MXPA98003217A - Method of mobilization of hematopoyetic mother cells - Google Patents

Abstract

A method of mobilizing hematopeytic stem cells from the bone marrow to the peripheral circulation is provided by administering to an animal an effective amount of natural, modified or multimeric forms of KC, groa, groalfa, or gr

Description

METHOD OF MOBILIZATION OF HEMATOPOVETIC STEM CELLS FIELD OF THE INVENTION In general terms, the present invention relates to methods for mobilizing hematopoietic stem cells.

BACKGROUND OF THE INVENTION All members of the intercrine or quinacline family are basic heparin-binding polypeptides having four cysteine residues that form two disulfide bridges. All these proteins, which have been functionally characterized, seem to be involved in proinflammatory and / or restorative functions. In clinical situations for the use of high doses of chemotherapy, the biomolecule of choice has been G-CSF. In general, in such treatment patients are prepared with a low dose of a chemotherapeutic agent such as cyclophosphamide. During remission, the patient is treated with a CSF, such as G-CSF, which causes the final mobilization of cells from the bone marrow to the peripheral circulation for leucopoietic blood collection. The patient is then given a high dose of chemotherapy to induce clinical remission of their cancer. The resulting failure of the bone marrow is treated by infusion of the stored blood cells that were previously collected. This procedure can be modified, for example, by the omission of the initial dose of chemotherapy and / or alternative blood collection protocols. Although the use of these hematopoietic stem cell transplantation techniques seems promising, multiple apheresis procedures are required to collect enough stem cells for successful grafting in the treatment of severe ileosuppression, when only G-CSF is used [see, v.gr ., Besinger and others, Blood. 81: 3158 (1993) and R. Haas, et al., Sem. in Oncology. 21:19 (1994)]. In this way, despite these significant advances, and the availability of certain regulatory biomolecules, late recovery of hematopoiesis remains an important source of morbidity and mortality for myelosuppressed patients. There is a continuing need in the art for compositions and methods for increasing hematopoietic recovery, particularly in cases of myelosuppression associated with chemotherapy.

BRIEF DESCRIPTION OF THE INVENTION In one aspect, the present invention provides the use of a chemokine in the preparation of a medicament for the stimulation of hematopoietic stem cells. This chemokine includes proteins derived from groja, groa, and grot. including mature, modified and multimeric forms of these chemokines. In a further aspect, the present invention provides a method for mobilizing hematopoietic stem cells in an animal, comprising administering to an animal an effective amount of a mature or modified or multimeric chemokine, as described herein. Other aspects and advantages of the present invention are further described in the following detailed description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph demonstrating the effect of roß (amino acids 1-73 of SEQ ID NO: 3) on the single agent mobilization test of example 1. Figure 2 is a graph showing the effect of groß ( amino acids 5-73 of SEO ID NO: 3) modified in the single agent mobilization test of example 1. Figure 3 is a bar graph demonstrating comparison of phosphate buffered saline (PBS), IL-8, roß (amino acids 1-73; SEQ ID NO: 3) and modified roß (amino acids 5-73 of SEQ ID NO: 3) in the single agent mobilization test.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides modified proteins, specifically chemokines, associated with inflammatory responses, hematopoiesis and myelopoiesis; these modified proteins are characterized in that they have increased biological activity compared to the corresponding mature unmodified or non-truncated proteins. The present invention provides methods for the treatment of myelosuppression by mobilizing hematopoietic stem cells from the bone marrow into the peripheral blood using the mature or modified or multimeric chemokines described herein.

