Anti-IL1RAP antibodies and their use for treating humans
11773174 · 2023-10-03
Assignee
Inventors
Cpc classification
C07K16/2866
CHEMISTRY; METALLURGY
C07K2317/732
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
A61P43/00
HUMAN NECESSITIES
A61K51/1033
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
A61K47/6849
HUMAN NECESSITIES
A61K51/1027
HUMAN NECESSITIES
C12N15/115
CHEMISTRY; METALLURGY
International classification
A61K39/00
HUMAN NECESSITIES
A61K47/68
HUMAN NECESSITIES
A61K51/10
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
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 cells associated with a solid tumour, 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 cells associated with a solid tumour, 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 for treating a solid tumor in a human comprising, administering to the human an effective amount of an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody with specificity for an extracellular domain of human IL1RAP wherein cells of the solid tumor express IL1RAP.
2. The method of claim 1, wherein the solid tumor is a skin cancer.
3. The method of claim 1, wherein the solid tumor is cancer of the urinary organs.
4. The method of claim 1, wherein the solid tumor is cancer of the uterus.
5. The method of claim 1, wherein the antibody is a monoclonal antibody.
6. The method of claim 5, wherein the monoclonal antibody is human or humanized.
7. The method of claim 1, wherein the antibody further comprises a cytotoxic or detectable moiety.
8. The method of claim 7, wherein the cytotoxic or detectable moiety comprises or consists of a radioisotope.
9. The method of claim 8, wherein the radioisotope is selected from the group consisting of astatine-211, bismuth-212, bismuth-213, iodine-131, yttrium-90, lutetium-177, samarium-153 and palladium-109.
10. The method of claim 7, wherein the cytotoxic moiety comprises or consists of a toxin.
11. The method of claim 7, wherein the cytotoxic moiety comprises or consists of a chemotherapeutic agent.
12. The method of claim 8, wherein the radioisotope is selected from the group consisting of technitium-99m, indium-111, gallium-67, gallium-68, arsenic-72, zirconium-89, iodine-12 and thallium-201.
13. The method of claim 1, wherein the antibody induces antibody-dependent cell-mediated cytotoxicity (ADCC).
14. The method of claim 1, wherein the antibody is of IgG1 isotype.
15. The method of claim 1, wherein the antibody is of IgG2a isotype.
16. The method of claim 1, wherein the antibody is administered parenterally.
17. The method of claim 16, wherein the parenteral administration is intravenous, subcutaneous, or intramuscular.
18. A method for reducing the size and/or reducing the growth of a solid tumor comprising administering to an individual an effective amount of an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody with specificity for an extracellular domain of human IL1RAP wherein cells of the solid tumor express IL1RAP.
19. The method of claim 18, wherein the solid tumor is skin cancer.
20. The method of claim 18, wherein the solid tumor is cancer of the urinary organs.
21. The method of claim 18, wherein the solid tumor is cancer of the uterus.
Description
(1)
(2) Flow cytometric analysis confirms that IL1 RAP expression is induced upon retroviral P210 BCR/ABL1 expression in cord blood CD34+ 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.
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(4) 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.
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(9) In FIGS. A-C, 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.
(10)
(11) 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.
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(15) In
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(18) 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.
(19)
(20) By using 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. 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.
(21)
(22) 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.
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(24) 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.
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(26) The AML cell line MONO-MAC-6 and the ALL cell line REH express IL1RAP.
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(28) 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.
(29)
(30) In the 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.
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(32) In the ADCC assay, a KMT-1 induced cell death was observed in primary AML CD34+CD38− cells.
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(39) Different cell lines derived from human solid tumours were stained with anti-human IL-1 RAcP/IL-1 R3-APC (cat no FAB676A, R&D system) (black lines) and isotype control (gray lines). Flow cytometry analysis show expression of IL1RAP on COL0829 (malignant melanoma), HCC1954 (breast ductal carcinoma), NCI-8228 (lung adenocarcinoma), NCI-H716 (colon cancer), OV-90 (ovarian adenocarcinoma), H716 (colon cancer), H2228 (lung adenocarcinoma), SH-4 (melanoma), SR (lymphoma) and SW 1783 (astrocytoma).
