Method of treatment of leukemia with anti-IL1RAP antibodies

Abstract

The present invention provides agents comprising or consisting of a binding moiety with specificity for interleukin-1 receptor accessory protein (IL1RAP) for use in inducing cell death and/or inhibiting the growth and/or proliferation of pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder, wherein the cells express IL1RAP. A related aspect of the invention provides agents comprising or consisting of a binding moiety with specificity for interleukin-1 receptor accessory protein (IL1RAP) for use in detecting pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder, wherein the cells express IL1RAP. Further provided are pharmacological compositions comprising the agents of the invention and methods of using the same.

Claims

1. A method of treatment of leukemia, the method comprising administering a therapeutically effective amount of an antibody with specificity for an extracellular domain of the interleukin-1 receptor accessory protein (IL1RAP), wherein the antibody inhibits IL1RAP binding activity associated signaling on pathological stem cells and/or progenitor cells expressing IL1RAP.

2. The method according to claim 1, wherein the pathological stem cells and/or progenitor cells comprise a BCR/ABL1 fusion gene.

3. The method according to claim 1, wherein the leukemia is selected from the group consisting of chronic myeloid leukemia (CIVIL), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

4. The method according to claim 1, wherein the leukemia is CML.

5. The method according to claim 1, wherein the leukemia is MDP.

6. The method according to claim 1, wherein the leukemia is MDS.

7. The method according to claim 1, wherein the leukemia is ALL.

8. The method according to claim 1, wherein the leukemia is AML.

9. The method according to claim 1, wherein the pathological stem cells and/or progenitor cells comprise a Ph chromosome.

10. The method according to claim 1, wherein the antibody blocks binding of one or more co-receptors to IL1RAP.

11. The method according to claim 10, wherein the one or more co-receptors are selected from the group consisting of IL1R1, ST2, C-KIT and IL1RL2.

12. The method according to claim 1, wherein the antibody induces apoptosis of the stem cells and/or progenitor cells.

13. The method according to claim 1, wherein the antibody further comprises a cytotoxic moiety.

14. The method according to claim 1, wherein the antibody blocks binding of IL1R1.

Description

(1) Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

(2) FIG. 1. P210 BCR/ABL1 Expression Induces IL1RAP Expression in Cord Blood CD34.sup.+ Cells

(3) Flow cytometric analysis confirms that IL1RAP expression is induced upon retroviral P210 BCR/ABL1 expression in cord blood CD34.sup.+ cells, three days post transduction. CD34.sup.+ GFP.sup.+ cells were gated according to the gates in the dot plots. The histogram shows the expression of IL1RAP for negative control staining (white), MIG control (light gray) and MIG-P210 (dark gray). The numbers in the dot plots show the percentage of cells within individual gates/quadrants. A representative experiment out of three is shown.

(4) FIGS. 2A-C. IL1RAP is Upregulated in Primitive CML Cells

(5) In FIG. 2A, FACS analysis on CD34.sup.+ cells from five CML patients and from 2 normal bm samples. FACS dot plot showing gating for CD34.sup.+CD38.sup.+ or CD34.sup.+CD38.sup. cells in a representative CML patient.

(6) FIG. 2B shows histograms of IL1RAP expression within CD34.sup.+CD38.sup.+ cells.

(7) FIG. 2C shows histograms of IL1RAP expression within CD34.sup.+CD38.sup. cells. White represent control stained samples and gray represent IL1RAP stained samples. The sorting gates for CD34.sup.+CD38.sup.IL1RAP.sup. and CD34.sup.+CD38.sup.IL1RAP.sup.+ cells are outlined in the histograms. The numbers in the dot plot and histograms show the percentage of cells within individual gates/quadrants.

(8) FIG. 3. IL1RAP Expression Distinguishes Ph.sup.+ from Ph.sup. CML Cells within the CD34.sup.+CD38.sup. Cell Compartment

(9) Flow-drop-FISH on CML CD34.sup.+CD38.sup.IL1RAP.sup. and CD34.sup.+CD38.sup.IL1RAP.sup.+ cells from 5 CML patient samples revealed an almost complete separation between BCR/ABL1.sup. and BCR/ABL1.sup.+ cells, respectively. Black bars represent BCR/ABL1 negative cells and white bars represent BCR/ABL1 positive cells. Outlined at the top of each bar is the number of Ph.sup.+ cells of the total nuclei scored.

(10) FIGS. 4A-B. IL1RAP Expression Distinguishes Ph.sup.+ CML Stem Cells from Normal HSC

(11) FIG. 4A shows the number of LTC-CFC derived from CD34.sup.+CD38.sup.IL1RAP.sup. and CD34.sup.+CD38.sup.IL1RAP.sup.+ cells. Black bars represent IL1RAP.sup. cells and white bars represent IL1RAP.sup.+ cells. Interphase FISH on LTC-CFC.

(12) In FIG. 4B black bars represent BCR/ABL1 negative cells and white bars represent BCR/ABL1 positive cells. Outlined at the top of each bar is the number of Ph.sup.+ cells of the total nuclei scored.

(13) FIGS. 5A-B.Killing of a CML Cell Line by Antibody Targeting of IL1RAP

(14) FIG. 5A shows histograms of IL1RAP expression on KU812 cells derived from a CML patient and containing a Philadelphia chromosome, compared to expression on KG-1 cells lacking a Philadelphia chromosome. White show control stained samples and gray show KMT-1 stained samples.