I. Definitions As defined herein, "hematopoietic synergistic factor" or "HSF" refers to a class of proteins, including naturally occurring chemokines and modified chemokines, which are characterized by having synergistic activity in the stimulation of hematopoiesis when administered in vivo and in vitro with another hematopoietic factor, such as a colony stimulating factor, or combined with natural circulating CSFs. The term "mature chemokines" also known as "intercourses", as used herein, defines the proteins conventionally referred to in the art as KC, roa. groß. and grot. For convenience, SEQ ID NO: 1 provides the amino acid sequences of the KC murine protein containing 72 residues. These sequences are available from the gene bank, Genbank, accession number J04596. The sequences of the human roa protein (aa 1-73) are provided in SEQ ID NO: 2. The sequences of the human roß protein (amino acids 1-73) are provided in SEQ ID NO: 3. The sequences of the human protein grot are provided in SEQ ID NO: 4. The cDNA and rota amino acid sequences are also provided in International Patent Application, Publication No. WO 92/00326 (January 9, 1992). These tag sequences have also been published in International Patent Application, Publication No. WO 94/29341 (December 22, 1994), which is incorporated herein by reference. The term "modified chemokines" is defined as in the International Application referred to above. The modified chemokines are derived from KC, groß, groa, and grot, preferably from groß, groa, and grot, and preferably from groß.

Modified chemokines include deaminated proteins characterized by the elimination of between about 2 to about 8 amino acids at the amino terminus of the mature protein. These deaminated chemokines useful in the method of the invention are preferably characterized by the removal of about 2 to about 8 amino acids from the amino terminus of the mature protein. Preferably, modified chemokines are characterized by removal of the first 4 amino acids at the amino (N-) end. Optionally, particularly when expressed recombinantly, the deaminated chemokines useful in this invention may contain an N-terminal Met embedded therein. The N-terminal methionine inserted into the protein for expression purposes can be excised, either during the processing of the protein by a host cell, or synthetically using known techniques. Alternatively, if desired, this amino acid can be cleaved by digestion with enzymes or other known means. Analogs or derivatives of these proteins that share the biological activity of the mature protein are also included with the term modified chemokine. As defined herein, said analogs and derivatives include modified proteins also characterized by alterations made in the known amino sequence of the proteins, for example, the proteins provided in SEQ ID NO: 1-4. Said analogs are characterized in that they have an amino acid sequence different from that of the mature protein by 8 or less amino acid residues, and preferably by approximately 5 or less residues. It may be preferable that any difference in the amino acid sequences of the proteins involve only conservative amino acids. Substitutions of conservative amino acids occur when an amino acid has substantially the same charge as the amino acid by which it is substituted and the substitution has no significant effect on the local conformation of the protein or its biological activity. Alternatively, changes such as the introduction of a certain amino acid into the sequence that can alter the stability of the protein, or allow it to be expressed in a desired host cell, may be preferred. Another characteristic of these modified proteins may be increased biological activity compared to the mature protein. The term "multimeric protein" or "multimer" here means multimeric forms of the mature and / or modified proteins useful in this invention., for example, dimers, trimers, tetramers and other aggregated forms. Such multimeric forms can be prepared by synthesis or recombinant expression and can contain chemokines produced by a combination of synthetic and recombinant techniques, as detailed below. Ultimers can be formed naturally by expression or they can be constructed in such multiple forms. Multimeric chemokines can include multimers of the same modified chemokine. Another multimer can be formed by the aggregation of different modified proteins. Another multimer is formed by the aggregation of a modified chemokine of this invention and a known mature chemokine. Preferably, a dimer or multimer useful in the invention would contain at least one deaminative chemokine protein and at least one other chemokine or other characterized protein for having the same type of biological activity. This other protein may be an additional deaminative chemokine, or another known protein.

