AU728977B2 - Models for haematopoiesis - Google Patents

Description

1 MODELS FOR HAEMATOPOIESIS This invention relates to model systems for haematopoiesis. In particular, the invention relates to in vitro and in vivo models which can be induced to produce neutrophils in the absence of G-CSF and/or GM-CSF. The model systems can be used to screen for putative haematopoietic, particularly neutrophil-promoting, activities. They may also be used to isolate, identify or characterise non-G-CSF, non-GM-CSF factors or cytokines, particularly neutrophilic stimulators.

BACKGROUND OF THE INVENTION It will be clearly understood that, although a number of prior art publications are referred to herein, "this reference does not constitute an admission that any of these documents forms part of the common general knowledge *in the art, in Australia or in any other country.

The biological regulators that govern the steady- 20 state production of cells in the blood are believed, in part, to be the same factors that, in an emergency situation, serve to promote the production of large numbers of functionally active and terminally differentiated cells (Demetri et al 1991). Two of the principal regulators that act on granulocyte-macrophage progenitor cells are granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF). Much of •the knowledge of these regulators has been obtained from experiments carried out using in vitro systems. The in vivo effects of CSFs have been studied in murine models by injecting pharmacological doses of CSF (Pojda et al 1990), by using transgenic mice (Lang et al 1987) and by studying mice with bone marrow cells which overproduce CSF (Marusic et al 1990). While these studies have confirmed the activity of CSFs in vivo, they have not provided H: \WendyS \Keep\ species\ 60831-98 Lu~dwig.doc 14/11/00 1A information about the physiological role of CSFs in steadystate or emergency haematopoiesis in animals, including man.

In order to try and resolve the complex issue of defining the precise role for GM-CSF and G-CSF, we have recently employed gene targeting in embryonic stem cells to create mice deficient in either GM-CSF (Stanley et al 1994) 9.o°9.

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H:\WendyS\Keep\species\60831-98 Ludwig.doc 14/11/00 WO 98/39413 PCT/AU98/00139 2 or G-CSF (Lieschke et al 1994). The naturally-occurring op/op mutation provides mice that are deficient in macrophage-colony stimulating factor (M-CSF), also known as colony stimulating factor-1 (CSF-1), (Wiktor-Jedrzejczak, 1990). Thus, mice deficient in one or more of these factors can be created by cross-breeding (International Patent Application No. W095/23862). Intriguingly, no striking and consistent haematopoietic abnormalities were detected in GM-CSF deficient mice, indicating either that GM-CSF is dispensable for steady-state haematopoiesis, or that GM-CSF plays some important role in haematopoiesis but that in its absence, mice have the capacity to recruit a functionally-related molecule capable of promoting the production of cells normally produced under the influence of GM-CSF. In contrast, mice deficient in G-CSF still have morphologically recognisable neutrophils in the blood, but are strikingly neutropaenic. They also show a reduction in bone marrow granulocyte-macrophage progenitor cells and maturing granulocytes. Thus, G-CSF plays a pivotal and indispensable role in maintaining normal numbers of neutrophils, although it is important to note that its presence is not mandatory for the production of mature neutrophils.

Production of neutrophilic granulocytes is one of the primary means whereby a host attempts to deal with acute infection by a wide range of pathogenic organisms (Babior 1978, 1984; Hurtel et al 1980). This is achieved principally through the capacity of neutrophils for phagocytosis, as well as the production of various bactericidal substances (Babior 1984; Johnson et al 1981; Till et al 1982). It has been shown that neutropaenic patients are at higher risk of infection than individuals with normal levels of neutrophils (Babior 1984). Moreover, higher numbers of neutrophils are often found in patients following infection (Kawakami 1990). Similar observations have been made in laboratory mice (Mosser et al 1980).

SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 3 In W095/23862, the entire disclosure of which is incorporated herein by reference, a G-CSF deficient (knockout) mouse was described as a model for chronic neutropaenia. Although neutropaenic, the G-CSF mice had essentially normal levels of monocytes and macrophages, supporting the idea that other factors are involved in maintaining the steady-state regulation of these cell types.

We have now found that G-CSF deficient mice infected with Candida albicans develop a striking neutrophilia. This observation was unexpected in view of the correspondence between the rise in G-CSF in normal mice infected with a range of pathogenic organisms, and of the documented capacity of injected G-CSF to promote neutrophil production in mice and man. Importantly, it was also found that the addition of preparations of heat-inactivated C. albicans to established cultures of bone-marrow derived stromal cells from G-CSF deficient mice promoted haematopoiesis, and in particular, the production of neutrophils. The present invention provides an in vitro model for haematopoiesis during infection.

SUMMARY OF THE INVENTION In one aspect, the invention provides an in vitro model of haematopoiesis, comprising a co-culture of bone marrow-derived stromal cells and target haematopoietic cells, which co-culture increases haematopoiesis following exposure to an inactivated microorganism. In particular, the invention provides a model for emergency haematopoiesis.

The co-culture may be a culture of stromal cells and bone marrow cells. These cells can be derived from a host having a disruption of a gene encoding a colony stimulating factor, such as G-CSF-deficient mice or G-CSF/GM-CSF double deficient mice. Other types of mice which can be used include those described in W095/23862, for example mice simultaneously deficient in GM-CSF, G-CSF SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 4 and CSF-1. Other animals or mice with gene disruptions may also be used, and the stromal cells and target haematopoietic cells may be derived from the same or different donor types.

Without wishing to be bound by any proposed mechanism, we believe that exposing irradiated stromal cells to haematopoietic cells in the presence of an inactivated microorganism results in the production of various combinations of cell types, depending on the nature and identity of the microorganism. The composition of the resulting cell population may depend on the combination of factors produced by the stromal cells in response to the inactivated microorganism.

