Cancer-killing cells

Abstract

The present invention relates to an in vitro culture of haematopoietic cells, wherein said haematopoietic cells differentiate to form granulocytes characterised by the ability to kill cancer cells. The invention also relates to said granulocytes, methods for identifying said haematopoietic cells and granulocytes, compositions and kits comprising the same, as well as uses of the same for treating cancer.

Claims

1. A method of formulating a cancer-cell killing formulation comprising: selecting hematopoietic stem and/or precursor cells derived from a donor that produces granulocytes with the ability to kill cancer cells; and formulating the selected hematopoietic stem and/or precursor cells within a carrier; thereby formulating the cancer-cell killing formulation.

2. The method of claim 1, wherein the granulocytes comprise neutrophils.

3. The method of claim 1, wherein the selected hematopoietic stem and/or precursor cells comprise hematopoietic stem cells, common myeloid progenitor cells, myeloblasts, N. promyelocytes, N. myelocytes, N. metamyelocytes, N. bands, or combinations thereof.

4. The method of claim 1, further comprising culturing, proliferating, and expanding the selected hematopoietic stem and/or precursor cells.

5. The method of claim 1, further comprising cryogenically freezing and storing the selected hematopoietic stem and/or precursor cells.

6. The method of claim 1, wherein the donor is an 18-25 year old human male.

7. The method of claim 1, further comprising measuring a surface charge of a granulocyte obtained from the donor.

8. The method of claim 1, wherein the carrier comprises a pharmaceutically-acceptable carrier, TNFα, a granulocyte-macrophage colony-stimulating factor (GM-CSF), a granulocyte colony-stimulating factor (G-CSF), a growth hormone; serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, an interleukin, Flt-3 ligand, thrombopoietin, fetal bovine serum (FBS), or combinations thereof.

9. The method of claim 8, further comprising administering the cancer-cell killing formulation to a subject who has cancer.

10. The method of claim 9, wherein the subject has a solid tumor cancer.

11. A cancer-cell killing formulation produced according to the method of claim 1.

12. The cancer-cell killing formulation of claim 11, wherein the selected hematopoietic stem and/or precursor cells comprise hematopoietic stem cells, common myeloid progenitor cells, myeloblasts, N. promyelocytes, N. myelocytes, N. metamyelocytes, N. bands, or combinations thereof.

13. The cancer-cell killing formulation of claim 11, wherein the carrier comprises a pharmaceutically-acceptable carrier, TNFα, a granulocyte-macrophage colony-stimulating factor (GM-CSF), a granulocyte colony-stimulating factor (G-CSF), a growth hormone; serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, an interleukin, Flt-3 ligand, thrombopoietin, fetal bovine serum (FBS), or combinations thereof.

14. The cancer-cell killing formulation of claim 11, in a cryogenically frozen form.

15. The method of claim 1, wherein the donor produces granulocytes which kill at least 5% of cancer cells in a cancer killing assay.

16. The method of claim 15, wherein the cancer killing assay comprises: a. admixing granulocytes obtained from a donor with a cancer cell line to form an admixture; b. incubating said admixture; and c. measuring the percent (%) of cancer cells killed in the incubated admixture.

17. The method of claim 1, wherein selecting hematopoietic stem and/or precursor cells derived from a donor that produces granulocytes with the ability to kill cancer cells comprises: a. admixing granulocytes obtained from the donor with a cancer cell line to form an admixture; b. incubating said admixture; c. measuring the percent (%) of cancer cells killed in the incubated admixture; and d. selecting hematopoietic stem and/or precursor cells from a sample from the donor when the granulocytes kill at least 5% of the cancer cells in the admixture.

18. The method of claim 17, wherein step d comprises selecting hematopoietic stem and/or precursor cells from a sample from the donor when the granulocytes kill at least 30% of the cancer cells in the admixture.

19. A method of formulating a cancer-cell killing formulation comprising: selecting hematopoietic stem and/or precursor cells derived from a donor that produces granulocytes with the ability to kill cancer cells; differentiating the selected hematopoietic stem and/or precursor cells into granulocytes, and formulating the granulocytes within a carrier; thereby formulating the cancer-cell killing formulation.

20. The method of claim 19, further comprising cryogenically freezing and storing the granulocytes.

Description

FIGURES

(1) Embodiments of the invention will now be described, by way of example only, with reference to the accompanying Figures, in which:

(2) FIG. 1 shows cytotoxicity of different neutrophil donors and different effector to target cell ratios.

(3) FIG. 2 shows cytotoxicity results of three CD34+ stem cell derived neutrophil populations from different donors.

