MXPA97009244A - Methods to increase hematopoyeti cells - Google Patents

Abstract

Methods for increasing hematopoietic cells, including platelets and erythrocytes, are described in patients receiving bone marrow or peripheral blood germ cell transplants. The methods comprise administering to a donor a sufficient amount of thrombopoietin to stimulate the proliferation of cells of the myeloid lineage, collect donor cells, and administer the collected cells to a recipient patient. The recipient patient can be treated with additional thrombopoietin. The sonutiles methods within the procedures of allogeneic transplants and autologo

Description

METHODS FOR INCREASING HEMATOPOYETIC CELLS BACKGROUND OF THE INVENTION Hematopoiesis is the process by which blood cells develop and differentiate from pluripotent germ cells in the bone marrow. This process involves a complex interaction of polypeptide growth factors (cytokines) that act via membrane binding receptors on their target cells. The action of the cytokine results in cell proliferation and differentiation, with a response to a particular cytokine frequently that is specific to linage and / or specific in period. The development of a single cell type, such as a platelet, from a germ cell may require the coordinated action of a plurality of cytokines that act in the proper sequence. It has been hypothesized for many years that the production of platelets can be regulated by specific humoral factors. Initial experiments have shown that the plasma or urine of thrombocytopenic animals contains an activity that promotes the formation of megakaryocytic colonies and increases the size of megakaryocytes of the marrow. This activity refers to, in the literature as "thrombopoietin" (reviewed REF: 25900 recently by McDonald, Exp. Hematol., 16: 201-205, 1988 and McDonald, A.J.P., Hematol., Oncol. 14: 8- 21, 1992). The low concentration of this activity and the lack of adequate bioassays greatly hinder the purification and characterization of the protein. Thrombopoietin has now been produced using cells grown in genetic engineering. See, de Sauvage et al., Nature 369: 533-538. 1994; Lok et al., Nature 369: 565-568, 1994; Kaushansky et al., Nature 369: 568-571, 1994; and Bartley et al., Cell 77: 1117-1124, 1994. Thrombopoietin has been shown to increase platelet numbers to normal (Lok et al., ibid.), and thrombocytopenic animals (Sprugel et al., Blood 84 (10). Suppl 1): 242a, 1994), and to stimulate the production of erythrocytes (Kaushansky et al., J. Clin. Invest., In Press).

In vi tro the TPO improves the survival and proliferation of CD34 + cells destined to become megakaryocytes (Papayannopoulou et al., Blood 84 (10 Suppl. 1): 324a, 1994). Although the cloning and characterization of TPO currently allows the investigation of its use in the stimulation of thrombopoiesis, thrombocytopenia and anemia remain as significant medical problems, such as in conjunction with radiation therapy and chemotherapy of cancer patients. There remains a particular need for methods to stimulate platelet production in patients receiving bone marrow transplants and peripheral blood germ cell transplants, including autologous transplants. There remains also a need to stimulate the production of erythrocytes. The present invention provides therapeutic methods that address these needs, and provide other related advantages.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides methods for increasing hematopoietic cells in a recipient patient in need of such an increase. The methods comprise the steps of (a) administering to a donor an amount of thrombopoietin (TPO) sufficient to stimulate the proliferation of myeloid lyase cells in the donor; (b) collecting the donor cells, wherein the cells are bone marrow cells or peripheral blood germ cells; and (c) administering bone marrow cells or peripheral blood germ cells to a recipient patient. The donor and the recipient can be different individuals or the same individual. Within one embodiment of the invention, the recipient patient has been treated with chemotherapy or radiation therapy. Within another embodiment, after or in conjunction with administration of bone marrow cells or peripheral blood germ cells, a sufficient amount of TPO is administered to elevate platelet recovery or red cell recovery to the recipient patient. Within another aspect, the present invention provides methods for preparing cells for transplantation comprising administering to a donor a sufficient amount of TPO to stimulate the proliferation of myeloid lyase cells in the donor, and collect cells from the donor, wherein the cells are bone marrow cells or peripheral blood germ cells. Within a third aspect, the present invention provides a method for stimulating platelet recovery or erythrocyte recovery in a patient receiving chemotherapy or radiation therapy comprising (a) administering to the patient an amount of TPO sufficient to stimulate the proliferation of myeloid lining cells in the patient; (b) collecting bone marrow cells or germ cells from the patient's peripheral blood prior to chemotherapy or radiation therapy; and (c) returning the cells collected to the patient after chemotherapy or radiation therapy. Within one embodiment, this method additionally comprises administering to the patient, after or in conjunction with the return of the harvested cells, a sufficient amount of TPO to improve platelet recovery or erythrocyte recovery. These and other aspects of the invention will become apparent from the reference to the following detailed description and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the effect of transplantation of bone marrow cells from donor mice treated with vehicle or TPO on platelet counts in recipient animals. In an experiment, marrow recipients treated with TPO are also treated with TPO (20 kU / day i.p.). The data are presented as means of 10-20 mice in two experiments. *, p < 0.05; **, p < 0.01. Figure 2 illustrates the effect of transplantation of bone marrow cells from donor mice treated with vehicle or TPO on erythrocyte counts in recipient animals. The data are expressed as the average of 20 mice in two experiments. *, p < 0.05; **, p < 0.005. Figure 3 illustrates the recovery of platelets in mice that received bone marrow transplants from donors treated with TPO or vehicle, with or without a post-transplant TPO treatment.