In a particularly preferred embodiment, stromal cells from bone marrow of G-CSF mice are co-cultured with bone marrow cells from mice of the same genotype.

However, as indicated above, bone marrow cells from other animals of other genotypes may be used.

The organism is preferably an infective microorganism, for example a microorganism such as a virus, a bacterium such as a mycobacterium, or a yeast. In principle, the infective organism can be any infectious agent that retains its antigenic properties following inactivation of its infectivity. In principle any such microorganism may be used. Preferably the microorganism is a bacterium such as Mycobacterium, for example Mycobacterium tuberculosis, Mycobacterium bovis and the like, or a yeast such as yeasts of the genus Candida.

The microorganism may be inactivated by any method which is capable of killing the microorganism, including but not limited to heat, chemical sterilants such as formaldehyde, glutaraldehyde or ethyleneimines, irradiation, ultrasonication, pressure disruption and the like. In addition, strains of microorganisms which are temperature-sensitive may be used at the non-permissive temperature. The person skilled in the art will be aware SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 5 of a wide variety of methods from which to choose the one which is most appropriate for a given micoorganism.

More preferably the microorganism is Candida albicans. Candida albicans which has been killed by heat or chemicals, preferably by heat, is especially preferred.

Haematopoiesis following exposure of the coculture to killed Candida albicans is preferably characterised by neutrophilia, ie. by a significant increase in the number of neutrophils.

In a second aspect, the invention provides an in vivo model of haematopoiesis, comprising a mouse deficient in one or more haematopoietic factors, and which has been injected with a sub-lethal dose of a living microorganism.

Preferably the microorganism is as described above.

Preferably the mouse is deficient in one or more of the colony-stimulating factors. More preferably the mouse is deficient in G-CSF or GM-CSF. Even more preferably the mouse is deficient in both G-CSF and GM-CSF.

As described above, other types of mice which may be used include those described in WO 95/23862.

In a third aspect, the invention provides a haematopoietic factor produced following exposure to a microorganism of bone marrow-derived stromal cells from a host animal which is deficient in a colony stimulating factor. In one preferred embodiment, a factor that promotes neutrophilia and which is produced by the in vitro model is contemplated. In an alternative embodiment, the factor promotes neutrophilia and is present in the serum or lymph of a mouse according to the in vivo model of the invention.

Preferably the factor has one or more of the following characteristics: a) ability to promote differentiation and multiplication of haematopoietic cells in the in vitro model of the invention; SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 6 b) the haematopoietic activity is not abrogated by antibody directed against known haematopoietic factors such as G-CSF, G-CSF, M-CSF (CSF-1) or S-CSF; c) has a molecular weight of greater than 10 kD; d) elutes as a peak on gel filtration using a Sephadex G-100 column.

Preferably the factor promotes differentiation of haematopoietic precursor cells into the granulocyte/macrophage and/or macrophage progenitor lineage. Most preferably the factor promotes differentiation into mature neutrophils. Most preferably the factor has the capacity to promote neutrophilia independent of the presence of G-CSF and/or GM-CSF.

It is believed that the factor of the invention is a protein or glycoprotein. Methods for purification of proteins and glycoproteins are extremely well-known in the art, and the person skilled in the art will be aware of a wide variety of suitable methods for purification of the factor to homogenity. Methods for identifying and isolating the gene encoding the factor are similarly wellknown. In addition, methods for preparing antibodies, including monoclonal antibodies, directed against the factor of the invention are very widely known in the art, and methods of preparation of antibodies, antibody fragments such as Fab or F(ab) 2 fragments, and antibody analogues such as ScFv fragments using recombinant methods are routine in the art.

Thus it will be clearly understood that the invention includes within its scope purified factors of the invention, nucleic acids encoding them, and antibodies, monoclonal antibodies, and ScFv constructs directed against them. Methods which may be used in the purification of factors, isolation of genes, and generation of antibodies include those set out in the U.S. provisional patent application by Ludwig Institute for Cancer Research, entitled "Dendritic Cell Factor" filed on 14 October 1997, SUBSTITUTE SHEET (RULE 26) WO98/39413 PCT/AU98/00139 7 the entire disclosure of which is incorporated herein by this reference.

The invention also includes within its scope a pharmaceutical composition comprising the factor of the invention, together with a pharmaceutically-acceptable carrier. Suitable carriers are widely known in the art.

See for example Remington's Pharmaceutical Sciences (Mack Publishing Easton, Pennsylvania).

In a fourth aspect, the invention provides a method of inducing an increase in neutrophil numbers in a mammal in need of such treatment, comprising the step of administering an effective amount of the factor of the invention to the mammal.

In an alternative embodiment of this aspect of the invention, there is provided a method of inducing neutrophil-promoting activity in a mammal in need of such treatment, comprising the step of administering an effective amount of the factor of the invention to the mammal.

The mammal may be a human, or may be a domestic mammal, including companion, farm and zoo mammals. The mammal may be at risk of infection, suffering from infection, or suffering from a condition which results in neutrophil deficiency.

Any suitable route of administration may be used.

The most suitable dosage and route of administration will depend on the route of the condition to be treated, and will be at the discretion of the attending physician or veterinarian.

Using the models of the invention and neutropaenic animals which are of the genotypes described above, the activities of one or more cytokines or haematopoietic factors can be studied. In particular, the in vitro and in vivo models of the invention can be used to identify neutrophilic factors.

Therefore, in a fifth aspect, the invention provides a method of screening for factors or agents which SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 8 are useful in the treatment of infection, comprising the step of testing a putative factor or agent for its ability to stimulate haematopoiesis in a model according to the invention. The activity of new factors can also be assessed, as well as combinations of such factors with known cytokines such as interleukins, known haematopoietic factors such as G-CSF or GM-CSF or the like. The in vitro and in vivo effects of such combinations on haematopoiesis can also be investigated using the invention. This method of screening is particularly useful in identifying agents useful for treatment of immunosuppressed patients or in clinical settings where elevated numbers of neutrophils are desired For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning. The word "inactivated" in reference to a microorganism means that the microorganism is either killed or has its ability to infect a host abolished by a treatment such as heat or exposure to chemicals, but that the antigenic properties of the microorganism are retained. The word "inactivation" has a corresponding meaning.