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(41) Histogram from flow cytometry analysis on cells from four different human cancer cell lines labeled with mab81.2, an antibody against IL1RAP, showing IL1RAP expression on H716 (colon cancer), H2228 (lung adenocarcinoma), HCC1954 (breast ductal carcinoma), and SH-4 (melanoma).
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(43) Graphs showing the degree of specific cell death induced by the anti-human IL1RAP antibody mab81.2, and human NK-cells in an ADCC assay. As isotype control, a non-specific human IgG1 antibody was included in the experiments.
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(45) MAb 81.2 was administered at 10 mg/kg body weight intraperitoneally twice weekly. Control mice were treated with equivalent volumes of PBS. Each experimental group contained ten mice. Results are presented as average tumour volume (mm3); error bars represent Standard Error of the Mean (SEM).
EXAMPLE 1
(46) IL1RAP is a cell surface biomarker for chronic myeloid leukemia stem cells
(47) Summary
(48) 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).
(49) Introduction
(50) 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 IL1 RAP is a cell surface biomarker for CML stem cells, for the first time allowing prospective separation of CML stem cells from normal HSC.
(51) Material and Methods
(52) Collection of CML Patient Cells
(53) Isolation and transduction of cord blood CD34.sup.+ cells
(54) 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.
(55) Microarray Analysis
(56) 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 Qlucore Omics Explorer 2.0 (Qlucore, Lund, Sweden).
(57) Flow Cytometric Analysis
(58) 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 IL1 RAP 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.
(59) Cell Sorting and Interphase FISH
(60) 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 μL 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.
(61) Long Term Culture—Initiating Cells (LTC-IC)
(62) M.sub.210b.sub.4 stroma cells were cultured in RPMI-1640 medium supplemented with 10% FCS as previously described.sup.24,25. 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 μL Myelocult medium (Stem Cell Technologies, Vancouver, Canada) containing 10.sup.−6M 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 μL medium was exchanged 3 h later. Once per week, the exchange of 100 μL 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.
(63) P210 BCR/ABL1 Expression in Cord Blood CD34.sup.+ Cells
(64) 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.
(65) Results and Discussion
(66) Global Gene Expression Analysis Identifies IL1RAP as Upregulated on CML CD34.sup.+ Cells
(67) 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.
(68) IL1RAP is upregulated on CD34.sup.+CD38.sup.− cells from CML patients and is induced as a consequence of ectopic P210 BCR/ABL1 expression
(69) 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.
(70) 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 (
(71) 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 (
(72) Flow-drop-FISH shows that IL1RAP expression separates normal and leukemic cells within CML CD34.sup.+CD38.sup.− cells
(73) To test whether the IL1 RAP expression distinguishes normal (Ph.sup.−) and leukemic (Ph.sup.+) 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,
(74) CML stem cells are CD34.sup.+CD38.sup.−IL1RAP.sup.+ whereas normal HSC are CD34.sup.+CD38.sup.−IL1RAP.sup.−/Low
(75) 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.−1L1RAP.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 culture—colony 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 (
(76) 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 IL1 RAP 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.
(77) References 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 I, 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, 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 0, 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, lwashita 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, 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.sup.+ 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 STATS-dependent expansion of the erythroid lineage. Exp Hematol. 2009; 37:367-375. 24. Hogge D E, et al. 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
(78) Antibody-targeting of IL1RAP on leukemia stem and progenitor cells cause antibody-dependent cell-mediated cytotoxicity (ADCC)
(79) Summary
(80) 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).
(81) 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.
(82) 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.
(83) Materials and Methods
(84) Generation of KMT-1; a polyclonal rabbit anti-human IL1RAP antibody
(85) Rabbits were immunized with the extracellular domain of IL1 RAP. 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.
(86) ADCC Assay
(87) 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 concentration—Percentage 7-AAD+ cells without antibody)/(0.01×Percentage 7-AAD—cells without antibody) from at least three independent experiments (except
(88) 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.