DETAILED DESCRIPTION OF THE INVENTION The term "germ cell" is used here to denote pluripotent hematopoietic germ cells and myeloid progenitor cells. The term "transplant" is used herein to denote the process of removing cells from a donor and subsequently administering the cells to a recipient. The term encompasses both allogeneic transplantation, where the donor and the recipient are different individuals of the same species; and an autologous transplant, where the donor and the recipient are the same individual. The term "increased hematopoietic cells" is used herein to denote restoration or improved recovery of hematopoietic cell levels after ablation, such as ablation resulting in r3 -the disease or therapeutic intervention. The term "thrombopoietin" encompasses proteins characterized by their ability to bind specifically to the MPL receptor of the same species and to stimulate platelet production in vivo. In animals for normal tests, TPO is able to increase platelet levels by 100% or more within 10 days after starting daily administration. A cDNA sequence of the representative human TPO is shown in SEQUENCE OF IDENTIFICATION NO: 1, and the corresponding amino acid sequence is shown in SEQUENCE OF IDENTIFICATION NO: 2. Analytical and experimental evidence indicates that the mature protein begins in the residue Ser-22. Those skilled in the art will recognize that the sequences illustrated correspond to individual alleles of the human TPO gene, and that allelic variation is expected to exist. Allelic variants include those that contain silent mutations and those in which mutations result in changes of amino acid sequences. It will also be apparent that a person skilled in the art will be able to create additional variants, such as by engineering sites that can facilitate manipulation of nucleotide sequences using alternative codons, by codon substitution to produce conservative changes in the amino acid sequence, etc. . The use of allelic and engineered TPO variants is contemplated by the present invention. In addition, amino-terminal TPO polypeptides of about 150 amino acids or more in length are known to be active (from Sauvage et al., Ibid.; Bartley et al., Ibid .; US Patent Application No. 08 / 346,999, co-pending, commonly assigned), and the use of such truncated forms of TPO are within the scope of the present invention. Thrombopoietins from non-human species have been described in the scientific literature (Lok et al., Ibid., De Sauvage et al., Ibid; Bartley et al., Ibid.). The present invention provides methods for increasing hematopoietic cells in patients, particularly patients suffering from radiation therapy and / or chemotherapy, such as in the treatment of cancer. Such therapies eliminate dividing progenitor cells in the marrow and peripheral blood, limiting therapy and often requiring transfusions to restore levels of circulation of platelets and other blood cells. Of particular interest are those patients receiving bone marrow transplants and / or peripheral blood germ cells after radiation therapy and patients suffering from congenital metabolic defects requiring bone marrow transplantation. Among these indications are bone marrow transplants associated with the treatment of breast cancer, leukemia, lymphoma, myel to multiple and congenital defects such as severe combined immune deficiency, thalassemia, and sickle cell anemia. Transplantation of peripheral blood germ cells may be preferred in conditions where the risk of tumor cells in the blood is not present. Methods for carrying out bone marrow and peripheral blood germ cell transplants are known in the art. For review, see Snyder et al., "Transfusion Medicine" in Benz and McArthur, eds., Hematoloqy 1994, American Society of Hematology, 96-106, 1994. Peripheral blood germ cells are collected by leukapheresis according to clinical procedures accepted Hematopoietic progenitor cells can be selected on the basis of cell surface markers (eg, CD34), allowing the enrichment of the desired cells and the depletion of contaminating tumor cells. The harvested cells are stored frozen in a suitable cryoprotectant (eg, dimethyl sulfoxide, hydroxyethyl starch) as necessary. The marrow cells are harvested from donors by a bone puncture under anesthesia. To reduce the volume, the collected marrow is usually processed to separate plasma from the cellular components. The removal of plasma can also eliminate the incompatibilities of red cells in allogeneic transplantation. The cellular fraction can be enriched by mononuclear cells using density gradient techniques or automated separation methods and reduction of T cells using various cytotoxic agents. The harvested marrow cells are cryopreserved according to established procedures that include controlled rate freezing and the use of cryoprotectants. The germ cells are thawed in a hot water bath immediately before use to minimize the losses associated with thawing. In the case of allogeneic transplants, the donors and recipients are matched in tissue to minimize the risk of graft-versus-host disease. An increase in the hematopoietic cells results from transplantation in a germ cell recipient patient, particularly myeloid lining cells, including the CD34 + germ cells and cells derived from the CD34 + germ cells. Of particular interest are cells in the megakaryocyte and erythrocyte lineages, which reconstitute the platelet and erythrocyte populations of the receptor, respectively. Within the present invention, a donor is treated, prior to the donation of marrow or peripheral blood cells, with TPO in an amount sufficient to stimulate the proliferation of myeloid lining cells. Such amount will generally be in the range of 0.5 lg / kg / day to 40 lg / kg / day, preferably l lg / kg / day at 20 lg / kg / day. The treatment of the donor will be carried out for a period of one week to several days, preferably approximately 2-5 days, for a period of 3 days to 2 weeks before harvesting of bone marrow or peripheral blood germ cells. It is preferred to treat the donor for a period of five to ten days before harvesting the cells. The increase in CD34 + germ cells and other myeloid lining cells in the donor will be manifested by an improved recovery of platelet and / or erythrocyte levels in the recipient of the transplant. Within one embodiment of the invention, the recipient is treated with TPO after transplantation to further improve platelet recovery. It has been found that post-transplant treatment with TPO improves the survival of lethally irradiated test animals having bone marrow from donors treated with TPO. "An amount of thrombopoietin sufficient to improve platelet recovery" is that amount that produces a statistically significant reduction in time for recovery of normal platelet levels or a statistically significant increase in platelet count compared to untreated patients. The doses of T? '~ >; used in the post-transplant treatment will generally be in the range of 0.5 lg / kg / day to 40 g / kg / day administered for approximately 3 to approximately 20 days. In general, patients who receive bone marrow transplants will require a longer post-transplant treatment than those who receive peripheral blood germ cell transplants. For use within the present invention, TPO can be prepared using cells grown by genetic engineering, according to methods generally known in the art. To summarize these methods, a DNA molecule encoding TPO is linked to other DNA sequences which are provided for maintenance and transcription in a host cell. The resulting expression vector is inserted into the host cell, and the resulting "transformed" or "transfected" cells are cultured in a suitable nutrient medium. Baby hamster kidney cells (BHK) are a preferred host. It is preferred to machine the cells to secrete the TPO into the medium, although the TPO of cell lysates can be recovered and processed in vi tro to give the active protein. See, in general, Sauvage et al., Ibid .; Lok et al., Ibid .; Kaushansky et al., Nature 369: 568-571, 1994; Wendling et al, Nature 369: 571-574, 1994; Bartley et al., Ibid .; and commonly assigned U.S. Patent Application Serial No. 08 / 366,859 and Serial No. 08 / 347,029, which are hereby incorporated by reference in their entirety. TPO can be purified from culture media conditioned in cells by a combination of chromatography and other techniques, including direct capture on an affinity matrix of ligands in dyes and ion exchange chromatography. The contaminating proteins can be eliminated by adsorption with hydroxyapatite.