(15) FIG. 5B shows the leukemic cell line KG-1 was devoid of IL1RAP expression, whereas KU812 express IL1RAP. As a consequence, low level of antibody induced cell death was observed in KG-1, while a dose-dependent ADCC effect was observed using KMT-1 on KU812 cells. As a control for unspecific ADCC effects, a rabbit IgG antibody was also used in the experiments. The graph shows the average and standard deviation of antibody induced cell death from three independent experiments.

(16) FIGS. 6A-B. Killing of CML Stem Cells by Antibody Targeting of IL1RAP

(17) FIG. 6A shows the use of KMT-1, normal bone marrow CD34+CD38 cells stained negative for IL1RAP, whereas CML CD34+CD38+ and CD34+CD38 cells expressed IL1RAP. Histograms on CML-1 are shown from a representative experiment. White show control stained samples and gray show KMT-1 stained samples.

(18) FIG. 6B shows that in, line with the level of IL1RAP expression, no obvious ADCC effect was seen using normal bone marrow CD34+CD38 cells, whereas KMT-1 induced a strong dose-dependent ADCC effect in both CML CD34+ and CD34+CD38 cells. As a control for unspecific ADCC effects, a rabbit IgG antibody was also used in the experiments. The graph shows the average and standard deviation of antibody induced cell death from three independent experiments using CML-1, CML-3, CML-4, and four normal bone marrow samples.

(19) FIGS. 7A-C. IL1RAP is Expressed Also on Primary ALL and AML Stem Cells

(20) In FIG. 7A, acute myeloid leukemia (AML) cells were received from patients at diagnosis. IL1RAP expression on CD34+CD38 and CD34+CD38+ cells from a representative AML patient is presented.

(21) FIG. 7B shows that the AML cell line MONO-MAC-6 and the ALL cell line REH express IL1RAP.

(22) In FIG. 7C, acute lymphoid leukemia (ALL) cells were received from patients at diagnosis. IL1RAP expression on CD34+CD38 and CD34+CD38+ cells from a representative Ph+ ALL patient is presented. White show control stained samples and gray show IL1RAP stained samples.

(23) FIG. 8. Killing of AML and ALL Cell Lines by Antibody Targeting of IL1RAP

(24) FIG. 8 shows, in ADCC assay, a KMT-1 dose dependent cell death was induced in both the MONO-MAC-6 and the REH cell line, suggesting that IL1RAP targeting antibodies may have a broader therapeutic window than just CML. As a control for unspecific ADCC effects, a rabbit IgG antibody was also used in the experiments. The graph shows the average and standard deviation of antibody induced cell death from three independent experiments.

(25) FIGS. 9A-B. Killing of AML and ALL Stem Cells by Antibody Targeting of IL1RAP

(26) FIG. 9A shows that a KMT-1 induced cell death was observed in both primary AML CD34+CD38. and FIG. 9B shows ALL CD34+CD38 cells, confirming that IL1RAP targeting antibodies also have a therapeutic effect in AML and ALL with upregulation of IL1RAP on their cell surface. As a control for unspecific ADCC effects, a rabbit IgG antibody was also used in the experiments. The graph shows the specific antibody induced cell death.

(27) FIGS. 10A-B. IL1RAP is Expressed on Leukemic Stem Cells from MPD and MDS Patients.

(28) FIG. 10A shows contour plots showing IL1RAP expression in CD34+CD38 cells of two MPD patients (MPD-1 and MPD-2), with and without the JAK2 mutation.

(29) FIG. 10B has histograms showing IL1RAP expression in an MDS patient progressed into AML. White show control stained samples and gray shows a sample stained with anti-IL1RAP antibodies.

EXAMPLE 1

(30) IL1RAP is a Cell Surface Biomarker for Chronic Myeloid Leukemia Stem Cells

(31) Summary

(32) Therapeutic strategies for chronic myeloid leukemia (CML) aiming at achieving a permanent cure of the disorder, will require a full eradication of the CML stem cells. The CML stem cells, sharing the capacity to self-renew with normal hematopoietic stem cells (HSCs), represent a small population of leukemic cells that so far have been indistinguishable from normal (HSCs) using cell surface markers. One strategy to target the CML stem cell would be to identify a cell surface biomarker for CML stem cells, to which future therapeutic antibodies could be directed. In this study, we identified IL1RAP as commonly upregulated both in primitive CML CD34+ cells and as a consequence of ectopic P210 BCR/ABL1 expression using global gene expression analyses. We further show that IL1RAP expression divides the rare CD34+CD38 cell population, harboring both CML and normal HSCs, into two fractions; one having low/absent expression, the other having higher IL1RAP expression. After establishing a protocol, allowing detection of BCR/ABL1 by FISH in small numbers of sorted cells, we observed that within the CML CD34+CD38 cells; the IL1RAP+ cells were BCR/ABL1+, whereas IL1RAP cells were almost exclusively BCR/ABL1. By further performing long term culture-initiating cell (LTC-IC) assays on the two cell populations, we found that candidate CML stem cells and normal HSC could be prospectively separated. This study thus identifies IL1RAP as the first cell surface biomarker distinguishing CML stem cells from normal HSC and opens up new avenues for therapeutic and diagnostic strategies in CML as well as in related disorders such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myeloproliferative disorders (MPDs) and myelodysplastic syndrome (MDS).

