Cancer-killing cells

Abstract

The present invention relates to in vitro cultures of hematopoietic cells that differentiate to form granulocytes characterized by the ability to kill cancer cells. The invention also relates to said granulocytes, methods for identifying said hematopoietic cells and granulocytes, compositions and kits containing same, and their use for treating cancer.

Description

[Technical field]

[0001] The present invention relates to cell-based therapies suitable for treating cancer. [Background technology]

[0002] Cancer is the leading cause of morbidity and mortality worldwide, and the incidence of cancer in developed countries is increasing year by year. The World Health Organization stated that in 2012 alone, there were approximately 14 million new cancer cases (with 8.2 million associated deaths), estimated to rise to 22 million cases over the next 20 years. Current treatment strategies include a combination of surgery, radiation, and cytotoxic chemotherapy, but many of these treatments are ultimately ineffective and associated with adverse side effects.

[0003] The safety and efficacy of hematopoietic stem cell transplantation (HSCT) as a therapeutic technique for treating certain cancers, such as renal cell carcinoma, is being evaluated. However, this treatment is still viewed as mainly experimental due to potentially fatal safety issues, and recipients exhibit graft-versus-host disease (GVHD) as a result of uncontrolled replication of pluripotent stem cells. Thus, improved and alternative cancer therapies are needed.

[0004] Despite the increasing incidence of cancer, it has been observed that approximately 50-60% of individuals will never develop cancer in their lifetime. In fact, in rare cases, some individuals will exhibit spontaneous cancer regression. This observation has led to the study of leukocytes derived from spontaneously regressing individuals and their use in Leukocyte Infusion Therapy (LIFT).

[0005] Traditional LIFT is performed using apheresis for the direct transfer of granulocytes (e.g., neutrophils) harvested from a donor into a cancer patient. Current techniques are not practical or scalable for use as a reliable cancer treatment. First, granulocytes, such as neutrophils, have a very limited shelf life (typically less than 24 hours), making their preservation difficult. Second, apheresis requires approximately five donors (which are very rare) to acquire the required cell volume. Third, to avoid immune responses derived from repeated exposure, the same donor cannot be used in subsequent administrations, thus necessitating an increased pool of suitable donors. Fourth, donors cannot be realistically expected to be available on demand or willing to provide an unending source of granulocytes for the LIFT procedure. Fifth, the cancer-killing efficacy of donor-derived granulocytes varies over time, resulting in inconsistent treatment outcomes. Summary of the Invention [Problem to be solved by the invention]

[0006] As yet, no viable alternative to conventional LIFT has been offered, nor have any solutions to the associated problems been provided, making conventional LIFT not viable as a scalable, safe, and reliable treatment technique.

[0007] The present invention provides a solution to at least one of the above problems. [Means for solving the problem]

[0008] The inventors have surprisingly found that it is possible to select hematopoietic cells capable of differentiating into granulocytes capable of killing cancer cells. Once such hematopoietic cells are selected from a donor, they can be stored for later therapeutic purposes or used directly as a drug, for example, in the treatment of cancer. Advantageously, hematopoietic cells obtainable by the method of the present invention can be immortalized, thus providing stable cell lines that can be stored and / or propagated indefinitely. Thus, the present invention reduces the need for multiple rare donors and / or direct transfer of donor-collected granulocytes into cancer patients. Thus, the present invention provides a viable, scalable, safe, and / or reliable therapy.

[0009] For the first time, the inventors have shown that the cancer-killing potency of granulocytes (e.g., neutrophils) is genetically, rather than epigenetically, defined. This is shown in Example 19, which shows that granulocytes derived from hematopoietic cells (e.g., hematopoietic stem cells) isolated from a donor have similar cancer-killing potency as mature granulocytes isolated directly from the same donor.

[0010] Advantageously, donors found to have granulocytes that exhibit high cancer-killing activity can be used as a source of hematopoietic cells (e.g., hematopoietic stem cells) that can differentiate into granulocytes that also exhibit high cancer-killing activity.

[0011] Advantageously, such hematopoietic cells can be stored and used to produce large quantities of granulocytes for use in the treatment of cancer, thus overcoming the problem of isolating sufficient quantities of fresh granulocytes from a donor.

[0012] Furthermore, hematopoietic cell-derived granulocytes have been found to kill cancer cells more rapidly than donor-isolated granulocytes, and hematopoietic cell-derived granulocytes may have better cancer-killing efficacy (e.g., against pancreatic cancer cells) than fresh donor-derived granulocytes.

[0013] Pancreatic cancer is known to be one of the most difficult cancers to treat. However, surprisingly, the present inventors have succeeded in isolating hematopoietic cells that differentiate into granulocytes and have specific efficacy against pancreatic cells.

[0014] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a. a surface potential defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec (or at least 1.0 μm.cm / volt.sec, e.g., at least 1.25 μm.cm / volt.sec, 1.5 μm.cm / volt.sec, or 1.75 μm.cm / volt.sec); and b. Ability to kill cancer cells The present invention provides an in vitro cell culture of hematopoietic cells which differentiate to form granulocytes, characterized by:

[0015] In a related aspect, the invention provides a method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. Measuring the surface potential of granulocytes obtainable from a donor; and b. selecting hematopoietic cells from said donor if the measured surface potential is defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec (or at least 1.0 μm.cm / volt.sec, e.g., at least 1.25 μm.cm / volt.sec, 1.5 μm.cm / volt.sec, or 1.75 μm.cm / volt.sec). The present invention provides a method comprising:

[0016] As used herein, the term "obtainable" also encompasses the term "obtained."