(89) Results
(90) Antibody-targeting of IL1RAP on CML stem and progenitor cells but also on a CML cell line directs NK-cells to ADCC
(91) 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.
(92) As expected, low levels of ADCC were observed in the IL1 RAP negative/low leukemia cell line KG-1, even at high KMT-1 concentrations (
(93) 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 anti-human IL1RAP antibody (Example 1,
(94) Antibodies targeting IL1RAP on AML and ALL cells direct NK-cells to ADCC
(95) IL1RAP expression was observed in AML CD34.sup.+CD38.sup.− cells in 9 out of 11 tested samples (
(96) 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 (
(97) 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 (
(98) Discussion
(99) 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 IL1 RAP 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.
(100) 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).
(101) 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.
(102) Interestingly, we also observed that IL1 RAP 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.
(103) In the ADCC experiments presented herein, a polyclonal anti-human IL1 RAP 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.
(104) References 1. Wilkinson R W, Lee-MacAry A E, Davies D, Snary D, Ross E L. Antibodydependent 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+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.
EXAMPLE 3
Gene Expression on Solid Tumours
(105) Materials and Methods
(106) Using the Oncomine search engine (www.oncomine.org), we identified all data sets containing various cell lines established from different tumour types. The largest data set identified was the data set “Wooster Cell Line2”. This data set contains 308 cancer cell lines, representing 20 different tumour types. The query term used was “IL1RAP” with the reporter setting “205277_at”.
(107) Results
(108) In total, we identified 17 different solid tumour types that were represented by cell lines meeting our criteria for an upregulated expression of IL1RAP (see Table 1). The percentage of cell lines within each tumour type showing upregulated IL1RAP ranged from 4% (colorectal cancer) to 67% (melanoma, prostate cancer). Among the tumour types, we identified some of the most common cancer entities in humans, including malignancies from breast, colon, lung, prostate and bladder. In addition, some tumour types associated with poor clinical outcomes, such as melanoma and brain tumours displayed highly upregulated expression of IL1RAP.
(109) Conclusions
(110) We conclude that several different tumour entities show an upregulated gene expression level of IL1RAP.
(111) These data indicate that treatment with antibodies directed against IL1RAP will provide a new therapeutic avenue in several different human cancer types.
(112) TABLE-US-00001 TABLE 1 Upregulation of IL1RAP in 308 cancer cell lines representing different tumour types* Number of tumours displaying Tumour type upregulation of IL1RAP** Bladder Cancer 3/9 (33%) Brain and CNS Cancer 7/16 (44%) Breast Cancer 4/19 (21%) Cervical Cancer 4/7 (57%) Colorectal Cancer 1/23 (4%) Esophageal Cancer 3/4 (75%) Gastric Cancer 1/5 (20%) Head and Neck Cancer 3/6 (50%) Kidney Cancer 1/8 (12%) Liver Cancer 3/9 (33%) Lung Cancer 14/73 (19%) Lymphoma 2/38 (5%) Melanoma 8/12 (67%) Ovarian Cancer 2/5 (40%) Pancreatic Cancer 3/9 (33%) Prostate Cancer 2/3 (67%) Sarcoma 5/13 (38%) *The Wooster data set on 308 cancer cell lines was searched using Oncomine (www.oncomine.org). The query term used was “IL1RAP” with the reporter setting “205227_at”. The platform used was Human Genome U133 Plus 2.0 Arrays (Affymetrix Inc.) **Only tumour cell lines displaying an equal or higher expression level of IL1RAP than in the Philadelphia-positive cell line KU812 were scored as “upregulated”. KU812 has previously been shown by us to have an upregulated protein expression of IL1RAP at the cell surface (Järås et al., 2010, PNAS 107(14): 16280-5).