II. Useful Proteins in the Invention In general, the chemokines useful in the method of the invention include the mature chemokines, or the modified and multimeric proteins derived therefrom, which are described in detail in International Patent Application Publication No. W094 / 29341 . Conveniently, these chemokines are selected from KC, groa, groß and grot. and preferably chemokine is groß. In a preferred embodiment, the method of the invention utilizes a deaminated chemokine protein of the invention. This protein comprises the amino acid sequence of mature chemokine useful in the invention truncated at its N-terminus between amino acid positions 2 and 8 of SEQ ID NOS: 1-4. Preferably, the deaminated protein of the invention has a protein sequence spanning amino acids 5 to 73 of SEQ ID NOS: 2-4, or amino acids 5 to 73 of SEQ ID NO: 1. Preferably, the method of invention is roß, deaminated, having the protein sequence spanning amino acids 5 to 73 of SEQ ID NO: 3. This deaminated roast is characterized by having at least about 2 trunks of biological activity higher than unmodified human groß. , determined in the HSF test of the previous references. As described in W094 / 29341, similar modifications can be made to the KC, roa and rot proteins, which are useful in the methods of the invention. All these proteins are described in the literature and are known to the person skilled in the art. Preferred multimeric proteins useful in this invention include, dimers or multimers containing at least one deamininated chemokine protein and at least one other chemokine and another protein characterized by having the same type of biological activity. This other protein may be an additional deaminative chemokine, or another known protein. For example, a suitable dimer useful in the methods of the invention comprises two deaminated proteins as described above, preferably linked by disulfide bonds. A convenient multimer may be an aggregate of two or more groß deaminated proteins, particularly two proteins consisting of amino acids 5 to 73 of SEQ ID NO: 3. Alternatively, another dimer of the invention may be a groß deaminated protein of the invention in combination with a mature groß protein. Similarly, various combinations of dimers or other multimeric forms may contain a combination of mature or modified groß and other chemokines, such as KC, groa and grot proteins. For example, a groß deaminated protein of the invention can form a dimer with an unmodified mature groa protein. The person skilled in the art can obtain other convenient multimers using the modified chemokines of the invention. However, the use of multimeric forms of two or more different modified proteins, as defined herein, is useful in the method of this invention. The chemokine used in this method can also be a multimeric form of a modified chemokine as discussed above and another known mature protein. These proteins and monomers have been described in detail in the literature and can be synthesized, or produced recombinantly using conventional techniques and / or the techniques described in the International Patent Publication.

III. Pharmaceutical Compositions Conveniently, the chemokines useful in the method of the invention are used in the preparation of medicaments and / or are useful in the form of a pharmaceutical composition. In this way, the chemokines can be formulated in pharmaceutical compositions and administered in the same manner described for example in International Patent Publication Publications No. W090 / 02762 (March 22, 1990) and Publication No. W094 / 29341 (December 22). 1994). These pharmaceutical compositions or medicaments useful in the mobilization of hematopoietic stem cells contain a therapeutically effective amount of a mature chemokine., modified or multimeric, as defined herein, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical" includes veterinary applications of the invention. The term "therapeutically effective amount" refers to that amount of a chemokine, either in monomeric or multimeric form, which is useful for mobilizing stem cells in sufficient quantities to achieve the desired physiological effect. In general, a mature, modified or deaminated chemokine useful in the invention (eg grg.) Is administered in an amount of between about 0.01 ng / kg of body weight to about 1 g / kg of body weight, and preference approximately 0.01 ng / kg body weight up to 10 mg / kg body weight per dose. Conveniently, when a multimeric chemokine is used in the method of the invention, the medically or composition contains amounts of the multimeric protein at the lower end of this scale. Preferably, these pharmaceutical compositions are administered by injection to human subjects and other mammals. However, administration may be by any appropriate internal route, and may be repeated as necessary, for example one to three times a day for one day to approximately one week. Suitable pharmaceutical carriers are well known to those skilled in the art and can be easily selected. Currently, the preferred vehicle is saline solution. Optionally, the pharmaceutical tests of the invention may contain other active ingredients or be administered in conjunction with other therapeutic agents. Suitable optional ingredients or other therapeutic agents include those conventional to treat conditions of this nature, for example, other anti-inflammatories, diuretics and immunosuppressants, among others. Conveniently, these modified chemokines are particularly suitable for administration in conjunction with the colony stimulating factor.

