EP0800402A1

EP0800402A1 - Mobilisation of haematopoietic cells

Info

Publication number: EP0800402A1
Application number: EP95940384A
Authority: EP
Inventors: Matthew John Mccourt; Lars Michael Wood; Michael George Hunter; Lloyd George Czaplewski
Original assignee: British Biotech Pharmaceuticals Ltd
Current assignee: Vernalis R&D Ltd
Priority date: 1994-12-22
Filing date: 1995-12-15
Publication date: 1997-10-15
Also published as: AU4185296A; WO1996019234A1; GB9426012D0

Abstract

The combination of CxC chemokine such as GRO and a haematopoiesis priming agent such as a colony stimulating factor promotes release and mobilisation of haematopoietic cells into the bloodstream.

Description

MOBILISATION OF HAEMATOPOIETIC CELLS

TECHNICAL FIELD

This invention relates to the combined use of CxC chemokines and haematopoiesis priming agents such as colony stimulating factors (CSFs), to promote the release and mobilisation of haematopoietic cells from the marrow. Such agents are useful in treatments where the enrichment of blood with haematopoietic stem cells and mature cells would be of value, for example in peripheral blood stem cell transplantation (PBSCT) and in enhancing immune responses.

BACKGROUND OF THE INVENTION

There is a demand for agents and methods for increasing the concentrations of stem cells and mature blood cells, for example neutrophils and monocytes, in the blood.

All mature blood cell types are derived from a single class of progenitor cell known as haematopoietic stem cells which are both pluripotent - that is they can give rise to all blood cell types, such as leukocytes and erythrocytes - and capable of self-renewal. Leukopaenia or depletion from the blood system of these leukocytes renders an individual susceptible to infection.This leukopaenia can occur during some viral infections, but clinically the most important cause is chemotherapy or radiotherapy used to treat malignant disease.

Many chemotherapy cancer treatments involve the administration of chemical agents which are highly toxic to cells undergoing division. This however, not only destroys the malignant dividing cells, but also destroys normal proliferating cells, particularly the stem cell of the haematopoietic system. As a consequence of this toxicity, the levels of circulating haematopoietic cells are drastically reduced and therefore the immune system is severely impaired rendering the subject susceptible to infection. These side effects restrict the dose of chemotherapy and thus limits the efficacy. More intensive chemotherapy treatments will require protection or replacement of the haematopoietic system.

Current clinical methods of protecting haematopoietic stem cells from chemical toxicity include autologous bone marrow transplantation. Prior to chemotherapy, bone marrow is surgically removed from the cancer patient and stored. Following the chemotherapy, and after the toxic chemical agent has been purged from the body, the bone marrow is returned, replacing the depleted stem cells. This technique is however time consuming, labour intensive and painful. An alternative is peripheral blood stem cell transplantation (PBSCT) which is less invasive, less intensive and less painful.

To achieve harvesting of cells in PBSCT, as there are insufficient numbers in normal circulating blood, stem cells and progenitor cells must be mobilised from the bone marrow. Following mobilisation, these stem cells must be collected and concentrated by apheresis procedures, before transplantation.

In the past the following agents have been used or proposed for use in stimulating the expansion and mobilisation of stem and progenitor cells:

G-CSF (granulocyte-CSF) (Johnsen et al. (1992) Bone Marrow Transplantation. 10:229-234)

G-CSF + chemotherapy (Teshima et al. (1992 ) Bone Marrow Transplantation. 10:215-220; Liu et al. (1993) Brit. J. Haemat. 84:31 -38; Craig et al. (1993) Brit. J. Haemat. 85:210-212)

GM-CSF (granulocyte-macrophage-CSF) (Mehta et al. (1993) Leukemia and Lymphoma. 1 1 :157-158; Mangan et al. (1993) Stem Cells. 11 :445-454)

GM-CSF + chemotherapy (Haas et al. (1992) Bone Marrow Transplantation. 9:459-465; Campos et al. (1993) Leukemia. 9(7):1409-1415) GM-CSF + IL-3 (Emerson et al. (1988) J. Clin. Invest. 82:1282-1287; Kanz et al. (1993) Eur. J. Cancer. 29A (Suppl 5) pp. s23-s26)

GM-CSF/IL-3 fusion protein (PIXY321) (Curtis et al. (1991 ) Proc. Natl. Acad. Sci. U.S.A. 88:5809-5813.

Stem cell factor (SCF)+Λ GM-CSF or IL-3 (multi-CSF) (Hoffmann et al. (1993) Stem Cells. 11 (suppl 2):76-82)

Stem cell factors and CSFs incl. IL-6.