Brief Description of the Figures Figure la shows the number of non-adherent cells recovered from the co-culture of G-CSF deficient stromal cells and G-CSF deficient bone marrow cells incubated for 7 days in the presence or absence of heat killed Candida albicans. 2.5 x 106 G-CSF deficient bone marrow cells were seeded on 1 x 106 irradiated G-CSF deficient stromal cells alone or along with heat-inactivated Candida albicans x 106). The cultures were incubated under the conditions described in Example 2, for seven days. The non-adherent cells were harvested and counted.

Figure lb shows the neutrophil population in the non-adherent cells, as enumerated by staining with Gr-1 SUBSTITUTE SHEET (RULE 26) WO98/39413 PCT/AU98/00139 9 antibody or isotype-matched control antibody and analysis by fluorescence-activated cell sorting (FACS).

Figure 2 shows the total number of colony-forming cells, observed after 14 days in semisolid agar cultures, recovered from the non-adherent fraction of the 7 day bone marrow and stromal cell co-cultures from wild type and G-CSF deficient mice in the presence or absence of Candida albicans.

Figure 3 shows the production of haematopoietic cells in Dexter cultures (Dexter et al, 1977) established using G-CSF-deficient stromal cells. Stromal cells were seeded with 2.5 x 106 G-CSF-deficient bone marrow cells together with 2.5 x 106 heat-inactivated Candida albicans (Figure 3a), or alone (Figure 3b). Haematopoietic cells are represented by bright cells in the foreground (Figure 3a), and stromal cells are represented by the dark cells in the background (Figure 3b).

Figure 4 shows levels of IL-6 in serum from G-CSF deficient mice and in wild-type control mice following infection by Candida albicans.

Figure 5 shows the effect of vari6us cytokines on the colony forming cells in bone marrows of G-CSF deficient mice (Figure 5a) and in wild-type, control mice (Figure 5b). Bone marrow from each type of mice was isolated on days 0, 3 and 7 after Candida albicans infection and assayed for progenitor cells by soft agar assay. Colonies were scored 7 days after plating, and each value represents the mean of observations on 3 mice for each genotype.

Figure 6 shows the results obtained when concentrated conditioned medium from G-CSF deficient stroma stimulated with Candida albicans was fractionated on a Sephadex G-100 column. Absorbance of the fractions was monitored at 280 nm, and then the fractions were tested on soft agar assay for colony-stimulating activity. Colonies were scored on day 7.

SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 10 Figure 7 shows the morphology of cells obtained when 2 x 104 metrizamide fractionated bone marrow cells were incubated in the presence of SCF (20 ng/ml) M-CSF ng/ml) Fraction no. 12 (from Figure 6) or medium alone at 37 0 C in a humidified atmosphere containing 5% CO 2 for four days. The slides were then stained with May-Grunwald Giemsa.

Figure 8 is another field from the same slide as that depicted in Figure 6, showing the presence of macrophages. The arrows indicate neutrophils.

Figure 9 shows results obtained when metrizamide-fractionated bone marrow cells were incubated in the presence of factors and conditioned medium from G-CSF/GM-CSF double deficient stroma a) M-CSF (10 ng/ml) b) SCF (20 ng/ml) c) conditioned medium (1:4 dilution) d) conditioned medium (1:4 dilution Ack-2 antibody e) conditioned medium (1:4 dilution) anti-M-CSF antibody f) medium alone for 4 days at 37 0 C in a humidified atmosphere containing

CO

2 The cells were stained with May-Grumwald Giemsa.

The arrows indicate neutrophils.

DETAILED DESCRIPTION OF THE INVENTION The invention will now be described by way of reference only to the following non-limiting examples, and to the figures.

General Methods Culture and Preparation of Candida albicans: Candida albicans (ATCC strain no. 14053) was grown in Sabouraud's 2% dextrose broth at 37 0 C with agitation. After 24 h in culture, cells were harvested by low speed centrifugation (1000 x washed twice in SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 11 saline, briefly sonicated and diluted to the desired density. Where heat-killed Candida albicans cells were to be used, the cells were killed by heating at 1210°C for minutes.

Candida albicans Infection in Mice: 8-10 week old G-CSF, G-CSF/GM-CSF double deficient and wild type sex-matched mice raised in microisolators were used for the study. Mice were bled by retro-orbital puncture 2 days before Candida challenge for baseline blood counts and for determining the levels of various cytokines. Mice were injected with 2.5 x 105 colony forming units (cfu) of Candida albicans through the lateral tail vein. Groups of mice were bled at various days after infection and thereafter sacrificed. Serum was separated immediately by centrifugation at 1000 x g for min, aliquoted and frozen at -20°C for further use.

Various organs (liver, spleen, lung and kidney) were collected under aseptic conditions in sterile phosphatebuffered saline (PBS) for quantification of Candida albicans load. For histological analysis, tissues were excised and immediately fixed in Bouin's solution.

Haematological Analysis: Blood samples were diluted immediately (1:4 in 2 mg/ml EDTA in mouse tonicity buffered saline) after collection, and blood cell counts were performed on a Sysmex-K1000 automated counter (Toa Medical Electronics Co.

Ltd, Kobe, Japan). Manual 100- cell leukocyte differential counts were performed on May-Grunwald/Giemsa stained smears. Semisolid agar cultures of bone marrow were prepared and scored as previously described (Metcalf 1984; Metcalf 1991). Colony formation was stimulated by the following recombinant growth factors at the concentrations specified: human G-CSF (10 ng./ml), murine GM-CSF ng/ml), murine IL-3 (10 ng/ml), murine M-CSF ng/ml), rat stem cell factor (SCF, 100 ng/ml).

SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 12 Mice with Targeted Gene Disruptions Mice deficient in expression of G-CSF (G-CSF GM-CSF (GM-CSF or both G-CSF and GM-CSF (G-CSF/GM-CSF double deficient) were produced by targeted gene disruption and appropriate subsequent inbreeding or cross-breeding as described in W095/23682. Mice deficient in both G-CSF and IL-6, were produced by appropriate crosses with IL-6 mice. Mice deficient in G-CSF and circulating Stem Cell Factor (SCF) are produced by appropriate crosses with Sl/Sld mutant mice.

Cytokine assays Bioassays for IL-6, G-CSF, IL-3, GM-CSF and SCF IL-6 assay IL-6 in serum or culture supernatant was measured by the 7TD1 proliferation assay as described in Brouckaert et al, 1989.

G-CSF assay The G-CSF bioactivity was assayed by a cellular proliferation assay using Ba/F3 cells expressing a transfected human G-CSF receptor (designated Ba/f 19.1), which confers on Ba/F3 cells the ability to proliferate in the presence of both human and mouse G-CSF (Layton, Ludwig Institute for Cancer Research, Melbourne). Briefly, test samples were serially diluted in 96-well flat bottomed plates and incubated for 48 h at 37 0 C in an atmosphere of

CO

2 in air with 10000 Ba/F19.1, in parallel with 10000 Ba/F3 cells. Dilutions of G-CSF were included as standards. After 48 h, the cells were pulsed with 0.5 gCi of 3 H]-thymidine, harvested after 4h, and incorporation of [3H]-thymidine assessed using a beta-scintillation counter.

GM-CSF and IL-3 assay To assay for bioactive GM-CSF, the FDC-P1 proliferative response 3 H]-thymidine incorporation) was used with adjustment for interleukin-3 (IL-3) bioactivity SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 13 on 32-D cells. IL-3 activity was assayed on 32-D cells (Kelso Gough, 1989).

SCF Assay SCF bioactivity was assayed by proliferation of Mo7e cells in the presence and absence of Ack-2 antibody.

Briefly, test samples and standard rat SCF were serially diluted in 96-well flat bottomed plates and incubated for 96 h at 37 0 C in an atmosphere of 5% CO 2 in air with 2000 Mo7e cells or with 2000 Mo7e cells which had been preincubated with Ack-2 antibody for 30 min at 37 0 C. At the end of the incubation period, cells were labelled with gCi of 3 H]-thymidine, harvested after 4 h, and incorporation of 3 H]-thymidine assessed using a betascintillation counter.

ELISAs for TNF-a and IL-la TNF-a and IL-la levels in the serum of Candida albicans infected and control mice and in the conditioned medium from the stromal cultures were measured using ELISA kits (Genzyme Co., Cambridge, USA) specific for murine cytokines, as recommended by the supplier.

Example 1 Effect of Candida albicans on peripheral blood neutrophil counts and bone marrow neutrophil counts in G-CSF-deficient, G-CSF/GM-CSF double deficient and wild-type mice Cohorts of wild-type, G-CSF deficient and G-CSF/GM-CSF double deficient mice were infected with a sub-lethal dose (2.5 x 10 5 cfu) of live Candida albicans by injection into the lateral tail vein. Peripheral blood neutrophil counts were obtained 2 days prior to infection (termed day 0) and 4 and 7 days after challenge with the micro-organism. A striking degree of neutrophilia was evident in day 4 in the G-CSF deficient group, and this was maintained at day 7, as shown in Table la.

SUBSTITUTE SHEET (RULE 26) Table la Blood Neutrophil counts Genotype Time 1Total white blood cell count Neutrophils (x 10 9/1) (x 10 1/1) Wild type day 0 6.2 ±2.15 0.6±0.35 7 6.9 0.94 1.5 0.6 G-CSF deficient day 0 4.86 ±1.1 0.13 0.06 7 4.0 1.2 1.92 0.8 G-CSF/GM-CSF deficient day 0 7.6 1.8 0.1 0.04 Iday 7 7.2 ±2.4 0.9 ±0.2 WO 98/39413 PCT/AU98/00139 15 The numbers of neutrophils present in the peripheral blood of both wild-type and G-CSF deficient mice on day 7 following Candida albicans infection were in the same range; this is rather surprising, since at the outset of the experiment, G-CSF deficient mice were neutropaenic, having only 20-30% of neutrophil numbers present in the wild-type control mice. Thus, the absolute numbers of neutrophils generated in Candida albicans infected G-CSF deficient mice were 4-5 times those in C. albicans infected control mice. This striking observation clearly indicates that one or more factors having the capacity to promote neutrophilia independent of the presence of G-CSF of CM-CSF is present.

This neutrophil-promoting activity may also contribute to infection-stimulated neutrophilia in wildtype control mice. G-CSF could not be detected in the serum of wild-type mice infected with Candida albicans.

The number of neutrophils in the bone marrow of these animals was also assessed at the same time points as the peripheral blood neutrophils for all three groups of mice (Table ib).