EXAMPLES

Example 1

(4) Recruitment of Donors

(5) Donors are pre-selected based on the probability of having neutrophils exhibiting high levels of Cancer Killing Activity (CKA) in a CKA assay described in Example 2. Pre-selection criteria include: no serious medical or psychiatric condition that effecting provision of consent or sample collection; no personal or family history of the cancer(s) being targeted for therapy; no history of chemotherapy or radiation therapy within three months prior to the sampling date; aged 18-24; optionally male (without wishing to be bound by theory, neutrophils from males are believed to exhibit the highest levels of CKA when tested in the CKA assay); and optionally blood groups O or rhesus negative.

(6) White Blood Cells (WBCs) are collected by drawing approximately 18 ml of human blood from a donor. The blood is split into three BD Vacutainer™ CPT tubes and centrifuged at 175×g for 35 minutes at 23° C. The mononuclear cell (MN) layer is collected and transferred to a 15 ml conical tube. The MN cells are centrifuged at 420×g for 5 minutes at 23° C., and washed with 10 ml Dulbecco's Modified Eagle's Medium (DMED) (Invitrogen, Carlsbad, Calif.) +10% foetal bovine serum (FBS) (Sigma. St. Louis, Mo.). Cells are counted and resuspended in medium to a final concentration of 1.6×10.sup.6 cells/ml.

Example 2

(7) Testing CKA of Extracted Granulocytes in a CKA Assay

(8) Cells are cultured in DMEM+10% FBS in a T25 flask to 80% confluence. The cell line is grown and maintained at 37° C., 8% CO.sub.2, in T75 cm.sup.2 cell culture flasks in DMEM supplemented with the following ingredients: 10% volume/volume FBS, penicillin (Sigma. St. Louis, Mo.), streptomycin (Sigma. St. Louis, Mo.), and supplemental L-glutamine (Sigma. St. Louis, Mo.). Cultured pancreatic cancer cells (e.g. Capan-2, ATCC HTB-80; Panc 10.05, ATCC CRL-2547; CFPAC-1, ATCC CRL-1918; HPAF-II, ATCC CRL-1997; SW 1990, ATCC CRL-2172; BxPC-3, ATCC CRL-1687; AsPC-1, ATCC CRL-1682; ATCC® TCP-1026™; SW1990, ATCC CRL-2172; SU.86.86, ATCC CRL-1837; BXPC-3, ATCC CRL-1687; Panc 10.05, ATCC CRL-2547; MIA-PaCa-2, ATCC CRL-1420; PANC-1, ATCC CRL-1469; or ATCC® TCP-2060™ commercially available from the American Type Culture Collection—United Kingdom (U.K.), Guernsey, Ireland, Jersey and Liechtenstein, LGC Standards, Queens Road, Teddington, Middlesex TW11 0LY, UK) are split and passaged before reaching 70% surface confluence in culture flasks.

(9) Cells are trypsinised, harvested and counted with Trypan Blue. Assay plates (24-well) are seeded with 8×10.sup.4 pancreatic cancer cells (e.g. pancreatic ductal adenocarcinoma cells) per well in 24-well flat bottom plates. Plates are incubated at 37° C. in 5% CO.sub.2 for 24 hours. Cells are labelled with 2.5 μM CellTracker™ Green for 45 minutes. Fresh medium is added to cells and they are returned to a CO.sub.2-incubator.

(10) The CKA assay is carried out by adding 500 μl of MN cell suspension (8×10.sup.5 granulocytes) to each well in which the pancreatic cancer cells are grown for 24 hours. The cells are mixed and placed in an incubator in 5% CO.sub.2 for 24 hours at 39° C. After a 24-hour incubation, cells are harvested by trypsinisation and centrifuged. Cells are resuspended in 100 μl cold phosphate-buffered saline (PBS), and 125 μl 0.4% Trypan Blue subsequently added. Cells are counted under microscope (using phase contrast and fluorescence microscopy).

(11) Granulocytes (e.g. neutrophils) capable of killing at least 70% or at least 80% of the cancer cells (i.e. having at least 70% CKA or 80% CKA, respectively) in the assay are considered particularly suitable for use in treating cancer.