[0017] The present invention provides a method for selecting hematopoietic cells, comprising measuring the surface potential of the hematopoietic cells; and selecting hematopoietic cells that can be differentiated into granulocytes that are suitable for treating cancer. Thus, in one embodiment, a method for selecting hematopoietic cells suitable for use in the treatment of cancer is provided, comprising: a. Measuring the surface potential of hematopoietic cells; and b. selecting hematopoietic cells that have a surface potential defined by an electrophoretic mobility of less than 2.0 μm.cm / volt.sec (or less than 1.0 μm.cm / volt.sec) and / or have a higher (e.g., more positive) surface potential than otherwise identical hematopoietic cells that differentiate to form granulocytes with reduced ability to kill cancer cells; A method is provided that includes:

[0018] A related embodiment provides the use of the surface potential of hematopoietic cells to select cells that can be differentiated into granulocytes suitable for treating cancer, the surface potential being higher (e.g., more positive) than the surface potential of otherwise identical hematopoietic cells that differentiate to form granulocytes that have a surface potential defined by an electrophoretic mobility of less than 2.0 μm.cm / volt.sec (or less than 1.0 μm.cm / volt.sec) and / or have a reduced ability to kill cancer cells.

[0019] The present invention provides an in vitro method for selecting a subject for treatment (e.g., a subject who would benefit from an agent described herein), comprising: a. mixing granulocytes from said subject with a cancer cell line; b. incubating the mixture; c. determining the percentage of cancer cells killed in said mixture; and d. If granulocytes from the subject kill less than 5% of the cancer cells in the mixture (suitably, if granulocytes from the subject kill less than 60%, preferably less than 80% or 90% of the cancer cells in the mixture), selecting the subject for treatment with an in vitro cell culture of hematopoietic cells, or granulocytes, or an in vitro cell culture of granulocytes, or a pharmaceutical composition of the invention. The present invention provides a method comprising:

[0020] In some embodiments, if the granulocytes from the subject kill less than 20% or 10% of the cancer cells in the mixture, the subject is selected for treatment with the in vitro cell culture of hematopoietic cells, or the granulocytes, or the in vitro cell culture of granulocytes, or the pharmaceutical composition of the present invention.Preferably, if the granulocytes from the subject kill less than 5% or 1% of the cancer cells in the mixture, the subject is selected for treatment with the in vitro cell culture of hematopoietic cells, or the granulocytes, or the in vitro cell culture of granulocytes, or the pharmaceutical composition of the present invention.

[0021] In vitro methods can also be used to monitor the ability of a subject's granulocytes to kill cancer cells.

[0022] In one aspect, the present invention provides an in vitro method for obtaining hematopoietic cells suitable for use in the treatment of cancer, comprising: a. contacting a cancer cell line with granulocytes obtainable from a donor to form a test sample, and incubating said test sample; and b. Obtaining hematopoietic cells from a sample derived from a donor if the percentage of cancer cells killed in the test sample is higher than the percentage of cancer cells killed in a control sample containing the same type of cancer cell line and granulocytes obtainable from a different donor. The present invention provides a method comprising:

[0023] The percentage of cancer cells killed in the control sample can be determined prior to performing the method of the invention or simultaneously (preferably simultaneously) with performing the method of the invention.

[0024] For example, in one embodiment, the method comprises: a. contacting a cancer cell line with granulocytes obtainable from a first donor to form a test sample; b. contacting the same type of cancer cell line with obtainable granulocytes from a different donor (e.g., control granulocytes) to form a control sample; c. incubating said sample; and d. Obtaining hematopoietic cells from a sample derived from said first donor if the % of cancer cells killed in the test sample is higher than the % of cancer cells killed in the control sample. Includes.

[0025] In some embodiments, the method may involve the use of multiple different test samples that include granulocytes from additional donors (eg, a second, third, fourth donor, etc.).

[0026] The referenced control sample may be a sample from a donor having granulocytes that do not kill cancer cells (e.g., granulocytes that do not kill at least 5% of cancer cells in the methods described herein). In other embodiments, the referenced control sample may be a sample from a donor having granulocytes that kill cancer cells (e.g., granulocytes that kill at least 5% of cancer cells in the methods described herein), in which case the method can be used to detect donors having granulocytes that exhibit optimal cancer-killing activity. In one embodiment, the control sample comprises granulocytes that kill up to 50%, 40%, 30%, 20% or 10% of cancer cells in the methods described herein. Preferably, the control sample comprises granulocytes that kill up to 5% of cancer cells in the methods described herein.

[0027] Preferably, hematopoietic cells are obtained when the percentage of cancer cells killed in the test sample is at least 5% higher than the percentage of cancer cells killed in the control sample. In some embodiments, the percentage of cancer cells killed in the test sample is at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% higher than the percentage of cancer cells killed in the control sample. More preferably, the percentage of cancer cells killed in the test sample is at least 35% higher than the percentage of cancer cells killed in the control sample.

[0028] In one embodiment, the method comprises using a 5:1 ratio of granulocytes to cancer cells and hematopoietic cells are obtained when the % of cancer cells killed in the test sample is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% higher than the % of cancer cells killed in the control sample. More preferably, the method comprises using a 5:1 ratio of granulocytes to cancer cells and hematopoietic cells are obtained when the % of cancer cells killed in the test sample is at least 30% higher than the % of cancer cells killed in the control sample.

[0029] In one embodiment, the method comprises using a 10:1 ratio of granulocytes to cancer cells and hematopoietic cells are obtained when the % of cancer cells killed in the test sample is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% higher than the % of cancer cells killed in the control sample. More preferably, the method comprises using a 10:1 ratio of granulocytes to cancer cells and hematopoietic cells are obtained when the % of cancer cells killed in the test sample is at least 20% higher than the % of cancer cells killed in the control sample.

[0030] In one aspect, the present invention provides an in vitro method for obtaining hematopoietic cells suitable for use in the treatment of cancer, comprising: a. mixing granulocytes obtainable from a donor with a cancer cell line to form a mixture; b. incubating the mixture; c. determining the percent of cancer cells killed in said test sample; and d. Obtaining hematopoietic cells from the donor-derived sample if the granulocytes kill at least 5% of the cancer cells in the test sample. The present invention provides a method comprising:

[0031] If the granulocytes kill at least 10%, 20%, 30%, 40% or 50% of the cancer cells in the mixture, hematopoietic cells can be obtained from the donor-derived sample.

[0032] In one embodiment, hematopoietic cells are obtained from said donor-derived sample if the granulocytes kill at least 60% of the cancer cells in the mixture.