(33) Introduction

(34) To identify a cell surface biomarker for CML stem cells, we performed global gene expression analyses and identified the interleukin 1 receptor accessory protein (IL1RAP) as the top candidate, being upregulated both in primitive CML patient cells and as a consequence of ectopic P210 BCR/ABL1 expression. Upon development of an assay for detecting BCR/ABL1 in low numbers of sorted cells, we show that the IL1RAP expression enables prospective separation of primitive leukemic and normal cells. Through long-term culturing-initiating cell assays, we further show that IL1RAP is a cell surface biomarker for CML stem cells, for the first time allowing prospective separation of CML stem cells from normal HSC.

(35) Material and Methods

(36) Collection of CML Patient Cells

(37) Isolation and Transduction of Cord Blood CD34.sup.+ Cells

(38) Blood and occasionally bone marrow samples from CML patients were obtained at diagnosis before treatment was initiated after informed consent according to a protocol approved by the local ethical board. Samples were received both from the Department of Hematology at Lund University Hospital, Sweden and from Rigshospitalet, Copenhagen, Denmark. Mononuclear cells (MNCs) were separated using Lymphoprep (Axis-Shield PoC AS, Oslo, Norway) according to the manufacturer's instructions and CD34.sup.+ cells were enriched using the CD34.sup.+ cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) as previously described.sup.22, on a regular basis, this yielded a purity of CD34.sup.+ cells above 95%. A subfraction of mononuclear cells was viably stored in liquid nitrogen before antibody staining was initiated. CD34.sup.+ cells were split in two fractions; one fraction was washed in PBS and resuspended in Trizol and frozen in 80 C, whereas the other fraction was frozen in liquid nitrogen. As reference samples, bone marrow samples from healthy volunteers were obtained after informed consent at the Lund University Hospital, followed by CD34-cell isolation as described above.

(39) Microarray Analysis

(40) Microarray analysis was performed using oligonucleotide slides from the Swegene DNA Microarray Resource Center at Lund University, Sweden. Hybridizationss were performed using the Pronto Universal Hybridization kit (Corning Inc, Corning, N.Y.). The RNA isolation and microarray analysis was performed essentially as previously described.sup.23. Data visualization was performed using the software QIucore Omics Explorer 2.0 (QIucore, Lund, Sweden).

(41) Flow Cytometric Analysis

(42) Flow cytometric analyses were performed in a FACS Canto and flow cytometric cell sorting was done in a FACS Aria (both from BD). Prior to cell staining, CD34.sup.+ cells were thawed according to standard procedures and washed once in PBS containing 2% FCS (washing medium). Biotin-labeled goat anti-human IL1RAP polyclonal antibody (batch 667, R&D Systems, Abingdon, UK) was used at a 1:100 dilution for staining the cells for 30 min on ice. Subsequently, the cells were washed and PE-conjugated streptavidin was used at a 1:200 dilution for 30 min. The APC-conjugated anti-CD34 and FITC-conjugated anti-CD38 monoclonal antibodies were used for co-staining (except IL1RAP all antibodies used were purchased from Beckton-Dickinson Immunocytometry Systems, Mountain View, Calif.). Before cell sorting, cells were washed twice to avoid unspecific binding of PE-conjugated streptavidin. Isotype matching control antibodies were used as negative controls.

(43) Cell Sorting and Interphase FISH

(44) Glass slides were treated with 0.01% poly L-lysine (Sigma-Aldrich, Stockholm, Sweden) for two hours while kept in a moist chamber, washed once in water, and dried on a hot plate at 37 C. until dry. Subsequently, a hydrophobic pen (Daido Sangyo Co., Ltd. Tokyo, Japan) was used to draw circles with a 96-well tissue culture plate as template. Prior to cell sorting, but after at least two hours drying in room temperature, 25 L PBS was applied to the rings to form drops. During cell sorting, 30 to 3000 cells were sorted simultaneously directly into two drops. To allow attachment of the cells to the surface and to avoid drying of the drops, slides were maintained in a moist chamber on ice for 30 min before cells were fixed in methanol:acetic acid (3:1) for 10 min. Subsequently, slides were incubated in a 70 C. oven over night, followed by FISH. Dual color probes for BCR/ABL1 (Abbot, Wiesbaden, Germany) were used.

(45) Long Term Culture-Initiating Cells (LTC-IC)

(46) M.sub.210B.sub.4 stroma cells were cultured in RPMI-1640 medium supplemented with 10% FCS as previously described.sup.24, 26. Two days prior to cell sorting, stroma cells were seeded into wells of a 96-well plate at density of 50,000 cells per mL in 200 L Myelocult medium (Stem Cell Technologies, Vancouver, Canada) containing 10.sup.6 M Hydrocortisone (Sigma-Aldrich, Stockholm, Sweden). Twenty-four hours before cell sorting, stroma cell were irradiated with 1000 Rad. During cell sorting, 100-500 cells were sorted directly into the stroma-precoated wells in duplicate and 100 L medium was exchanged 3 h later. Once per week, the exchange of 100 L culture medium was repeated. After 5-6 weeks culture, cells were washed and plated in methylcellusose medium (MethoCult H44435; Stem Cell Technologies) in a 24-well plate. Two weeks later, the number of colonies was scored. Colonies from individual wells were pooled, washed, applied to PBS drops on slides, and followed by FISH analysis as described above.