(Metcalf (1993) Stem Cells. 11 (suppl. 2):1-11.)

IL-11 and G-CSF (Cairo et al. (1993) Blood. 81 , No.1 :27-34)

IL-7 (Perry et al. (1994) abstract Haematopoiesis, Keystone Symposium, Breckenridge, Jan 4 - 1 1.)

IL-12 (Jackson et al. (1994) abstract No. 97, ISEH, Minneapolis Aug 21 -25.)

The above references are incorporated by way of example.

Despite the past proposals mentioned supra, alternative or better ways of mobilising stem cells and of increasing the concentrations of mature haematopoietic cells are sought.

The administration of colony stimulating factors, anti-VLA antibodies (Papayannopoulou and Nakamoto (1993) Proc. Natl. Acad. Sci. USA. 90:9374-9378.) and cytotoxic drugs, such as high dose cyclophosphamide, Ara-C and 5-fluorouracil, are known to prime the differentiation and expansion of haematopoietic stem and progenitor cells to mature blood cells and their eventual release from the bone marrow into the circulating blood system. This invention is based on the discovery that members of the CxC chemokine sub¬ family can act in synergy with colony stimulating factors to promote the mobilisation of stem cells and mature haematopoietic cells to circulating blood, resulting in an increase of circulating stem cells and mature blood cell types, such as neutrophils and monocytes. The levels of stem cell mobilisation and increased mature blood cell concentrations obtained are greater with the combined administration of a CSFs and a CxC chemokine, than by treatment with either of the individual components.

G-CSF- or GM-CSF-induced release of stem cells from the bone marrow is slow, unpredictable and occurs over a long period of time, taking for example between 4 and 14 days. In addition, prolonged administration of a CSF can lead to undesirable side effects such asiπter alia bone pain and fever, and successful engraftment can require multiple apheresis procedures to accumulate sufficient haematopoietic stem cells to ensure re-population. The timing of the apheresis and the stem cell yield are variable.

Therefore, a reduction in the duration of CSF administration, improvements in stem cell yield and more predictable harvesting time would improve current clinical procedures.

One of the benefits of this invention is that haematopoietic stem cell mobilisation and increased mature blood cell concentrations in the blood stream, achievable by haematopoietic priming agents such as colony stimulating factors, can be increased rapidly by administration of a CxC chemokine, and the time course of stem cell and mature blood cell release is highly predictable.

The invention therefore has application inter alia in the treatment of cancer patients undergoing chemotherapy, in the treatment of patients with acute leukopaenia and in patients suffering from the effects of acute infection.

CxC chemokines such as IL-8, NAP-1 and GRO-aipha are in themselves known. Haematopoietic priming agents such as cytotoxic chemical compounds, anti-VLA₄ antibodies (Papayannopoulou and Nakamoto, ibid) and colony stimulating factors for example G-CSF, GM-CSF, are also known. However, treatment with both a CxC and a haematopoiesis priming agent to achieve haematopoietic stem and progenitor cell mobilisation or to achieve increases in the number of mature blood cells does not appear to have been proposed previously.

BRIEF DESCRIPTION OF THE INVENTION.

This invention follows from the surprising discovery that members of the CxC chemokine sub-family, can act in synergy with a haematopoiesis priming agent (HPA) for the mobilisation of haematopoietic stem cells and for increasing the mature blood cell concentrations, predominantly of neutrophils, monocytes and basophils. These effects, obtained from the synergistic administration of HPAs (e.g. CSFs) and CxCs, are greater than the individual or additive levels obtained from these components.

DETAILED DESCRIPTION OF THE INVENTION.

According to the present invention there is provided the use of a CxC chemokine and a haematopoiesis priming agent (HPA) in the preparation of an agent for promoting the release and mobilisation of haematopoietic cells into the bloodstream.

The invention is thus useful in a method for inducing the release of haematopoietic stem cells from the marrow of an animal, and for increasing the concentration of mature haematopoietic blood cell subsets, the method comprising the simultaneous, sequential or individual administration to an animal, of an effective dose of a HPA and a CxC chemokine.

Haematopoietic cells include stem cells, progenitor cells and differentiated cells of the haematopoietic system including platelets, erythrocytes and leukocytes (white blood cells), which include inter alia neutrophils, basophils, monocytes and lymphocytes.

The term haematopoiesis priming agent (HPA) as used throughout the specification refers to any agent capable of mobilising haematopoietic stem cells or progenitor cells or leukocytes into the bloodstream. Examples of HPAs include but are not limited to colony stimulating factors (CSF), cytotoxic chemical compounds (for example cyclophosphamide, Ara C or 5-fluorouracil) and anti-VL- antibodies.