[0033] In one embodiment, hematopoietic cells are obtained from said donor-derived sample if the granulocytes kill at least 70% of the cancer cells in the mixture.

[0034] Preferably, hematopoietic cells are obtained from said donor-derived sample if the granulocytes kill at least 80% or 90% of the cancer cells in the mixture.

[0035] In one embodiment, the method comprises using a 5:1 ratio of granulocytes to cancer cells and hematopoietic cells are obtained from said donor-derived sample when the granulocytes kill at least 30% of the cancer cells in the mixture.Preferably, the method comprises using a 5:1 ratio of granulocytes to cancer cells and hematopoietic cells are obtained from said donor-derived sample when the granulocytes kill at least 40% of the cancer cells in the mixture.

[0036] In one embodiment, the method comprises using a 10:1 ratio of granulocytes to cancer cells and hematopoietic cells are obtained from the donor-derived sample when the granulocytes kill at least 45%, 50% or 60% (preferably at least 60%) of the cancer cells in the mixture. More preferably, the method comprises using a 10:1 ratio of granulocytes to cancer cells and hematopoietic cells are obtained from the donor-derived sample when the granulocytes kill at least 80% of the cancer cells in the mixture.

[0037] In some embodiments, the cancer cell line is a cervical cancer cell line (e.g., HeLa).

[0038] In one embodiment, the method comprises using a 5:1 ratio of granulocytes to cervical cancer cells and hematopoietic cells are obtained from said donor-derived sample when the granulocytes kill at least 30% of the cervical cancer cells in the mixture.Preferably, the method comprises using a 5:1 ratio of granulocytes to cervical cancer cells and hematopoietic cells are obtained from said donor-derived sample when the granulocytes kill at least 40% of the cervical cancer cells in the mixture.

[0039] In one embodiment, the method comprises using a ratio of granulocytes to cervical cancer cells of 10:1, and hematopoietic cells are obtained from said donor-derived sample when the granulocytes kill at least 45%, 50% or 60% (preferably at least 60%) of the cervical cancer cells in the mixture. More preferably, the method comprises using a ratio of granulocytes to cervical cancer cells of 10:1, and hematopoietic cells are obtained from said donor-derived sample when the granulocytes kill at least 80% of the cervical cancer cells in the mixture.

[0040] In some embodiments, the cancer cell line is a pancreatic cancer cell line (e.g., PANC-1).

[0041] In one embodiment, the method comprises using a 5:1 ratio of granulocytes to pancreatic cancer cells and hematopoietic cells are obtained from the donor-derived sample when the granulocytes kill at least 50% or 60% of the pancreatic cancer cells in the mixture.Preferably, the method comprises using a 5:1 ratio of granulocytes to pancreatic cancer cells and hematopoietic cells are obtained from the donor-derived sample when the granulocytes kill at least 65% or 70% of the pancreatic cancer cells in the mixture.

[0042] In one embodiment, the method comprises using a ratio of granulocytes to pancreatic cancer cells of 10:1 and hematopoietic cells are obtained from the donor-derived sample when the granulocytes kill at least 70% of the pancreatic cancer cells in the mixture.Preferably, the method comprises using a ratio of granulocytes to pancreatic cancer cells of 10:1 and hematopoietic cells are obtained from the donor-derived sample when the granulocytes kill at least 80% or 90% of the pancreatic cancer cells in the mixture.

[0043] In one embodiment, an in vitro method for selecting granulocytes that selectively kill cancer cells is provided, comprising: a. contacting a cancer cell line with granulocytes obtainable from a donor to form a test sample, and incubating said test sample; and b. selecting said granulocytes as selective for cancer cells if the % of cancer cells killed in the test sample is higher than the % of non-cancerous cells killed in a control sample containing a non-cancerous cell line and granulocytes obtainable from the same donor. A method is provided that includes:

[0044] The method preferably comprises the further step of obtaining hematopoietic cells from a sample derived from said donor if granulocytes are selected.

[0045] The percentage of non-cancerous cells killed in the control sample can be determined prior to performing the method of the invention or simultaneously (preferably simultaneously) with performing the method of the invention.

[0046] For example, in one embodiment, the method comprises: a. contacting a cancer cell line with granulocytes obtainable from a donor to form a test sample; b. contacting the non-cancerous cell line with obtainable granulocytes from the same donor (e.g., control granulocytes) to form a control sample; c. incubating said sample; and d. selecting said granulocytes as selective for cancer cells if the % of cancer cells killed in the test sample is higher than the % of non-cancerous cells killed in the control sample. Includes.

[0047] In some embodiments, the method may involve the use of multiple different test samples that include granulocytes from additional donors (eg, a second, third, fourth donor, etc.).

[0048] In one embodiment, granulocytes are considered selective for cancer cells if the % of cancer cells killed in the test sample is at least 2%, 5%, 10%, 15%, 20%, 30%, 40% or 50% higher than the % of non-cancerous cells in the test sample.

[0049] In one embodiment, the method involves using a 5:1 ratio of granulocytes to cancer cells, and hematopoietic cells are obtained when the % of cancer cells killed in the test sample is at least 10%, 20% or 30% higher than the % of cancer cells killed in the control sample.

[0050] In one embodiment, the method involves using a 10:1 ratio of granulocytes to cancer cells, and hematopoietic cells are obtained when the % of cancer cells killed in the test sample is at least 10% or 20% higher than the % of cancer cells killed in the control sample.

[0051] Preferably, the granulocytes kill less than 35%, 25%, 15%, 10%, 5% or 1% of non-cancerous cells in the methods described herein.

[0052] Any non-cancerous cell line can be used in the method. In one embodiment, the non-cancerous cell line is an epithelial cell, such as a breast epithelial cell. Preferably, the non-cancerous cell line is the MCF-12F non-cancerous cell line (commercially available as ATCC® CRL-10783™ from American Type Culture Collection, 10801 University Boulevard. Manassas, VA 20110 USA).