IV. Methods for Mobilizing Hematopoietic Stem Cells The invention provides improved methods of treating conditions characterized by immunosuppression or lower number of hepatopoietic stem cells and differentiated cells thereof, including, without limitation, inflammation, fever, viral, fungal and bacterial infections, cancer, myelopoietic malfunction, disorders of hematopoiesis, aplastic anemia and autoimmune diseases, and conditions characterized by low production and / or differentiation of hematopoietic cells and / or bone marrow. This method includes administering to a selected mammal a pharmaceutical composition of the invention. Preferably, this composition is administered together with, or contains, a colony stimulating factor. Suitable sources of colony stimulating factor are well known and include, for example, GM-CSF, M-CSF, G-CSF and IL-3, natural, synthetic and recombinant. In another preferred embodiment, a deamininated chemokine useful in the invention can be administered in vivo, and allowed to act synergistically with natural colony stimulating factors found in a selected patient. In a preferred embodiment, the method of the invention utilizes the deaminated chemokines described herein in conjunction with GM-CSF (or G-CSF). The use of a modified chemokine, such as a roß deaminated, according to the method of the invention, in combination with CSF (this combination has been observed to be synergistic) allows the administration of lower doses of CSF to a patient, reducing the extremely unpleasant side effects caused by GM-CSF (G-CSF). Mature chemokines and modified or multimeric chemokines useful in the method of the invention are characterized by the ability to mobilize hematopoietic stem cells when administered alone, or by having synergistic activity in the stimulation of hematopoiesis when administered in vivo. in vitro with another hematopoietic factor, such as a colony stimulating factor or a growth factor, or combined with natural circulation CSFs, or administered in a protocol with chemotherapy. In one embodiment, the invention provides a method for mobilizing hematopoietic stem cells in an animal, administering to an animal an effective amount of the composition or medicament containing a mature chemokine selected from human roß [SEQ ID NO: 3], human roa [ SEQ ID N0: 2], human grot [SEQ ID NO: 4], and murine KC [SEQ ID NO: 1]. In another embodiment of this invention, a method for mobilizing hematopoietic stem cells in an animal includes administering to an animal an effective amount of a modified protein derived from a chemokine selected from groß, groa, rot, and KC. A method for mobilizing haematopoietic stem cells in an animal is provided as a preferred embodiment, by administering to an animal an effective amount of a modified protein derived from human roß chemokine [SEQ ID NO: 3]. In another aspect, the present invention provides a method for mobilizing hematopoietic stem cells in an animal, comprising administering to an animal an effective amount of a multimeric protein, comprising an association of at least one chemokine as described above and a second one. chemokine In the practice of the hematopoietic stem cell mobilization method, the term "effective amount" of these proteins can be defined as that amount which, when administered to a patient by an appropriate means, mobilizes hematopoietic stem cells and increases the number of stem cells hematopoietic in the peripheral blood. It is expected that this amount is higher than the amount required to stimulate the growth or development of hematopoietic progenitor cells. The effective amount increases in the circulation the amount of cells that differ from the hematopoietic stem cells in applicable clinical or veterinary situations. A convenient effective amount may be from about 0.01 ng / kg to 10 mg / kg body weight per dose. Suitable means of administration for mobilizing stem cells include, without limitation, bolus injection or increasing administration of the effective amount by injection, intravenous drip, or any other appropriate internal route. The dosages may be repeated as necessary, for example one to three times a day for one day to approximately one week. Additionally, the method of this invention, which employs the mature chemokines or the modified or multimeric chemokines identified above, can be used in transplantation regimens of peripheral blood hematopoietic stem cells. For example, following an optional initial dose of a chemotherapeutic agent, the mature chemokines or the modified or multimeric chemokines identified above are administered instead of the CSFs now used to mobilize hematopoietic stem cells from the bone marrow into the peripheral circulation for collection. , as well as for its readministration after high doses of chemotherapy. Suitable chemotherapy agents include, without limitation, well-known agents such as cyclophosphamide ida, cisplatin, ARA-C, 5-fluorouracil, ethopside, epirubicin, carboplatin, busulfan, mitoxantrone and carmustine.

