AU712235B2 - Method of mobilizing hematopoietic stem cells - Google Patents

Description

0 WO 97/15595 PCT/US96/17074 METHOD OF MOBILIZING HEMATOPOIETIC STEM CELLS Field of the Invention The present invention relates generally to methods for mobilizing hematopoietic stem cells.

Background of the Invention All the members of the intercrine or chemokine family are basic heparin-binding polypeptides which have four cysteine residues which form two disulfide bridges.

All these proteins which have been functionally characterized appear to be involved in proinflammatory and/or restorative functions.

In clinical situations for the use of high dose chemotherapy, the biomolecule of choice has been G-CSF.

Generally, in such treatment, patients are primed with a low dose of a chemotherapeutic agent like cyclophosphamide. During the remission, the patient is treated with a CSF, such as G-CSF, which causes eventual mobilization of cells from the bone marrow to the peripheral circulation for harvesting of leukophoresed blood. The patient is thereafter administered a high dose of chemotherapy to induce clinical remission of their cancer. The resultant bone marrow failure is treated by infusion of the stored blood cells collected previously.

This procedure may be modified, by the omission of the initial dose of chemotherapy and/or alternate blood collection protocols.

While the use of these hematopoietic stem cell transplantation techniques looks promising, multiple apheresis procedures are required to harvest sufficient stem cells for successful engraftment to treat severe WO 97/15595 PCT/US96/17074 2 myelosuppression when G-CSF is used alone [see, e.g., Bensinger et al, Blood, 81:3158 (1993) and R. Haas et al, Sem. in Oncology, 21:19 (1994)]. Thus, despite these significant advances and the availability of certain regulatory biomolecules, delayed recovery of hematopoiesis remains an important source of morbidity and mortality for myelosuppressed patients.

There exists a continuing need in the art for compositions and methods to enhance hematopoietic recovery, particularly in cases of chemotherapy associated myelosuppression.

Summary of the Invention In one aspect, the present invention provides for the use of a chemokine in the preparation of a medicament for the stimulation of hematopoietic stem cells. This chemokine includes proteins derived from KC, groB, groa, and groy, including mature, modified, and multimeric forms of these chemokines.

In yet 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 described further in the following detailed description of the preferred embodiments thereof.

Brief Description of the Drawings Fig. 1 is a graph demonstrating the effect of groB (amino acids 1-73 of SEQ ID NO: 3) in the single agent mobilization assay of Example 1.

Fig. 2 is a graph demonstrating the effect of modified groB (amino acids 5-73 of SEQ ID NO: 3) in the single agent mobilization assay of Example 1.

WO 97/15595 PCT/US96/17074 3 Fig. 3 is a bar graph demonstrating the comparison of phosphate buffered saline (PBS), IL-8, groB (amino acids 1-73; SEQ ID NO:3) and modified groo (amino acids 5-73 of SEQ ID NO:3) in the single agent mobilization assay.

Detailed Description of the Invention The present invention provides modified proteins, specifically chemokines, associated with inflammatory responses, hematopoiesis and myelopoiesis, which modified proteins are characterized by having enhanced biological activity as compared to the corresponding unmodified or untruncated mature 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 the naturally occurring chemokines and modified chemokines, which are characterized by having synergistic activity in stimulating hematopoiesis when administered in vivo and in vitro with another hematopoietic factor, such as a colony stimulating factor, or combined with naturally circulating CSFs.

The term "mature chemokines" also known as "intercrines", as used herein defines the proteins conventionally referred to in the art as KC, groa, gro, and gro7. For convenience, the amino acid sequences of the murine protein KC which contains 72 residues is WO 97/15595 PCT/US96/17074 4 provided in SEQ ID NO:1. These sequences are available from Genbank, accession number J04596. The sequences of the human protein groa (aa 1-73) are provided in SEQ ID NO:2. The sequences of the human protein groB (amino acids 1-73) are provided in SEQ ID NO: 3. The sequences of the human protein groy are provided in SEQ ID NO:4.

The cDNA and amino acid sequences of gro7 are also provided in International Patent Application, Publication No. WO 92/00326 (Jan. 9, 1992). These groy sequences have further been published in International Patent Application, Publication No. WO 94/29341 (December 22, 1994), which is incorporated by reference herein.