[0053] In one embodiment, a method for selecting granulocytes that selectively kill cancer cells is provided, comprising: a. contacting a cancer cell line with granulocytes obtainable from a donor to form a test sample, and incubating said test sample; b. The granulocytes. i. the % of cancer cells killed in the test sample is higher than the % of cancer cells killed in a first control sample containing the same type of cancer cell line and granulocytes obtainable from a different donor; and ii. The % of cancer cells killed in the test sample is higher than the % of non-cancerous cells killed in a second control sample comprising a non-cancerous cell line and granulocytes obtainable from the same donor (as in step a.) (e.g., control granulocytes). To choose A method is provided that includes:

[0054] In some embodiments, the method may include comparing the % of non-cancerous cells killed by (cultures of) two or more granulocytes derived from different donors, thus allowing selection of the granulocytes (and donor) that exhibit the lowest % killing of non-cancerous cells.

[0055] In one aspect, the present invention provides an in vitro method for selecting suitable granulocytes for use in the treatment of pancreatic cancer, comprising: a. mixing granulocytes with a pancreatic cancer cell line to form a mixture; b. incubating the mixture; c. determining the percentage of pancreatic cancer cells killed in said mixture; and D. Selecting granulocytes that kill at least 5% of pancreatic cancer cells in the mixture. The present invention provides a method comprising:

[0056] The pancreatic cancer cell line may be a pancreatic ductal adenocarcinoma cell line.

[0057] The granulocytes may kill at least 10%, 20%, 30%, 40% or 50% of the pancreatic cancer cells in the mixture.

[0058] In one embodiment, the granulocytes kill at least 60% of the pancreatic cancer cells in the mixture.

[0059] In one embodiment, the granulocytes kill at least 70% of the pancreatic cancer cells in the mixture.

[0060] Preferably, the granulocytes kill at least 80% or 90% of the pancreatic cancer cells in the mixture.

[0061] In one embodiment, the method includes using a 5:1 ratio of granulocytes to pancreatic cancer cells, where the granulocytes kill at least 50% or 60% of the pancreatic cancer cells in the mixture.Preferably, the method includes using a 5:1 ratio of granulocytes to pancreatic cancer cells, where the granulocytes kill at least 65% or 70% of the pancreatic cancer cells in the mixture.

[0062] In one embodiment, the method includes using a ratio of granulocytes to pancreatic cancer cells of 10:1, where the granulocytes kill at least 70% of the pancreatic cancer cells in the mixture.Preferably, the method includes using a ratio of granulocytes to pancreatic cancer cells of 10:1, where the granulocytes kill at least 80% or 90% of the pancreatic cancer cells in the mixture.

[0063] In some embodiments, the cancer cell line is a pancreatic cancer cell line (e.g., PANC-1).

[0064] A related aspect is an in vitro method for selecting suitable granulocytes for use in the treatment of cancer, comprising: a. mixing granulocytes with a plurality of different cancer cell lines to provide a plurality of mixtures; b. incubating the mixture; c. determining the percentage of cancer cells killed in said mixture; and d. If the granulocytes kill at least 5% of the cancer cells in the mixture, selecting said granulocytes as suitable for use in treating a cancer of the same type / subset as the cancer cell line. The present invention provides a method comprising:

[0065] Advantageously, such methods allow for rapid screening of granulocytes for their ability to kill multiple cancer types / subsets. In some embodiments, the granulocytes are then classified according to the cancer type / subset for which they are suitable for use in treating.

[0066] The term "type" as used in this context refers to cancer of the same organ or tissue as the cancer cell line. For example, if the cancer cell line is a pancreatic ductal adenocarcinoma cell line, the granulocytes that kill at least 5% of the pancreatic ductal adenocarcinoma cells in the mixture are considered suitable for use in treating all pancreatic cancers.

[0067] The term "subset" as used in this context means not only that the cancers are of the same organ or tissue, but also that the cancers share additional characteristics with the cancer cell line (e.g., both are carcinomas, sarcomas, etc. of the same organ or tissue). For example, if the cancer cell line is a pancreatic ductal adenocarcinoma cell line, granulocytes that kill at least 70% of the pancreatic ductal adenocarcinoma cells in the mixture would be suitable for use in treating all pancreatic ductal adenocarcinoma variants.

[0068] The in vitro method may further comprise measuring and / or selecting granulocytes based on surface potential (e.g., cell surface charge) as disclosed herein. The in vitro method may further comprise measuring and / or selecting granulocytes based on cell density (e.g., at least 1.077 g / ml) as disclosed herein. The in vitro method may further comprise measuring and / or selecting granulocytes based on the expression or activity of toll-like receptors; and / or the absence or inactivity of expression of programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2 on granulocytes.

[0069] The in vitro method according to said embodiment may be representative of a cancer killing activity (CKA) assay (eg claim 6).

[0070] As used herein, the term "mixing" refers to mixing one or more components together in any order, whether sequentially or simultaneously. In one embodiment, "mixing" refers to contacting a first component with a second component (e.g., granulocytes with a cancer cell line).

[0071] The term "plurality" means at least 2. In one embodiment, "plurality" means at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. "Plurality" may mean at least 30, 40, 50, 60, 70, 80, 90, or 100. In one embodiment, "plurality" means 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another embodiment, "plurality" means 30, 40, 50, 60, 70, 80, 90, or 100.

[0072] In one embodiment, granulocytes can be obtained from a donor, for example a human donor. Alternatively, or in addition, granulocytes can be obtained from a subject with a different type / subset of cancer than the cancer cell line used in the method of the present invention. Advantageously, the inventors have found that a subject with a certain type / subset of cancer may have granulocytes capable of killing cancer cells of a different cancer type / subset. This is particularly surprising when the subject has a particularly low concentration of granulocytes capable of killing cells of the cancer that the subject has been diagnosed with.

[0073] Cancer cell lines for use in the methods of the present invention may be one or more selected from pancreatic cancer cell lines, liver cancer cell lines, esophageal cancer cell lines, gastric cancer cell lines, cervical cancer cell lines, ovarian cancer cell lines, lung cancer cell lines, bladder cancer cell lines, kidney cancer cell lines, brain tumor cell lines, prostate cancer cell lines, myeloma cancer cell lines, non-Hodgkin's lymphoma (NHL) cell lines, laryngeal cancer cell lines, uterine cancer cell lines, or breast cancer cell lines.