When administered with the chemokines according to this invention, the amounts of chemotherapeutics are the amounts used conventionally, ie, approximately 1.2 g / m2 of ethoside, 800 μg / m2 of ARA-C, 200 mg / kg of cyclophosphamide, etc. . For such dosages, see Hass et al., Seminars in Oncol., 21_: 19-24 (1994), incorporated herein by reference. The chemokines identified above can be used to complement the CSFs conventionally used in treatment regimens. Alternatively, the chemokines identified above can be used in combination with therapies with other hematopoietic regulatory biomolecules, such as the molecules involved in hematopoiesis referred to above, or with growth factors, conventional pharmaceutical agents and / or drugs, for the same purposes. Suitable sources of said growth factors are well known and include, without limitation, natural, synthetic and recombinant GM-CSF, G-CSF, stem cell factor, and Flt-3 ligand. Other suitable biomolecules include (S) -5-oxo-L-prolyl-a-glutamyl-La-aspartyl-Nß- (5-amino-l-carboxypentyl) -8-oxo-N7- [N-CN- (5- oxo-L-prolyl) -La-glutamuil} -L-α-aspartyl] -L-threo-2,7,8-triaminooctanoyl-lysine [(pGlu-Glu-Asp) 2-Sub ~ (Lys) 2] [Pelus et al., Exp. Hematol., 22: 239-247 (1994)]. The person skilled in the art can easily select other pharmaceutical agents and other drugs for their co-administration.

The advantages of the use of this invention in replacement, or in conjunction with the traditional methods of transplantation of hematopoietic stem cells from peripheral blood, is that a faster recovery of PMNs and / or platelets occurs than with bone marrow transplantation; the risk of infection is reduced and the method allows for potentially higher curative doses of chemotherapy, or to administer a series of intensified dose chemotherapies. The following examples are illustrative only and do not limit the scope of the invention.

EXAMPLE 1 PROOF OF MOBILIZATION Using known techniques, chemokines derived from KC [SEQ ID N0: 1], groß [SEQ ID N0: 3] and grot [SEQ.

ID N0: 4], including modified and multimeric chemokines. See, for example W094 / 29341 for further discussion regarding the preparation of said chemokines. The ability of these chemokines to mobilize hematopoietic stem cells in mice is analyzed. Each chemokine is analyzed in concentrations of 50, 10 and 2 μg / mouse and is administered by subcutaneous, intramuscular, intraperitoneal, intravenous or oral route. The chemokine mobilization kinetics of the hematopoietic stem cells are monitored at 15-minute intervals for a period of 60 minutes, extracting blood samples by cardiac puncture of the mice. Mobilized hematopoietic stem cells are fractionated and collected by separation on a Lympholite-Mtm density gradient. The cells are washed for future use. The elements of the mature blood cells are enumerated using a Technicon ™ Hl hematology analyzer, equipped with veterinary software. The mobilization of mature inflammatory cells, such as polymorphonuclear cells (PMN), eosinophils and basophils, is taken into account when assessing the overall potential inflammatory profile. To monitor the premature and late hematopoietic progenitor cells, a UFC-GM test is carried out on the blood samples collected during the mobilization phase, that is, the colony forming units (CFU-GM) are determined on days 7 and 14. Cells are adjusted to 10 6 cells / ml in McCoys medium without serum. A single layer agar system consisting of McCoys medium enriched with nutrients (NaHCO3, pyruvate, amino acids, and HEPES buffer), 0.3% Bactoagar, and 15% fetal bovine serum is used. Cells from the blood samples (final concentration 105 cells / ml). The agar plates were incubated at 37 ° C, 7.5% CO2 for 7 to 14 days. Colonies of proliferating cells (UFC-GM) are counted using a microscope. In addition, highly proliferative potential hematopoietic progenitors (PPH) are counted in CFU cultures on day 14. Included in these studies is the IL-8 chemokine that mobilizes hematopoietic stem cells as a single factor, as a positive control. Preliminary experiments have shown that the administration of roß [SEQ ID NO: 3] results in a dose-dependent mobilization of GM-CFU, similar to the results with the control, modified groß, the truncation protein of 4 terminal amino acids ( aa 5-73) de groß, mobilized significantly greater numbers of hematopoietic progenitor cells than groß (amino acids 1-73) or IL-8. No significant changes (more than 3-fold) were seen in mature cell elements in mice treated with groß, indicating specific mobilization of hematopoietic progenitor cells. This result demonstrates that modified deaminated chemokines may have increased mobilization characteristics compared to mature proteins.