The term "modified chemokines" is defined as in the above-referenced International Application. The modified chemokines are derived from KC, gro3, groa, and groy, more preferably from groo, groa, and groy, and most preferably from groo. The modified chemokines include desamino proteins characterized by the elimination of between about 2 to about 8 amino acids at the amino terminus of the mature protein. These desamino chemokines useful in the method of the invention are preferably characterized by removal of about 2 to about 8 amino acids from the amino terminus of the mature protein. Most preferably, the modified chemokines are characterized by removal of the first 4 amino acids at the amino- terminus. Optionally, particularly when expressed recombinantly, the desamino chemokines useful in this invention may contain an inserted N-terminal Met.

The N-terminal methionine which is inserted into the protein for expression purposes, may be cleaved, either during the processing of the protein by a host cell or synthetically, using known techniques. Alternatively, if so desired, this amino acid may be cleaved through enzyme digestion or other known means.

WO 97/15595 PCT/US96/17074 Also included by the term modified chemokine are analogs or derivatives of these proteins which share the biological activity of the mature protein. As defined herein, such analogs and derivatives include modified proteins also characterized by alterations made in the known amino sequence of the proteins, the proteins provided in SEQ ID NOS: 1-4. Such analogs are characterized by having an amino acid sequence differing from that of the mature protein by 8 or fewer amino acid residues, and preferably by about 5 or fewer residues.

It may be preferred that any differences in the amino acid sequences of the proteins involve only conservative amino acid substitutions. Conservative amino acid substitutions occur when an amino acid has substantially the same charge as the amino acid for 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 in the sequence which may alter the stability of the protein, or permit it to be expressed in a desired host cell may be preferred. Another characteristic of these modified proteins may be enhanced biological activity in comparison to the mature protein.

By the term "multimeric protein" or "multimer" is meant herein multimeric forms of the mature and/or modified proteins useful in this invention, 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. Multimers may form naturally upon expression or may be constructed into such WO 97/15595 PCT/US96/17074 6 multiple forms. Multimeric chemokines may include multimers of the same modified chemokine. Another multimer may be formed by the aggregation of different modified proteins. Still 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 desamino chemokine protein and at least one other chemokine or other protein characterized by having the same type of biological activity. This other protein may be an additional desamino chemokine, or another known protein.

II. Proteins Useful 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. Desirably, these chemokines are selected from KC, groa, gro3 and groy, and most preferably the chemokine is grop.

In one preferred embodiment, the method of the invention utilizes a desamino 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 desamino protein of the invention has a protein sequence spanning amino acids 5 to 73 of SEQ ID NOS: 2-4, or amino acids to 72 of SEQ ID NO:1. Most preferably, the method of the invention is desamino gro3, which has the protein sequence spanning amino acids 5 to 73 of SEQ ID NO:3.

WO 97/15595 PCT/US96/17074 7 This desamino-grog is characterized by having at least about two logs higher biological activity than unmodified, human grog, as determined in the abovereferences HSF assay.

As described in WO94/29341, similar modifications can be made to the KC, groa and groy proteins which are useful in the methods of the invention. These proteins are all described in the literature and are known to those of skill in the art.

Preferred multimeric proteins useful in this invention include, dimers or multimers containing at least one desamino chemokine protein and at least one other chemokine or other protein characterized by having the same type of biological activity. This other protein may be an additional desamino chemokine, or another, known protein. For example, a desirable dimer useful in the methods of the invention comprises two desamino proteins as described above, preferably linked by disulfide bonds. A desirable multimer may be an aggregate of two or more desamino groB proteins, particularly two proteins consisting of amino acids 5-73 of SEQ ID NO:3. Alternatively, another dimer of the invention may be a desamino groB protein of the invention in combination with a mature groB protein. Similarly, various combinations of dimers or other multimeric forms may contain a combination of the mature or modified groB and other chemokines, such as the KC, groa and groy proteins. For example, a desamino grop protein of the invention may form a dimer with an unmodified mature groa protein. One of skill in the art may obtain other desirable multimers using the modified chemokines of the invention. However, the use of multimeric forms of two or more different modified proteins as defined herein are WO 97/15595 PCT/US96/17074 8 useful in the method of this invention. The chemokine employed in this method may 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 may be synthesized, or produced recombinantly, using conventional techniques and/or the techniques described in International Patent, Publication No. W094/29341.