[0074] Suitable cell lines are commercially available from the American Type Culture Collection United Kingdom (UK), Guernsey, Ireland, Jersey and Liechtenstein, LGC Standards, Queens Road, Teddington, Middlesex, TW11 0LY, UK. For example, pancreatic cell lines include 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™.

[0075] Preferably, the cancer cell line is a pancreatic cancer cell line, such as PANC-1.

[0076] In one embodiment, the cancer cell line is a cervical cancer cell line, such as a HeLa cell.

[0077] The incubation step may be carried out for 1 hour to 100 hours. Preferably, the incubation step may be carried out for 5 hours to 75 hours, for example, 10 hours to 20 hours.

[0078] The incubation step may be carried out for 6 hours to 6 days. Preferably, the incubation step may be carried out for 6 hours to 2 days, for example, for 12 hours to 36 hours, for example, for 16 to 24 hours. In one embodiment, the incubation step is carried out for 24 hours. The incubation step may be carried out at any temperature suitable for cell growth and survival, for example, at a temperature of 35°C to 42°C, preferably at a temperature of 37°C to 39°C. Preferably, the incubation step is carried out at 37 or 39°C for 24 hours. Preferably, the incubation step is carried out at 30 to 40°C (for example, 37°C) for 16 to 24 hours.

[0079] The percentage of killed cancer cells can be measured by referring to the total number of starting cancer cells. The number of killed cancer cells can be measured using any suitable means, for example, by viability staining (e.g., trypan blue staining) and microscopic observation, or by other automated means, for example, by a cell electrical detection instrument, such as the RT-CES™ system available from ACEA Biosciences, Inc. (11585 Sorrento Valley Rd., Suite 103, San Diego, CA 92121, USA). In some embodiments, the percentage of killed cancer cells can be determined within 24 hours (e.g., of incubation of cancer cell lines and granulocytes). The percentage of killed cancer cells is preferably the maximum number of killed cancer cells when carrying out the method of the present invention.

[0080] The number of killed cancer cells can also be measured using the ACEA Biosciences xCELLigence RTCA DP Analyzer system. The xCELLigence System is a real-time cell analyzer that can continuously and dynamically monitor changes in cell phenotype without the use of labels by measuring electrical impedance. Such measurements can be performed as detailed in Example 11. The system is commercially available from ACEA Biosciences 6779 Mesa Ridge Road #100, San Diego, CA 92121 USA.

[0081] The method of the invention may involve the use of a ratio of granulocytes to cancer cells of at least 1:1, 5:1 or 10:1. Preferably, the method involves the use of a ratio of granulocytes to cancer cells of 5:1. More preferably, the method involves the use of a ratio of granulocytes to cancer cells of 10:1.

[0082] Those skilled in the art will appreciate that when the method of the present invention includes a comparison step between two samples (e.g., between a "test sample" and a "control sample"), the conditions (e.g., assay conditions during the method) should be kept constant. For example, the concentration ratio of granulocytes to cancer cells should be the same, the time conditions, etc. should also be the same. When a comparison is made between two samples herein, preferably the samples are equivalent. For example, the samples being compared may be of the same sample type (e.g., blood) and subjected to the same processing steps. In some embodiments, the only difference between the samples is the donor from which the samples are obtained. For example, in embodiments in which the percentage of cells having a particular characteristic is determined, the total number of cells in each sample may be the same and a suitable comparison can be made.

[0083] In one embodiment, the mixture is 8x10 5 granulocytes and 1.5x10 4 cancer cells, or preferably, 8x10 5 granulocytes and 8x10 4 Contains cancer cells.

[0084] In one embodiment, the granulocytes are capable of killing at least 5% of the cancer cells in the methods described herein. The granulocytes are capable of killing at least 10%, 20%, 30%, 40% or 50% of the cancer cells in the methods described herein. In one embodiment, the granulocytes are capable of killing at least 60% of the cancer cells in the methods described herein. In one embodiment, the granulocytes are capable of killing at least 70% of the cancer cells in the methods described herein. Preferably, the granulocytes are capable of killing at least 80% or 90% of the cancer cells in the methods described herein.

[0085] In embodiments where the method includes using a 5:1 ratio of granulocytes to cancer cells, the granulocytes can kill at least 30% of the cancer cells in the methods described herein. Preferably, when the method includes using a 5:1 ratio of granulocytes to cancer cells, the granulocytes can kill at least 40% of the cancer cells in the methods described herein. In embodiments where the method includes using a 10:1 ratio of granulocytes to cancer cells, the granulocytes can kill at least 45%, 50% or 60% (preferably at least 60%) of the cancer cells in the methods described herein. More preferably, when the method includes using a 10:1 ratio of granulocytes to cancer cells, the granulocytes can kill at least 80% of the cancer cells in the methods described herein.

[0086] In some embodiments, the cancer cell line is a cervical cancer cell line (e.g., HeLa). In embodiments where the method includes using a 5:1 ratio of granulocytes to cervical cancer cells, the granulocytes can kill at least 30% of cervical cancer cells in the methods described herein. Preferably, when the method includes using a 5:1 ratio of granulocytes to cervical cancer cells, the granulocytes can kill at least 40% of cervical cancer cells in the methods described herein. In embodiments where the method includes using a 10:1 ratio of granulocytes to cervical cancer cells, the granulocytes can kill at least 45%, 50% or 60% (preferably at least 60%) of cervical cancer cells in the methods described herein. More preferably, when the method includes using a 10:1 ratio of granulocytes to cervical cancer cells, the granulocytes can kill at least 80% of cervical cancer cells in the methods described herein.

[0087] In some embodiments, the cancer cell line is a pancreatic cancer cell line (e.g., PANC-1). In embodiments where the method includes using a 5:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can kill at least 50% or 60% of the pancreatic cancer cells in the methods described herein. Preferably, when the method includes using a 5:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can kill at least 65% or 70% of the pancreatic cancer cells in the methods described herein. In embodiments where the method includes using a 10:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can kill at least 70% of the pancreatic cancer cells in the methods described herein. Preferably, when the method includes using a 10:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can kill at least 80% or 90% of the pancreatic cancer cells in the methods described herein.

