WO1997015595A1

WO1997015595A1 - Method of mobilizing hematopoietic stem cells

Info

Publication number: WO1997015595A1
Application number: PCT/US1996/017074
Authority: WO
Inventors: Louis Martin Pelus; Andrew Garrison King
Original assignee: Smithkline Beecham Corporation
Priority date: 1995-10-24
Filing date: 1996-10-24
Publication date: 1997-05-01
Also published as: EP0866806A1; NO981818D0; CZ120298A3; BR9611173A; KR19990066997A; AU712235B2; HUP9802531A3; ZA968896B; MX9803217A; AR004093A1; NO981818L; EP0866806A4; JPH11512747A; HUP9802531A2; PL326364A1; NZ321789A; TR199800742T2; AU7520996A

Abstract

A method of mobilizing hematopoietic stem cells from the bone marrow to the peripheral circulation is provided by administering to an animal an effective amount of nature, modified or multimeric forms of KC, groβ, groα or groη.

Description

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 cyclophospha¬ mide. 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, e.g. , 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 engraft ent to treat severe myelosuppression when G-CSF is used alone [see, e.g. , Bensinger et al, Blood. 81:3158 (1993) and R. Haas et al , Sem. in Oncoloσγ. £1:19 (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, groβ , groα , and gr o , 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 groβ (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 groβ (amino acids 5-73 of SEQ ID NO: 3) in the single agent mobilization assay of Example 1. Fig. 3 is a bar graph demonstrating the comparison of phosphate buffered saline (PBS), IL-8, groβ (amino acids 1-73; SEQ ID NO:3) and modified groβ (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.

J. 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 , groβ , and groy . For convenience, the amino acid sequences of the murine protein KC which contains 72 residues is provided in SEQ ID NO:l. These sequences are available from Genbank, accession number J04596. The sequences of the human protein ςrroα (aa 1-73) are provided in SEQ ID NO:2. The sequences of the human protein groβ (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 groy 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, groβ , groa , and grroγ, more preferably from groβ , groa , and groy , and most preferably from groβ . 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- (N-) 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. 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, e.g., 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, e.g., 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 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.

JJ. 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 , groβ and groy , and most preferably the chemokine is groβ .

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 5 to 72 of SEQ ID N0:1. Most preferably, the method of the invention is desamino groβ , which has the protein sequence spanning amino acids 5 to 73 of SEQ ID NO:3. This desamino-gro3 is characterized by having at least about two logs higher biological activity than unmodified, human groβ , as determined in the above- references HSF assay. As described in W094/29341, similar modifications can be made to the KC, ςrroα 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 groβ 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 groβ protein of the invention in combination with a mature groβ protein. Similarly, various combinations of dimers or other multimeric forms may contain a combination of the mature or modified groβ and other chemokines, such as the KC, grro and groy proteins. For example, a desamino groβ 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 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.

JJJ. 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. Generally, a mature, modified or desamino chemokine useful in the invention (e.g., groβ) 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.

JV. 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 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, e.g., 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 groβ , 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. 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 groβ [SEQ ID NO: 3] , human grroα [SEQ ID N0:2], human groy [SEQ ID N0:4], and murine KC [SEQ ID NO:l] .