For pharmaceutical use, TPO is formulated for parenteral delivery, particularly intravenous or subcutaneous, according to conventional methods. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, the pharmaceutical formulations will include a hematopoietic protein in combination with a pharmaceutically acceptable carrier, such as saline, buffered saline, 5% dextrose in water or the like. The formulations may additionally include one or more excipients, preservatives, solubilizers, buffering agents (eg, phosphate buffer), albumin or a non-ionic detergent to prevent protein losses on the surfaces of flasks, etc. In addition, TPO may be combined with other cytokines, particularly cytokines that act early such as germ cell factor IL-3, IL-6, IL-11 or GM-CSF. When such a combination therapy is used, the cytokines can be combined in an individual formulation or they can be administered in separate formulations. Formulation methods are well known in the art and are described, for example, in Reminaton's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton PA, 1990, which is incorporated herein by reference.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES Example 1 The mouse thrombopoietin is prepared using transfected baby hamster kidney cells (cells 570 from BHK, ATCC CRL 10314). The serum free medium contains 145 kU / ml of TPO activity, where 10 units are o-defined as the amount of TPO that gives the maximum average stimulation in a mitogenesis assay (incorporation of 3H-thymidine) using the transfected BaF3 cells with an expression vector encoding the human MPL receptor (Vigon et al., Proc. Nati, Acad. Sci. USA 89: 5640-5644, 1992) as target cells. BaF3 is an interleukin-3 dependent on the pre-lymphoid cell line derived from murine bone marrow (Palacios and Steinmetz, Cell 41: 747-734, 1985, Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986). The cells are exposed to test samples in the presence of 3H-thymidine. The amount of 3 H-thymidine incorporated into the cellular DNA is quantified by comparison with a standard curve of human TPO. The samples of mouse TPO are effective in colony formation assays in a range of approximately 100-400 U / ml. In vivo activities are observed in the range of 20-40 kU / day in mice. For in vivo experiments, TPO is diluted to the desired concentration in an endotoxin-free phosphate buffered solution (PBS) and administered as intraperitoneal or subcutaneous injections. Female Balb-C mice (in the age range of 8-12 weeks) are obtained from Broekman B.V. (Someren, The Netherlands) and fed with commercially available rodent food and provided with acidified water ad libi tum. Transplant recipients are maintained in a pathogen-free environment and provided with water containing ciprofloxacin at a concentration of 1 mg / ml, polymyxine-B at 70 lg / ml, and sucrose at 2 g / 100 ml. The recipient mice are placed in a box of polymethyl acetate-acetate and irradiated lethally (8.5 Gy) using a linear accelerator of Philips SL 75-5 / 6 mV (Philips Medical Systems, Best, The Netherlands). The irradiation is divided into two parts in a posterior-anterior and anterior-posterior position, at a dose rate of 4 Gy / minute. The mice are transplanted with 105 bone marrow cells from donor mice in ready state. The transplant takes place within four hours of harvesting the marrow. Groups of 5 recipient mice are treated with TPO in a dose of 20 kU / day intraperitoneally (i.p.) on days 1-5, 3-8 or 3-12 after transplantation. The control animals are transplanted with an equal amount of marrow cells and given saline at similar time intervals after transplantation. Compared with control receptors treated with saline, the administration of TPO does not result in accelerated platelet reconstitution. A dose of 30 kU / day administered subcutaneously (s.c.) on days 1-14 is also ineffective in the acceleration of platelet recovery. No effect is observed in the reconstitution of white blood cells or red blood cells. In a second set of experiments, the donor mice are treated with TPO for five consecutive days in a dose of 20 kU / day i.p. per mouse. On day 5 the mice are sacrificed, and, bled, the bone marrow and spleens are harvested. The red blood cells, the white blood cells and the platelets are counted in a Sysmex 800 counter (TOA Medical Electronics Company, Kobe, Japan). Treatment with TPO induces an increase of 2.5 times the number of platelets, but does not have an effect on the number of white blood cells or red blood cells. The levels of progeny cells are also determined in the donor mice treated with TPO. Bone marrow cells are harvested by flowing the femurs under sterile conditions with RPMI 1640 containing 450 μg / ml penicillin, 250 μg / ml streptomycin, and 2% fetal bovine serum (FBS) (GIBCO BRL, Gaithersburg, MD). Suspensions of individual spleen cells are prepared by macerating the organs and washing them once with RPMI 1640 containing 2% FBS. To determine the colony-forming units, CFU-GM are grown according to published procedures (Fibbe et al., J. Immunol., 148: 417, 1992). Briefly, bone marrow cells are cultured in microtiter plates containing 10 4 cells / well in a semi-solid medium in the presence of murine GM-CSF (1.25 ng / ml). Peripheral blood mononuclear cells and spleen cells are grown in 3.5 cm boxes containing 5x105 cells / ml and 106 cells / ml, respectively. Cells are cultured in a totally humid atmosphere at 37 ° C containing 5% C02 • After 6 days of culture the number of colonies (defined as aggregates of >) is counted; 20 cells) using an inverted microscope. The CFU mixture assay is performed in an identical mode in 3.5 cm boxes in the presence of a combination of 1.25 ng / ml of recombinant murine GM-CSF, 2 U / ml of recombinant human EPO, 25 ng / ml of IL -3 recombinant murine, 5% of transferin, 5% of bovine serum albumin, 5% of 10"3 b-mercaptoethanol, and 7.5% of modified Dulbecco's medium of Iscove (IMDM) After 6 to 7 days of culture at 37 ° C in a totally humid atmosphere at 5% C02, the number of colony-forming cells is counted using an inverted microscope.TPO treatment results in an increased number of colony forming units (CFU) and BFU- It is in the bone marrow or spleen compared to controls treated with saline (Table).