[0088] Granulocytes that kill less than 5% of the cancer cells are preferably discarded.

[0089] Suitably, granulocytes may be selected if they kill at least 80%, 85%, 90% or 95% of cancer cells. Granulocytes that kill less than 70% (suitably less than 80%, 85%, 90% or 95%) of cancer cells are preferably discarded.

[0090] In one embodiment, an in vitro method for selecting suitable granulocytes for use in the treatment of cancer comprises: a. Mix granulocytes with a cancer cell line (preferably a pancreatic cancer cell line or multiple different cancer cell lines) and culture at 8x10 5 granulocytes and 8x10 4 providing a mixture (or mixtures) each comprising a cancer cell and a b. incubating said mixture (or mixtures) at 39° C. for 24 hours; c. determining the % of cancer cells killed in said mixture (or mixtures); and d. If the granulocytes kill at least 5% of the cancer cells in the mixture, selecting said granulocytes as suitable for use in treating a cancer of the same type / subset as the cancer cell line. Includes.

[0091] In one embodiment, the in vitro method according to the above aspect may also comprise simultaneously assaying granulocytes of the subject to be treated.

[0092] The in vitro method according to the invention may further comprise obtaining hematopoietic cells from a donor from which the selected granulocytes are available or have been obtained. Thus, the in vitro method of the invention may also constitute a method for selecting hematopoietic cells suitable for use in the treatment of cancer.

[0093] In another embodiment, an in vitro method for selecting hematopoietic cells suitable for use in the treatment of cancer comprises: a. mixing available granulocytes from a donor with a plurality of different cancer cell lines to provide a plurality of mixtures; b. incubating the mixture; c. determining the percentage of cancer cells killed in said mixture; and d. If the granulocytes kill at least 5% of the cancer cells in the mixture, selecting hematopoietic cells from said donor as suitable for use in treating the same type / subset of cancer as the cancer cell line killed by said granulocytes. Includes.

[0094] The in vitro method may further include measuring and / or selecting hematopoietic cells based on the surface potential disclosed herein and / or based on the surface potential of granulocytes differentiated from hematopoietic cells. The in vitro method may further include measuring and / or selecting hematopoietic cells based on the cell density disclosed herein (e.g., at least 1.077 g / ml) and / or based on the cell density of granulocytes differentiated from hematopoietic cells (e.g., at least 1.077 g / ml). The in vitro method may further include measuring and / or selecting hematopoietic cells based on the expression or activity of toll-like receptors; and / or the absence or inactivity of expression of programmed cell death 1 (PD-1) receptors; CD115; CD224; CXCR1; and / or CXCR2 on granulocytes differentiated from hematopoietic cells.

[0095] In one aspect, there is provided a hematopoietic cell obtainable (eg, obtained) by the method of the invention.

[0096] In one aspect, there is provided a granulocyte obtainable (eg obtained) by the method of the invention.

[0097] Hematopoietic cells selected according to the present invention can be differentiated into granulocytes (e.g., neutrophils) with advantageous properties. For example, the granulocytes can kill cancer cells more quickly than granulocytes obtained directly from a donor. In one embodiment, the granulocytes obtained from the hematopoietic cells described herein have a maximum half-life cancer kill% within 15 hours of contact with cancer cells. Preferably, the granulocytes obtained from the hematopoietic cells described herein have a maximum half-life cancer kill% within 10 hours of contact with cancer cells.

[0098] Suitably, such values ​​are obtained when the ratio of granulocytes to cancer cells used in the methods described herein is 10:1.

[0099] The term "maximal half-life cancer killing %" used in this context means half of the total cancer cells that can be killed by granulocytes.For example, if granulocytes kill 50% of the total cancer cells used in the method described herein, then the maximum half-life cancer killing % will be 25% of the total cancer cells used in the assay.

[0100] In some embodiments, granulocytes that kill less than 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% of cancer cells may be discarded.

[0101] The in vitro method of the present invention may include a combination of techniques described herein to improve the selection of cells suitable for treating cancer. For example, when multiple hematopoietic cells or granulocytes are selected because they meet a defined density threshold, cell surface potential / electrophoretic mobility can be evaluated to help identify hematopoietic cells that produce granulocytes (e.g., neutrophils) with high CKA or select granulocytes with improved CKA. Advantageously, such a combination of techniques improves the ability to select hematopoietic cells or granulocytes of the present invention. Furthermore, by applying such a combination of techniques to granulocytes (e.g., neutrophils), cells with improved CKA can be detected and hematopoietic cells obtained from the donor.

[0102] The invention also provides an in vitro cell culture of hematopoietic cells of the invention, or a differentiation method comprising differentiating hematopoietic cells obtainable according to the method of the invention, into granulocytes. In a related aspect, an in vitro cell culture of granulocytes obtainable (e.g. obtained) by such a method is provided. In one embodiment, the in vitro cell culture comprises: a. a surface potential defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec (or at least 1.0 μm.cm / volt.sec, e.g., at least 1.25 μm.cm / volt.sec, 1.5 μm.cm / volt.sec, or 1.75 μm.cm / volt.sec); and b. Ability to kill cancer cells The antibody is enriched for granulocytes having

[0103] In one aspect, the present invention provides a pharmaceutical composition comprising hematopoietic cells; and granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone; serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, fetal bovine serum (FBS), or a combination thereof.

[0104] In one embodiment, the pharmaceutical composition comprises hematopoietic cells; and granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF), and growth hormone, and serotonin, and vitamin C, and vitamin D, and glutamine (Gln), and arachidonic acid, and AGE-albumin, and interleukin, and TNF-alpha, and Flt-3 ligand, and thrombopoietin, and fetal bovine serum (FBS).

[0105] Suitably, the growth hormone may be human growth hormone. The hematopoietic cells comprised in the composition may be obtainable (e.g., obtained) by the method of the invention or may be part of an in vitro cell culture of hematopoietic cells of the invention.

