WO1999036441A2

WO1999036441A2 - Haemopoietic stem cell inhibitor(s)

Info

Publication number: WO1999036441A2
Application number: PCT/EP1999/000318
Authority: WO
Inventors: Eckart Wunder; Philipp Henon
Original assignee: Telistar International
Priority date: 1998-01-17
Filing date: 1999-01-18
Publication date: 1999-07-22
Also published as: AU3026999A; WO1999036441A3

Abstract

A protein or proteins contained in a supernatant inhibiting recruitment of haemopoietic progenitor or stem cells, preferably of the myeloid lineages, is described. Furthermore, a process for the preparation of the protein or proteins in the supernatant and the enrichment of such an acidic protein or proteins by means of affinity and ion exchange chromatography and an assay to determine the activity of such an acidic protein or such acidic proteins are disclosed.

Description

Inhibition of Cells

The invention concerns a protein or proteins contained in a supernatant, characterized in that the protein or proteins inhibit recruitment of haemopoietic progenitor or stem cells preferably of the myeloid lineages.

The invention also concerns a process for the preparation of a protein or proteins contained in a supernatant, comprising stimulation of plastic adherent mononuclear cells from multiple myeloma patients with the aggressive form of the disease with colony stimulating factors, preferably with granulocyte - colony stimulating factor(G-CSF) or granulocyte macrophage-colony stimulating factor(GM-CSF), and incubating these cells between 3 to 100 hours, preferably for 48 hours, and passing the supernatant obtained through a Sepharose-Blue affinity column at low salt concentrations followed by anion exchange chromatography in a manner known per se.

Furthermore, the invention relates to an assay for determining the activity of a protein or proteins inhibiting haemopoietic progenitor or stem cell recruitment, comprising incubating highly purified CD34+ cells with colony stimulating factors, preferably G-CSF or GM-CSF, whereby the resulting number of colonies is reduced as compared to controls not containing the inhibitory protein or proteins.

Deregulated generation of monoclonal B cells leads to an impressive accumulation of malignant plasma cells in bone marrow of patients with multiple myeloma (MM), inducing marked damage on bones and inner organs. Symptoms and clinical course show large variations, and heterogeneity was observed at chromosomal and DNA levels. Since in some patients peripheral blood cell counts are decreased (Hematology, 1026-1031 , 1991 ), more often in erythrocytes than in granulocytes and platelets, one may wonder which marrow cell disfunction is responsible for this decrease.

In a precedent study, the capacity of marrow cells from patients with MM were analyzed and granulo-monocytic colonies (CFU-GM) in short term cultures supplemented with growth factors (Stem Cells 13,suppl. 2, 51-55,1995) were evaluated; it was found that 37 out of 96 cases gave rise to low numbers of colonies (in average 82 vs 269 CFU-GM/2x10⁵ MNC (mononuclear cells) for MM-LO (Multiple Myeloma with low colony growth) and MM-HI(high colony growth), respectively). Since patients were studied at diagnosis and before any treatment, differences in CFU-GM growth are part of the natural history of the disease, and represent two phenotypes. In addition, in the MM-LO patient subset total MNC per ml aspirate were 40% less numerous than in the MM-HI subset, the percentage of plasmocytes was heigher (50% vs 24%), and the disease was always severe (stage II - III according to the criteria of Salmon and Durie). Measurement by flow cytometry of the percentage of BM (bone marrow)CD34⁺ cells showed in average similar values in the MM-LO and MM-HI subsets and in normal controls (1 ,45% vs 1 ,40% and 1 ,41 %). These observations underscore that abnormality at the CFU-GM level may accompany the pathognomonic B cell defect; it was of major interest to find out whether this disturbance is due to cells from the B-lymphoid or another lineage.

Recruitment of myeloid progenitors or stem cells after stimulation with GM-CSF or placental cell conditioned medium (PCM) in short term cultures of normal bone marrow is modulated by mature "accessory" cells, more specifically by monocytes (Peripheral Blood Stem Cell Autografts, Ed. Wunder E., Henon P., p.58- 66, Springer Verlag,1993). Compromised colony growth in MM-LO patients could therefore either be due to impairment in myeloid progenitor proliferation, or to impairment in accessory lymphoid or non lymphoid cells interfering with the CFU- GM recruitment. In the present invention hematopoietic precursors and mature accessory cells from bone marrow of MM-LO patients were isolated by cell sorting, in order to test their contribution to CFU-GM formation separately. Moreover it was tested whether impairment is induced by soluble factors, or by direct cell- to cell interaction. Finally, a simplified assay was developed, which is applicable under clinical and laboratory routine conditions for identifying patients that carry this particular defect.