(47) P210 BCR/ABL1 Expression in Cord Blood CD34.sup.+ Cells

(48) Umbilical cord blood samples were collected from normal deliveries after obtaining informed consent according to a protocol approved by the local ethical board. CD34.sup.+ cells were enriched as previously described.sup.22, yielding a purity of CD34.sup.+ cells above 95%. The RD114 pseudotyped MSCV-IRES-GFP (MIG) and MIG-P210 viral vectors were used in this study.sup.23. CD34.sup.+ cells were cultured and transduced in SFMM medium (Stem Cell Technology, Vancouver, Canada) supplemented with thrombopoietin (TPO; 50 ng/mL), stem cell factor (SCF; 100 ng/mL), and Flt-3-ligand (FL; 100 ng/mL) as previously described.sup.23.

(49) Results and Discussion

(50) Global Gene Expression Analysis Identifies IL1RAP as Upregulated on CML CD34.sup.+ Cells

(51) Much effort has been put into investigations aimed at identifying a cell surface biomarker for Ph.sup.+ CML stem cells (reviewed by C Eaves.sup.14). Leukemic and normal cells can rather easily be identified retrospectively in CML following detection of the leukemia specific BCR/ABL1 fusion gene by FISH, making it an ideal disorder for evaluating attempts to prospectively separate leukemic and normal cells. However, so far, no cell surface marker has been identified that allows prospective separation of CML stem cells from normal HSC. Global gene expression analyses have proven to be a powerful strategy in searching for new HSC markers such as the SLAM receptors distinguishing hematopoietic stem and progenitor cells.sup.15. To search for upregulated genes encoding candidate cell surface biomarkers for CML stem cells, the transcriptional profiles of CD34.sup.+ cells from 11 CML patient samples and 5 normal bone marrow (bm) samples were compared. The identified upregulated genes in CML were matched to the Gene Ontology (GO) category integral to plasma membrane that had been manually curated to include all known CD molecules (see Material and Methods for details). In total, 13 upregulated genes in CML CD34.sup.+ cells matched to the integral to plasma membrane gene category (data not shown). To further link the upregulated genes more directly to P210 BCR/ABL1 expression, we in parallel generated a list of upregulated genes as a consequence of P210 BCR/ABL1 expression in cord blood CD34.sup.+ cells. This analysis resulted in 23 upregulated genes matching to the same GO category gene list (data not shown). Interestingly, only one gene, the Interleukin 1 receptor accessory protein (IL1RAP), showed a strong upregulation both in CD34.sup.+ CML cells and in cord blood CD34.sup.+ cells as a consequence of P210 BCR/ABL1 expression. The findings that IL1RAP was present on both gene lists suggest that its upregulation on primitive CML cells is closely coupled to the P210 BCR/ABL1 expression and indicate that IL1RAP is a novel leukemia-associated antigen on primitive CML cells.

(52) IL1RAP is Upregulated on CD34.sup.+CD38.sup. Cells from CML Patients and is Induced as a Consequence of Ectopic P210 BCR/ABL1 Expression

(53) IL1RAP is a member of the Toll-like receptor superfamily and is a well-known co-receptor to Interleukin 1 receptor type 1 (IL-1R1).sup.16. IL1RAP is thus crucial in mediating the effect of the pro-inflammatory cytokine IL-1, but it is also involved in mediating the signal of IL-33, a cytokine that activates T-cells and mast cells through binding its receptor ST2, which subsequently dimerizes with IL1RAP.sup.17. IL-1R1 activation has previously been shown to stimulate colony growth of interferon sensitive CML cells.sup.18, however, IL1RAP has to our knowledge not previously been linked directly to CML.

(54) As P210 BCR/ABL1 is present in CML cells as a hallmark of the disease, ideally, a reliable cell surface biomarker in CML, should be directly coupled to the presence and expression of P210 BCR/ABL1. In agreement with the microarray data, IL1RAP expression was indeed upregulated on the cell surface on CB CD34.sup.+ cells following retroviral P210 BCR/ABL1 expression (FIG. 1). This suggests that P210 BCR/ABL1 regulates IL1RAP expression, either directly or through an indirect effect, strengthening its candidature as a CML biomarker.

(55) We next investigated the cell surface IL1RAP expression on CML CD34.sup.+CD38.sup.+ cells, representing the majority and more mature CD34.sup.+ cells. In this cell population, an upregulation of IL1RAP was observed compared to the expression in corresponding normal bm cells (FIGS. 2A, B). The normal CD34.sup.+CD38.sup.+ cells displayed a lower IL1RAP expression that partially overlapped with the expression on CML cells. We then turned to the CD34.sup.+CD38.sup. cell compartment of normal cells, containing the HSCs. In agreement with a previous study, this population displayed a low/absent IL1RAP expression (FIG. 2C).sup.19. Strikingly, the CD34.sup.+CD38.sup. cells from CML patients, harboring both Ph.sup.+ CML stem cells and normal HSCs were divided into two populations; one having low/absent IL1RAP expression, the other having higher IL1RAP expression (FIG. 2C). In the peripheral blood (PB) of five CML patients, the IL1RAP positive cell fraction constituted between 75% and 95% of the CD34.sup.+CD38.sup. cells (n=5). Based on these findings, we speculated that the IL1RAP expression might distinguish normal and leukemic cells within the CD34.sup.+CD38.sup. cell compartment in CML. As all CML stem cells and normal HSC exclusively are found within the CD34.sup.+CD38.sup. cells, such separation between normal and leukemic cells, would allow a prospective separation of CML stem cells from normal HSC.