EXAMPLE 4
Analysis of IL1RAP Expression on Human Cell Lines by Flow Cytometry
(113) Materials and Methods
(114) Reagents Fc-receptor blockers from BD Biosciences anti-human CD16 (cat no 555404) anti-human CD32 (cat no 555447) APC-mouse IgG1 k Isotype control (cat no 555751) from BD Biosciences Anti-human IL-1 RAcP/IL-1 R3-APC (cat no FAB676A) from R&D system.
(115) Cell Lines
(116) TABLE-US-00002 TABLE 2 Cell line Description ATCC/DSMZ Catalog No. KG-1 Human acute myeloid ACC 14 leukemia (used as a negative control) KU-812 Human chronic myeloid ACC 378 leukemia in myeloid blast crisis (used as a positive control) NCI-H2228 Lung Adenocarcinoma CRL-5935 NCI-H716 Colon Cancer CCL-251 HCC1954 Breast Ductal Carcinoma CRL-2338 SR Lymphoma CRL-2262 OV-90 Ovarian Adenocarcinoma CRL-11732 COLO 829 Malignant Melanoma CRL-1974 SH-4 Melanoma CRL-7724 SW 1783 Astrocytoma HTB-13
(117) The cell lines were cultured under standard conditions in medium recommended by the suppliers.
(118) FACS Analysis
(119) Cells (350 000) were resuspended in 2 ml FACS buffer (PBS without calcium and magnesium supplemented with 0.5% BSA), and centrifuged for 4 min at 300 x g. The supernatant was discarded and Fc-receptors were blocked by incubating cells with anti-CD16/CD32 mAbs at a concentration of 3 μg/ml in a volume of 30 μl for 5 minutes at room temperature. Then, 55 μl FACS buffer and 4 μl APC-labeled isotype antibody or 5 p| APC-labeled monoclonal antibody directed against human IL1RAP were added to the cells and incubated for 30 minutes at +4° C. The cells were washed with 3 ml FACS buffer, centrifuged for 4 minutes at 300 x g and the supernatant was discarded. Cells were finally resuspended in 200 μl FACS buffer and flow cytometric analysis was performed according to standard settings on a FACS Cantoll flow cytometer (BD Biosciences).
(120) Results
(121) IL1RAP expression levels on the solid tumour cell lines tested are shown in Table 3 below and in
(122) TABLE-US-00003 TABLE 3 Expression of IL1RAP on different human cell lines. Values represent mean fluorescence intensity. Cell line Blank Isotype Anti-IL1RAP KG-1 46 52 113 KU-812 62 69 451 NCI-H2228 80 96 587 NCI-H716 60 96 2043 HCC1954 112 119 410 SR 51 54 2257 OV-90 78 89 1921 COLO 829 77 82 3732 SH-4 40 51 5189 SW 1783 119 153 341
(123) Conclusions
(124) Expression of IL1RAP was observed on the solid tumour cell lines NCI-H2228, NCI-H716, HCC1954, SR, OV-90, COLO 829, SH-4 and SW 1783. The expression on these cell lines was comparable or higher than that on the human chronic myeloid leukemia cell line KU-812.
EXAMPLE 5
Antibody-Targeting of IL1RAP on Solid Tumour Cells Causes Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
(125) Materials and Methods
(126) Development and production of the chimeric monoclonal antibody 81.2 hlgG 1.
(127) A murine hybridoma cell line which was secreting monoclonal antibodies specific to the extracellular part of human IL1RAP was generated by standard procedures. Briefly BALB/c mice were immunized with a fusion protein consisting of the extra cellular part of IL1RAP and the Fc-part of human IgG1 (Pro100-Lys330). Splenocytes were fused with the mouse myeloma cell line Sp2/0 and clones producing and antibodies directed against the extracellular part of IL1RAP were isolated by screening with the fusion protein used for the immunisations and counter-screened with human IgG1.
(128) The antibody produced by the hybridoma cell line clone 81.2 was of IgG1/kappa type and was found to have a high specificity to IL1RAP-positive cells and the recombinant protein human IL1RAP (21-367). From this cell line, total RNA was isolated and cDNA representing the variable regions of the heavy and light chains, VH and VK, were amplified by PCR, cloned and sequenced.