Table Treatment of the donor TPO Saline solution Femur Nucleated cells (xlO6) 18.4 ± 4.7 19.9 ± 4.3 CFU (XlO3) 55.3 ± 12.5 * 38.6 ± 5.2 BFU-E (XlO3) 24.0 ± 4.9 * 16.4 ± 5.3 Spleen Nucleated cells (xlO6) 71.8 ± 35.0 78.4 ± 42.5 CFU (XlO3) 27.3 ± 16.9 16.3 ± 11.4 BFU-E (xlO3) 10.2 ± 2.3 1.9 ± 0.7 Results are expressed as absolute cell numbers (mean ± SD, n = 7) per organ (femur or spleen ). CFU represents the total number of colonies grown in the CFU mix assay. * p < 0.05.

The recipient animals irradiated lethally with 105 bone marrow cells are transplanted from donors that have been treated with TPO in a dose of 20 kU / day i.p. for five consecutive days, or control donors treated with saline. Blood samples are taken after the transplant of individual recipients every 3 days due to bleeding from the tail vein. No visible difference is observed in the bleeding tendency between bone marrow cell receptors modified with TPO or unmodified. Cellular accounts are analyzed using student's T tests. In the MANOVA analysis, the groups are compared with respect to their course over time. Analyzes are performed on the logarithm values of the data. The values of < 0.05 are considered statistically significant. The curves are compared using the MANOVA test. The results show that reconstitution of platelets in marrow recipients treated with TPO is significantly altered in comparison with control animals with transplantation with an equal number of bone marrow cells from control donors treated with saline (Figure 1). In addition, platelet nadir counts are higher in animals receiving marrow treated with TPO than those receiving control marrows (88 x 109 versus 30 x 109 on day 12 after transplantation, average of 20 mice).

As shown in Figure 1, post-transplant treatment with 20 kU / day TPO i.p. on days 1-5 it does not result in an additional acceleration of platelet reconstitution in mice receiving marrows from donors treated with TPO. In addition, to accelerate the reconstitution of platelets, TPO-modified bone marrow cell receptors also exhibit accelerated erythrocyte reconstitution (Figure 2). The nadir counts of erythrocytes are also significantly higher in these animals than in the controls with a transplant with an equal number of unmodified bone marrow cells. Experiments are carried out to further verify that this effect is due to a direct activity of TPO on erythropoiesis and not related to differences in platelet counts and bleeding tendency. In this experiment, recipient animals are not bled until day 12 after transplantation, at which time the recipient mice are sacrificed, and the numbers of bone marrows and progenitor cells derived from the blood are evaluated. Bone marrow cell receptors modified with TPO have a higher number of BFU-E / femur colonies (770 ± 386 vs. 422 ± 320, mean ± SD, n = 5) and higher reticulocytes in the blood (44% vs. 8). %, mean of 5 mice) than controls transplanted with an equal number of unmodified bone marrow cells, although these differences do not reach statistical significance. The post-transplant treatment with TPO does not result in an additional acceleration of the reconstitution of erythrocytes at the doses tested.