For the purpose of this invention a HPA acts to prime the marrow by shifting the stem cells and progenitor cells out of their quiescent steady state, thereby allowing the CxC chemokine to rapidly mobilise the haematopoietic cells into the bloodstream.

The term "colony stimulating factor" or "CSF as used throughout the specification refers to cytokines which induce progenitor cells found in the bone marrow to differentiate into mature blood cell types.

Colony stimulating factors which find use in the invention include, but are not limited to: neutrophil granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (M.-CSF, CSF-1 ) , multi-CSF (IL-3), erythropoietin (EPO), eosinophil CSF (Eos-CSF, IL-5), stem cell factor (SCF), erythroid potentiating activity (EPA), interleukin-7 (IL-7), interleukin-11 (IL-11 ) and interleukin-12 (IL-12), or functional variants thereof.

Any other cytokine which has the attributes of affecting differentiation or proliferation of haematopoietic stem and progenitor cells have utility in this invention.

In a preferred embodiment of the invention, the haematopoiesis priming agent for use in the invention is a colony stimulating factor.

In a more preferred embodiment of the invention, the CSF for use in the invention is G- CSF, or variants thereof.

The invention encompasses the use of naturally occurring, or recombinant CSFs, including murine and human, or protein engineered forms ("variants" and "analogues") which may have improved biophysical or biological properties. Variants or analogues with improved biophysical properties include those recombinant proteins capable of being more easily expressed or purified. Variants may also possess less toxicity when administered to the patient.

The term "CxC chemokines" as used throughout the specification refers to a family of cytokines classified in the CxC sub-family of chemokines by amino acid homology and in possessing an amino acid separating the first two cysteine groups.

Any form of CxC chemokine protein can be used in synergy with CSFs to induce the release of haematopoietic cells. The invention encompasses the use of the various naturally occurring, or recombinant CxC chemokine proteins that have been described, including murine or human, as well as protein engineered forms which may have improved biophysical or biological properties ("variants" and "analogues"). Variants or analogues with improved biophysical properties include those recombinant proteins capable of being more easily expressed or purified. Variants may also possess less toxicity when administered to the patient.

CxC chemokines which find use in the invention include inter alia: IL-8, GRO-α , GRO- β , GRO-γ, neutrophil-activating protein 2 (NAP-2), epithelial-cell-derived neutrophil- activating protein (ENA 78), platelet factor (PF4), γlP-10 and granulocyte chemotactic protein 2 (GCP-2), or functional variants thereof.

In a preferred embodiment of the invention, the CxC chemokines for use in the invention are IL-8 and GRO-alpha, or functional variants thereof.

The term "variant" (or its synonym for present purposes "analogue") is used, broadly, in a functional sense. As a practical matter though, most variants will have a high degree of homology with the prototype molecule if biological activity is to be substantially preserved. It will be realised that the nature of changes from the prototype molecule is more important than the number of them. As guidance, though, at the amino acid level, it may be that (in increasing order of preference) at least, 50% 60%, 70%, 80%, 90% and preferably 95% of the residues will be the same as the native molecule; at the nucleic acid level, nucleic acid coding for an analogue may for example hybridise under stringent conditions (such as at approximately 35°C to 65°C in a salt solution of approximately 0.9 molar) to nucleic acid coding for the prototype molecule, or would do so but for the degeneracy of the genetic code.

Individual CSFs or CxC chemokines can be purified from media conditioned by particular cell types, or expressed by recombinant organisms or cells. Methods for expression and purification of CSFs or CxCs by recombinant techniques are known in the art.

CxC chemokines and HPAs in particular CSFs, which are defined as above and which are yet to be identified, also find utility in the invention.

In a preferred embodiment of the invention there is provided the use of IL-8 and G- CSF in the preparation of an agent for promoting the release and mobilisation of haematopoietic stem, progenitor and mature blood cells into the bloodstream.

In another preferred embodiment of the invention there is provided the use of GRO- alpha and G-CSF in the preparation of an agent for promoting the release and mobilisation of haematopoietic stem, progenitor and mature blood cells into the bloodstream.

The invention is useful in inducing the release, into the bloodstream, of haematopoietic stem cells and committed progenitor cells, and in increasing the concentration in the blood of mature blood cells. Mature blood cells include neutrophils, eosinophils, basophils, monocytes, leukocytes, macrophages, lymphocytes, platelets and erythrocytes. The increase in the circulating number of mature blood cells may be particularly useful in potentiating the body's response to infection and tumours.

