PRL can improve the development and function of the
B-cell
lineage from hematopoietic progenitor cells after SBMT.
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WO 98/31385 PCT/US98/00887
16
Example VIII. Treatment with~rolactin after BMT improves T-cell lineage
development.
Animals treated with r-hPRL after SBMT were evaluated for their T-cell
progenitor
content (CD4+CD8+ cell) in the thymus as well as T-cell function. T-cell
progenitor cell content
was analyzed by double-color flow cytometry analysis as described above.
Reagent used
included FITC-labeled anti-Lyt-2 (CD8) and PE labeled anti-L3T4 (CD4) obtained
from Becton
Dickinson. T-cell progenitor content (CD4+CD8+ cell) in thymus was increased
when examined
at day 14 and day 21 after SBMT. The mature T cell content (CD4+CD8 or CD4
CD8+ cell) in
the spleen and lymph node were also increased.
The effect of r-hPRL administration on T-cell function was also evaluated. The
effect of
PRL on antigen-specific T cells during a primary immune response was evaluated
by examining
the splenic T-cell proliferation to KLH in KLH-immunized mice after SBMT.
Mice were immunized subcutaneously with 100pg of KLH in complete Freund's
adjuvant
at day 7 after SBMT. At day 21 (i.e. two weeks after KLH immunization), the
spleens were
harvested and the cell suspension was used to assess KLH-specific
proliferation. Briefly,
KL.H( 100pg/mL) and splenocytes ( 1 x 106/200mL/well) were added to flat-
bottomed 96-well plates
(Costar). Four days later, proliferation was assayed by pulsing with lmCi of
3H-thymidine (6.7
Ci/mmol, New England Nuclear, Boston, MA) for 8 hours followed by harvesting
the cells using a
MASH II apparatus (Microbiological Associates. Bethesda, MD). Incorporated
labeled DNA was
counted using a scintillation counter. The results of this experiment is shown
in Table 9.
Table 9
Effects
of r-hPRL
on anti
en-s ecific
res onse
of s lenic
T cells
after
SBMT
Groups
Immunization
Cellularit~
3H-Thymidine
Incorporation
in vitro
(x 10 )
(-) KLH (+) KLH
BMT (--) 9.4+/-1.2 1,467+/-4861,602+/-456
BMT+r-hPRL (--) 13.5+/-1.3*1,399+1-3671,708+/-437
BMT KLH 12.5+/-1.1 1,536+/-50570,483+/-12,677
BMT+r-hPRL KLH 16.8+/-2.2*1,488+/-772117,396+/-13,199**
~= <0.05.
* * <0.01
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The data demonstrate that r-hPRL administration exerted significant
immunopotentiating
effects as demonstrated by the significantly increased in vitro proliferation
to KLH in the mice
receiving r-hPRL treatment. The data verify that r-hPRL may improve the
development and
function of T-cell lineage from hematopoietic progenitor cells after SBMT.
Example IX. Prolactin improves NK recovery after BMT
NK cells are lymphoid cells that mediate MHC-unrestricted killing of tumors
and virally-
infected cells. Recently, it was reported that NK cells play an important role
in hematopoiesis.
Studies were therefore undertaken to examine the development and function of
NK cells after
SBMT with and without r-hPRL treatment. The effect on total NK cell (SE6+
cells)content was
evaluated using flow cyometry as described previously. As shown in Table 10
below, the NK
(SE6+ cells) content in both BM and spleen increased in mice receiving r-hPRL
administration
after SBMT. Because the cellularity of both BM and spleen also increased (BM
group vs. BM+r-
hPRL group: 7.8x 106 vs. 12.6x 106 for BM; 54.Sx 106 vs. 78.Sx 106 for
spleen)) the absolute
numbers of NK cells in BM and Spleen increased 5.41 and 4.83 fold at day 14.
These values
increased 2.34 and 1.78 fold at day 21.
Table 10
r-hPRL improves
NK development
after SBMT
SE6+ Cells
Treatment Percent Number ( 10)
Spleen HBSS 1.43+/-0.27 0.53+/-0.12
r-hPRL 2.76+/-0.45* 2.56+/-0.35**
BMC HBSS 1.53+/-0.27 0.22+/-0.03
r-hPRL 3 .13+/-0.31 1.19+/-0.19*
* *
* <0.05. * *
<0.01
The functionality of the NK cells was also examined by assessing NK
cytotoxicity.
YAC-1 (NK sensitive target cells) were labeled by incubation for 1 hour at
37°C with Nas 1 Cr04
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WO 98/31385 PCT/US98/00887
18
(New England Nuclear) Boston, MA, specific activity approximately 400~tCi/pg).
After
incubation, the target cells were washed 3 times in RPMI 1640 supplemented
with 2% FCS
before used in the assay. Effector cells (splenocytes) and target cells were
added to round
bottomed 96-well plates (Costar) to obtain effector/target (E/T) cell ratio of
40/ I , 20/ 1, 10/ 1, and
5/1. Four replicate wells were used. After a standard 4 hour incubation) the
supernatants were
harvested and analyzed using a gamma counter (Model 5500. Beckman Instrument,
Irvine, CA).
The percent specific lysis was calculated as follows: %specific lysis = CPMexp
-
CPMspontaneous/ CPMmaximun - CPMspontaneous x 100%.
Using this method, it was observed that r-hPRL did increase the NK function,
as assessed
by cytotoxicity, in mice receiving SBMT using standard YAC- I cell targets
(Figure 9).
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