USE OF ALPHA-1-MICROGLOBULIN FOR PROTECTION OF BONE MARROW CELLS

Abstract

The present invention relates to alpha-1-microglobulin (A1M) for use in the protection of bone marrow cells in a subject.

Claims

1. Alpha-1-microglobulin (A1M) for use in the protection of bone marrow cells in a subject.

2. A1M for use in the protection of one or more of hematopoietic stem cells and progenitor cells residing in the bone marrow or other hematological niches of a subject.

3. A1M for use according to claim 1 or 2, wherein the protection is for damage caused by exposure to ionizing radiation, chemotherapy or genotoxic substances.

4. A1M for use according to claim 3, wherein the radiation is ionizing radiation, which emanates from a source external to the body such as in external beam radiotherapy or X-ray radiography.

5. A1M for use according to claim 3, wherein the radiation is ionizing radiation, which emanates from a source internalized in the body such as unsealed source radionuclide therapy (RNT); peptide receptor nuclide radiation therapy (PRRT); radioimmunotherapy (RIT) or brachytherapy.

6. A1M for use according to any of the preceding claims, wherein the protection is for ionizing radiation used for diagnostic purposes.

7. A1M for use according to claim 5, wherein a compound labelled with radionuclide is selected from the group consisting of receptor ligands, Affibody molecules, Diabodies, antibody fragments, and/or other small molecules.

8. A1M for use according to claims 5-7, wherein a somatostatin analogue is labelled with radionuclide.

9. A1M for use according to claim 8, wherein the somatostatin analogue is selected from the group consisting of octreotide, lanreotide, Tyr.sup.3-octrotide, Tyr.sup.3-octrotate, DOTADOC, DODATATE, DOTA-lanreotide, pasireotide, dopastatin and octreotide LAR.

10. A1M for use according to any of claims 4-9, wherein A1M is administered before, at the same time or after exposure to radiation.

11. A1M for use according to claim 10, wherein A1M is infused over a period of time.

12. A1M for use according to any of claims 4-11, wherein radiation and administration of A1M are essentially simultaneous.

13. A1M for use according to any of claims 4-11, wherein A1M is administered on more than one occasion before or after radiation.

14. A1M for use in the treatment of bone marrow cell damages.

15. A1M for use in the treatment of one or more of hematopoietic stem cells and progenitor cells residing in the bone marrow and other hematological niches.

16. A1M for use according to any of claims 3-15, wherein the damages result in a decreased production of one or more of hematopoietic stem cells and progenitor cells, such as but not limited to, reticulocytes, compared with a control or a sample taken from the same subject before any exposure to radiation, chemotherapy or genotoxic substances.

Description

LEGENDS TO FIGURES

[0102] FIG. 1: Three dimensional structure of A1M with high-lighted C34 residue and marked N- and C-termini.

[0103] FIG. 2: A1M confers protection to reticulocytes within the bone marrow and peripheral blood cells following exposure to .sup.177Lu-DOTATATE.

[0104] A. Single cell suspension from bone marrow were obtained by crushing femur in PBS containing 2% FCS and passing them though a 70 um cell strainer to obtain single cell suspension. Cells were blocked by incubation with mouse Fc receptor binding inhibitor and then stained with monoclonal antibodies against Ter119, CD44, CD71 and CD45. To exclude dead cells DAPI was used.

[0105] B. Peripheral blood was collected from vena saphena and reticulocytes were determined using LSR Fortessa or Canto II flow cytometry using Retic-Count.

[0106] Data is presented as meanStd and individual data points. Differences in groups were analyzed using one-way ANOVA with post hoc Tukey.

[0107] FIG. 3: A1M confers protection of the proerythroblasts following .sup.177Lu-DOTATATE. A. Single cell suspension from bone marrow were obtained by crushing femur in PBS containing 2% FCS and passing them though a 70 um cell strainer to obtain single cell suspension. Cells were blocked by incubation with mouse Fc receptor binding inhibitor and then stained with monoclonal antibodies against Ter119, CD44, CD71 and CD45. To exclude dead cells DAPI was used.