SUBSTITUTE SHEET (RULE 26) Table lb Percentage of Neutrophils in the Bone Marrow of Mic~e Before and After Candida Infection

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r a, Genotype Time Total cell count/femur I Promyelocyte/myelocyte Metainyelocyte/Polymorph x 10')1 lWild type day 0 1.67±0.29 11 24 7 2.7 ±0.8 23 37 G-CSF deficient day 0 1.5± 0.41 5 7 2.5± 0.7 35 18 G-CSF/GM-CSF day 0 1.8±0.2 15 deficiencies 7 12.3t 0.42 139 125 WO 98/39413 PCT/AU98/00139 17 Example 2 In vitro Model for Emergency Haematopoiesis Stromal cell cultures (Dexter et al, 1977) from bone marrow of G-CSF-deficient G-CSF/GM-CSF double deficient mice and wild type mice were established using conditions slightly modified from those described by Dexter et al (1977). Cultures were established in growth medium (Iscove's Modified Dulbecco's medium: IMDM)/RPMI-1640 [50:50]; 1 x non-essential amino acids; 1 x vitamins; heat inactivated FCS) at 37 0 C in an atmosphere of 5% 02: 10% C0 2 :85% N 2 and allowed to become confluent. After 3 weeks, the adherent cells were trypsinized, washed and sub-cultured into 25 cm 2 flasks at a density of 1 x 106 cells per flask. After 3 days, the cells were irradiated (1100 rad gamma-irradiation) and then seeded with bone marrow cells from mice deficient in G-CSF, mice deficient in both G-CSF and GM-CSF, or from control mice (1 x 106 or x 106 cells per flask). In test cultures, 2.5 x 106 heat-killed Candida albicans cells were added. The cultures were replenished with fresh culture medium each week by removing half of the medium and replacing this with the same volume of fresh medium. Cultures were terminated after 7 days, 14 days or 21 days, and the total number of non-adherent cells was determined for each of these time points. Figure la shows the number of non-adherent cells recovered after 7 days. The cell population in the nonadherent fractions was analysed for granulocytes by preparing cytospins of the samples and performing a 100 cell differential count; the results are shown in Figure lb. In some experiments, the cells were subjected to FACS analysis by staining the cells with antibodies against the granulocyte cell surface marker Gr-1; the results of these analyses are shown in Figure 5b. In addition, the colony-forming cells in the non-adherent fractions were analysed by plating the cells in soft agar in the presence of IL-3 and GM-CSF, and scoring the colonies after 14 days. These results are shown in Figure 2.

SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 18 Figure 3 shows haematopoietic cells in Dexter cultures established using G-CSF deficient stromal cells seeded with G-CSF-deficient bone marrow cells in the presence or absence of heat-inactivated C. albicans (Figure 3a and Figure 3b respectively).

Example 3 Cytokine levels in G-CSF and wild-type mice infected with Candida albicans It is well established that mice experimentally infected with Candida albicans express a range of cytokines, including TNF, IL-1, IL-4, IL-6, IFN-y and TGF-P. It is also known that administration of particular cytokines, eg., GM-CSF, G-CSF, IL-1, IL-6, IL-11 and SCF to mice can result in neutrophilia. As a first step to identifying the regulators that mediate neutrophilia in Candida albicans-infected G-CSF deficient mice, we have measured the activities of IL-6, GM-CSF and IL-3 in serum alone, in spleen conditioned medium (SCM) and lung conditioned medium (LCM) from these mice by using specific bioassays. The levels of IL-6 in serum of G-CSF deficient and of control, wild-type (WT) mice are shown in Figure 4.

Levels of cytokines in other conditioned media and in serum of Candida albicans-infected and control mice are shown in Table 2.

The effects of various cytokines on formation of colonies in soft agar bone marrow assays following C. albicans infection were also examined, and the results are shown in Figure 5. This indicates that there are more granulocyte/macrophage and macrophage progenitors in Candida albicans-infected G-CSF deficient mice than in similarly-infected wild-type mice. These progenitors also remain elevated for longer in Candida albicans-infected G-CSF deficient mice than in wild-type mice. This effect was greatest when the bone marrow was harvested 7 days after infection; in contrast, for the wild type mice the effect was greatest at 3 days after infection.

SUBSTITUTE SHEET (RULE 26) Table 2 Levels of various cytokines in the spleen conditioned medium, lung conditioned medium and serum of the uninfected G-CSF-deficient and wild type mice and in mice 7d after Candida infection. T indicates an increase in the level of the cytokine, and the number of arrows indicates the level of increase of the particular cytokine Cytokine Source Time Wild type G-CSF-/- GM-CSF Serum Baseline not detectable not detectable Candida infected not detectable not detectable Lung conditioned medium Baseline 1T Candida infected TTT TTT Spleen conditioned medium Baseline not detectable not detectable Candida infected TT not detectable IL-3 Serum Baseline not detectable not detectable Candida infected T not detectable Lung conditioned medium Baseline not detectable not detectable Candida infected ITT TTT Spleen conditioned medium Baseline not detectable not detectable Candida infected T not detectable IL-6 Serum Baseline not detectable not detectable Candida infected I TTT WO 98/39413 PCT/AU98/00139 20 Example 4 Neutralisation of Growth Factors or Cytokines in the In Vitro Model for Emergency Haematopoiesis High levels of a particular cytokine in G-CSF deficient mice exposed to Candida albicans when compared to control mice would indicate the possibility that this cytokine was mediating the induced neutrophilia either wholly or in part. However, since it is possible that there may be more than one candidate among the known factors with cytokine activity, the in vitro system provides a tool which is very useful in identifying the factor(s) responsible for the observed neutrophilia in the G-CSF deficient and/or G-CSF/GM-CSF double deficient mice.

Firstly, neutralising antibodies corresponding to known haematopoietic regulators are added to conditioned medium recovered from co-cultures of stromal cells and target bone marrow cells which have been exposed to heat inactivated C. albicans. This antibody-treated conditioned medium is subsequently used to supplement liquid and semisolid agar cultures that include fresh bone'marrow cells or enriched preparations of granulocyte-macrophage colony forming cells (GM-CFCs) derived from cyclophosphamidetreated mice. GM-CFCs are initially enriched by densitygradient-centrifugation and subsequently by elutriation centrifugation, using the procedure of Lord and Spooner (1986). Liquid culture assays are used to determine whether the presence of the antibody suppresses haematopoiesis, and in particular neutrophil production.