The invention also has a clear clinical indication as an adjunct to the use of CSFs in peripheral blood cell transplantation.

Presently, only G-CSF is licensed for clinical use to promote accelerated granulocyte recovery following chemotherapy and to release stem cells from marrow so that they can be harvested before chemotherapy. This G-CSF induced stem cell release and expansion treatment can take between 4-14 days. Thus the optimum time for initiating leukapheresis is extremely unpredictable and requires routine monitoring of blood cell count. GM-CSF is licensed for use in conjunction with autologous bone marrow transplantation. Clinical trials with other CSFs are underway

Current G-CSF treatment generally takes 4 to 7 days, but can take up to 14 days. According to the present invention there is provided a means of mobilisation of haematopoietic stem, progenitor and mature cells to the blood stream, and an increase in the predictability of the harvest time, by the synergistic action of a CxC chemokine and a HPA (e.g. a CSF). Mobilisation of stem ceils and progenitor stem cells from the marrow and increased blood concentrations of the various leukophils occurs more rapidly when CxC chemokines synergise with CSFs, producing a predictable, fast and enhanced yield of haematopoietic cells, potentially obviating the need for repeated leukapheresis.

According to an aspect of the invention there is provided for the use of a CxC chemokine and a haematopoiesis priming agent, in the preparation of an agent for promoting the release and mobilisation of haematopoietic stem and progenitor cells and/or for increasing the concentration in the blood of mature blood cells, prior to harvesting for peripheral blood stem cell transplantation, or peripheral blood mature cell transplantation.

According to a preferred aspect of the invention there is provided for the use of a CxC chemokine and a colony stimulating factor (CSF), in the preparation, of an agent for promoting the release and mobilisation of haematopoietic stem and progenitor cells and/or for increasing the concentration in the blood of mature blood cells, prior to harvesting for peripheral blood stem cell and/or mature cell transplantation. Haematopoietic stem ceils and/or mature blood cells so harvested may then be returned to the subject from whom they were removed either in whole blood, or by concentration by leukapheresis and apheresis procedures, following myeloablative treatment, e.g. chemotherapy.

Engraftment of stem cells in this way may also be applicable for gene therapy.The ability of multipotent haematopoietic stem cells for self-renewal, the ability to genetically manipulate cells and tissues generally, and the ability of circulating stem cells to permeate tissues, re-enter and lodge in the bone marrow makes the self- populating haematopoietic stem cells useful targets for gene therapy. The synergistic use of a HPA (e.g. a CSF) and a CxC chemokine expands the number of harvestable stem cells available for gene therapy.

According to another aspect of the invention there is provided for the genetic manipulation of haematopoietic stem ceils harvested following their mobilisation from bone marrow into the blood by the synergistic action of a CxC chemokine and a HPA. The harvested haematopoietic stem cells may be purified and enriched by apheresis or leukapheresis prior to genetic manipulation, and may then also be subjected to ex vivo expansion.

According to a preferred aspect of the invention there is provided for the genetic manipulation of haematopoietic stem cells harvested following their mobilisation from bone marrow into the blood by the synergistic action of a CxC chemokine and a CSF. The harvested haematopoietic stem cells may be purified and enriched by apheresis and/or leukapheresis prior to genetic manipulation, and may then also be subjected to ex vivo expansion.

According to another aspect of the invention there is provided for the administration to a patient, of these genetically manipulated haematopoietic stem cells to achieve long term engraftment. The invention also has use in preventing or treating leukopaenia, particularly neutropenia.

The invention is particulariy useful in inducing neutrophilia in various disease states. For example neutropenia may arise as a result of microbial (such as bacterial) infection; the neutropenia can be addressed by inducing neutrophilia. Other diseases of which neutropenia is a symptom or cause may also be treated by means of the invention, possibly in conjunction with cytotoxic agents. Such diseases are exemplified by, but not limited to, the following:

congenital neutropenias such as Kostmann's syndrome and

Schwachman-Diamond syndrome; childhood and adult cyclic neutropenia; post-infective neutropenia; myelo-dysplastic syndrome; and neutropenia associated with chemotherapy and radiotherapy.

In summary, combined CxC/HPA administration to aπ animal gives an enhanced yields of mobilised stem cells and progenitor cells and an increase in the blood of mature haematopoietic cell concentrations, predominantly white blood cells; and this offers a more predictable and manageable regime for peripheral blood ceil transplantation.

The ability to induce leukophiiia will find clinical and veterinary application in all utilities where the raising of haematopoietic cell levels is important. For example, to enhance immune responses against acute or chronic infections, particularly parasitic and bacterial infections, for example in severe chronic neutropenia. It may also have a role in promoting wound healing.