In one third of the patients with multiple myeloma in bone marrow aspirates low numbers of myeloid colonies are found after stimulation with growth factors like

GM-CSF or G-CSF.

In Stem Cells 13(suppl.2) 51-55,1995 it is described that contrary to the teaching in earlier publications the amount of bone marrow stem cells CD34+ is in the normal range and not reduced. The reason for this reduced colony formation was ascribed in this publication to an insufficient stimulus which is provided by accessory mature cells. Contrary to this prior art teaching it has been found that the reduced colony formation is due to a protein or proteins which inhibit the formation of colonies.

The present invention relates to a protein or proteins contained in a supernatant, characterized in that the protein or proteins inhibit recruitment of haemopoietic progenitor or stem cells, preferably of the myeloid lineages.

The invention also concerns a process for the preparation of a protein or proteins contained in a supernatant, comprising stimulation of plastic adherent mononuclear cells from multiple myeloma patients with the aggressive form of the disease with colony stimulating factors, preferably with granulocyte - colony stimulating factor(G-CSF) or granulocyte macrophage-colony stimulating factor(GM-CSF), and incubating the cells between 3 to 100 hours, preferably for 48 hours. The supernatant is purified by passing it through a Sepharose-Blue affinity column(Cibacron-Blue, Pharmacia) at low salt concentration. The activity containing fraction is further purified by anion exchange chromatography. The behavior of the chromatography fractions shows that they contain an acidic protein or proteins and these fractions can be stored in frozen form without significant loss of activity.

Furthermore, the invention relates to an assay for determining the activity of a protein or proteins inhibiting haemopoietic progenitor or stem cells recruitment, comprising incubating highly purified CD34+ cells with colony stimulating factors, preferably G-CSF or GM-CSF, whereby the resulting number of colonies is reduced as compared to controls.

So far only growth factors are known which promote the production of certain cells. They have found their place as valuable pharmaceuticals in the treatment of a number of diseases. Combating diseases with (a) factor(s) which inhibit ceil growth opened new avenues for the treatment and control of abnormal bone marrow cell proliferation in diseases such as chronic myeloid leukemia(CML), Polycythemia Vera and primary thrombocythemia, which all are diseases being frequently encountered and never cured so far, except in the case of allografting, in CML; sequential administration of the inhibitor before and after aggressive chemotherapy courses in the treatment of malignant diseases is putting at rest the hematopoietic stem cells, thus avoiding their chemotherapy-related damage, and consequently preserving the hematopoietic bone marrow potential of the patients undergoing chemotherapy. This hematopoietic tolerance opens the long desired possibility to substantially increase the dosage of pharmaceuticals in the treatment of malignant diseases and hereby substantially increases the efficacy of chemotherapeutics.

Examples The following examples serve to further illustrate the invention. Bone marrow aspirates

Samples were collected, after informed consent, from sternum aspirates of patients and healthy volunteers, and put into glass flasks containing 1 ml RPMI with 0.2% heparin. After 1 :2 dilution in RPMI, the light density fraction was obtained by Ficoll centrifugation at room temperature for 30 min on a Beckman

TJ 7 centrifuge at 3000rpm. The MNC fraction was washed twice in RPMI.

Cytofluorimetric analysis and separation

10⁸ MNC from MM BM and healthy volunteers were incubated with anti- CD34 MAb (HPCA-2-PE, BD, France); PE(phycoerythrin)-conjugated isotype control was used. Cells were then washed twice in RPMI with 2% (w/v) FCS(fetal calf serum), analyzed and sorted, under aseptic conditions, on a FACStar (BD, CA) equipped with an argon laser operating at 488 nm and 250 mW. The red (PE) fluorescence was detected using a 588/40 nm band pass filter. Three droplet sorting was performed at 2000 to 4000 events per second. Sorted cells were collected in 1.5 ml tubes containing 200 μl fetal calf serum (FCS) into two fractions of CD34⁺ and CD34^" cells. After one wash CD34 positive and negative cells were resuspended in Iscove's medium (Difco, France) and kept on ice.