Example 2 A second experiment is carried out to compare platelet counts in lethally irradiated mice receiving markers from untreated donors with TPO, and to determine the effect of treatment with post-transplant TPO of the recipient animals. Fl B6D2 mice obtained from Taconic (Germantown, NY) and housed under specific pathogen-free conditions. The mice are housed five per cage and receive acid water and food ad libi tum. Forty female mice are used as receptors, and five male mice are used as donors. Recombinant human TPO is prepared using transfected BHK 570 cells. The main molecular species have a band of 70 kD. The preparation has a specific activity of 5641 U / lg. The protein is prepared in potassium phosphate buffer at 29 mM, pH 6.0, containing 0.05% polysorbate 80 and 0.13 M NaCl and stored and frozen in aliquots of 20 kU. The TPO and vehicle solutions are thawed directly before use and injected into mice once a day, subcutaneously. Two donor mice are each treated with 20 kU of TPO per day for four days, then sacrificed by cervical dislocation on the fifth day. The control donors are treated with vehicle only. The femoral parts are removed aseptically, and the bone marrow is extracted with F2 from Ham (Fred Hutchinson Cancer Research Center, Seattle, WA) containing 2% fetal bovine serum by inserting a 25 g needle attached to a syringe. The cell suspension is flowed twice through an 18 g needle, a 20 g needle, and a 22 g needle to produce a suspension of individual cells. The nucleated cells are counted in a hemocytometer. On day -2, the recipient mice are exposed to 1200 cGy of total body irradiation from a 137Cs source (Gammacell 40 Irradiator, Atomic Energy of Canada Radiochemical Company, Kanata, Canada). Bone marrow transplants are performed in hours two to four after irradiation. Twenty mice receive bone marrow (lxlO5 cells) from donors treated with TPO, and twenty mice receive lxlO5 cells from vehicle-treated donors. The recipients are treated with TPO (20 kU / day) starting on day 1 (2 days after transplantation) and continuing for 14 days.