Semi-solid agar cultures are used to determine whether the antibody suppresses the generation of colonies containing neutrophils. The absence of neutrophilpromoting activity in conditioned media would suggest the possibility that an indispensable component of the neutrophil-promoting activity is cell-associated. The same assays using the same conditioned media (vide supra) supplemented by sonicated preparations of stromal cells SUBSTITUTE SHEET (RULE 26) WO 98/39413 WO 9839413PCT/AU98/00139 21 from cultures exposed to heat-inactivated Candida albicans and target bone marrow cells can then be utilised to test whether the activity is associated with stromal cells.

In a second strategy, antibodies are added directly to cultures of stromal cells according to the in vitro model described in Example 2, that include heatinactivated Candida albicans and target bone marrow cells to see whether the antibodies suppress haematopoiesis and neutrophil production.

Example 5 Bone Marrow Liquid Assay for Neutrophil- Promoting Activity Conditioned medium was generated from G-CSFdeficient stroma incubated with Candida albicans for 24 h.

Initially this conditioned medium was tested for its colony stimulating activity (CSA) in a soft agar assay using bone marrow cells isolated from C57B1/6 mice. 25,000 bone marrow cells were plated in the presence of 200 il of conditioned medium in a soft agar culture in 1 ml final volume. Colonies were scored on day 7 after plating, and the results are shown in Figure 7. The colonies were stained and further typed depending on cell morphology.

The results are summarised in Table 3.

Table 3 Amount of Final volume Dilution Total number conditioned of colonies medium 200 p.l iml 5x The conditioned medium was further tested on FDCP-1 and 32D cells for GM-CSF/IL-3 activity, and on Mo7 e cells in the presence or absence of an antibody against SCF receptor (also known as c-kit), designated Ack-2 (Dr Andreas Strasser, Walter and Eliza Hall Institute of SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 22 Medical Research, Melbourne, Australia) to measure the SCF activity. These results are shown in Table 4.

Table 4 Cells Assay Activity in Conditioned Medium FDCP-1 GM-CSF/IL-3 not detected 32-D IL-3 not detected M07 e (+Ack-2) SCF <1.25 ng/ml Conditioned medium was fractionated in the following manner. 20 ml of conditioned medium was concentrated 10-fold on a YM10 membrane, which has a molecular weight cut-off at 10 kD, and then fractionated using a Sephadex G-100 column (Pharmacia).

Sephadex G-100 (fine grade) was packed in a column 33 cm long and 1.5 cm internal diameter. For conditioned medium from G-CSF deficient mice the elution buffer was 20 nM Tris/0.1 M NaCl, pH 7.4. The elution was performed under standard conditions, using dextran blue as a marker. Absorbance at 280 nm was measured for each of the fractions collected. The fractions were further tested for neutrophil-promoting activity in soft agar colony assay and bone marrow liquid assay. The results are summarised in Figure 6.

Neutrophil-promoting activity (NPA) in the conditioned medium and the fractions was assayed on bone marrow cells enriched for GM-CFCs as described by Lord and Spooner (1986). C57B1/6 mice were injected i.p. with cyclophosphamide at 200 mg/kg 3 days prior to being killed.

Bone marrow cells from femurs of cyclophosphamide-treated mice were flushed and subjected to metrizamide fractionation. The band of low density cells at the interphase was collected, washed and resuspended in RPMI- 1640 with 10% FCS. Cells were plated at 2 x 104 cells/well SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 23 in 96-well round-bottomed plates in the presence of different concentrations of various growth factors [SCF, GM-CSF, M-CSF; 0.1-10.0 ng/ml], conditioned medium fractions at 1:8 final dilution, or in the presence of medium alone, for 96 h. To measure relative DNA synthesis, cells were labelled with 3 H]-thymidine 18 h prior to the end of the incubation period. At the end of the incubation period cells were harvested, lysed and the amounts of radioactivity incorporated counted in a beta-counter. The results are shown in Table Table Sample 3 H] -Thymidine Incorporation (cpm) Fraction #8 400 Fraction #9 408 Fraction #10 380 Fraction #11 280 Fraction #12* 230 Fraction #13 230 control 100 M-CSF 10 ng/ml 700 M-CSF 1 ng/ml 241 SCF 20 ng/ml 2952 SCF 2 ng/ml 136 GM-CSF 10 ng/ml 20460 GM-CSF 1 ng/ml 4058 For morphological analysis cells were plated in chamber slides and at the end of 96h, cells were stained with May-Grunwald-Giemsa. These results are illustrated in Figure 7.

SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 24 Example 6 Neutralisation of Growth Factors or Cytokines In Vi vo The results represented in Figures 1 and 3 show that the in vitro system mimics the in vivo situation.

Therefore this in vitro model is useful for the study of the effect of neutralisation of cytokines on neutrophil production. In cases where an antibody against a known factor is shown to neutralise the neutrophil-promoting activity in the in vitro model, this antibody is used in mice to study its effect in vivo. In the event that the granulocytic promoting activity induced by the Candida albicans infection is not abolished by neutralising the activity of any known factor, identification and characterisation of the stromal cell-derived activity is undertaken using purification, eg. by using standard physical and chromatographic separation procedures, sequencing and molecular cloning. Partial sequencing of tryptic peptides provides the basis of constructing oligonucleotides for screening gene/cDNA libraries, and enabling production of a molecular clone corresponding to the factor.

G-CSF-deficient, G-CSF/GM-CSF deficient and control animals are treated with anti-cytokine neutralising antibodies prior to, and following, intravenous injection of preparations of Candida albicans. The precise antibody treatment protocol depends on the nature and characteristics of the antibody preparation. Antibodytreated mice are examined 4 and 7 days following infection with Candida albicans, and the extent of infection-driven neutrophilia is monitored by determining white blood cell (WBC) counts in a Sysmex 1000 analyser in combination with manual differential counts on May-Grunwald/Giemsa smears of peripheral blood.

SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 25 Example 7 Conditioned Medium from Irradiated G-CSF/GM-CSF Double-Deficient Stromal Cells Stimulated with Heat-Inactivated Candida albicans Mice deficient in both G-CSF and GM-CSF were produced as described in our earlier PCT application, WO 95/23682.

Conditioned medium was harvested from irradiated stromal cell cultures derived from bone marrow of G-CSF/GM-CSF double deficient mice and stimulated with heat killed Candida albicans. 50 .l of conditioned medium was added to 5,000 metrizamide-fractionated bone marrow cells in a total volume of 200 gl in 96 well round-bottomed plates and incubated for 96 h at 370C in a humidified atmosphere containing 5% CO 2 Cells were also incubated with M-CSF, SCF, IL-3, or medium alone under identical conditions. Parallel cultures were also established in 16 well chamber slides. 3 H]-thymidine was added to the cultures in 96 well round-bottom plates 18 h before the end of the incubation period. At the end of incubation relative proliferation was measured by 3 H]-thymidine uptake, and the proportion of neutrophils in the culture determined by microscopic examination of May-Grunwald- Giemsa stained cells in chamber slides.

In parallel samples in the same experiment the activities of M-CSF and SCF activity in conditioned medium were neutralised using anti-M-CSF and Ack-2 anti-SCF receptor antibodies. The Ack-2 antibody was as described in Example 5, and the anti-M-CSF antibody was a gift from Dr Richard Stanley (Albert Einstein College of Medicine, Bronx, Standard murine M-CSF and rat SCF preparations were also incubated with anti-M-CSF and Ack-2 antibodies respectively. Briefly, M-CSF activity was neutralised by co-incubating conditioned medium and standard cytokines with an equal amount of neutralising anti-M-CSF antibody at 37°C for half an hour. These samples were then added to 5,000 metrizamide-fractionated SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 26 bone marrow cells and the cells were incubated as described above. For blocking SCF activity, cells were incubated in the presence of Ack-2 antibody prior to the addition of either conditioned medium or SCF. The results are summarized in Table 6.

SUBSTITUTE SHEET (RULE 26) Table 6 Stimulus cpm cpm in the presence of cpm in the presence of anti-M-CSF antibody Ack-2 antibody M-CSF 10 ng/ml 15926 160 not done SCF 20 ng/ml 7330 7267 100 conditioned medium 786 319 200 (1:4 dilution) control 100 44 100 WO 98/39413 PCT/AU98/00139 28 SCF activity in the conditioned medium was beyond the detection limit in the Mo7 e cell assay, which was performed as described in Example The chamber slide results, shown in Figure 9, show that neutrophils are present only in the cultures incubated in the presence of conditioned medium, and that neither antibody directed against M-CSF nor antibody directed against SCF receptor abrogates the stimulatory effect of conditioned medium from the stromal G-CSF/GM-CSF double deficient mice. This indicates that the factor elicited by Candida albicans in the stromal cell cultures is neither G-CSF nor SCF.

These results show that the neutrophilia observed in the previous examples using G-CSF deficient mice was not the result of trace amounts of GM-CSF or CSF-1.

Since G-CSF deficient mice have 20-30% of neutrophil numbers seen in wild type mice, the generation and maintenance of this proportion of neutrophils is independent of G-CSF, providing a clear indication of the action of another factor(s) for neutrophil production. In normal mice infected with a range of microorganisms, neutrophil numbers are elevated, and this is thought to be under the control of G-CSF.

In view of the fact that initially the G-CSF deficient animals have only 20-30% of the neutrophil numbers present in wild-type mice, the increase observed in Candida albicans-infected G-CSF deficient animals corresponds to a 4- to 5-fold increment compared with the infected controls.

This response was unexpectedly reproduced in an in vitro system comprising stromal cells, which are thought to produce soluble or membrane-associated cytokines or growth factors. Co-cultures of stromal and bone marrow cells obtained from G-CSF deficient mice showed a very striking increase in the numbers of non-adherent cells in SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 29 the presence of heat-inactivated Candida albicans. At least 50% of these cells were neutrophils.

Similar increases in neutrophil numbers are observed in G-CSF/GM-CSF double deficient mice infected with Candida albicans in vivo. This indicates that GM-CSF is not likely to be the factor responsible for promoting neutrophil production in G-CSF deficient mice. The present invention provides a means to understand the basis of infection-driven neutrophilia in G-CSF deficiency. It is also useful for identifying an activity that may have therapeutic value in the treatment of infections.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and are incorporated herein by this reference.

SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 30

REFERENCES

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N. Engl. J. Med., 1978 298 659 Babior B.M.

Blood, 1984 64 959 Brouckaert P. et al J. Exp. Med., 1989 169 2257 Cook N. et al The EMBO Journal, 1989 8:2967 Demetri G.D. and Griffin J.D.

Blood, 1991 78 2791 Dexter, Allen T.D. and Lajtha L.G.

J. Cell. Physiol., 1977 91 335-338 Hurtel B. et al Ann. Immunol., 1980 131C 105 Johnson K.J. and Ward P.A.

J. Immunol., 1981 126 2365 Kawakami M.

Blood, 1990 76 1962 Kelso A. and Gough N.M.

Growth Factors, 1989 1(2) 165-177 Lang R.A. et al Cell, 1987 51 Lieschke et al SUBSTITUTE SHEET (RULE 26) WO 98/39413 WO 9839413PCT/AU98/00139 31 Blood, 1994 84 1737-1746 Lord, B.I. and Spooner, E.

Lymphokine Research, 1986 5 59-72 Marusic A. et al Agents Actions, 1990 31 280 Mosser S.A. and Domer J.E.