III. Pharmaceutical Compositions Desirably, 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. Thus, the chemokines can be formulated into pharmaceutical compositions and administered in the same manner as described in, e.g., International Patent Applications, Publication No. WO 90/02762 (Mar. 22, 1990) and Publication No. W094/29341 (Dec. 22, 1994).

These medicaments or pharmaceutical compositions useful in the mobilization of hematopoietic stem cells contain a therapeutically effective amount of a mature, modified or multimeric chemokine as defined herein and an acceptable pharmaceutical 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, whether in monomeric or multimeric form, which is useful for mobilizing stem cells in sufficient amounts to achieve the desired physiological effect.

WO 97/15595 PCT/US96/17074 9 Generally, a mature, modified or desamino chemokine useful in the invention grog) is administered in an amount between about 0.01 ng/kg body weight to about 1 g/kg body weight and preferably about 0.01 ng/kg body weight to 10 mg/kg body weight per dose.

Desirably, when a multimeric chemokine is used in the method of the invention, the medicament or composition contains amounts of the multimeric protein at the lower end of this range. Preferably, these pharmaceutical compositions are administered to human or other mammalian subjects by injection. However, administration may be by any appropriate internal route, and may be repeated as needed, e.g. one to three times daily for between 1 day to about one week.

Suitable pharmaceutical carriers are well known to those of skill in the art and may be readily selected.

Currently, the preferred carrier is saline, Optionally, the pharmaceutical assays of the invention may contain other active ingredients or be administered in conjunction with other therapeutics. Suitable optional ingredients or other therapeutics include those conventional for treating conditions of this nature, e.g.

other anti-inflammatories, diuretics, and immune suppressants, among others. Desirably, these modified chemokines are particularly well suited for administration in conjunction with colony stimulating factor.

IV. Methods for Mobilizing Hematopoietic Stem Cells The invention provides improved methods of treating conditions characterized by immunosuppression or low numbers of hematopoietic stem cells and cells differentiated therefrom, including, without limitation, inflammation, fever, viral, fungal, and bacterial WO 97/15595 PCT/US96/17074 infections, cancer, myelopoietic dysfunction, hematopoiesis disorders, aplastic anemia, and autoimmune diseases, and conditions characterized by low production and/or differentiation of hematopoietic and/or bone marrow cells. This method involves 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, natural, synthetic and recombinant GM-CSF, M-CSF, G-CSF and IL-3. In another preferred embodiment, a desamino chemokine useful in the invention can be administered in vivo, and permitted to act in synergy with the natural colony stimulating factors found in a selected patient.

In one preferred embodiment, the method of the invention uses the desamino chemokines described herein in conjunction with GM-CSF (or G-CSF). The use of a modified chemokine, such as a desamino gro,, according to the method of the invention in combination with CSF (this combination has been observed to have synergy) permits lower doses of CSF to be administered to a patient, reducing the extremely unpleasant side effects caused by GM-CSF (G-CSF).

The mature chemokines and the 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 stimulating hematopoiesis when administered in vivo and in vitro with another hematopoietic factor, such as a colony stimulating factor or a growth factor, or combined with naturally circulating CSFs, or administered in a protocol with chemotherapy.

WO 97/15595 PCT/US96/17074 11 In one embodiment, the invention provides a method for mobilizing hematopoietic stem cells in an animal by administering to an animal an effective amount of the composition or medicament containing a mature chemokine selected from human grof [SEQ ID NO: human groa [SEQ ID NO:2], human groy [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 involves administering to an animal an effective amount of a modified protein derived from a chemokine selected from gro3, groa, groy, and KC. As preferred embodiment there is provided a method for mobilizing hematopoietic stem cells in an animal by administering to an animal an effective amount of a modified protein derived from chemokine human grog [SEQ ID NO: 3].

In still 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, which comprises an associated of at least one chemokine as described above and a second chemokine.

In the practice of the method of mobilizing hematopoietic stem cells, the term "effective amount" of these proteins may be defined as that amount which, when administered to a patient by suitable means, mobilizes hematopoietic stem cells and increases the number of hematopoietic stem cells in the peripheral blood. This amount is expected to be higher than the amount required to stimulate the growth or development of hematopoietic progenitor cells. The effective amount increases in the circulation the cells which are differentiated from the hematopoietic stem cells in applicable clinical or veterinary situations. A desirable effective amount may be about 0.01 ng/kg to 10 mg/kg body weight per dose.