EXAMPLE 2 MOBILIZATION TEST IN COMBINATION WITH HEMATOESTIMULANTS Hematoestimulants are analyzed in combination with the chemokines previously identified as mobilization factors. Hematoestimulants include G-CSF, GM-CSF, (S) -5-oxo-L-prolyl-a-glutamyl-La-aspartyl-N8- (5-amino-l- ca rboxipen tyl) -8-oxo-N7 - [N- £ N- (5-bear-L-prolyl) -La-glu tamil} -L-α-aspartyl] -L-reo-2,7,8-triaminooctanoyl-lysine [(pGlu-Glu-Asp) 2-Sub- (Lys) 2] [Pelus, cited above] and ligand of FLT-3. Any G-CSF imine, i.e., a hematoestimulant other than a CSF such as G-CSF or GM-CSF, but having haematopoietic activity can be used. In combination tests, the hematoestimulant is administered, for example G-CSF, at 50 μg / kg to mice, four days before the chemotains or modified or multimeric chemokines derived from KC [SEQ ID N0: 1], roß [SEQ ID N0: 3] and grot [SEQ ID N0: 4], As in Example 1, the dose of chemokine and the time of blood collection are varied. A CFU-GM test is carried out as described in Example 1, with SCF, IL-1 and GM-CSF as the source of colony stimulating activity. As in example 1, mature cell elements of the blood, premature and late progenitors were measured. Combination studies with pretreatment with hematoestimulant use MlP-la as a positive control.

EXAMPLE 3 MODEL OF TRANSPLANT OF PERIPHERAL BLOOD STEM CELLS MURINA A) Mobilization of primitive stem cells from repopulation in the long term. The following experiment was carried out in vivo in a stem cell transplant model to determine whether ro-truncated N-terminally [aa 5-73 of SEQ ID N0: 3; called roß-T] mobilizes primitive stem cells from repopulation in the long term. In this model, mice treated with gamma radiation are bone marrow cell receptors. The mice are followed for 100 days to determine their survival. The capacity of blood stem cells collected from mice treated with PBS, groß-T (50 μ to 15-30 min.), G-CSF (1 μ / mouse twice daily x 4), or G-CSF, then roß-T to rescue the mice, otherwise irradiated lethally. Blood mononuclear cells (up to 1E + 6) are collected from mice treated with PBS protein 0-10% of the mice 100 days after transplantation. Mice that received marrow cells as a positive control test had a 100% survival at day 100. Mobilized blood cells (1E + 6 cells / mouse) collected from mice treated with groß-T alone, protected 70% of the receivers. Mobilized blood cells (1E + 6 cells / mouse) collected from donors treated with G-CSF protected 80% of the receptors. Mobilized blood cells collected from donors treated with G-CSF and groß-T mobilize larger numbers of repopulation cells than G-CSF alone. B) Mobilization of peripheral blood stem cells. The rate at which the peripheral blood stem cells mobilized by groß-T recovered mature blood cell lines in an irradiated host was evaluated. 1E + 6 low density peripheral blood cells (LDPBC) were injected into irradiated recipients and bled by cardiac puncture on days 7 to 19 after radiation. LDPBC from different groups were collected under optimal conditions for mobilization of UFC-GM. The groups compared in this experiment were PBS, roß-T alone (50 ag, 15 min), G-CSF (twice daily x 5 days, 1 μ / mouse, alone), and groß-T + G-CSF . Normal mice were bled daily for comparison with the transplanted animals. Mice that received a transplant from donors treated with PBS failed to recover their mature blood cell elements and died. The rate of neutrophil recovery in the mice that received cells mobilized by trunked groß was faster than that of those receiving mobilized G-CSF cells. Mice transplanted with LDPBC mobilized by the combination of groß-T + G-CSF resulted in a higher neutrophil recovery rate than cells mobilized with groß-T. Recovery of the platelet count in these same mice followed the same pattern: groß-T + G-CSF > groß-T > G-CSF > > PBS. However, on day 19, the platelet counts were still far from returning to normal values. These data indicate that blood stem cells mobilized with groß-T are grafted into recipient mice, with resultant rates of neutrophil and platelet recovery equal to or better than for stem cells mobilized with G-CSF. Many modifications and variations of the present invention are included in the specification mentioned above and are expected to be obvious to the person skilled in the art. Said modifications and alterations to the compositions and methods of the present invention are considered covered within the scope of the appended claims.