[0108] Data is presented as meanStd. Differences in groups were analyzed using one-way ANOVA with post hoc Tukey.

[0109] FIG. 4: A1M treatment improves expansion of erythroid cells from a murine DBA model and patients. CD117 (Kit)+ bone marrow cells were treated with Doxycycline to induce Rps19 deficiency. Twenty four hours after Doxycycline administration, cells were treated with drugs interfering with iron or heme availability. The ATP measuring platform CellTiter Glo was used to monitor viable cells after 5 days of expansion. In A) a schematic picture of the drug screen is shown. B) Proliferation of bone marrow cells (as described in A) from Rps19 inducible mice treated with respective drug compounds interfering with iron or heme availability is presented. Shown is also a schematic picture of drug activity where A1M is shown to have intracellular effects by reducing unbound heme in erythroblast. The cells were cultured in erythroid promoting media for 5 days. Kruskal-Wallis non parametric test with Dunn's multiple comparisons test was used for statistical analysis, and genotypes were compared to respective control, separately. p-values: *005, **001, ***0001, ****00001. C) Concentration of free unbound intracellular heme in Kit+ bone marrow cells from the Rps19 deficient mouse expanded in erythroid culture with A1M or vehicle for 72 hours. Mann Whitney non parametric analysis was employed for statistical analysis within each genotype. D) Expansion of CD34+ erythroid precursors from peripheral blood of DBA patients with mutations in RPS19, RPL35a and RPS26, or healthy controls treated with A1M or vehicle in erythroid promoting media for 7 days. A1M-treated or vehicle treated values from each donor is presented pairwise.

EXPERIMENTAL

Example 1A1M Protects Against Radiation-Induced Damage to the Bone Marrow and Peripheral Blood Cells

[0110] In this study, we show that human recombinant A1M (A1M) confers protection against radiation-induced damage to the bone marrow and peripheral blood cells following .sup.177Lu-DOTATATE (150 MBq) exposure in BALB/c mice.

Methods

Recombinant Human A1M

[0111] Recombinant human A1M (A1M, variant RMC-035 corresponding to A1M (R66H+N17,96D)) were supplied by A1M Pharma AB (Lund, Sweden).

Radiopharmaceuticals

[0112] Conjugation of radiopharmaceutical precursor's lutetium (.sup.177Lu)-chloride (LuMark, IDB, Holland) and DOTA-(Tyr.sup.3)-Octreotate (ANMI, Belgium), denoted .sup.177Lu-DOTATATE, were performed at Lund University Hospital (Lund, Sweden). Quality control of the .sup.177Lu-DOTATATE conjugate was performed at Lund University Radionuclide Centre (Lund, Sweden).

Animal Studies

[0113] Female BALB/c mice (Taconic, Denmark) at the age of 12 weeks were used in this study. Two groups (n=5-10) received a subcutaneous administration of either A1M (20 mg/kg) or vehicle buffer (10 mM Na-phosphate pH 7.4+0.15 M NaCl+12 mM histidine) followed by an intravenous (i.v.) injection, 30 minutes later, of .sup.177Lu-DOTATATE (150 MBq). A control group (n=5-10) received a subcutaneous administration of vehicle, followed by, 30 minutes after, an i.v. injections of NaCl. Animals were sacrificed 4 days post-injections.

[0114] After 4 days (post .sup.177Lu-DOTATATE administration), blood, for peripheral blood cell and reticulocyte count, was sampled from vena saphena, on non-anesthetized animals, in EDTA pre-coated vials (Microvette CB 300 K2E, Sarstedt, Nmbrecht, Germany) and placed on a rocking mixer in room temperature followed by analysis as described below. Thereafter the animals were anaesthetized using isoflurane, sacrificed by cervical dislocation and femur (left and right) sampled and placed in PBS, pH 7.4 in a 24 well plate standing on wet ice.