WO 97/15595 PCT/US96/17074 12 Suitable means of administration for mobilizing stem cells include, without limitation, bolus injection or incremental administration of the effective amount by injection, i.v. drip, or any other appropriate internal route. Dosages may be repeated as needed, e.g. one to three times daily for between 1 day to about one week.

Additionally, the method of this invention employing the mature chemokines, or modified or multimeric chemokines identified above may be used in peripheral blood hematopoietic stem cell transplantation regimens. For example, following an optional initial dose of a chemotherapeutic agent, the mature chemokines or modified or multimeric chemokines identified above are administered in place of the CSFs now used to mobilize hematopoietic stem cells from the bone marrow to the peripheral circulation for harvesting, as well as for readministration following high doses of chemotherapy.

Suitable chemotherapy agents include, without limitation, the well-known agents such as cyclophosphamide, cisplatinum, ARA-C, 5-fluorouracil, etopside, epirubicin, carboplatin, busulfan, mitoxantrone and carmustine. When administered with the chemokines according to this invention, the amounts of the chemotherapeutics are those amounts conventionally employed, about 1.2g/m 2 etopside, 800 gg/m 2 ARA-C, 200 mg/kg cyclophosphamide, etc. See for such dosages Hass et al, Seminars in Oncol., 21:19-24 (1994), incorporated herein by reference.

The chemokines identified above may be used to complement the conventionally used CSFs in treatment regimens. Alternatively, the chemokines identified above may be used in combination therapies with other hematopoietic regulatory biomolecules, such as the molecules involved in hematopoiesis above-referenced, or WO 97/15595 PCT/US96/17074 13 with growth factors, conventional pharmaceuticals and/or drugs, for the same purposes. Suitable sources of such 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 aspartyl-N- (5-amino-l-carboxypentyl)-8-oxo-N 7 oxo-L-prolyl)-L-a-glutamuyl}-L-a-aspartyl]-L-threo-2,7,8triaminooctanoyl-lysine [(pGlu-Glu-Asp)2-Sub-(Lys)2] [Pelus et al, Exp. Hematol., 22:239-247 (1994)]. Still other pharmaceuticals and drugs for co-administration may be readily selected by one of skill in the art.

The advantages of the use of this invention in replacement or in conjunction with traditional methods of peripheral blood hematopoietic stem cell transplantation are that more rapid recovery of PMNs and/or platelets occur than with bone marrow transplantation, the risk of infection is reduced and the method permits potentially higher curative doses of chemotherapy, or a series of dose intensified chemotherapy to be administered.

The following examples are illustrative only and do not limit the scope of the invention.

Example 1 Mobilization Assay Chemokines derived from KC [SEQ ID NO:1, groB [SEQ ID NO:3] and groy [SEQ ID NO:4] including modified and multimeric chemokines are prepared using known techniques. See, WO94/29341 for additional discussion relating to the preparation of such chemokines. These chemokines are tested for the ability to mobilize hematopoietic stem cells in mice. Each chemokine is assayed in concentrations of 50, 10 and 2 gg/mouse and administered via subcutaneous, WO 97/15595 PCT/US96/17074 14 intramuscular, intraperitoneal, intravenous, or oral route. The kinetics of chemokine mobilization of hematopoietic stem cells are monitored in 15 minute intervals over a period of 60 minutes by collecting blood samples by cardiac puncture from the mice. The mobilized hematopoietic stem cells are fractionated and collected by separation over a Lympholyte-M" density gradient.

Cells are washed for future use.

Mature blood cell elements are enumerated using a Technicon HI hematology analyzer, equipped with veterinary software. Mobilization of mature inflammatory cells, such as polymorphonuclear (PMN) cells, eosinophils, and basophils are taken in to account when evaluating the overall potential inflammatory profile.

To monitor early and later hematopoietic progenitor cells, a CFU-GM assay is performed, i.e. blood samples collected during the mobilization phase are assessed for colony forming units (CFU-GM) at days 7 and 14. Cells are adjusted to 106 cells/ml in McCoys medium without serum. A single layer agar system consisting of McCoys medium enriched with nutrients (NaHCO 3 pyruvate, amino acids, vitamins and HEPES buffer), 0.3% Bacto agar, and 15% fetal bovine serum is used. Cells from the blood samples (final concentration of 105 cells/ml) are added to the agar system. The agar plates are incubated at 37 0 C, 7.5% CO 2 for 7-14 days. Colonies of proliferating cells (CFU-GM) are counted utilizing a microscope.