Dosage of the agent (HPA and CxC) in accordance with any aspect of the invention will be such as to be effective and will be under the control of the physician or clinician. As general but not exclusive guidance, though, doses may be in the range of from 0.001 to 1 mg/kg, preferably from 0.01 to 0.2 mg/kg. Doses may be administered repeatedly, for example from 1 to 6 times per day, preferably from 1 to 3 times per day. It may be preferable to administer the colony stimulating factor HPA daily for between 1 and 14 days prior to administering the CxC chemokine. Within minutes or 1 - 3 hours after administration of the CxC chemokine it may be appropriate for a unit of blood to be taken and stored, or apheresis or leukapheresis procedures carried out.

Therapeutic or prophylactic administration of the agent can be by injection, preferably via intra-venous, intra-peritoneal, intra-muscular or sub-cutaneous routes in a clinically acceptable formulation. Other routes such as transdermal, oral, intranasal or by inhalation may also be possible.

G-CSF is currently in clinical use. Therefore, identical or similar administration and formulation as that used for G-CSF can also be used for administration and/or formulation of the agent of this invention.

Preferred features for each aspect of the invention are as for each other aspect, mutatis mutandis.

The invention will now be illustrated by the following examples. The examples refer to the accompanying figures, in which:

FIGURE 1 is a histogram showing the leukophilic effect in mice, of IL-8 and rhGRO following initial G-CSF treatment, measured as total white blood cell (WBC) count.

FIGURE 2 is a histogram showing the effect in mice, of IL-8 and rhGRO on neutrophil count following initial G-CSF treatment.

FIGURE 3 is a histogram showing the effect in mice, of IL-8 and rhGRO on lymphocyte count following initial G-CSF treatment. FIGURE 4 is a histogram showing the effect in mice, of IL-8 and rhGRO on monocyte count following initial G-CSF treatment.

FIGURE 5 is a histogram showing the effect in mice, of IL-8 and rhGRO on basophil count following initial G-CSF treatment.

FIGURE 6 FIGURE 5 is a histogram showing the effect in mice, of IL-8 and rhGRO on eosinophil count following initial G-CSF treatment.

FIGURE 7 is a histogram showing the effect of IL-8 and rhGRO on total white blood cell count in naive and G-CSF primed mice.

FIGURE 8 is a histogram showing the effect of IL-8 and rhGRO on neutrophil count in naive and G-CSF primed mice.

FIGURE 9 is a histogram showing the effect of IL-8 and rhGRO on lymphocyte count in naive and G-CSF primed mice.

FIGURE 10 is a histogram showing the effect of IL-8 and rhGRO on monocyte count in naive and G-CSF primed mice.

FIGURE 11 is a histogram showing the effect of IL-8 and rhGRO on eosinophil count in naive and G-CSF primed mice.

FIGURE 12 is a histogram showing the effect of IL-8 and rhGRO on basophil count in naive and G-CSF primed mice.

FIGURE 12 is a histogram showing the effect of IL-8 on haematopoietic progenitor mobilisation in G-CSF primed mice.

FIGURE 14 is a histogram showing the effect of rhGRO on haematopoietic progenitor mobilisation in G-CSF primed mice. 1 4

The following test compounds were used diluted in phosphate buffered saline (PBS; Gibco Dulbecco's 'B') for administration:

G-CSF (neupogen, Amgen/Roche) lot No. B0343 MFD. 02 93. exp. 02 95.

Recombinant human GRO (MGSA; Serotec, product code PHP056; GRO-alpha) Batch No. 9701. exp. 08 95.

Recombinant human lnterleukin-8 (R&D Systems; Cat. No. 208-llJCF)

EXAMPLE 1 Mature blood cell mobilisation in response to selected chemokines. in mice primed with G-CSF.

Male C57BLJ6J mice (6 - 8 weeks;n=5) were dosed on days 0, 1 and 2 with G-CSF (neupogen, Amgen/Roche), 100μg/kg sub-cutaneous (s.c.) b.i.d. @ 0 and 7 hours. On day 3 mice received either phosphate buffered saline (PBS; 10Oμl) IL-8 or rhGRO (MGSA; melanoma growth stimulatory activity), 100μg/kg s.c. @ t=0hr. At t=0.5hr (30 mins) mice were terminally anaesthetised with halothane and blood (0.5ml) was withdrawn by cardiac puncture using a 21 G needle and 2.0ml syringe. Blood samples were immediately anticoagulated with EDTA, present in 0.5ml sample cups (Teklab, UK). Total and differentiated white blood cell counts were performed using a Technicon H1 (Bayer, UK) according to the manufacturers instructions, with FDA approved software (see Figures 1 - 6).