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 groβ , 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 groβ [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. 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, i.e., about 1.2g/m² etopside, 800 μg/m² ARA-C, 200 mg/kg cyclophosphamide, etc. See for such dosages Hass et al , Seminars in Oncol.. 1:19-24 (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 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 (S)-5-oxo-L-prolyl-α-glutamyl-L-α- aspartyl-N⁸-(5-amino-l-carboxypentyl)-8-oxo-N-[N-{N-(5- oxo-L-prolyl)-L-α-glutamuyl}-L-α-aspartyl]-L-threo-2,7,8- triaminooctanoyl-lysine [ (pGlu-Glu-Asp)₂-Sub-(Lys)₂] [Pelus et al, EXP. Hematol.. 21:239-247 (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:l, groβ [SEQ ID NO:3] and groy [SEQ ID N0:4] including modified and multimeric chemokines are prepared using known techniques. See, e.g., W094/29341 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 μg/mouse and administered via subcutaneous, 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 i __^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 IO⁶ cells/ml in McCoys medium without serum. A single layer agar system consisting of McCoys medium enriched with nutrients (NaHC0₃, 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°C, 7.5% C0₂ 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. Preliminary experiments have shown that administration of groβ [SEQ ID NO: 3] results in a dose dependent mobilization of CFU-GM, similar to the results with the control. Modified groβ , the N-terminal 4 amino acid truncation protein (aa5-73) of groβ mobilized significantly greater numbers of hematopoietic progenitor cells than groβ (amino acids 1-73) or IL-8. No significant changes (> 3 fold) in mature cell elements were observed in groβ 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, (S)-5-oxo-L-prolyl-α-glutamyl-L-α-aspartyl-N⁸-(5-amino-l- carboxypentyl)-8-oxo-N⁷-[N-{N-(5-oxo-L-prolyl)-L-α- glutamuyl}-L-α-aspartyl]-L-threo-2,7,8-triaminooctanoyl- lysine [ (pGlu-Glu-Asp)₂-Sub-(Lys)₂] [Pelus, cited above] and FLT-3 ligand. Any G-CSF mimetic, i.e., 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, e.g., G-CSF, is administered at 50 μg/kg to mice four days prior to the chemokines or modified or multimeric chemokines derived from KC [SEQ ID NO:l], groβ [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. 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 pre¬ treatment utilizes MlP-lα 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 groβ [aa 5-73 of SEQ ID NO:3; termed groβ-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, groβ-T (50 μg at 15 - 30 min.), G-CSF (1 μg/mouse BID x 4) , or G-CSF, then groβ-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 groβ-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 groβ-T mobilize greater numbers of repopulating cells than G-CSF alone. B. Mobilization of Peripheral Blood Stem Cells

The rate at which peripheral blood stem cells mobilized by groβ-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, groβ-T alone (50 μg, , 15 min) , G-CSF (BID x 5 days, 1 μg/mouse alone) , and groβ-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 groβ was faster than those who received G-CSF mobilized cells. Mice transplanted with LDPBC mobilized by the combination of groβ-T + G-CSF resulted in a faster neutrophil recovery rate than groβ-T mobilized cells.

The recovery of platelet counts in these same mice followed the same pattern: groβ-T + G-CSF > groβ-T > G-CSF > > PBS. However, on day 19, platelet counts are still far from returning to normal values. These data indicate that groβ-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. 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.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) 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:

(A) ADDRESSEE: SmithKline Beecham Corporation -

Corporate Patents

(B) STREET: 709 Swedeland Road

(C) CITY: King of Prussia

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19406-2799

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: WO

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/547,262

(B) FILING DATE: 24-OCT-1995

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Hall, Linda E.

(B) REGISTRATION NUMBER: 31,763

(C) REFERENCE/DOCKET NUMBER: SBC P50161-2PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-270-5015

(B) TELEFAX: 215-270-5090

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 72 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Ala Pro lie Ala Asn Glu Leu Arg Cys Gin Cys Leu Gin Thr Met 1 5 10 15

Ala Gly lie His Leu Lys Asn lie Gin Ser Leu Lys Val Leu Pro

20 25 30

Ser Gly Pro His Cys Thr Gin Thr Glu Val lie Ala Thr Leu Lys

35 40 45

Asn Gly Arg Glu Ala Cys Leu Asp Pro Glu Ala Pro Leu Val Gin

50 55 60

Lys lie Val Gin Lys Met Leu Lys Gly Val Pro Lys

65 70

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 73 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Ala Ser Val Ala Thr Glu Leu Arg Cys Gin Cys Leu Gin Thr Leu 1 5 10 15

Gin Gly lie His Pro Lys Asn lie Gin Ser Val Asn Val Lys Ser

20 25 30

Pro Gly Pro His Cys Ala Gin Thr Glu Val lie Ala Thr Leu Lys

35 40 45

Asn Gly Arg Lys Ala Cys Leu Asn Pro Ala Ser Pro lie Val Lys

50 55 60

Lys lie lie Glu Lys Met Leu Asn Ser Asp Lys Ser Asn

65 70 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 73 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Ala Pro Leu Ala Thr Glu Leu Arg Cys Gin Cys Leu Gin Thr Leu 1 5 10 15