[0115] All animal experiments were conducted in compliance with the national legislation on laboratory animals' protection and with the approval of the Ethics Committee for Animal Research (Lund, Sweden).

Bone Marrow Flow Cytometry Analysis:

[0116] Bone marrow cells were isolated by crushing femur in PBS containing 2% FCS (GIBCO, Waltham, Mass., USA) and passing them though a 70 um cell strainer to obtain single cell suspension. Single-cell suspensions were blocked by incubation with mouse Fc receptor binding inhibitor (eBioscience, Waltham, Mass., USA) and then stained with the following specific mouse monoclonal antibodies (mAb) purchased from BD Biosciences (Stockholm, Sweden), Ter119, CD44, CD71 and CD45. To exclude dead cells 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI) was used. All experiments were performed on LSRFortessa (BD Biosciences) flow cytometry and analyzed with FlowJo software.

[0117] Bone marrow cellularity was counted from the single cell suspension using hematology analyzer SYSMEX KX-21 N.

Blood Analysis

[0118] Following collection of peripheral blood SYSMEX KX-21N hematology analyzer was used for determining Blood parameters. Reticulocyte count was determined using LSR Fortessa or Canto II flow cytometry using Retic-Count (BD Biosciences).

Statistical Analysis

[0119] Results were evaluated by comparisons of all experimental groups using analysis of variance (ANOVA). All statistical calculations were made in GraphPad Prism (GraphPad Prism 7.0; GraphPad Software; GraphPad, Bethesda, Md., USA).

Results

[0120] Analysis of hematopoietic cells in bone marrow and peripheral blood

[0121] The effects of radiation on the bone marrow and peripheral blood cells were evaluated 4 days after injection of 150 MBq .sup.177Lu-DOTATATE using flow cytometry and Sysmex hematological analyzer. It was observed that the percentage of viable reticulocytes was significantly reduced in both bone marrow and peripheral blood following exposure to .sup.177Lu-DOTATATE (FIG. 2). Subcutaneous co-administration of A1M, deposited 30 minutes prior to the .sup.177Lu-DOTATATE administration, maintained a completely preserved reticulocyte population at the level of the non-radiation exposed control animals (FIG. 2).

Effects of Radiation on Terminal Erythroid Differentiation

[0122] The terminal erythroid differentiation within the bone marrow was evaluated 4 days after the injection of 150 MBq .sup.177Lu-DOTATATE. In addition to the effects seen on reticulocytes (in line with that of FIG. 2) a clear effect, although not statistically significant, was also seen on the proerythroblast population (FIG. 3, population denoted I). No effect of radiation was observed in any of the other progenitor populations (denoted population II-IV). Subcutaneous co-administration of A1M, deposited 30 minutes prior to the .sup.177Lu-DOTATATE administration, displayed protection of the proerythroblast population, in addition to the reticulocytes, and maintained them at the level of the non-radiation exposed control animals (FIG. 3).

Example 2A1M Reduces Excess Intracellular Heme and Improves Proliferation in Diamond-Blackfan Anemia

[0123] In this study, we show that human recombinant A1M (A1M) is the only compound tested which lead to an increased proliferation of murine Rps19 deficient erythroid precursors along with an ability to reduce the level of unbound heme.

INTRODUCTION

[0124] Diamond-Blackfan anemia (DBA) is a congenital disorder where patients show macrocytic anemia and a scarcity of erythroid precursors in the bone marrow. Around 70% of all patients have mutations in ribosomal proteins, most commonly in RPS19. Protein translation in general and translation of certain mRNA in particular are altered in DBA, contributing to the disease phenotype. The tumor suppressor p53 is hyperactivated in DBA, resulting in decreased proliferation and increased apoptosis in erythroid precursors. Current treatments for DBA are glucocorticoids, blood transfusions, or allogenic bone marrow transplantation. Unfortunately, all available therapies have side effects impairing the quality of life for the patients. For this reason, there is an urgent need for disease specific treatments for DBA.