Short term culture for colony growth of myeloid progenitors

Colonies were grown in short term culture in methylcellulose ( 0,8% final conc.,Eurobio, France) dissolved with Iscove's medium; growth factors were procured by 0.5 ml pretested placental cell conditioned medium (PCM) (Exp. Hematol., 8, 2, 177-184, 1989), (equivalent to 10 ng GM-CSF), and 20% (v/v) fetal calf serum. Two to four dishes were set up in parallel. In standard assays 2x10^ bone marrow MNC in a final volume of 1 ml were seeded per 3.5 cm Petri dish. After incubation for seven days at 37°C and 5% CO2, colonies were evaluated with an inverted microscope by counting ceil aggregates of more than 50 cells. To test whether MM-plasmocytes modified progenitor growth, different proportions (0-

16%) of these cells were added to 2x10⁵ MNC recovered from a cytapheresis product after mobilization with high dose cytostatics and G-CSF. Mixed reconstitution of normal and MM bone marrow progenitors and accessory cells

MNC fractions from MM-LO bone marrow (Pat. Bl or BE) and from a healthy volunteer of similar age were sorted as described above. Positive and negative fraction cells were kept on ice and cell densities adjusted. Normal CD34 cells were mixed with MM CD34 negative ceils, and conversely, MM CD34+ cells were mixed with normal CD34 negative cells in such way, that in the suspension 1x1 O^ (or 2x10⁴) hematopoeitic cells and 1x10§ (or 2x10⁶) accessory cells were present per ml ; these cells were inoculated in Petri dishes , stimulated and cultured in methylcellulose as above, and colonies were evaluated (Fig.1b)1+2).

Assessment of impaired colony formation in normal BM CD34⁺ cells in presence of cell free supernatants of MM-LO adherent cells

In order to find out whether impairment in progenitor or stem cell growth is mediated via cell- to cell interaction with mature MM-LO cells or by conditioned medium from mature MM-LO cells, supernatants from MM-LO cells were collected and tested with purified normal BM CD34⁺ cells in short term culture assays.

In normal and MM-LO bone marrow MNC fractions the monocyte content was evaluated after staining with May-Grϋnwald/Giemsa; 2x10^ monocytes (calculated in the MNC fraction) were then suspended in 1 ml colorless RPMI supplemented with 10% (v/v) fetal calf serum, and incubated in 3.5 cm Petri dishes. After 2h at 37°C at 5% CO2, nonadherent cells are removed, adherent cells carefully rinsed with prewarmed RPMI, and 1ml RPMI medium supplemented with 10% fetal calf serum and 10ng GM-CSF was added; after incubation for 24h the supernatant was collected, centrifuged at 600g for 10 min and stored at -20°C. Supernatants from blood MNC were obtained in a similar way.

Five x10³ flow sorted (monocyte free) CD34⁺ cells from normal bone marrow were suspended in 0,9ml methylcellulose dissolved in Iscove's medium with 20% (v/v) fetal calf serum, 0.1 ml Iscove's medium, and 0,1 ml of either normal or MM-

LO monocyte supernatant . To assay both supernatants simultaneously, the 0,1 ml Iscove's medium aliquot was replaced by supernatant, an equivalent volume of both normal and MM-LO supernatants being therefore present in each culture.

Two different doses of MM-LO supernatant were tested : in cultures as above for stimulation 0,15 ml PCM was used throughout, and 0,15 ml or 0,075 ml MM-LO supernatants and respective aiiquots of Iscove's medium were added per dish.

Routine testing of MM patients with supernatants from adherent blood and bone marrow cells

Effect of blood MNC cell supernatants on CFU-GM growth was assessed in a simplified way. Umbilical cord blood CD34⁺ cells were obtained by immunomagnetic separation using microspheres (system MiniMACS, Miltenyi) ((Hematopoietic Stem Cells, The Mulhouse Manual, Ed. E.Wunder, H. Sovalat,

P.R. Henon, S. Serke, p.201-211 , AlphaMed Press.Dayton, Ohio 1993)). Cultures with 5x10³ CD34⁺ cells were set up as above in the presence of 0,1 ml of normal and 0,1 ml of MM-LO or MM-HI patient blood or bone marrow MNC supernatant (Table 1 ;page 9).

Statistical treatments

In parallel cultures average and standard deviation of colony numbers were calculated. For comparison between inhibition in adherent cell supernatants of MM-LO and MM-HI patients the two-tailed Mann Whitney test was applied.

Preparation of starting materials and control experiments.

To obtain fresh CD34⁺ precursors and CD34" accessory cells at sufficient numbers, MM-LO and normal bone marrows were processed on the same day. Approximately 10^ MNC were flow-sorted within 4-6 hours; pure cell fractions were then mixed to reconstitute heterologous cell populations of standardized composition (compare page 6), i.e. 10⁴ (or 2x10⁴) CD34+ per 10⁶ (or 2x10⁶) CD34" cells per ml of suspension. Viability as measured by Trypan blue exclusion was above 97% in all fractions. MM-LO patients Bl (name of patient) and BE (name of patient) were tested. CD34⁺ cells from both patients, gave rise to high colony numbers in the presence of normal CD34- cells. In contrast, normal CD34+ cells in the presence of MM CD34- cells gave rise to reduced colony numbers (48% in Bl; 45% in BE) (Fig. 1 b)1 and 1 b2). These results suggest that CD34- cells from MM-LO patients are responsible for the reduced growth of CD34+ cells from these patients. To exclude cell damage due to sorting, CD34+ and CD34- cells were mixed from the same volunteer; colony growth efficiency was similar to that found when plating the unseparated marrow cells.