In addition, early hematopoietic high proliferative potential (HPP) progenitors, are counted in the day 14 CFU cultures.

The chemokine IL-8, which mobilizes hematopoietic stem cells as a single factor, is included in these studies as a positive control.

WO 97/15595 PCT/US96/17074 Preliminary experiments have shown that administration of groB [SEQ ID NO: 3] results in a dose dependent mobilization of CFU-GM, similar to the results with the control. Modified groB, the N-terminal 4 amino acid truncation protein (aa5-73) of groB mobilized significantly greater numbers of hematopoietic progenitor cells than groB (amino acids 1-73) or IL-8. No significant changes 3 fold) in mature cell elements were observed in groB treated mice, indicating specific mobilization of hematopoietic progenitor cells. This result demonstrates that the modified desamino chemokines may have enhanced mobilization characteristics compared to the mature proteins.

Example 2 Mobilization Assay in Combination with Hematostimulants Hematostimulants are assayed in combination with the chemokines identified above as mobilization factors. The hematostimulants include G-CSF, GM-CSF, carboxypentyl)-8-oxo-N 7 glutamuyl}-L-a-aspartyl]-L-threo-2,7,8-triaminooctanoyllysine [(pGlu-Glu-Asp) 2 -Sub-(Lys) 2 [Pelus, cited above] and FLT-3 ligand. Any G-CSF mimetic, a hematostimulant which is not a CSF like G-CSF or GM-CSF, but has hematopoietic activity, may be used.

In combination assays, the hematostimulant, G-CSF, is administered at 50 gg/kg to mice four days prior to the chemokines or modified or multimeric chemokines derived from KC [SEQ ID NO:1], groB [SEQ ID NO:3] and groy [SEQ ID NO:4]. As in Example 1, the dose of chemokine and time of blood collection is varied.

M -1 WO 97/15595 PCT/US96/17074 16 A CFU-GM assay is performed as described above in Example 1, with SCF, IL-1 and GM-CSF as the source of colony stimulating activity. Mature blood cell elements, early and later progenitors are measured as for Example 1.

Combination studies with hematostimulant pretreatment utilizes MIP-la as the positive control.

Example 3 Murine Peripheral Blood Stem Cell Transplantation Model A. Mobilization of Primitive Long Term Repopulating Stem Cells The following experiment was performed in an in vivo stem cell transplantation model to determine if N-terminally truncated groS [aa 5-73 of SEQ ID NO:3; termed groo-T] mobilizes primitive long term repopulating stem cells. In this model, gamma irradiated mice are recipients of bone marrow cells. Mice are followed for 100 days for survival.

The ability of blood stem cells collected from mice treated with either PBS, groo-T (50 gg at 15 min.), G-CSF (1 Ag/mouse BID x or G-CSF, then groo-T to rescue otherwise lethally irradiated mice.

Blood mononuclear cells (up to 1E+6) collected from PBS treated mice protein 0-10% of the mice 100 days post transplant. Mice receiving marrow cells as the assay positive control were at 100% survival as of day 100.

Mobilized blood cells (1E+6 cells/mouse) collected from mice treated with gro-T alone protected 70% of recipients. Mobilized blood cells (1E+6 cells/mouse) collected from G-CSF treated donors protected 80% of recipients. Mobilized blood cells collected from donors treated with G-CSF and gro3-T mobilize greater numbers of repopulating cells than G-CSF alone.

-0 WO 97/15595 PCT/US96/17074 17 B. Mobilization of Peripheral Blood Stem Cells The rate at which peripheral blood stem cells mobilized by gro$-T recovered mature blood cell lineages 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 7-19 post irradiation. LDPBC from the different groups were collected under optimal conditions for CFU-GM mobilization. The groups compared in this experiment were PBS, grof-T alone (50 Ag,, 15 min), G-CSF (BID x days, 1 pg/mouse alone), and groS-T G-CSF. Normal mice were bled daily for comparison to the transplanted animals.

Mice which received a transplant from PBS treated donors failed to recover mature blood cell elements and died. The rate of neutrophil recovery in the mice which received cells mobilized by truncated grop was faster than those who received G-CSF mobilized cells.