LIST OF SEQUENCES (l) GENERAL INFORMATION (i) APPLICANT: SmithKine Beecham Corporation Pelus, Louis M. King, Andrew G. (ii) TITLE OF THE INVENTION: Method of Hematopoietic Stem Cell Mobilization (iii) SEQUENCE NUMBER: 4 (iv) DIRECTION FOR CORRESPONDENCE: A) ADDRESS: SmithKine Beecham Corporation - Corporate Patents B) STREET: 709 Swedeland Road C) CITY: King of Prussia D) STATE: Pennsylvania E) COUNTRY: EU F) POSTAL CODE: 19406-2799 (V) METHOD OF READING ON THE COMPUTER: A) TYPE OF MEDIUM: Flexible disk B) COMPUTER: PC compatible with IBM C) OPERATING SYSTEM: PC-DOS / MS-DOS D) SOFTWARE: Patentln Reléase # 1.0, version # 1.30 (vi) CURRENT APPLICATION DATA: A) APPLICATION NUMBER: WO B) DATE OF SUBMISSION: C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: A) NUMBER OF APPLICATION: US 08 / 547,262 B) DATE OF SUBMISSION: October 24, 1995 (viii) INFORMATION ON POWDER / AGENT A) NAME: Hall, Linda E. B) REGISTRATION NUMBER: 31,763 C) REFERENCE / DOCUMENT NUMBER: SBC P50161-2PCT (ix) INFORMATION ON TELECOMMUNICATIONS: A) TELEPHONE: 215-270-5015 B) TELEFAX: 215-270-5090 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 72 amino acids B) TYPE: amino acid C) TYPE OF CHAIN: D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: protein ( xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Pro Xle? As "Clu Leu ^ ^ ^ Leu Gin ^ Het 15 Wing 61 and Ile His L | u Lys? sn llß ßln ß« Lßu, ys ^ ^ ^ «« «Ly Pro His cy Thr ßlB ^ Gau ^? Le ^ ^ ^ ° A n «and Ar, ßlu ^ ^ Leu Asp pro ^ ^ ^ ^ ^ ^ Lys lie val Gln Lys M.t Leu Lys Qly ^ pro ^ 2) INFORMATION FOR SEO ID NO: 2: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 73 amino acids B) TYPE: amino acid C) TYPE OF CHAIN: D) TOPOLOGY: unknown ii) TYPE OF MOLECULE: protein xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: Ala Ser Val Ala Thr Glu Leu Arg Cys ßln Cys Leu Gln Thr Leu 5 10 15 Gln Gly lie His Pro Lys Asn He Gln Ser Val Asn Val Lys Ser 20 25 30 Pro Gly Pro His Cys Wing Gln Thr Glu Val He Wing Thr Leu Lys 35 40 45 Asn Gly Arg Lys Lys He He Glu Met Leu Asn Ser Asp Lys Ser Asn 65 70) INFORMATION FOR SEQ ID NO: 3: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 73 amino acids B) TYPE: amino acid C) TYPE OF CHAIN : D) TOPOLOGY: unknown ii) TYPE OF MOLECULE: protein xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Ala Pro Leu Ala Thr Glu Leu Arg Cys Gln Cys Leu Gln Thr Leu 5? O 15 Gln Gly He His Leu Lys Asn He Gln Ser Val Lys Val Lys Ser 20 25 3o Pro Gly Pro His Cys Wing Gln Thr Glu Val He Wing Thr Leu Lys 35 40 45 Asn Gly Gln Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Lys 50 55 6th Lys He He Glu Lys Met Leu Lys Asn Gly Lys Ser Asn 65 70 ) INFORMATION FOR SEQ ID NO: 4: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 73 amino acids B) TYPE: amino acid C) TYPE OF CHAIN: D) TOPOLOGY: unknown ii) TYPE OF MOLECULE: protein i) DESCRIPTION OF SEQUENCE : SEQ ID NO: 4: Ala Ser Val Val Thr Glu Leu Arg Cys Gln Cys Leu Gln Thr Leu 1 5 10 15 Gln Gly He His Leu Lys Asn He Gln Ser Val Asn Val Arg Ser 20 25 30 Pro Gly Pro His Cys Wing Gln Thr Glu Val He Wing Thr Leu Lys 35 40 45 Asn Gly Lys Lys Wing Cys Leu Asn Pro Wing Being Pro Met Val Gln 50 55 60 Lys He He Glu Lys He Leu Asn Lys Gly Ser Thr Asn 65 70