(56) Flow-Drop-FISH Shows that IL1RAP Expression Separates Normal and Leukemic Cells within CML CD34.sup.+CD38.sup. Cells

(57) To test whether the IL1RAP expression distinguishes normal (Ph.sup.) and leukemic (Ph+) cells within the CML CD34.sup.+CD38.sup. cell compartment, we established a new protocol for doing fluorescent in situ hybridization (FISH) on small numbers of sorted cells (see Material and Methods). The first steps in this protocol is partly based on a method for sorting cells into drops on slides followed by single cell immuno-staining.sup.20. By applying this new protocol involving cell sorting directly into drops on slides followed by FISH, hereafter referred to as Flow-drop-FISH, we sorted as few as 30 cells into a drop, from which 15 nuclei were successfully scored by FISH (CML-5, FIG. 3). Interestingly, we found by Flow-drop-FISH that the CML CD34.sup.+CD38.sup.IL1RAP.sup.+ cells were BCR/ABL1.sup.+, whereas CML CD34.sup.+CD38.sup. IL1RAP.sup. cells were almost exclusively Ph.sup. (n=5, FIG. 3). These data show that IL1RAP expression separates leukemic and normal cells within the CML CD34.sup.+CD38.sup. cell compartment, indicating that CML stem cells and normal HSC can be prospectively separated

(58) CML Stem Cells are CD34.sup.+CD38.sup.IL1RAP.sup.+ Whereas Normal HSC are CD34.sup.+CD38.sup.IL1RAP.sup./Low

(59) Studies on chronic phase CML stem cells has so far relayed on access to rare CML patients in which the stem cells compartment have been dominated by leukemic cells following long-term assays.sup.14. As CML stem cells generally show poor engraftment in immuno-deficient mice, the long-term culture initiating cell (LTC-IC)-assay is widely used as a surrogate assay for detection of candidate CML stem cells. To test whether CML CD34.sup.+CD38.sup.IL1RAP.sup.+ and CD34.sup.+CD38.sup.IL1RAP.sup./low uniquely contain candidate CML stem cells and normal HSC, respectively, we tested the two cell populations in the LTC-IC assay. For bone marrow CD34.sup.+ cells from normal controls, long term culturecolony forming cells (LTC-CFC) were found at an >100-fold higher frequency among CD34.sup.+CD38.sup.IL1RAP.sup. cells compared to CD34.sup.+CD38.sup.IL1RAP.sup.+ cells (FIG. 4A, n=2), indicating that normal CD34.sup.+CD38.sup.IL1RAP.sup. are hierarchically on top of CD34.sup.+CD38.sup.IL1RAP.sup.+ cells. In CML, we observed on average a 3.6-fold higher frequency of LTC-CFC within the CD34.sup.+CD38.sup.IL1RAP.sup. cells compared to the CD34.sup.+CD38.sup.IL1RAP.sup.+ cells (n=5, FIG. 4A), suggesting that CML CD34.sup.+CD38.sup.IL1RAP.sup. cells are more enriched for primitive cells. Importantly, although a higher number of LTC-IC were found among CD34.sup.+CD38.sup.IL1RAP.sup. cells than within CD34.sup.+CD38.sup.IL1RAP.sup.+ cells from both CML patient samples and from normal controls, FISH on CML LTC-colonies revealed an almost complete discrimination between Ph.sup. and Ph.sup.+ cells in the two groups (FIG. 4B). CML LTC-colonies derived from CD34.sup.+CD38.sup.IL1RAP.sup. cells were almost exclusively Ph.sup., whereas CD34.sup.+CD38.sup.IL1RAP.sup.+ were almost exclusively Ph.sup.+. These data suggest that IL1RAP is a novel cell surface biomarker that can be used to separate CML stem cells from normal HSC.

(60) Herein, we identified through global gene expression analysis a novel cell surface antigen, IL1RAP, that following challenging in multiple assays fulfilled the criteria for being a novel cell surface biomarker for Ph.sup.+ CML stem cells. Based on this discovery, future directed therapies in CML could be designed to target the CML stem cells while preserving normal HSC by using a therapeutic antibody directed towards IL1RAP. In addition, an antibody cocktail containing anti-CD34, anti-CD38 and anti-IL1RAP antibodies can be used for diagnostic purposes and for follow-up studies of CML patients under different treatments. Importantly, a prospective separation of normal and CML stem cells will enable future mechanistic studies of these two cell populations. Moreover, we here also show that Flow-drop-FISH could serve as a useful method in characterizing genetic aberrations in small numbers of sorted cells, such as leukemic stem cells, a cell type that has been purified to increasingly smaller and purer cell populations.sup.21. For future studies, this method would for example allow detection of genetical aberrations in various small leukemic stem and progenitor cell populations, findings that are likely to provide novel insights into which orders the various aberrations have been acquired, key knowledge to understand leukemogenesis. In addition, Flow-drop-FISH could be used to monitor therapeutic effects on leukemic stem cells during treatment. Importantly, we here identified by using Flow-drop-FISH that IL1RAP is the first cell surface biomarker that distinguishes CML stem cells from normal HSCs, a finding that opens up new therapeutic opportunities for CML and other neoplastic hematologic disorders associated with upregulation of IL1RAP on stem cells and/or progenitor cells.