This data suggest that the synergistic action between G-CSF and a CxC chemokine increases the numbers of mature haematopoietic cells in the bloodstream.

Control experiments testing the ability of the CxC chemokines to increase the concentration of mature blood cells independently, are present in example 2 infra. EXAMPLE 2 - Mature blood cell mobilisation in response to selected chemokines compared in naive and G-CSF primed mice.

In order to establish that the leukophilic effects obtained with the two CxC chemokines; IL-8 and rhGRO, following G-CSF pre-treatment were the result of the synergistic action with G-CSF and not due to the chemokines alone, five male C57BIJ6J mice (6 - 8 weeks;25 - 30kg) were either dosed; a) solely with phosphate buffered saline (PBS;100μl), IL-8 or rhGRO 100μg/kg s.c. @ t=0hr, or; b) as in example 1 supra, on days 0, 1 and 2 with G-CSF (neupogen, Amgen/Roche), 100μg/kg sub cutaneous (s.c.) b.i.d. @ 0 and 7 hours, and then on day 3 mice received either phosphate buffered saline (PBS; 100μl), IL-8 or rhGRO-alpha 100μg/kg s.c. @ t=0hr.

At t=0.5hr (30 min.) mice were terminally anaesthetised with halothane and blood (0.5ml) was withdrawn by cardiac puncture using a 21 G needle and 2.0ml syringe. Blood samples were immediately anticoagulated with EDTA, present in 0.5ml sample cups (Teklab, UK). Total and differentiated white blood cell counts were performed using a technicon H1 (Bayer.UK) according to the manufacturers instructions, with FDA approved software (See Figures 7 - 12).

The results demonstrate that G-CSF pre-treatment only generates an approximate two-fold increase in total white blood cells concentration compared to the PBS control. IL-8 and rhGRO (GRO-alpha) did not increase total white blood cell concentration to any great extent when administered on their own. However, following G-CSF pre- treatment, a 2 - 4-fold increase in total white blood cell numbers was obtained. The neutrophil, lymphocyte, monocyte, basophil and eosinophil counts were all significantly elevated in the co-administration experimental regimes.

This data demonstrates the synergistic action of G-CSF and the two CxC chemokines IL-8 and GRO-alpha, in increasing the concentration of mature haematopoietic cells, particularly neutrophils, in the blood stream. Example 3. Progenitor mobilisation bv IL-8 in G-CSF primed mice

This experiment investigates the ability of IL-8 and GRO-alpha (rhGRO) to augment the effects of G-CSF after 3 days of G-CSF treatment on progenitor cell mobilisation.

Groups of C57BL 6J mice (n=5/group) were dosed with G-CSF 100μg/kg s.c. diluted appropriately in PBS (injection volume 40μl) b.i.d. at 0 and 7 hours on days 0, 1 and 2. After the last G-CSF treatment (days +2,) groups received PBS 40μl s.c, or IL-8, 100μg kg s.c. Blood was removed 30 minutes later by cardiac puncture from mice under terminal halothane anaesthesia. Blood samples were immediately pooled and anticoagulated with heparin (Multiparin, 5,000 units/ml) 100 - 200units per ml blood. Low density mononuclear cells were prepared over ficoll gradients. The number of haematopoietic progenitors in each sample was estimated by plating the low density mononuclear cells in methyicellulose plates containing appropriate nutrients and growth factors (commercially available from Stem Cell Technologies, Vancouver, Canada). The plates were incubated at 37°C in 5% O2, 5% CO₂ for 7 days and the colonies were scored using a low magnification microscope. The number of progenitors mobilised into the peripheral blood is presented in colony forming units (CFU) per ml .

The effect of IL-8 on mobilisation of multi-potent progenitors to peripheral blood in G- CSF primed mice, is shown in Figure 13. IL-8 improves the yield of peripheral blood progenitors after 3 days of G-CSF priming with an approximate 3.5-fold increases over the G-CSF treatment alone.

Example 4. Progenitor mobilisation bv GRO-alpha in G-CSF primed mice

This experiment investigates the ability GRO-alpha (rhGRO) to augirient the effects of G-CSF after 3 days of G-CSF treatment on progenitor cell mobilisation.