In another experiment, malignant plasmocytes were added at increasing proportion (up to 16% of total cell content ) to MNC cells from mobilized blood. No influence on colony growth was found (Fig. 2).

In other experiments the supernatants from adherent marrow cells were tested as described above. While 0.1 ml supernatant from normal bone marrow adherent cells stimulated colony growth by sorted BM-CD34⁺ cells as efficiently as PCM or 10ng GM-CSF, the same volume of the MM-LO BM supernatant (Pat. Bl) only produced 19% of the colonies obtained in presence of PCM. When both supernatants were present simultaneously (normal + MM-LO), the colony number was found 48% of that obtained in presence of PCM or normal supernatant alone. The supernatant of another MM-LO patient also interfered with stimulation by PCM, and dose dependance could be demonstrated : whereas 76 colonies were obtained with PCM alone, 55% thereof were obtained in the presence of 0,075 ml, and 27% with 0,15 ml supernatant, respectively.

The effect of supernatants derived from peripheral blood (PB)adherent cells of MM patients was assessed; a simplified assay using enriched and monocyte- depleted CD34⁺ cell fractions from fresh cord blood was employed. A series of blood (MM-LO and MM-HI) and bone marrow (MM-LO) derived supernatants were tested in the same experiment and compared to normal supernatants. All of five

MM-LO BM supernatants showed inhibition at varying degrees averaging 24% reduction of the growth in presence of PCM alone; and so did all four PB supernatants (averaging 14%). On the contrary, supernatants from MM-HI patients blood, showed no significant inhibitory activity, and the difference between MM-LO and MM-HI PB ac (adherent ceils) was highly significant (Table 1 ; p= 0,028; below).

Table 1 :

Patient subset % growth inhibition using adherent cell population from peripheral blood from bone marrow

MM-LO

SI -14 CE -22

BE -21 Zl -28

ME -12 FL -14

KN - 7 SE -32

GR -24 mean (+ SD) -14 % + 7 mean (+ SD)

MM-HI

BR +8

BO +8

FR 0

FE +7 p = 0.028

GE -4

KU +2

BIR -17

SIM -7

OB +7 mean (+ SD) - 0.3 % + 7.5

Legend : Routine assay for colony growth disturbance by MM-LO adherent cell supernatants stemming from blood and bone marrow. Growth reduction in comparison to normal adh.c. supernatant tested in the same experiment (= 100% growth) with enriched and monocyte depleted umbilical cord blood CD34⁺ is given in %; together with average values in the different groups the range is indicated. Incubation with 0,1ml supernatant of different MM-LO and MM-HI patients. Legends:

Fig.1 b)1 : Patient Bl, at the exacerbating phase of the disease, two separate cultures with 1 and 2X 10⁵ total cells per dish.

Fig. 1 b)2: Patient BE (range indicated by bars )

Fig. 2 : Effect of malign plasmocytes on colony growth of MNC from a leucapheresis product after stimulation in short term culture.

In each dish 2 X 10^s MNC were present, and increasing amounts of plasmocytes from Patient JA were added.

Claims

1. Protein or proteins contained in a supernatant, characterized in that the protein or proteins inhibit recruitment of haemopoietic progenitor or stem cells.

2. Protein or proteins according to claim 1 , characterized in that the protein or proteins inhibit progenitor or stem cells of the myeloid lineage.

3. A process for the preparation of a protein or proteins contained in a supernatant, comprising stimulation of plastic adherent mononuclear cells from multiple myeloma patients with the aggressive form of the disease with colony stimulating factors, incubating these cells between 3 to 100 hours and passing the supernatant obtained through a Sepharose-Blue affinity column at low salt concentration followed by anion exchange chromatography in a manner known per se.

4. A process according to claim 3, comprising using granulocyte-colony stimulating factor as stimulator.

5. A process according to claim 3, comprising using granulocyte macrophage- colony stimulating factor as stimulator.

6. A process according to any one of claims 3 to 5, comprising incubating the cells between 30 to 66 hours.

7. A process according to any one of claims 3 to 5, comprising incubating the mixture for 48 hours.

8. Assay for determining the activity of a protein inhibiting haemopoietic progenitor recruitment, comprising incubating highly purified CD34+ cells with colony stimulating factors.

9. Assay according to claim 8, comprising using granulocyte-colony stimulating factor as stimulator.

10. Assay according to claim 8, comprising using granulocyte macrophage-colony stimulating factor as stimulator.