Mice are bled from sinusoidal retroorbital under ether anesthesia. Fifty blood samples 11 are collected in heparinized micropipettes (VWR Scientific, Seatle, WA) and are added in the form of drops in the microtainer tubes with EDTA (Becton Dickinson, San José, CA). Blood drops are placed on slides, and smears are prepared. The blood is analyzed in a Cell Dyn 3500 hematology analyzer (Abbott, Santa Clara, CA). Hematocrits, RBC accounts, WBC accounts and platelet counts are determined. In mice that receive marrow from control donors, platelet counts fall on day 8 at low levels (below 6% of normal) and recovery begins in animals treated with TPO or control on day 12 ( Figure 3). There is no difference between the two groups in platelet recovery. However, in controls treated with vehicle only 3 out of 10 animals, while in groups treated with TPO 7 out of 9 they survive, death is related to hemorrhage, standard deviations are larger within the group treated with TPO and that some animals with very low platelet counts are able to survive.Mice receiving marrow from donors treated with TPO also have platelet numbers that are below 6% of normal on day 8. Animals that are treated with TPO during 14 days have, in general, a faster recovery in platelet counts Eight of nine animals treated with TPO survive, while only four of nine vehicle-treated mice survive RBCs recover faster in mice receiving bone marrow pretreated with TPO and treated with TPO compared to controls.There is no influence of TPO treatment on blood cell recovery It will be appreciated from the foregoing that, although the specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: ZymoGenetics, Inc. 1201 Eastlake Avenue East Seattle WA USA 98102 (ii) TITLE OF THE INVENTION: METHODS FOR INCREASING HEMATOPOYETIC CELLS (iii) SEQUENCE NUMBER: 2 (iv) DIRECT CORRESPONDENCE: (A) RECIPIENT: ZymoGenetics, Inc. (B) STREET: 1201 Eastlake Avenue East (C) CITY: Seattle (D) STATE: WA (E) COUNTRY: UNITED STATES OF AMERICA (F) CODE POSTAL (ZIP): 98102 (v) READING FORM ON THE COMPUTER: (A) TYPE OF MEDIA: Soft disk (B) COMPUTER: Compatible with an IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patent In Reread # 1.0, Version # 1.25 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (viii) EMPLOYEE / AGENT INFORMATION: (A) NAME: Parker, Gary E (B) REGISTRATION NUMBER: 31-648 (C) REFERENCE / FILE NUMBER: 95-04 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 206-442-6600 ext. 6673 (B) TELEFAX: 206-442-6678 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1062 base pairs (B) TYPE: nucleic acid (D) ) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1..1059 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 1 ATG GAG CTG ACT GAA TTG CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA 48 Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Wing 1 5 10 15 AGG CTA ACG CTG TCC AGC CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC 96 Arg Leu Thr Leu Ser Ser Pro Pro Pro Wing Ala Cys Asp Leu Arg Val 20 25 30 CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC 144 Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser 35 40 45 CAG TGC CCA GAG GTT CAC CCT TTG CCT ACÁ CCT GTC CTG CTG CCT GCT 192 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Wing 50 55 60 GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG 240 Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys 65 70 75 80 GCA CAG GAC ATT CTG GGA GCA GTG ACC CTT CTG GG GGA GTG ATG 288 Wing Gln Asp lie Leu Gly Wing Val Thr Leu Leu Leu Glu Gly Val Met 85 90 95 GCA GCA CGG GGA CAA CTG GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG 336 Wing Wing Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly 100 105 lio CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT GGG GCC CTG CAG AGC CTC 384 Gln Leu Ser Oly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu 115 120 125 CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG ACC ACCT GCT CAC AAG GAT 432 Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp 130 135 140 CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CTG CTC CGA GGA AA GTG 480 Pro Asn Ala lie Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val 145 150 155 160 CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC 528 Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Wing 165 170 175 CCA CCC ACC AC GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC ACÁ CTG 576 Pro Pro Thr Thr Wing Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu "180 185 190 AAC GAG CTC CCA AAC AGG ACT TCT GGA TTG TTG GAG HERE AAC TTC ACT 624 Aßn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr 195 200 205 GCC TCA GCC AGA ACT ACT GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA 672 Wing Being Wing Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly 210 215 220 TTC AGA GCC AAG ATT CCT GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG 720 Phe Arg Ala Lys lie Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu 225 230 235 240 GAC CAA ATC CCC GGA TAC CTG AAC AGG ATA CAC GAA CTC TTG AAT GGA 768 Asp Gln lie Pro Gly Tyr Leu Asn Arg lie His Glu Leu Leu Asn Gly 245 250 255 ACT CGT GGA CTC TTT CCT GGA CCC TCA CGC AGG ACC CTA 3GA GCC CCG 816 Thr Arg Gly Leu Phß Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro 260 265 270 GAC ATT TCC TCA GGA ACÁ TAC GAC ACÁ GGC TCC CTG CCA CCC AAC CTC 864 Asp Lie Ser Ser Gly Thr Be Aßp Thr Gly Ser Leu Pro Pro Asn Leu 275 280 285 CAG CCT GGA TAT TCT CCT TCC CCA ACC CAT CCT CCT ACT GGA CAG TAT 912 Gln Pro Gly Tyr Pro Pro Pro Pro Thr Pro Pro Thr Gly Gln Tyr 290 295 300 ACG CTC TTC CCT CTT CCA CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC 9S0 Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu 305 310 315 320 CAC CCC CTG CTT CCT GAC CCT TCT GCT CCA ACG CCC ACC CCT ACC AGC 1008 Pro His Pro Leu Leu Pro Asp Pro Pro Pro Pro Wing Pro Thr Pro Ser 325 330 335 CCT CTT CTA AAC ACÁ TCC TAC ACC CAC TCC CAO AAT CTG TCT CAG GAA 1056 Pro Leu Leu Asn Thr Ser Tyr Thr His SS Gln Asn Leu Ser Gln Glu 340 345 350 GGG TAA 1062 Gly (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 353 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 2 Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala 1 5 10 15 Arg Leu Thr Leu Ser Ser Pro Pro Pro Wing Cys Asp Leu Ars Val 20 25 30 Leu Ser Lys Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser 35 40 45 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Wing 50 55 60 Val Asp Phe Sex Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys 65 70 75 80 Wing Gln Asp He Leu Gly Wing Val Thr Leu Leu Leu Glu Gly Val Met 85 90 95 Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly 100 105 no Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu 115 120 125 Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp 130 135 140 Pro Asn Ala He Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val 145 150 155 160 Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val. Arg Arg Ala 165 170 175 Pro Pro Thr Thr Wing Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu 180 185 190 Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr 195 200 205 Wing Being Wing Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly 210 215 220 Phe Arg Wing Lys He Pro Gly Leu Leu Asn Gln Thr Being Arg Ser Leu 225 230 235 240 Asp Gln He Pro Gly Tyr Leu Asn Arg lie His Glu Leu Leu Asn Gly 245 250 255 Thr Arg Gly Leu Phe Pro Gly Pro Be Arg Arg Thr Leu Gly Wing Pro 260 265 270 Asp Be Ser Gly Thr Be Asp Thr Gly Ser Leu Pro Pro Asn Leu 275 280 285 Gln Pro Gly Tyr Ser Pro Pro Pro Thr His Pro Pro Thr Gly Gln Tyr 290 295 300 Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu 305 310 315 320 His Pro Leu Leu Pro Asp Pro Be Ala Pro Thr Pro Thr Pro Thr Ser 325 330 335 Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln siu 340 345 350 Gly It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers. Having described the invention as above, property is claimed as contained in the following:

Claims (4)

CLAIMS 1. The use of bone marrow cells or pluripotent or germinal peripheral blood cells in the manufacture of a medicament for increasing hematopoietic cells in a recipient patient in need of such an increase, for example, to stimulate the recovery of platelets or erythrocytes in a patient receiving chemotherapy or radiation therapy, in which the cells have been harvested from a donor who has been administered thrombopoietin (TPO), for example human TPO to stimulate the proliferation of cells of the myeloid line. 2. The use according to claim 1, characterized in that the medicament is for use with a recipient patient treated with chemotherapy or radiation therapy. 3. The use according to claim 1, characterized in that the donor and the recipient patient are the same individual, optionally in which the recipient patient is treated with chemotherapy or radiation between the collection stages and the second administration. 4. The use according to any of claims 1 to 3, characterized in that it additionally comprises that the medicament allows the administration of such cells concurrently with or before the administration of thrombopoietin sufficient to improve the recovery of platelets or the recovery of erythrocytes . 5. A method for preparing cells for transplantation, characterized in that it comprises: administering to a donor an amount of thrombopoietin (TPO), for example human TPO, sufficient to stimulate the proliferation of cells of the myeloid line in the donor; Collect donor cells, in which the cells are bone marrow cells or peripheral blood pluripotential cells. 6. The use of thrombopoietin (TPO), for example human TPO in an amount to stimulate the proliferation of cells of the myeloid line, in the preparation of bone marrow cells or peripheral blood pluripotent cells. 7. The use of thrombopoietin (TPO), for example human TPO, in the manufacture of a medicament for the stimulation of proliferation of cells of the myeloid line in a donor of bone marrow cells or peripheral blood cells. SUMMARY OF THE INVENTION Methods for increasing hematopoietic cells, including platelets and erythrocytes, are described in patients receiving bone marrow transplants or peripheral blood germ cells. The methods comprise administering to a donor a sufficient amount of thrombopoietin to stimulate the proliferation of cells of the myeloid lineage, collect donor cells, and administer the collected cells to a recipient patient. The recipient patient can be treated with additional thrombopoietin. The methods are useful within the procedures of allogeneic and autologous transplants. 33

1. The use of bone marrow cells or pluripotent or germinal peripheral blood cells in the manufacture of a medicament for increasing hematopoietic cells in a recipient patient in need of such an increase, for example, to stimulate the recovery of platelets or erythrocytes in a patient receiving chemotherapy or radiation therapy, in which the cells have been harvested from a donor who has been administered thrombopoietin (TPO), for example human TPO to stimulate the proliferation of cells of the myeloid line.

2. The use according to claim 1, characterized in that the medicament is for use with a recipient patient treated with chemotherapy or radiation therapy.

3. The use according to claim 1, characterized in that the donor and the recipient patient are the same individual, optionally in which the recipient patient is treated with chemotherapy or radiation between the collection stages and the second administration.

4. The use according to any of claims 1 to 3, characterized in that it additionally comprises that the medicament allows the administration of such cells concurrently with or before the