(129) The genetic element coding for the murine VK in frame with the constant part of human kappa gene was synthesised and cloned in to a plasmid mammalian expression vector.
(130) The PCR fragment coding for the murine VH were combined with the constant parts of human IgG1 and cloned in to a plasmid mammalian expression vector.
(131) HEK 293 cells were co-transfected with both plasmids and the cells were cultured in serum-free medium supplemented with 100 ng/ml kifunensine. The chimeric antibody 81.2 hlgG1 was purified from the culture medium by Protein G chromatograhy.
(132) Flow Cytometry
(133) Cells from four different human solid cancer cell lines, H2228 (adenocarcinoma; non-small cell lung cancer), H716 (colorectal adenocarcinoma), HCC1954 (ductal breast carcinoma), and SH-4 (melanoma), were harvested and stained with mab81.2, an anti-human IL1RAP antibody (Cantargia AB, Lund, Sweden). For detection, cells were stained with a secondary anti-human IgG PE-conjugated antibody (Thermo-Fisher, Waltham, Mass.), and cells were analyzed using a FACS CANTO flow cytometer (BD Immunocyteometry Systems, Mountain View, Calif.).
(134) ADCC Assay
(135) The ADCC assay was based on a protocol previously described (see Example 2 above). In brief, target cells were labeled with PKH26 (Sigma-Aldrich, St Louis, Mo.) according to manufacturer's instructions, and seeded into a 96-well plate at a density of 10,000 cells per well. Subsequently, antibodies were added to wells in different concentrations and incubated for 30 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). A non-specific human IgG1 antibody was used as control in the experiments (Eureka Therapeutics, Emeryville, Calif.).
(136) The degree of cell death was assessed by detection of 7-AAD positive cells using a FACS CANTO flow cytometer (BD). The level of antibody induced cell death was calculated according to the following equation: Percentage 7-AAD+ cells at defined antibody concentration—Percentage 7-AAD+ cells without antibody.
(137) Results
(138) An antibody against human IL1RAP labels human non-leukemic cancer cells in flow cytometry, and directs NK-cells to ADCC resulting in killing of human cancer cells
(139) We have shown that KMT1, a polyclonal antibody against human IL1 RAP, could direct NK-cells to ADCC, and induce cell death of the IL1RAP-high expressing CML cell line KU812, but not on IL1RAP-low expressing KG1 cells (see Example 2 above).
(140) The results from the present study show that not only leukemic cells are sensitive to ADCC mediated by IL1RAP, but also cells from solid human cancers. Four different human cancer cell lines, representing four different solid human cancer types, were studied, and all showed expression of IL1RAP on the cell surface (
(141) All four cell lines tested were also shown to be sensitive to ADCC mediated by mab81.2, an antibody against human IL1RAP, in what seems to be a dose-dependent way (
(142) Conclusion
(143) The present study confirms that IL1 RAP is expressed on the cell surface of several human cancer types, including lung cancer, colon cancer, breast cancer, and malignant melanoma.
(144) Using an antibody directed against IL1RAP, the cells of all four cell solid tumour lines tested were shown to be targeted by specific NK-mediated killing in an ADCC-assay.
EXAMPLE 6
Efficacy of Monoclonal Antibody 81.2 In Vivo in a Human Melanoma Sk-MEL-5 Xenograft Mouse Model
(145) Materials and Methods
(146) The development and production of the mouse monoclonal antibody 81.2 of IgG1 and IgG2a isotype.
(147) A murine hybridoma cell line which was secreting monoclonal antibodies specific to the extracellular part of human IL1 RAP was generated by standard procedures. Briefly BALB/c mice were immunized with a fusion protein consisting of the extra cellular part of IL1RAP and the Fc-part of human IgG1 (Pro100-Lys330). Splenocytes were fused with the mouse myeloma cell line Sp2/0 and clones producing and antibodies directed against the extracellular part of IL1 RAP were isolated by screening with the fusion protein used for the immunisations and counter-screened with human IgG1.