Groups of C57BU6J mice (n=5/group) were dosed with G-CSF 100μg/kg s.c. diluted appropriately in PBS (injection volume 40μl) b.i.d. at 0 and 7 hours on days 0, 1 and 2. After the last G-CSF treatment (days +2,) groups received PBS 40μl s.c, or GRO- alpha, 10Oμg/kg s.c. Blood was removed 30 minutes later by cardiac puncture from mice under terminal halothane anaesthesia. Blood samples were immediately pooled and anticoagulated with heparin. Low density mononuclear cells were prepared over ficoll gradients. The number of haematopoietic progenitors in each sample was estimated by plating the low density mononuclear cells in methylcellulose plates containing appropriate nutrients and growth factors (commercially available from Stem Cell Technologies, Vancouver, Canada). The plates were incubated at 37°C in 5% O2, 5% CO2 for 7 days and the colonies were scored using a low magnification microscope. The number of progenitors mobilised into the peripheral blood is presented in colony forming units (CFU) per ml .

The effect of GRO-alpha on mobilisation of multi-potent progenitors to peripheral blood in G-CSF primed mice, is shown in Figure 14. GRO-alpha improves the yield of peripheral blood progenitors after 3 days of G-CSF priming with an approximate 3.5- fold increases over the G-CSF treatment alone.

The mature cell and progenitor data from the above examples suggests that the use of CxC chemokines in conjunction with G-CSF, in a clinically relevant setting, could achieve a significant improvement over the currently used G-CSF treatment. That translates to a reduction in the number of treatments in hospitals required to collect progenitors and/or a greater success rate of transplantation. Synergy with other haematopoiesis priming agents such as cytotoxic chemical agents, anti-VLA-j antibodies or other colony stimulating factors, such as GM-CSF, IL-3, SCF; and other CxC chemokines, such as GRO-beta , GRO-gamma, neutrophii-activating protein 2 (NAP-2), epithelial-cell-derived neutrophii-activating protein (ENA 78), platelet factor (PF4), γlP-10 and granulocyte chemotactic protein 2 (GCP-2) would be expected.

Claims

1. The use of a CxC chemokine and a haematopoiesis priming agent (HPA) in the preparation of an agent for promoting the release and mobilisation of haematopoietic cells into the bloodstream.

2. The use as claimed in claim 1 , wherein the haematopoiesis priming agent (HPA) is a colony stimulating factor (CSF).

3. The use as claimed in claim 1 , wherein the haematopoiesis priming agent (HPA) is a cytotoxic chemical compound.

4. The use as claimed in claim 1 , wherein the haematopoiesis priming agent (HPA) is an anti VLA₄ antibody.

5. The use as claimed in any of claims 1 to 4, wherein the haematopoietic cells are white blood cells .

6. The use as claimed in any of claims 1 to 4, wherein the haematopoietic cells are neutrophils.

7. The use as claimed in any of claims 1 to 4, wherein the haematopoietic ceils are lymphocytes.

8. The use as claimed in any of claims 1 to 4, wherein the haematopoietic cells are basophils.

9. The use as claimed in any of claims 1 to 4, wherein the haematopoietic cells are monocytes.

10. The use as claimed in any of claims 1 to 4, wherein the haematopoietic cells are eosinophils.

11. The use as claimed in any of claims 1 to 4, wherein the haematopoietic cells are erythrocytes.

12 The use as claimed in any of claims 1 to 4, wherein the haematopoietic cells are progenitor cells.

13. The use as claimed in any of claims 1 to 4, wherein the haematopoietic cells are stem cells.

14. The use of a CxC chemokine and a HPA as claimed in any of claims 1 to 4, in the preparation of an agent for preventing or treating leukopaenia.

15. The use of a CxC chemokine and a HPA as claimed in claim 1 to 4, in the preparation of an agent for preventing or treating congenital neutropenia.

16. The use of a CxC chemokine and a HPA as claimed in any of claims 1 to 4, in the preparation of an agent for preventing or treating childhood or adult cyclic neutropenia.

17. The use of a CxC chemokine and a HPA as claimed in any of claims 1 to 4, in the preparation of an agent for preventing or treating post-infective neutropenia.

18. The use of a CxC chemokine and a HPA as claimed in any of claims 1 to 4, in the preparation of an agent for preventing or treating myelo-dysplastic syndrome.

19. The use of a CxC chemokine and a HPA as claimed in any of claims 1 to 4, in the preparation of an agent for preventing or treating neutropenia associated with chemotherapy.

20. The use of a CxC chemokine and a HPA as claimed in any of claims 1 to 4, in the preparation of an agent for treating acute or chronic microbial, fungal or parasitic infection.