Mice transplanted with LDPBC mobilized by the combination of gro3-T G-CSF resulted in a faster neutrophil recovery rate than grop-T mobilized cells.

The recovery of platelet counts in these same mice followed the same pattern: grop-T G-CSF gro3-T G-CSF PBS. However, on day 19, platelet counts are still far from returning to normal values.

These data indicate that groS-T mobilized blood stem cells engraft in recipient mice, with resultant neutrophil and platelet recovery rates equal to or better than G-CSF mobilized stem cells.

WO 97/15595 PCT/US96/17074 18 Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

WO 97/15595 PCT/US96/17074 19 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: SmithKline Beecham Corporation Pelus, Louis M.

King, Andrew G.

(ii) TITLE OF INVENTION: Method of Mobilizing Hematopoietic Stem Cells (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: SmithKline Beecham Corporation Corporate Patents STREET: 709 Swedeland Road CITY: King of Prussia STATE: PA COUNTRY: USA ZIP: 19406-2799 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: WO FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/547,262 FILING DATE: 24-OCT-1995 (viii) ATTORNEY/AGENT INFORMATION: NAME: Hall, Linda E.

REGISTRATION NUMBER: 31,763 REFERENCE/DOCKET NUMBER: SBC P50161-2PCT (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 215-270-5015 TELEFAX: 215-270-5090 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 72 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: unknown WO 97/15595 PCT/US96/17074 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Ala Pro Ile Ala Asn Glu Leu Arg Cys Gin Cys 1 5 10 Ala Gly Ile His Leu Lys Asn Ile Gin Ser Leu 25 Ser Gly Pro His Cys Thr Gin Thr Glu Val Ile 40 Asn Gly Arg Glu Ala Cys Leu Asp Pro Glu Ala 55 Lys Ile Val Gin Lys Met Leu Lys Gly Val Pro INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 73 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Ala Ser Val Ala Thr Glu Leu Arg Cys Gin Cys 1 5 10 Gin Gly Ile His Pro Lys Asn Ile Gin Ser Val 25 Pro Gly Pro His Cys Ala Gin Thr Glu Val Ile 40 Asn Gly Arg Lys Ala Cys Leu Asn Pro Ala Ser 55 Lys Ile Ile Glu Lys Met Leu Asn Ser Asp Lys Leu Gin Thr Met Lys Val Leu Pro Ala Thr Leu Lys Pro Leu Val Gin Lys Leu Asn Ala Pro Ser Gin Thr Leu Val Lys Ser Thr Leu Lys Ile Val Lys Asn WO 97/15595 PCT/US96/17074 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 73 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Ala Pro Leu Ala Thr Glu Leu Arg Cys Gin Cys 1 5 10 Gin Gly Ile His Leu Lys Asn Ile Gin Ser Val 25 Pro Gly Pro His Cys Ala Gin Thr Glu Val Ile 40 Asn Gly Gin Lys Ala Cys Leu Asn Pro Ala Ser 55 Lys Ile Ile Glu Lys Met Leu Lys Asn Gly Lys INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 73 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Ala Ser Val Val Thr Glu Leu Arg Cys Gin Cys 1 5 10 Gin Gly Ile His Leu Lys Asn Ile Gin Ser Val 25 Pro Gly Pro His Cys Ala Gin Thr Glu Val Ile 40 Asn Gly Lys Lys Ala Cys Leu Asn Pro Ala Ser 55 Lys Ile Ile Glu Lys Ile Leu Asn Lys Gly Ser Leu Lys Ala Pro Ser Leu Asn Ala Pro Thr Gin Thr Leu Val Lys Ser Thr Leu Lys Met Val Lys Asn Gin Val Thr Met Asn Thr Leu Arg Ser Leu Lys Val Gin 22 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

*00 *0 0 0 *o .1 of 0* 0 31/08 '99 TUE 16:55 [TX/RX NO 98021

Claims (2)