REFERENCES

(61) 1. Deininger M W, Goldman J M, Melo J V. The molecular biology of chronic myeloid leukemia. Blood. 2000; 96:3343-3356. 2. Fialkow P J, Denman A M, Jacobson R J, Lowenthal M N. Chronic myelocytic leukemia. Origin of some lymphocytes from leukemic stem cells. J Clin Invest. 1978; 62:815-823. 3. Kavalerchik E, Goff D, Jamieson C H. Chronic myeloid leukemia stem cells. J Clin Oncol. 2008; 26:2911-2915. 4. Jiang X, Zhao Y, Smith C, et al. Chronic myeloid leukemia stem cells possess multiple unique features of resistance to BCR-ABL targeted therapies. Leukemia. 2007; 21:926-935. 5. Copland M, Hamilton A, Elrick L J, et al. Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction. Blood. 2006; 107:4532-4539. 6. Jin L, Hope K J, Zhai Q, Smadja-Joffe F, Dick J E. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat Med. 2006; 12:1167-1174. 7. Tavor S, Petit I, Porozov S, et al. CXCR4 regulates migration and development of human acute myelogenous leukemia stem cells in transplanted NOD/SCID mice. Cancer Res. 2004; 64:2817-2824. 8. Jin L, Lee E M, Ramshaw H S, et al. Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell. 2009; 5:31-42. 9. Majeti R, Chao M P, Alizadeh A A, et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell. 2009; 138:286-299. 10. Hosen N, Park C Y, Tatsumi N, et al. CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia. Proc Natl Acad Sci USA. 2007; 104:11008-11013. 11. van Rhenen A, van Dongen G A, Kelder A, et al. The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells. Blood. 2007; 110:2659-2666. 12. Eisterer W, Jiang X, Christ O, et al. Different subsets of primary chronic myeloid leukemia stem cells engraft immunodeficient mice and produce a model of the human disease. Leukemia. 2005; 19:435-441. 13. Bhatia M, Wang J C, Kapp U, Bonnet D, Dick J E. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci USA. 1997; 94:5320-5325. 14. Jiang X, Zhao Y, Forrest D, Smith C, Eaves A, Eaves C. Stem cell biomarkers in chronic myeloid leukemia. Dis Markers. 2008; 24:201-216. 15. Kiel M J, Yilmaz O H, Iwashita T, Terhorst C, Morrison S J. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell. 2005; 121:1109-1121. 16. Subramaniam S, Stansberg C, Cunningham C. The interleukin 1 receptor family. Dev Comp Immunol. 2004; 28:415-428. 17. Ali S, Huber M, Kollewe C, Bischoff S C, Falk W, Martin M U. IL-1 receptor accessory protein is essential for IL-33-induced activation of T lymphocytes and mast cells. Proc Natl Acad Sci USA. 2007; 104:18660-18665. 18. Estrov Z, Kurzrock R, Wetzler M, et al. Suppression of chronic myelogenous leukemia colony growth by interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptors: a novel application for inhibitors of IL-1 activity. Blood. 1991; 78:1476-1484. 19. Hystad M E, Myklebust J H, Bo T H, et al. Characterization of early stages of human B cell development by gene expression profiling. J Immunol. 2007; 179:3662-3671. 20. Ema H, Morita Y, Yamazaki S, et al. Adult mouse hematopoietic stem cells: purification and single-cell assays. Nat Protoc. 2006; 1:2979-2987. 21. Dick J E. Stem cell concepts renew cancer research. Blood. 2008; 112:4793-4807. 22. Nilsson M, Karlsson S, Fan X. Functionally distinct subpopulations of cord blood CD34+ cells are transduced by adenoviral vectors with serotype 5 or 35 tropism. Mol Ther. 2004; 9:377-388. 23. Jaras M, Johnels P, Agerstam H, et al. Expression of P190 and P210 BCR/ABL1 in normal human CD34(+) cells induces similar gene expression profiles and results in a STAT5-dependent expansion of the erythroid lineage. Exp Hematol. 2009; 37:367-375. 24. Hogge D E, Lansdorp P M, Reid D, Gerhard B, Eaves C J. Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor. Blood. 1996; 88:3765-3773. 25. Castor A, Nilsson L, Astrand-Grundstrom I, et al. Distinct patterns of hematopoietic stem cell involvement in acute lymphoblastic leukemia. Nat Med. 2005; 11:630-637.

EXAMPLE 2

(62) Antibody-Targeting of IL1RAP on Leukemia Stem and Progenitor Cells Cause Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

(63) Summary

(64) Therapeutic strategies for leukemias aimed at achieving a permanent cure will require a full eradication of the leukemia stem cells. The leukemia stem cells, representing a small population of leukemic cells, have so far have been indistinguishable from normal hematopoietic stem cells (HSCs) using cell surface markers. A new concept for targeting leukemia stem cells would be to identify a cell surface biomarker for leukemia stem cells, to which future therapeutic antibodies could be directed (see Example 1).

(65) In this study, we generate an anti-IL1RAP antibody and provide proof of concept that anti-IL1RAP antibodies targeting chronic myeloid leukemia (CML) stem cells, Acute myeloid leukaemia (AML) stem cells, and Acute lymphoblastic leukaemia (ALL) stem cells can be used to induce antibody-dependent-cell-mediated cytotoxicity (ADCC), whereas no cytotoxic effect was observed on normal HSC. Furthermore, we demonstrate a dose-dependent IL1RAP targeting ADCC in the IL1RAP positive cell lines KU812 (CML), MONO-MAC-6 (acute myeloid leukemia; AML) and REH (acute lymphoblastic cell line; ALL). We also demonstrate that MDS and MPD stem cells have increased IL1RAP expression, indicative that future therapeutic anti-IL1RAP targeting antibodies will be effective also in these disorders.