Claims (12)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of a protein derived from a mammalian chemokine selected from the group consisting of (a) roa SEQ ID NO: 2, (b) groß SEQ ID NO: 3, (c) grot SEQ ID NO: 4; and (d) KC SEQ ID NO: 1, in the preparation of a medicament useful for mobilizing hematopoietic stem cells.

2. The use according to claim 1, characterized in that said chemokine is selected from the group consisting of: (a) mature groß; (b) amino acids 5 to 73 of SEQ ID NO: 3; (c) a multimeric chemokine protein comprising an association of two or more of (a) or (b); and (d) a multimeric chemokine protein comprising an association of (a) or (b) with a second chemokine.

3. The use according to claim 1, characterized in that said chemokine is selected from the group consisting of: (a) mature groa; (b) amino acids 5 to 73 of SEQ ID NO: 2; (c) a multimeric chemokine protein comprising an association of two or more of (a) or (b); and (d) a multimeric chemokine protein comprising an association of (a) or (b) with a second chemokine.

4. The use according to claim 1, characterized in that said chemokine is selected from the group consisting of: (a) mature grot; (b) amino acids 5 to 73 of SEQ ID NO: 4; (c) a multimeric chemokine protein comprising an association of two or more of (a) or (b); and (d) a multimeric chemokine protein comprising an association of (a) or (b) with a second chemokine.

5. The use according to claim 1, characterized in that said chemokine is selected from the group consisting of: (a) mature KC; (b) amino acids 5 to 72 of SEQ ID NO: 1; (c) a multimeric chemokine protein comprising an association of two or more of (a) or (b); and (d) a multimeric chemokine protein comprising an association of (a) or (b) with a second chemokine.

6. The use according to claims 1 to 5, characterized in that said chemokine comprises between about 0.01 ng to about 1 g of said medicine.

7. The use according to claims 1-5, characterized in that said medicament is coadministered with a growth factor or another regulatory biomolecule and atopoietic.

8. The use according to claim 7, characterized in that said growth factor is selected from the group consisting of GM-CSF, G-CSF, mother cell factor, and Flt-3 ligand.

9. The use according to claim 7, characterized in that said biomolecule is (S) -5-oxo-L-prolyl-a-glutamyl-La-as? Artil-N- (5-amino-l-carboxypentyl) -8-oxo-N7- [N-. { N- (5-oxo-L-p ropil) -L-a-glutamyl} -La-aspartyl] -Lt reo-2, 7,8-triaminooctanoyl-lysine [(pGlu-Glu-Asp) 2 -Sub- (Lys) 2] - 10.- The use according to claim 1, characterized in that The drug is used in the treatment of a patient receiving transplantation of peripheral blood hematopoietic stem cells. 11. The use according to claim 10, characterized in that the patient receives a dose of a chemotherapeutic agent selected before treatment with the drug; hematopoietic stem cells are collected from the peripheral blood of the treated patient; a chemotherapeutic agent is administered to the patient; and the collected cells are reinfused to the patient. 12. The use according to claim 11, characterized in that said chemotherapeutic agent is selected from the group consisting of cyclophosphamide, cisplatin, ARA-C, 5-fluorouracil, ethopside, epirubicin, carboplatin, busulfan, mitoxantrone and carmustine.