1. 9-9 16!4 5 1# /1 P:outDD\.x-62 W81WI -23 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. Use of a chemokine selected from the group consisting of: mature gro3, amino acids 5 to 73 of SEQ ID NO: 3; a rnultimeiric chemokine protein which comprises an association of two or more of(a) or and a multimeric cliemokine protein which comprises an association of or (b) with a second chemokine; in the preparation of a medicament useful for mobilizing hematopoietic stem cells. 2. Use of chemokinue selected from the group consisting of: mature groa- amino acids 5 to 73 of SEQ ID NO: 2; a multimeric chemokine protein which comprises an association of two and/more :of or and a inultimeric chemokine protein which comprises an association of(a) or with a second chemokine; in the preparation of a medicamnent useful for mobilizing hemnatopoietic stem cells. 3. Use of chemokine selected from the group consisting of: mature gro ry amino acids 5 to 73 of SEQ ID NO: 4; a multimeric chemokine protein which comprises an association of two or more of and/or and a multimeric chemokine protein which comprises an association of or (b) with a second chemokine;, in the preparation of a medicamnent useful for mobilizing hematopoietic stem cells. 4. Use of a chemokine selected from the group consisting of:, mature KC; 31/08 '99 TUE 16:55 [TX/RX NO 9802]
2. 31- 8-S3fl5!4B 7/ 18 al- B-99:16!46 7/ 18 PjRDCD%75296.23V 26/81W9 -24 amino acids 5 to 72 of SEQ ID NO: 1; a multixneric chemokine protein Which comprises an association of two or more of and/or and a multimeric chemokine protein which comprises an association of or with a second chemokine; in the preparation of a medicamnent useful for mobilizing hematopoietic stein cells. Use according to claims 1-4 wherein said chemokine comprises between about O.0lng to about 1 g of said medicament. Use according to claims 1-4, wherein said medicament is co-administered with a 0 growth factor or other hcmatopoietic regulatory biomolecule. 7. Use according to claim 6 wherein said growth factor is selected from the group consisting of GM-CSF, G-CSF, stemn cell factor, and Flt-3 ligand. 8. Use according to claim 6 where said biomolecule is c-aspartyl-N 8 -(5-amino- 1-caboxypentyl)-8-oxo-N 7 C*C**C L-cc-aspartyl]-L-threo-27,8-trianfloocfanoyl-lyilC [(pGlu-Glu-Asp)Z-Sub-(Lys) 2 9. Use according to any one of claims 1 to 4 wherein the medicament is used in the treatment of a patient receiving peripheral blood hematopoietic stem cell transplantation. Use according to claim 9 wherein the patient has received a dose of a selected chemotherapeutic agent prior to treatment with the medicament; the hematopoietic stemn cells from the peripheral blood of the patient treated are harvested; a chemotherapeutic agent is administered; and the patient is reitifused with the harvested cells. 11. Use according to claim 10 wherein said chemotherapeutic agent is selected from the group consisting of cyclophosphamirle, cisplatinum, ARA-C, 5-fluorouracil, etopside, epirubicin, carboplatin, busulphan. initoxantrone and carmustine. 31/08 '99 TUE 16:55 [TX/RX NO 9802] 31- 8-8315!45 8/ 18 31- B-99:16:46 B/ 18 r \0P~kDCWSV996XM- 7AIIII99 12. The use according to claim 6 wherein said growth factor is G-CSF. 13. The use according to claim 1 wherein the chemokine is amino acids 5 to 73 of SEQ ID NO: 3. 14. The use according to claim 2 wherein the chemnokine is a multimeric chemokine protein comprising an association of two or more of amino acids 5 to 73 of SEQ ID NO: 3. Method of mobilizing hematopoietic stem cells comprising administering to a patient :10 an effective amount of a chemokine selected from the group consisting of: mature gro/i;, amino acids 5 to 73 of SEQ ID NO: 3; a xnultimeric chemokine protein which comprises an association of two or more of and/or and a multimeric chemokine protein which comprises an association of or (b) with a second chemokine. *020 16, Use of a chcmokine according to claim 1 substantially as hereinbefore described with reference to the Examples. 17. Use of a chemokixie according to claim 2 substantially as hereinbefore described with 0 25 reference to the Examples. 31/08 '99 TUE 16:55 [TX/RX NO 9802] 3 1- 8 99 1 !4 6 /1 26 19. Use of a chemokine according to claim 4 substantially as hereinbefore described with reference to the Examples. DATED this 26TH day of AUGUST, 1999 SmithKline Beecham Corporation by DAVIES COLLISON CAVE :Patent Attorneys for the applicant(s) 0 POP POP:% soC *Pgo 31/08 '99 TUE 16:55 [TX/RX NO 9802]