(66) This study thus opens up for a novel therapeutic opportunity in CML, AML, ALL, MDS, and MPD by antibody targeting of IL1RAP on leukemic stem cells.

(67) Materials and Methods

(68) Generation of KMT-1; A Polyclonal Rabbit Anti-Human IL1RAP Antibody

(69) Rabbits were immunized with the extracellular domain of IL1RAP. Serum from rabbits were purified according to standard procedures, except that an additional step was added, in which antibodies binding to the immunoglobulin domain, present on the immunizing protein for increased half-life, was discarded through binding to immunoglobulin loaded columns. Purified antibodies were confirmed in ELISA to bind the extracellular domain of IL1RAP and to be devoid of antibodies binding the human immunoglobulin domain. When used in flow cytometry, a PE-conjugated goat anti-rabbit IgG antibody was used as secondary reagent.

(70) ADCC Assay

(71) The ADCC assay was based on a protocol previously described.sup.1. In brief, target cells were labelled with PKH26 (Sigma-Aldrich, St Louis, Mo.) according to manufacturer's instructions and either cells were put directly into wells of a 96-well plate, or seeded into the wells following sorting of CD34.sup.+CD38.sup. cells. The KU812 and KG-1 cell lines and primary CD34.sup.+ cells were seeded at 10,000 cells per well, whereas primary CD34.sup.+CD38.sup. cells were seeded at 2,000-3,000 cells per well. Subsequently, antibodies were added to wells in different concentrations and incubated for 20 min before 100,000 NK-effector cells were added to each well. NK-cells were extracted from healthy volunteers after informed consent by using a NK-cell negative cell isolation kit according to manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). Rabbit IgG antibodies purified from a non-immunized rabbit was used as control antibody in the experiments (R&D Systems Abingdon, UK). 7-AAD positive cells for detection of cell death were measured using a FACS CANTO flow cytometer (BD). The average and standard deviation of antibody induced cell death was calculated according to the following equation: (Percentage 7-AAD+ cells at defined antibody concentrationPercentage 7-AAD+ cells without antibody)/(0.01Percentage 7-AAD cells without antibody) from at least three independent experiments (except FIG. 9; 1 experiment only).

(72) Samples from eleven AML patients and two Ph+ ALL patients were received from Lund University hospital and the expression of IL1RAP was analyzed in the CD34.sup.+CD38.sup.+ and CD34.sup.+CD38.sup. cell populations using the same settings as for the analysis of CML cells. The AML cell line MONO-MAC-6 and the ALL cell line REH were also tested in ADCC assays using the same setup as the for the KG-1 and KU812 cell lines.

(73) Results

(74) Antibody-Targeting of IL1RAP on CML Stem and Progenitor Cells but Also on a CML Cell Line Directs NK-Cells to ADCC

(75) Antibody-dependent-cell-mediated cytotoxicity (ADCC) is a conserved mechanism of the innate immune system, through which several therapeutic antibodies, such as Rituximab directed against CD20, are believed to at least partially exert their therapeutic effect.sup.2. To test whether ADCC could be achieved using IL1RAP as a target, we generated a polyclonal rabbit anti-human IL1RAP antibody hereafter referred to as KMT-1, as the Fc domain of rabbit antibodies in contrast goat antibodies are recognized by cells of the human immune system.

(76) As expected, low levels of ADCC were observed in the IL1RAP negative/low leukemia cell line KG-1, even at high KMT-1 concentrations (FIGS. 5A, B). In contrast, in the CML cell line KU812 expressing IL1RAP, a natural killer (NK)-cell mediated ADCC was observed in the presence of KMT-1 (FIGS. 5A, B), demonstrating that KMT-1 has the potential to induce ADCC by recruiting cytotoxic immune cells to IL1RAP+ target cells.

(77) On primary cells from CML patients and from normal controls, KMT-1 showed a slightly weaker, but similar staining pattern as the previously used polyclonal goat antihuman IL1RAP antibody (Example 1, FIG. 6A). Immature cells from CML-1, CML-3 and CML-4 (no more cells remained from CML-2 and CML-5) were tested in ADCC assays in parallel to cells from healthy control samples. In CML CD34.sup.+ cells, the binding of KMT-1 resulted in ADCC at higher levels than in normal CD34.sup.+ control cells, correlating to the expression level of IL1RAP, in particular at lower antibody concentrations (FIG. 6B). More strikingly, among the stem cell enriched CD34.sup.+CD38.sup. cells, KMT-1 did not induce ADCC of normal CD34.sup.+CD38.sup. cells, whereas a clear dose dependent ADCC effect was observed in CML CD34.sup.+CD38.sup. cells (FIG. 6B), again showing strong correlation to the expression pattern of ILA RAP on these cell types.

