CA2277482A1 - Enhancement of hematopoietic cells - Google Patents

Abstract

The invention relates to a method for enhancing hematopoiesis by contacting hematopoietic stem 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-suppresion 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-suppresion and a prolactin.

Description

WO 98/31385 _ PCT/US98/00887 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.

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.

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.

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.
_.._ .___ ~ _ i 5 ~ PCT/US98/00887 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, 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 (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.
__ ___.._...T_..___.. .. _. .

WO 98!31385 7 PCT/US98/00887 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 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 i T __.__~-__._ _.. _ ..T._ _ i 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 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 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 WO 98/31385 ~ 0 PCTILTS98/00887 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 __._ ._.__?_ 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 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 .........~.._......w._._....... T . .....

WO 98/31385 ~ 3 PCT/US98/00887 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.

WO 98/31385 ~ 4 PCT/US98/00887 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 _ ~..__ . . ~. .... i 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.

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 ~__._..~ _ _ _.~. i 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 (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).
_..~__~._.-___... ___.r._._._._...

Claims (21)

THE INVENTION CLAIMED IS:

1.~A method for enhancing hematopoiesis comprising adding a composition containing prolactin to hematopoietic stem cells or progenitor cells.

2. The method of claim l, wherein the prolactin is recombinant prolactin.

3. The method of claim 1, wherein the prolactin is human prolactin.

4. A method for treating an animal to improve hematopoiesis, comprising administering an effective amount of a pharmaceutically acceptable composition containing prolactin.

5. The method of claim 4, wherein the prolactin is recombinant prolactin.

6. The method of claim 4, wherein the prolactin is human prolactin.

7. The method of claim 4, wherein the animal suffers from hematopoietic-suppression.

8. The method of claim 7, wherein the hematopoietic-suppression is caused by a therapeutic treatment selected from the group consisting of AZT, irradiation exposure, cytoreductive treatment, chemotherapy, cytolytic therapy, immunocytolytic therapy, and combinations thereof.

9. A method for treating an animal to prevent hematopoietic-suppression, comprising administering an effective amount of a pharmaceutically acceptable composition containing prolactin.

10. The method of claim 9, wherein the prolactin is recombinant prolactin.

11. The method of claim 9, wherein the prolactin is human prolactin.

12. The method of claim 9, wherein the hematopoietic-suppression is caused by a therapeutic treatment selected from the group consisting of the administration of AZT, irradiation exposure, cytoreductive treatment, chemotherapy treatment, cytolytic therapy, immunocytolytic therapy, and combinations thereof.

13. A composition comprising a cytokine capable of enhancing hematopoiesis and a prolactin.

14. The composition of claim 13, wherein the prolactin is recombinant prolactin.

15. The composition of claim 13, wherein the prolactin is human prolactin.

16. The composition of claim 13, wherein the cytokine is selected from the group consisting of interleukin-1, interleukin -2, interleukin 3, interleukin-4, interleukin-6, interleukin-7, interleukin-9, interleukin-11, interleukin -15, c-Kit ligand, granulocyte-monocyte colony-stimulating factor, monocyte-colony-stimulating factor, granulocyte-colony-stimulating factor, Flt3 ligand, Mpl ligand, Epo, Tpo, GH, IGF, TGF-.beta., and mixtures thereof.

17. A composition comprising a therapeutic that can cause hematopoietic-suppression and a prolactin.

18. The composition of claim 17, wherein the prolactin is human prolactin.

19. The composition of claim 17, wherein the prolactin is recombinant prolactin.

20. The composition of claim 17, wherein the prolactin is recombinant human prolactin.

21. The composition of claim 17, wherein the therapeutic is selected from the group consisting of AZT, a chemotherapeutic agent, an anticytolytic agent, an immunocytolytic agent and mixtures thereof.