[0125] It has already been demonstrated that erythroid precursor cells from a DBA patient contain pathologically high intracellular heme levels, which could explain poor erythroid cell proliferation. Since unbound heme is toxic, the increase in intracellular heme needs to be met by equivalent amounts of globin to generate hemoglobin, the essential oxygen carrying molecule of red blood cells. In DBA however translation is impaired and in erythroid cells the main synthesized proteins are globins. This finding suggest that drugs reducing heme toxicity are potential treatment strategies for DBA, by either enhancing globin mRNA translation, which is the rationale behind an ongoing clinical trial with Leucin, or by reducing intracellular heme levels.

[0126] In this example, we screened for novel therapeutic strategies for reducing toxic unbound intracellular heme in DBA. While drugs inhibiting heme synthesis failed to improve proliferation of Rps19 deficient erythroid progenitor cells, treatment with the heme scavenger A1M resulted in reduction of elevated intracellular heme levels and increased proliferation of erythroid cells from both a murine model for DBA, and DBA patients.

Methods

Drug Treatment of Murine Bone Marrow Cells

[0127] Ethical permission was granted by a local ethical committee for all animal research. Bone marrow from inducible Rps19 deficient mice of 8-14 weeks was enriched for CD117 (Kit) expression using magnetic beads (Miltenyi, Germany) according to manufacturer's instructions. Cells were cultured in StemSpan serum free expansion medium (SFEM) (Stem cell technologies, Canada), 1% penicillin/streptomycin (GE Healthcare, US), 10% fetal bovine serum (ThermoFisher Scientific, US), 100 ng/ml mSCF (Peprotech, US), 300 g/ml h-holo-transferrin (Sigma-Aldrich, US), and 2 U/ml hEpo (Johnson-Johnson, US) with 0.2 g/ml Doxycycline (Sigma-Aldrich).

[0128] The drugs administered 24 hours after Doxycycline administration were A1M (supplied by A1M Pharma AB, Sweden), N-methyl mesoporphyrin IX (AH Diagnostics, Denmark), Succinylacetone, hemin, Deferoxamine, Ferrostatin and N-acetyl-L-cystein and hemopexin (Sigma-Aldrich). Cell expansion 5 days after drug administration was measured using CellTiter Glo (Promega, US), which measures the number of viable and metabolically active cells in culture based on quantitation of the ATP present. Plates were read on Victor 3 Multilabel counter (PerkinElmer, US).

A1M Treatment of Human Samples

[0129] Peripheral blood samples were collected at Lund University hospital using informed consent according to ethical permission granted by the Swedish ethical review board. The three DBA patients had mutations in RPS19, RPS26 and RPL35a respectively, and all received blood transfusions with chelation therapy. Mononuclear cells from DBA patients and healthy subjects were obtained using lymphoprep (Fresenius Kabi, Germany) and enriched for CD34 expression using magnetic beads (Miltenyi, Germany) according to manufacturer's instructions. Cells were cultured in SFEM (Stem cell technologies), 1% penicillin/streptomycin (GE Healthcare), 100 ng/ml hSCF (Peprotech), 2 U/ml Epo (Johnson-Johnson). 5 M A1M or vehicle (Tris-HCl/NaCl) were added every 2-3 days for a total of 7-8 days. Cell count was performed manually using a hemocytometer.

Results and Discussion

A1M Increases Proliferation of Erythroid Rps19 Deficient Cells

[0130] Since increased intracellular heme levels may contribute to impaired erythroid precursor proliferation in DBA patients, we performed a drug screen for compounds affecting iron or heme availability in erythroid cells from a DBA mouse model (FIG. 4A). None of the compounds affecting heme synthesis or iron availability improved proliferation of Rps19 deficient cells. Strikingly, A1M (at 5 and 10 M) was the only compound tested showing increased proliferation of Rps19 deficient erythroid precursors. No effect on proliferation was seen in WT cells, indicating a specific effect of A1M only in Rps19 deficient erythroid precursors (FIG. 4B).