Infec. Inunun., 1980 27 37 Pojda Z. et al Exp. Hernatol., 1990 18 27 Stanley et al Proc. Natl. Acad. Sci USA, 1994 91 5592-5596 Till G.O. et al J. Clin. Invest., 1982 69 1126 Wiktor-Jedrzejczak

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Proc. Natl. Acad. Sci USA, 1990 87 4828-4832 SUBSTITUTE SHEET (RULE 26)

Claims (29)

1. An in vitro model for haematopoiesis, comprising a co-culture of bone marrow-derived stromal cells and target haematopoietic cells, which co-culture manifests haematopoiesis following exposure to an inactivated microorganism.

2. A model according to Claim 1, in which the co- culture is a culture of stromal cells and bone marrow cells.

3. A model according to Claim 1 or Claim 2, in which the stromal cells and/or the bone marrow cells are derived from a host having a disruption of a gene encoding a colony-stimulating-factor.

4. A model according to Claim 3, in which the colony-stimulating-factor of G-CSF, G-CSF and GM-CSF, G-CSF or CSF-1. A model according to any one of Claims 1 to 4, in which both the stromal cells and the bone marrow cells are derived from G-CSF deficient mice.

6. A model according to any one of Claims 1 to 5, in which the microorganism is an infective microorganism.

7. A model according to any one of Claims 1 to 5, in which the microorganism is a virus, a bacterium, or a yeast.

8. A model according to Claim 7, in which the microorganism is a bacterium of the generus Mycobacterium, or a yeast of the genus Candida.

9. A model according to Claim 8, in which the microorganism is Mycobacterium tuberculosis or Mycobacterium bovis. A model according to Claim 8, in which the microorganism is Candida albicans.

11. A model according to any one of Claims 1 to in which the microorganism is inactivated by heat, a chemical sterilant, irradiation, ultrasonication or pressure disruption. SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 33

12. An in vivo model of haematopoiesis, comprising a mouse deficient in one or more haematopoietic factors, and which has been injected with a sub-lethal dose of a living microorganism.

13. An in vivo model according to Claim 12, in whci-h the mouse is deficient in one or more haematopoietic factors of the colony stimulating factor family.

14. An in vivo model according to Claim 13, in which the mouse is deficient in G-CSF and/or GM-CSF.

15. An in vivo model according to Claim 14, in which the mouse is deficient in both G-CSF and GM-CSF.

16. An in vivo model according to any one of Claims 13 to 15, in which the microorganism is as defined in any one of Claims 6 to 11.

17. A haematopoietic factor produced following exposure to a microorganism of bone marrow-derived stromal cells from an animal which is deficient in a colony- stimulating factor.

18. A factor according to Claim 17 which promotes differentiation of haematopoietic precursor cells into the granulocyte/macrophage and/or macrophage progenitor lineage.

19. A factor according to Claim 17 or Claim 18 which promotes differentiation of haematopoietic precursor cells into mature neutrophils. A factor according to any one of Claims 17 to 19 which has the capacity to promote neutrophilia independent of the presence of G-CSF and/or GM-CSF.

21. A haematopoietic factor according to Claim 17 to 20, which: a) promotes neutrophilia; and b) is produced in an in vitro model according to any one of Claims 1 to 11.

22. A haematopoietic factor according to any one of Claims 17 to 21 which promotes neutrophilia and which is SUBSTITUTE SHEET (RULE 26) WO 98/39413 PCT/AU98/00139 34 present in the serum or lymph of an in vivo model according to any one of Claims 12 to 16.

23. A factor according to any one of Claims 17 to 22 which has one or more of the following characteristics: a) ability to promote differentiation and multiplication of haematopoietic cells in the in vitro model of the invention; b) the haematopoietic activity is not abrogated by antibody directed against known haematopoietic factors such as G-CSF, GM-CSF, M-CSF or SCF; c) has a molecular weight of greater than 10 kD; d) elutes as a peak on gel filtration using a Sephadex G-100 column.

24. A pharmaceutical composition comprising a factor according to any one of Claims 17 to 23, together with a pharmaceutically-acceptable carrier. A method of inducing an increase in neutrophil numbers in a mammal in need of such treatment, comprising the step of administering an effective amount of the factor of the invention to the mammal.

26. A method of inducing neutrophil-promoting activity in a mammal in need of such treatment, comprising the step of administering an effective amount of the factor of the invention to the mammal.

27. A method according to Claim 25 or Claim 26, in which the mammal is at risk of infection, suffer from infection, or suffering from a condition which results in neutrophil deficiency.

28. An isolated nucleic acid molecule encoding a factor according to any one of Claims 17 to 23.

29. An antibody, antibody fragment or antibody analogue directed against a factor according to any one of Claims 17 to 23. A method of screening for a factor or agent useful in the treatment of infection, comprising the step of testing a putative factor or agent for its ability to SUBSTITUTE SHEET (RULE 26) 35 stimulate haematopoiesis in a model according to any one of Claims 1 to 16.

31. A method according to Claim 30, in which the factor is a cytokine or haematopoietic factor.

32. A method according to Claim 30 or Claim 31, in which the factor is a neutrophilic factor.

33. A method according to any one of Claims 30 to 32, in which a combination of two or more factors is tested.

34. An in vitro model according to Claim 1, substantially as herein described with reference to the examples and drawings. An in vitro model according to Claim 12, substantially as herein described with reference to the examples and drawings. 15 36. A haematopoietic factor according to Claim 17, substantially as herein described with reference to the examples and drawings. go 37. A method of screening according to Claim substantially as herein described with reference to the 20 examples and drawings. Dated this 14th day of November 2000 LUDWIG INSTITUTE FOR CANCER RESEARCH By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia Trade Mark Attorneys of Australia