Gin Gly lie His Leu Lys Asn lie Gin Ser Val Lys Val Lys Ser

20 25 30

Pro Gly Pro His Cys Ala Gin Thr Glu Val lie Ala Thr Leu Lys

35 40 45

Asn Gly Gin Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Lys

50 55 60

Lys lie lie Glu Lys Met Leu Lys Asn Gly Lys Ser Asn

65 70

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 73 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Ala Ser Val Val Thr Glu Leu Arg Cys Gin Cys Leu Gin Thr Leu

1 5 10 15

Gin Gly lie His Leu Lys Asn lie Gin Ser Val Asn Val Arg Ser

20 25 30

Pro Gly Pro His Cys Ala Gin Thr Glu Val lie Ala Thr Leu Lys

35 40 45

Asn Gly Lys Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Gin

50 55 60

Lys lie lie Glu Lys lie Leu Asn Lys Gly Ser Thr Asn

65 70

Claims

WHAT IS CLAIMED IS:

1. Use of a protein derived from a mammalian chemokine selected from the group consisting of (a) groa SEQ ID NO:2, (b) groβ SEQ ID NO: 3, (c) groy SEQ ID NO:4; and (d) KC SEQ ID NO:l, in the preparation of a medicament useful for mobilizing hematopoietic stem cells.

2. Use according to claim 1 wherein said chemokine is selected from the group consisting of:

(a) mature groβ ;

(b) amino acids 5 to 73 of SEQ ID NO: 3;

(c) a multimeric chemokine protein which comprises an association of two or more of (a) or (b) ; and

(d) a multimeric chemokine protein which comprises an association of (a) or (b) with a second chemokine.

3. Use according to claim 1 wherein said chemokine is selected from the group consisting of:

(a) mature grroα ;

(b) amino acids 5 to 73 of SEQ ID NO: 2;

4. Use according to claim 1 wherein said chemokine is selected from the group consisting of:

(a) mature groy ;

(b) amino acids 5 to 73 of SEQ ID NO: 4; (c) a multimeric chemokine protein which comprises an association of two or more of (a) or (b) ; and

5. Use according to claim 1 wherein said chemokine is selected from the group consisting of:

(a) mature KC;

(b) amino acids 5 to 72 of SEQ ID NO: 1;

6. Use according to claims 1-5 wherein said chemokine comprises between about 0.01 ng to about 1 g of said medicament.

7. Use according to claims 1-5, wherein said medicament is co-administered with a growth factor or other hematopoietic regulatory biomolecule.

8. Use according to claim 7 wherein said growth factor is selected from the group consisting of GM-CSF, G-CSF, stem cell factor, and Flt-3 ligand.

9. Use according to claim 7 where said biomolecule is (S) -5-oxo-L-prolyl-α-glutamyl-L-α- aspartyl-N⁸-(5-amino-l-carboxypentyl) -8-oxo-N⁷-[N-{N-(5- oxo-L-prolyl) -L-α-glutamuyl}-L- -aspartyl]-L-threo-2 ,7,8- triaminooctanoyl-lysine [ (pGlu-Glu-Asp)₂-Sub-(Lys)₂] .

10. Use according to claim 1 wherein the medicament is used in the treatment of a patient receiving peripheral blood hematopoietic stem cell transplantation.

11. Use according to claim 10 wherein the patient has received a dose of a selected chemotherapeutic agent prior to treatment with the medicament; the hematopoietic stem cells from the peripheral blood of the patient treated are harvested; a chemotherapeutic agent is administered; and the patient is reinfused with the harvested cells.

12. Use according to claim 11 wherein said chemotherapeutic agent is selected from the group consisting of cyclophosphamide, cisplatinum, ARA-C, 5- fluorouracil, etopside, epirubicin, carboplatin, busulphan, mitoxantrone and carmustine.