[0106] In one aspect, the present invention provides a pharmaceutical composition comprising granulocytes; granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone; serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha (e.g., UniProt Accession No. P01375), Flt-3 ligand, thrombopoietin (e.g., UniProt Accession No. P40225), fetal bovine serum (FBS), or a combination thereof. The granulocytes contained in the composition are obtainable (e.g., obtained) by the method of the present invention.

[0107] In one embodiment, the pharmaceutical composition comprises granulocytes; and granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF), and growth hormone, and serotonin, and vitamin C, and vitamin D, and glutamine (Gln), and arachidonic acid, and AGE-albumin, and interleukin, and TNF-alpha, and Flt-3 ligand, and thrombopoietin, and fetal bovine serum (FBS).

[0108] The interleukin may be interleukin-3 (IL-3) (e.g., UniProt Accession No. P08700), interleukin-8 (IL-8) (e.g., UniProt Accession No. P10145), interleukin-4 (IL-4) (e.g., UniProt Accession No. P05112), interleukin-6 (IL-6) (e.g., UniProt Accession No. P05231), interleukin-18 (IL-18) (e.g., UniProt Accession No. Q14116), or a combination thereof. Suitably, the interleukin may be interleukin-3 (IL-3), interleukin-8 (IL-8), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-18 (IL-18).

[0109] In one embodiment, the granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone, serotonin, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, or thrombopoietin may be human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone, serotonin, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, or thrombopoietin.

[0110] All the above reagents are commercially available from Sino Biological Inc., Suite B-310 (also Suite B-209, B-203), 14 Zhong He Street, BDA, Beijing 100176, PRChina.

[0111] The present invention also relates to the hematopoietic cells, in vitro cell cultures of hematopoietic cells, granulocytes, in vitro cell cultures of granulocytes, pharmaceutical compositions or kits described herein for use as a medicament. The medicament may be for use in the treatment of cancer, and thus in a related aspect there is provided a hematopoietic cell, in vitro cell cultures of hematopoietic cells, granulocytes, in vitro cell cultures of granulocytes, pharmaceutical compositions or kits for use in the treatment of cancer. Corresponding methods for treating cancer are also provided, comprising administering the in vitro cell cultures of hematopoietic cells, granulocytes, in vitro cell cultures of granulocytes, pharmaceutical compositions or kits of the invention to a subject in need thereof.

[0112] In another aspect, the invention provides a cell bank comprising the hematopoietic cells, the in vitro cell culture of hematopoietic cells, the granulocytes, the in vitro cell culture of granulocytes, or the pharmaceutical composition of the invention.

[0113] In a further aspect, a. an in vitro cell culture of hematopoietic cells, hematopoietic cells, granulocytes, an in vitro cell culture of granulocytes, or a pharmaceutical composition of the invention; and b. Instructions for its use in medicine A kit is provided comprising:

[0114] The term "hematopoietic cells" as used herein refers to cells that can differentiate into granulocytes, preferably neutrophils. The term "hematopoietic cells" thus encompasses hematopoietic stem cells as well as progenitor cells (e.g., differentiated from hematopoietic stem cells) that can differentiate into granulocytes, preferably neutrophils. Progenitor cells may be referred to herein as "granulocyte progenitor cells." Hematopoietic cells according to the present invention may relate to hematopoietic stem cells, granulocyte progenitor cells or a combination thereof. Preferably, the term "hematopoietic cells" as used herein does not encompass human embryonic stem cells. In one embodiment, the hematopoietic cells are cells of the hematopoietic pathway or equivalent cells. In one embodiment, the hematopoietic cells are induced pluripotent stem cells (iPSCs) or equivalent cells. In one embodiment, the iPSCs are obtainable from somatic cells of a donor. Generation of iPSCs is a technique well known in the art, see Yu et al. (2007), Science, 318:1917-1920, the teachings of which are incorporated herein by reference.

[0115] In one embodiment, the hematopoietic cells are nuclear transfer embryonic stem cells (NT-ESCs) or equivalent cells. In one embodiment, NT-ESCs can be obtained by injecting the nucleus of a donor-derived cell into an egg cell from which the original nucleus has been removed. The generation of NT-ESCs is a technique well known in the art, see Tachibana M, Amato P, Sparman M et al. (2013), Cell, 154(2):465-466, the teachings of which are incorporated herein by reference.

[0116] In one embodiment, where hematopoietic cells are obtained from a donor-derived sample, the hematopoietic cells can be isolated from the sample. In another embodiment, where hematopoietic cells are obtained from a donor-derived sample, the sample is a sample containing somatic cells, and the hematopoietic cells are obtained by inducing pluripotency of cells (e.g., somatic cells) in the sample to obtain iPSCs.

[0117] In another embodiment, in which hematopoietic cells are obtained from a donor-derived sample, the sample is a sample containing somatic cells, and the hematopoietic cells are obtained by injecting the nucleus of a cell (e.g., a somatic cell) in the sample into an egg cell (e.g., from which the original nucleus has been removed) to obtain NT-ESCs.

[0118] In one embodiment, the hematopoietic cells are hematopoietic stem cells. Hematopoietic stem cells can be selected based on cell surface polypeptide markers selected from, for example, CD34 (e.g., UniProt Accession No. P28906), CD59 (e.g., UniProt Accession No. P13987), Thy1 (e.g., UniProt Accession No. P04216), CD38 (e.g., UniProt Accession No. P28907), C-kit (e.g., UniProt Accession No. P10721), and lin. In one embodiment, hematopoietic stem cells can be selected based on cell surface polypeptide markers selected from, for example, CD34 (e.g., UniProt Accession No. P28906), CD59 (e.g., UniProt Accession No. P13987), Thy1 (e.g., UniProt Accession No. P04216), CD38 (e.g., UniProt Accession No. P28907), C-kit (e.g., UniProt Accession No. P10721), and lin. + , CD59 + , Thy1 + , CD38 low / - , C-kit low / - , and lin - Preferably, the hematopoietic cells express CD34. Antibodies for detecting the presence or absence of said markers are commercially available and can be obtained, for example, from BD Biosciences Europe, ebioscience, Beckman Coulter and Pharmingen.