Example 3

(12) Testing Surface Potential of Haematopoietic Cells and Neutrophils

(13) Electrophoresis is used to investigate the surface potential variation in haematopoietic cells (e.g. haematopoietic stem cells, and/or precursor cells) and neutrophils by measuring the electrophoretic mobility. The suspended cells are collected from culture, by mechanical detachment and collection from the culture substrate. Collected cells are redistributed in an electrophoresis buffer solution containing 10 mM Tris-HCl and 291 mM glucose, and are introduced into a rectangular glass electrophoresis chamber. 200V DC is applied across the electrophoresis chamber. The electrophoretic velocity of cells, u, is measured by recording the time needed for cells passing a fixed length with 3 mA under a microscope with a CCD camera. The electrophoretic mobility, μ, is calculated by μ=ugS/I, where g is the conductivity of medium, S is the cross-sectional area of the electrophoresis chamber, and I is the current. For each condition typically at least 9 readings are performed to calculate cell electrophoretic mobility.

Example 4

(14) Extracting Haematopoietic Stem Cells from Peripheral Blood

(15) Upon giving consent the donors are given a granulocyte-colony stimulating factor (G-CSF) and/or a granulocyte-macrophage colony-stimulating factor (GM-CSF), e.g. Neupogen® (commercially available from Amgen Inc. USA) to help harvest peripheral haematopoietic stem cells with minimal possible discomfort to donors. Cell surface polypeptide markers are used for identifying long-lasting multipotent stem-cells. Suitably markers may include CD 34.sup.+, CD59.sup.+, Thy1.sup.+, CD38.sup.low/−, C-kit.sup.−/low, and lin.sup.−.

Example 5

(16) Expansion and Differentiation of Haematopoietic Cells

(17) The haematopoietic cells (e.g. haematopoietic stem cells) are stimulated using a supernatant growth factor suspension, to either develop more stem cells or differentiate into precursor cells (e.g. myeloid or granulocyte progenitor cells) or granulocytes. Suitable neutrophil synthesis methods are disclosed in Lieber et al, Blood, 2004 Feb. 1; 103(3):852-9, and Choi et al, Nat. Protoc., 2011 March; 6(3):296-313.

(18) The protocol is composed of four major stages: culturing and proliferation of haematopoietic cells; short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor (GM-CSF), a granulocyte colony-stimulating factor (G-CSF), a human growth hormone (HGH); serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin-3 (IL-3), interleukin 8 (IL-8), Interleukin-4 (IL-4), Interleukin-6 (IL-6), interleukin-18 (IL-18), TNF-alpha, Flt-3 ligand, thrombopoietin, foetal bovine serum (FBS), or combinations thereof; and directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells (DCs), Langerhans cells (LCs), macrophages and osteoclasts.

Example 6

(19) Preparation of Cell Banks

(20) Haematopoietic stem cells, granulocyte precursor cells and granulocytes obtainable therefrom, are cryogenically frozen and stored in appropriate cell banks.

Example 7

(21) Use in Patients for Treating Solid Tumours

(22) Stored haematopoietic cells (e.g. haematopoietic stem cells or granulocyte precursor cells obtainable therefrom), and granulocytes (e.g. neutrophils) differentiated therefrom are matched to cancer patients based on their cancer type, blood type (ABO, rhesus and HLA), and/or genetics. Patients may also be matched based on human leukocyte antigen (HLA) similarity.

(23) Patients are treated using: IV infusion of haematopoietic cells (including haematopoietic stem cells, and granulocyte precursor cells) together with granulocyte-colony stimulating factor, human growth hormone, serotonin, and interleukin into the patient; or IV infusion of stimulated granulocyte precursor cells (obtainable from haematopoietic stem cells) into the patient. Without wishing to be bound by theory, it is believed that said cells naturally differentiate into granulocytes (e.g. neutrophils) having a high CKA in a CKA assay in vivo; or direct IV infusion of granulocytes (e.g. neutrophils) having a high CKA in a CKA assay which have been differentiated from haematopoietic cells (e.g. haematopoietic stem cells).

(24) Typically, cells are infused once weekly for 8 weeks with a cell volume of 2×10.sup.11 administered per week. Progress of the therapy is monitored and dosing is adapted accordingly.

Example 8

(25) Treatment of a Patient with Pancreatic Cancer

(26) Mary is diagnosed with metastatic pancreatic ductal adenocarcinoma (PDAC) at age 69. Surgery is no longer an option (un-resectable), gemcitabine inadequate in preventing disease progression, and Abraxane or Folfirinox unsuitable on her Oncologist's recommendation due to the side-effects that will render her incapable of enjoying the time she has left with her family. Mary's prognosis is 3-6 months to live, and she is desperate to live long enough to see her newly-expected grandchild.