(148) The antibody produced by the hybridoma cell line clone 81.2 was of IgG1/kappa type and was found to have a high specificity to IL1RAP-positive cells and the recombinant protein human IL1RAP (21-367). From this cell line, total RNA was isolated and cDNA representing the variable regions of the heavy and light chains, VH and VK, were amplified by PCR, cloned and sequenced.
(149) The genetic element coding for the murine VK in frame with the constant part of murine kappa gene was synthesised and cloned in to a plasmid mammalian expression vector.
(150) The PCR fragment coding for the murine VH were combined with the constant parts of murine IgG2a and cloned in to a plasmid mammalian expression vector.
(151) HEK 293 cells were co-transfected with both plasmids and the cells were cultured in serum-free medium. The mouse antibody 81.2 of IgG2a isotype was purified from the culture medium by Protein G chromatograhy.
(152) Flow Cytometry
(153) In order to confirm the IL1RAP expression on the human malignant melanoma cell line, SK-MEL-5, and compare expression to the human CML cell line KU812, both cell lines were cultured according to standard procedures and maintained in logarithmic growth phase. At cell harvest 3.5−5.0×10.sup.5 cells/mL were labeled with the mouse IgG1 81.2 monoclonal antibody at 1-50 μg/mL. An IgG1 isotype control antibody was used as control. The staining was analyzed using the Accuri C6 Flow Cytometer.
(154) Drugs and Treatment
(155) TABLE-US-00004 TABLE 4 Drug/Testing Agent Group n Agent mg/kg Route Schedule Control 10 Vehicle (PBS) — ip biwk × 6 Treated 10 mAb 81.2 10 ip biwk × 6
(156) In vivo administration of human IL1RAP specific mAb 81.2 or vehicle
(157) Eight to 12-week-old female CD.17 SCID mice were injected with 1×10.sup.7SK-M EL-5 tumour cells in 50% Matrigel per animal, subcutaneously in the flank. Treatment was started approximately one week after melanoma cell injection when tumours had reached a size of 108-128 mm.sup.3. A paired match of tumour size in 20 animals was done giving 10 mice each in the two treatment groups.
(158) 81.2, a mouse IgG2a monoclonal antibody, was prepared at a dose of 10 mg/kg and with a volume of 10 mL/kg in PBS. Control animals were given equal volumes of PBS. Treatments were given via the intra-peritoneal route. Tumour volume by calliper measurement and total weights were monitored twice weekly.
(159) The endpoint of the study is tumour growth delay.
(160) Results
(161) Flow Cytometry
(162) TABLE-US-00005 TABLE 5 Expression of IL1RAP on SK-MEL-5 human melanoma cell line and human CML cell line KU812. Values represent mean fluorescence intensity IL1RAP Cell line Sample Name Concentration expression SK-MEL-5 No label N/A 473 81.2 1 μg/mL 501 81.2 10 μg/mL 19010 81.2 50 μg/mL 17560 Isotype Control 10 μg/mL 605 Isotype Control 50 μg/mL 548 Secondary Only 1 μg/mL 497 KU812 No label N/A 291 81.2 1 μg/mL 1188 81.2 10 μg/mL 2156 81.2 50 μg/mL 1868 Isotype Control 10 μg/mL 715 Isotype Control 50 μg/mL 463 Secondary Only 1 μg/mL 309
(163) In Vivo Activity of Exemplary mAb 81.2
(164) Analysis of the study at day 33 from start of dosing showed a statistically significant delay in tumour growth in the treatment group compared to the control group on days 22 (p<0.05), 26 and 29 (p<0.001) and day 33 (p<0.0001) (see
(165) Conclusion
(166) Expression of IL1RAP was confirmed by flow cytometry on the melanoma tumour cell line SK-MEL-5 and showed an expression which was higher than that on the human chronic myeloid leukemia cell line KU-812.
(167) The in vivo data indicate that the human IL1RAP specific monoclonal antibody, 81.2, administered twice weekly at a dose of 10 mg/kg, caused inhibition in tumour cell growth of the IL1RAP expressing human melanoma cell line, SK-MEL-5.