A1M Lowers Elevated Heme Levels in Rps19 Deficient Cells

[0131] A1M protects against heme induced cell and tissue damage by scavenging and degrading heme. In Rps19 deficient erythroid cells A1M was shown to significantly reduce the level of unbound intracellular heme back to WT levels (FIG. 4C). Since treatment with the mainly extracellular heme scavenger hemopexin had no effect on DBA cells (FIG. 4B), A1M likely functions intracellularly on erythroid DBA cells.

Early Erythroid Cells from DBA Patients Increase Proliferation at A1M Treatment

[0132] Purified erythroid precursors from three DBA patients cultured with 5 M A1M all showed improved expansion, while no such trend was observed in cells from healthy subjects (FIG. 4D).

[0133] In summary, this study identifies the heme binding protein A1M to increase proliferation in erythroid cells from DBA patients, by normalizing the levels of unbound intracellular heme. Our findings suggest that A1M has the potential to reduce heme toxicity in anemic conditions caused by ribosomal protein deficiency, such as del 5q-myelodysplastic syndrome. Taken together, this study has identified that A1M can be used to treat cells from DBA patients. It also serves as a proof of concept study that targeting heme levels could be used in developing more disease specific DBA therapies.

Example 3Evaluation of Hematopoietic Recovery after Several Different Inducers of Bone Marrow Damage

[0134] Study the effect of A1M treatment on bone marrow recovery after a number of different damage, including exposure to whole body irradiation, genotoxic and cytotoxic molecules (such as 5-FU, cisplatin etc.), hemolysis induced by agents such as Phenylhydrazine or damage caused by genetic defects such as RPS19-deficiency in Diamond-Blackfan anemia.

[0135] The above will be evaluated by the following means:

1. Serial Transplantations in Mice for Evaluating Stem/Progenitor Recovery.

[0136] Gold standard experiment is to perform serial transplantations as well as limited-dilution experiments (Frisch et al. 2014, Rundberg Nilsson et al. 2015). Serial transplantations means that bone marrow from damaged mice (see the different damage above) that were A1M treated or non-treated will be re-transplanted to irradiated mice together with competitor cells at least twice. This is to demonstrate that long-term stem cells are preserved in A1M treated mice.

2. Limited-Dilution Experiments in Mice.

[0137] Limited-dilution experiments are used to functionally quantify stem cells (Bonnefoix et al. 2010). Different numbers of bone marrow cells from A1M treated and non-treated mice will be re-transplanted to irradiated mice together with healthy competitor cells. Based on the level of hematopoietic reconstitution from treated mice compared to healthy cells it is possible to estimate the number of stem cells that survived the damage.

3. FACS and Colony Assays after Treatment in Mice.

[0138] Standard experiments to be performed to evaluate number of progenitor cells. FACS analysis will determine if A1M therapy leads to increased survival of hematopoietic progenitor cells and stem cells.

4. A1M Uptake in Stem/Progenitor Cells.

[0139] A1M will be injected into healthy and damaged (see above) mice. FACS will then be used to sort stem and progenitor cells from the animals and determine the uptake of A1M.

5. A1M Knockout Mice.

[0140] The above experiments are performed in animals with normal endogenous levels of A1M and are performed to evaluate the potential of A1M as a drug. To more clearly determine mechanism of A1M in protecting hematopoietic stem/progenitor cells during bone marrow damage the above experiments will be performed on A1M knockout mice that are deficient of A1M.

REFERENCES

[0141] Bonnefoix, T. and M. Callanan (2010). Accurate hematopoietic stem cell frequency estimates by fitting multicell Poisson models substituting to the single-hit Poisson model in limiting dilution transplantation assays. Blood 116(14): 2472-2475. [0142] Frisch, B. J. and L. M. Calvi (2014). Hematopoietic stem cell cultures and assays. Methods Mol Biol 1130: 315-324. [0143] Rundberg Nilsson, A., C. J. Pronk and D. Bryder (2015). Probing hematopoietic stem cell function using serial transplantation: Seeding characteristics and the impact of stem cell purification. Exp Hematol 43(9): 812-817 e811.