(27) Mary is invited to try Leukocyte Infusion Therapy (LIFT). To assess the potential suitability of the therapy, the hospital extracts 20 ml of blood from Mary and sends it for analysis using the Cancer Killing Activity Assay, the assay identifies that the pancreatic cancer killing activity of her granulocytes is less than 5%. Such a low reading demonstrates the inadequacy of her own innate immune system to fight off her cancer which will kill her if the efficacy of the granulocytes in her body is not improved.

(28) Mary's patient notes and assay result are used to find a suitable cancer killing granulocyte match. Mary is blood group A. Mary's profile is processed using a cell database for a cell bank and suitable granulocytes (that prior to cryogenic freezing exhibit a 70-90% Cancer Killing Activity (CKA) in the Cancer Killing Activity assay of Example 2) are identified. Cryogenically freezing granulocytes helps preserve the CKA and so the cells are able to be dispatched directly to the hospital (The Royal Marsden) with no further testing. Mary is due to visit that week for her first treatment. The hospital appropriately stores the cells. Mary receives her first infusion of 2×10.sup.9 granulocytes having CKA on the 13.sup.th December under close supervision. Mary is invited back to the hospital 3 days later where she is given an ultrasound scan which reveals significant tumour lysis and no signs of tumour lysis syndrome. The medical team decide to increase the granulocyte dose incrementally over 3 successive treatment sessions until it reaches 2×10.sup.11.

(29) An ultrasound is carried out on the 17.sup.th January; one week after the 4-week course of four treatments is completed, and shows complete tumour destruction and conversion into scar tissue with good healing taking place. 20 ml of Mary's blood is taken: i) to assess the presence of metastatic cancer cells in her blood (to confirm complete clearance of the cancer) alongside a biopsy; and ii) to test the Cancer Killing Activity of Mary's granulocytes (to indicate risk of remission). Mary receives regular check-ups first monthly and then 6-monthly.

(30) Two years later a new tumour is discovered on Mary's pancreas. Her clinicians treat the tumour with radiotherapy and administer a single high dose of LIFT to ensure destruction of any cancer cells that may be present in the blood. Mary enjoys the life that is given back to her and the family she gets to see grow up.

(31) After the therapy, the haematopoietic cells (e.g. haematopoietic stem cells) from the cell bank are stimulated to produce more granulocytes (having desired CKA as tested using the assay of Example 2) to replenish stocks. Thus sufficient stock of the required granulocytes for similar patient situations is ensured.

Example 9

(32) MTT “CKA Assay”

(33) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a yellow tetrazole which is positively charged and readily penetrates viable eukaryotic cells. Viable cells with active metabolism convert MTT into a purple coloured formazan product (1(E,Z)-5-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan) through NAD(P)H-dependent oxidoreductase mitochondrial enzymes, with an absorbance maximum near 570 nm. When cells die, they lose the ability to convert MTT into formazan, thus colour formation serves as a useful and convenient marker of only the viable cells. A solubilization solution is added to dissolve the insoluble purple formazan product into a coloured solution. The absorbance of this coloured solution can be quantified by measuring at wavelength 570 nm by a spectrophotometer. The absorption of a reference wavelength of 690 nm is subtracted from the absorption of the 570 nm wavelength. Therefore, the MTT assay was used to measure how many live cells remained as a way to determine Cancer Killing Actvity (CKA)—cytotoxicity to cancer cells.

(34) Method for Preparing Hela Target Cells

(35) Day 1: 1) HeLa cells (a robust type of cervical cancer cell) were cultured and harvested when they reached log phase 2) 10000 HeLa cells (target cells) were added to each well of a 96 Flat bottom plate at a final volume of 100 uL 3) Target cells were left to adhere overnight before leukocyte (effector cells) addition and all experimental conditions were set in triplicates

(36) Day 2: 4) Effector cells were added to the target cells at different ratios (e.g. 1:1, 5:1, 10:1, 50:1, effector to target cells) 5) The cells were left to incubate for 16-24 hours at 37° C. 6) Target cells alone and target cells in the presence of Triton X were also plated in triplicates as controls for 0% and 100% cytotoxicity, respectively.

(37) After the desired incubation time: 1) The wells were washed with PBS twice to remove effector cells and dead target cells 2) MTT solution was prepared by dissolving the kit solution 10 times with culture medium, i.e: for 100 wells: take 1000 uL (1 mL) of MTT stock (provided in the kit) and 9000 uL(9 mLs) of culture medium (RPMI-1640) added 3) Add 100 ul/well of the prepared MTT solution at step 2 4) This was incubated for 4 h 5) MTT Solution was removed from all wells and 100 ul/well of the solvent was added (provided in the kit). 6) The formazan crystals were solubized when needed by pipetting and the plate read at 570 and 690 nm. Background absorbance measured at 690 nm was substracted from absorbance measured at 570 nm.