(78) Antibodies Targeting IL1RAP on AML and ALL Cells Direct NK-Cells to ADCC

(79) IL1RAP expression was observed in AML CD34.sup.+CD38.sup. cells in 9 out of 11 tested samples (FIG. 7A). In the CD34.sup.+CD38.sup.+ cell population, a similar IL1RAP expression pattern was observed (FIG. 7A). In addition, IL1RAP was expressed in the AML cell line MONO-MAC-6 and the ALL cell line REH (FIG. 7B). IL1RAP expression was also observed in Ph+ ALL CD34.sup.+CD38.sup. cells in 2 out of 2 tested samples (FIG. 7C). Using IL1RAP as target, the MONOMAC-6 and REH cell lines were also tested in ADCC assays. In both these cell lines, a dose dependent IL1RAP targeting ADCC effect was observed (FIG. 8), demonstrating that therapeutic anti-IL1RAP targeting antibodies have a broader application than just CML.

(80) We also performed ADCC experiments on primary AML and ALL CD34+CD38 cells and demonstrated proof of principle that also in these disorders, an increased cell death could be achieved using KMT-1 (FIG. 9).

(81) In addition, CD34+CD38 cells from one MDS patient at progression into AML and two MPD patients (one of them JAK2 mutation+) were stained with an IL1RAP targeting antibody. An increased IL1RAP expression was observed in comparison to normal bone marrow CD34+CD38 cells (FIG. 10, FIG. 2C).

(82) Discussion

(83) In the present study, we have identified IL1RAP as the first cell surface biomarker that distinguishes candidate CML stem cells from normal HSCs and used this knowledge to induce an antibody-dependent cell killing of CML stem cells. Further, we identified IL1RAP as upregulated on AML stem cells, ALL stem cells, MPD stem cells and MDS stem cells and showed that both AML and ALL stem cells can be killed using an IL1RAP-targeting antibody, whereas normal stem cells were unaffected. Based on the finding that CML, ALL and AML stem cells can be killed by IL1RAP targeting antibodies, it is expected that also MPD and MDS stem cell would be killed in the ADCC assay. These findings opens up a new concept for treatments of leukemia patients by direct targeting of the leukemia stem cells, a concept that is distinct from the tyrosine kinase inhibitors currently used, which preferentially target cells downstream of the CML stem cells.sup.3, 4.

(84) The reason why CML stem cells are resistant to drugs such as Glivec is partially unclear, but factors that may contribute are features such as quiescence and relatively high level of BCR/ABL1 expression, but also combinatorial expression of specific membrane transporter proteins in these cells.sup.3, 5, 6. Given these features of the CML stem cells, it is highly desirable to find novel treatment approaches to ultimately eradicate the CML stem cells. An antibody-based therapy directly targeting CML stem cells would serve in such a strategy as the antibodies mode of action is independent of the known resistant mechanisms causing CML stem cells to be unresponsive to kinase inhibitor treatments. The major limitations for such developments have been the complete lack of a cell surface receptor distinguishing CML Ph+ from normal, healthy (Ph) stem cells. We herein identified IL1RAP as such a target from global gene expression analyses and importantly linked its expression to BCR/ABL1 expression (see Example 1 above).

(85) Importantly, by generation of an antibody targeting IL1RAP, we here, for the first time, provide proof of concept that candidate CML stem cells can be targeted while preserving normal HSC. Importantly, as the antibodies mode of action in ADCC is to direct immunological cells to target cell killing, the therapeutic mechanisms is independent of the known mechanisms causing kinase inhibitor resistance in CML using current treatments. Hence, antibody targeting of CML stem cells has the capacity to eradicate CML stem cells, either alone or in combination with current regimens, ultimately leading to a permanent cure for CML patients.

(86) Interestingly, we also observed that IL1RAP targeting antibodies can cause ADCC of AML stem cells; the most common type of acute leukemia among adults having a poor prognosis, and also ALL stem cells; the most common type of childhood leukemia. Collectively, the finding of IL1RAP expression on leukemic stem cells having a CD34.sup.+CD38.sup. immuno-phenotype in CML, AML, ALL, MDS, and MPD, and the ADCC experiments demonstrating cell killing in an IL1RAP dependent manner, indicates that these disorders can be treated with anti-IL1RAP therapeutic antibodies.

(87) In the ADCC experiments presented herein, a polyclonal anti-human IL1RAP antibody was used (which is essentially a mixture of several different monoclonal antibodies). However, it will be appreciated by persons skilled in the art that individual monoclonal antibodies targeting IL1RAP can also be identified which have ADCC potential.

REFERENCES

(88) 1. Wilkinson R W, Lee-MacAry A E, Davies D, Snary D, Ross E L. Antibody dependent cell-mediated cytotoxicity: a flow cytometry-based assay using fluorophores. J Immunol Methods. 2001; 258:183-191. 2. Morris J C, Waldmann T A. Antibody-based therapy of leukaemia. Expert Rev Mol Med. 2009; 11:e29. 3. Copland M, Hamilton A, Elrick L J, et al. Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction. Blood. 2006; 107:4532-4539. 4. Jorgensen H G, Allan E K, Jordanides N E, Mountford J C, Holyoake T L. Nilotinib exerts equipotent antiproliferative effects to imatinib and does not induce apoptosis in CD34.sup.+ CML cells. Blood. 2007; 109:4016-4019. 5. Graham S M, Jorgensen H G, Allan E, et al. Primitive, quiescent, Philadelphia positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. Blood. 2002; 99:319-325. 6. Jiang X, Zhao Y, Smith C, et al. Chronic myeloid leukemia stem cells possess multiple unique features of resistance to BCR-ABL targeted therapies. Leukemia. 2007; 21:926-935.