21. The use of a CxC chemokine and a HPA as claimed in any of claims 1 to 4, in the preparation of an agent for promoting the release and mobilisation of stem cells and /or increasing the concentration in the blood of mature blood cells prior to harvesting for peripheral blood cell transplantation.

22. The use as claimed in claim 21 , wherein harvested cells are administered to a patient after the patient has undergone cytotoxic chemo- or radio-therapy.

23. The use as claimed in claim 21 , wherein harvested stem cells are administered to a patient after genetic manipulation of at least some of the cells.

24. The use as claimed in any one of claims 1 to 21 , wherein the CxC chemokine is a natural mammalian or recombinant protein selected from the group: IL-8, GRO-α , GRO-β , GRO-γ, neutrophii-activating protein 2 (NAP-2), epithelial-cell-derived neutrophii-activating protein (ENA 78), platelet factor (PF4), γlP-10 and granulocyte chemotactic protein 2 (GCP-2), or functional variants thereof.

25. The use as claimed in claim 24, wherein the mammalian CxC chemokine is a murine protein.

26. T e use as claimed in claim 24, wherein the mammalian CxC chemokine is a human protein.

27. The use as claimed in claims 24, 25 and 26, wherein the CxC chemokine is GRO-alpha, or a functional variant thereof.

28. The use as claimed in claim 24, 25 and 26 wherein the CxC chemokine is IL-8, or a functional variant thereof.

29. The use as claimed in any one of claims 1 , 2 or 5 to 16, wherein the colony stimulating factor (CSF) and is a natural or recombinant protein selected from the group: neutrophil granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM- CSF), macrophage CSF (M-CSF, CSF-1 ) , multi-CSF (IL-3), erythropoietin (EPO), eosinophil CSF (Eos-CSF, IL-5), stem cell factor (SCF), erythroid potentiating activity (EPA), interleukin-7 (IL-7), interleukin-1 1 (IL-1 1 ) and interleukin-12 (IL-12) or functional variants thereof.

30. The use as claimed in any one of claims 1 , 2 or 5 to 16, wherein the colony stimulating factor (CSF) is G-CSF, or a functional variant thereof.

31. A product comprising a CxC chemokine and a haematopoiesis priming agent (HPA) for separate, simultaneous or sequential administration for: promoting the release and mobilisation of haematopoietic cells; preventing or treating leukopaenia; preventing or treating congenital neutropenia; preventing or treating childhood or adult cyclic neutropenia; preventing or treating post-infective neutropenia; preventing or treating myelo-dysplastic syndrome; preventing or treating neutropenia associated with chemotherapy; treating acute or chronic microbial, fungal or parasitic infection; or promoting release and mobilisation of stem cells prior to harvesting from a patient.

32. A method for promoting the release and mobilisation of stem cells and progenitor stem cells, or for increasing the concentration of mature haematopoietic cells in the blood in a human or non-human animal, the method comprising the separate, simultaneous or sequential administration to the animal of an effective dose of a haematopoiesis priming agent (HPA) and a CxC chemokine.

33. A method for promoting the release and mobilisation of stem cells and progenitor stem cells, or for increasing the concentration of mature haematopoietic cells in the blood in a human or non-human animal, the method comprising the separate, simultaneous or sequential administration to the animal of an effective dose of a colony stimulating factor (CSF) and a CxC chemokine.

34 A method as claimed in claim 33, wherein the colony stimulating factor is selected from the group: neutrophil granulocyte CSF (G-CSF), granulocyte- macrophage CSF (GM-CSF), macrophage CSF (M-CSF, CSF-1) , multi-CSF (IL-3), erythropoietin (EPO), eosinophil CSF (Eos-CSF, IL-5), stem cell factor (SCF), erythroid potentiating activity (EPA), interleukin-7 (IL-7), interleukin-11 (IL-11) and interleukin-12 (IL-12) or functional variants thereof.

35. A method as claimed in claim 34, wherein the colony stimulating factor is neutrophil granulocyte CSF (G-CSF), or a functional variant thereof.

36. A method as claimed in any of claims 32 to claim 35, wherein the CxC chemokine is selected from the group:IL-8, GRO-α , GRO-β , GRO-γ , neutrophii-activating protein 2 (NAP-2), epithelial--cell-derived neutrophii-activating protein (ENA 78), platelet factor (PF4), γlP-10 and granulocyte chemotactic protein 2 (GCP-2), or functional variants thereof.

37. A method as claimed in claim 36, wherein the CxC chemokine is IL-8, or a functional variant thereof.

38. A method as claimed in claim 36, wherein the CxC chemokine is GRO-alpha, or a functional variant thereof.