(38) Demonstrating Variable CKA in Donor Neutrophils

(39) Leukocyte cones from anonymous blood donors were selected and neutrophils were isolated by Ficoll-Hypaque separation (Oh H, Siano B, Diamond S. Neutrophil Isolation Protocol. Journal of Visualized Experiments: JoVE. 2008; (17):745). These neutrophils were used in the aforementioned MTT assay in ratios of effector to target cell of 1:1, and 5:1.

(40) FIG. 1 shows the percentage cytotoxicity recorded by MTT for the different donors. There is a difference between the donors at ratios 1:1 and 5:1. In conclusion, the MTT assay, is able to demonstrate differences in CKA between neutrophils from different donors.

Example 10

(41) Demonstrating CKA of Stem Cell Derived Neutrophils

(42) Culturing Neutrophils from CD34+ Stem Cells

(43) We cultured neutrophils from umbilical cord blood derived stem cells expressing the CD34 protein, using the protocol as described by Timmins N E, Palfreyman E, Marturana F, Dietmair S, Luikenga S, Lopez G, et al. Clinical scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells. Biotechnology and bioengineering. 2009; 104(4):832-40.

(44) The resulting cultures were tested for neutrophil content using CD11b+ and CD15 markers by Fluorescence-activated cell sorting (FACS). We also measured the production of Reactive Oxygen Specimens (ROS), more specifically the production of superoxide anion (O.sub.2−) by use of the nitroblue tetrazolium (NBT) assay (kit and protocol commercially-available from Sigma-Aldrich, Catalogue No. 840W-1KT).

(45) Since differences in ROS activity were found based on the age of the stem cell derived neutrophils (data not shown), we enumerated three stem cell batches on the same day for consistency/comparability. The results of the FACS derived counting of the proportion of CD11b+ and CD15+ cells are listed in Table 1.

(46) TABLE-US-00001 TABLE 1 Percentage of CD11b+/CD15+ positive neutrophils CD11b+ CD15+ CD34+ FACS on 26.09.2017 (% of all cells) (% of all cells) CD34+ 14.09.2017 batch 008A 77.8 4.45 (12 days in culture) CD34+ 14.09.2017 batch 709A 76.0 5.1 (12 days in culture) CD34+ 14.09.2017 915 (12 days in 69.2 10.0 culture)

(47) Demonstrating CKA of CD34+ Derived Neutrophils

(48) Stem cell derived neutrophils (batches 008A, 709A and 915) were used as effector cells in the CKA MTT assay using HeLa target cells (see Example 9). The effector to target cell ratio is based on CD11b+/CD15+. The results are summarized in FIG. 2, which demonstrate that CD34+ stem cell derived neutrophils demonstrate cytotoxicity in a HeLa cell CKA assay and that the results are different between different donors. Batch 008A shows consistently lower cytotoxicity than batches 915 and 709A up to 10:1 effector to target ratio, despite being prepared at the same time as the other batches and cultured in the same way.

(49) The results demonstrate that stem cells from different donors can a) be differentiated in vitro to produce neutrophils that demonstrate cancer killing abilities, and b) that this cancer killing activity varies by the source donor.

(50) The results support the fact that the cancer killing activity (CKA) by the innate immune system varies by individual and that the same innate variance in CKA seen in neutrophils taken directly from donors via leukocyte cones, is also shown in a donor's stem cells. By selecting donors with proven high cancer killing activity of their innate immune system, and using their haematopoietic cells (i.e. haematopoietic stem cells) for ex vivo expansion and differentiation, a cell bank can be created with leukocytes with high cancer killing activity to be used in the treatment of cancer.

Example 11

(51) Isolation of High-Density Neutrophils

(52) 10 ml of heparinized (20 U/ml) human blood is mixed with an equal volume of 3% Dextran T500 in saline and incubated for 30 minutes at room temperature to sediment erythrocytes. A 50 ml conical polypropylene tube is prepared with 10 ml sucrose 1.077 g/ml and slowly layered with a leukocyte-rich supernatant on top of the 1.077 g/ml sucrose layer prior to centrifuging at 400×g for 30 minutes at room temperature without brake. The high-density neutrophils (HDN) appear in the pellet. Low-density neutrophils (LDN) co-purify with monocytes and lymphocytes at the interface between the 1.077 g/ml sucrose layer and plasma.

(53) The HDNs may be tested in a CKA assay described herein. Haematopoietic cells are suitably obtained from a donor having HDNs.

(54) All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.