[0119] In another embodiment, the hematopoietic cell is a granulocyte progenitor cell. The granulocyte progenitor cell may be one or more selected from common myeloid progenitor cells, myeloblasts, N. promyelocytes, N. myelocytes, N. metamyelocytes, N. neutrophil band cells, or combinations thereof.

[0120] Hematopoietic cells (such as hematopoietic stem cells or granulocyte progenitor cells) or cell cultures can be differentiated into granulocytes. Differentiation can be carried out using any suitable method, such as the method based on the disclosures in Lieber et al., Blood, 2004 Feb 1;103(3):852-9, and / or Choi et al., Nat.Protoc., 2011 Mar;6(3):296-313, and / or Timmins et al., Biotechnology and bioengineering.2009;104(4):832-40, which are incorporated herein by reference.

[0121] In one aspect, the invention provides a method of differentiating hematopoietic cells, comprising mixing said hematopoietic cells with granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone; serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, fetal bovine serum (FBS), or a combination thereof.

[0122] In one embodiment, the invention provides a method of differentiating hematopoietic cells comprising mixing said hematopoietic cells with granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF), and growth hormone, and serotonin, and vitamin C, and vitamin D, and glutamine (Gln), and arachidonic acid, and AGE-albumin, and interleukin, and TNF-alpha, and Flt-3 ligand, and thrombopoietin, and fetal bovine serum (FBS).

[0123] The hematopoietic cells may be part of a hematopoietic cell culture.

[0124] In one embodiment, differentiation of hematopoietic cells comprises culturing said hematopoietic cells with one or more feeder cells. Suitably, the feeder cells may be OP9 cells. OP9 cells (ATCC® CRL-2749™) are commercially available from American Type Culture Collection United Kingdom (UK), Guernsey, Ireland, Jersey and Liechtenstein, LGC Standards, Queens Road, Teddington, Middlesex, TW11 0LY, UK. In one embodiment, hematopoietic cells may be cultured with one or more feeder cells and Flt-3 ligand, thrombopoietin, fetal bovine serum (FBS), or a combination thereof.

[0125] Thus, in one embodiment, the pharmaceutical composition or cell culture of the invention may further comprise feeder cells, such as OP9 cells.

[0126] The term "granulocyte" encompasses the following cell types: neutrophils, basophils, and eosinophils. Preferably, the granulocyte is a neutrophil. Granulocytes may express the cell surface polypeptide markers CD11b (e.g., UniProt Accession No. P11215) and CD15. Granulocytes also produce reactive oxygen species (O 2 - ) may be produced.

[0127] The present invention encompasses granulocytes suitable for use in the treatment of cancer. Preferably, said granulocytes comprise a surface potential defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec (preferably at least 2.25 μm.cm / volt.sec or at least 2.5 μm.cm / volt.sec). In one embodiment, said granulocytes comprise a surface potential defined by an electrophoretic mobility of at least 2.75 μm.cm / volt.sec, or at least 3.0 μm.cm / volt.sec. Preferably, said granulocytes may comprise a surface potential defined by an electrophoretic mobility of at least 3.25 μm.cm / volt.sec, or at least 3.5 μm.cm / volt.sec. Preferably, said granulocytes may comprise a surface potential defined by an electrophoretic mobility of at least 3.75 μm.cm / volt.sec, or at least 4.0 μm.cm / volt.sec. Granulocytes also have the ability to kill cancer cells.

[0128] Alternatively, the granulocytes may comprise a surface potential defined by an electrophoretic mobility of at least 1.0 μm.cm / volt.sec or at least 1.25 μm.cm / volt.sec. For example, the granulocytes may comprise a surface potential defined by an electrophoretic mobility of at least 1.5 μm.cm / volt.sec or at least 1.75 μm.cm / volt.sec. The granulocytes are also capable of killing cancer cells.

[0129] "Cancer cell killing ability" can be determined by mixing cells (e.g., granulocytes such as neutrophils) with cancer cells and measuring the viability of the cancer cells (e.g., after incubation). If the cancer cells are no longer viable (i.e., killed), the cells exhibit the ability to kill cancer cells. In one embodiment, the ability to kill cancer cells is determined using a cancer killing activity (CKA) assay as described herein.

[0130] In one embodiment, the CKA assay comprises: a. contacting a cancer cell line with granulocytes to form a test sample (preferably at a ratio of granulocytes to cancer cells of 10:1); b. incubating said test sample; and c. Determining the percentage of cancer cells killed in said test sample. Includes.

[0131] In one embodiment, the CKA assay comprises: a. mixing granulocytes with a cancer cell line to provide a mixture (preferably at a ratio of granulocytes to cancer cells of 10:1); b. incubating the mixture; and c. Measuring the percentage of cancer cells killed in said mixture. Includes.

[0132] In a preferred embodiment, the CKA assay comprises: a. Granulocytes and cancer cell lines are mixed and incubated at 8x10 5 granulocytes and 8x10 4 providing a mixture comprising: b. incubating the mixture at 39° C. for 24 hours; c. Measuring the percentage of cancer cells killed in said mixture. Includes.

[0133] In a preferred embodiment, the CKA assay comprises: a. mixing granulocytes with a cancer cell line to provide a mixture comprising a ratio of granulocytes to cancer cells of at least 1:1 (e.g., 5:1 or 10:1); b. incubating the mixture for 16-24 hours (e.g., at 30-40° C.); c. Measuring the percentage of cancer cells killed in said mixture. Includes.

[0134] In one embodiment, a granulocyte can be considered to kill cancer cells if it kills at least 5% of the cancer cells in the methods described herein. A granulocyte can be considered to kill cancer cells if it kills at least 10%, 20%, 30%, 40% or 50% of the cancer cells present. In one embodiment, a granulocyte can be considered to kill cancer cells if it kills at least 60% of the cancer cells present. In one embodiment, a granulocyte can be considered to kill cancer cells if it kills at least 70% of the cancer cells present. Preferably, a granulocyte can be considered to kill cancer cells if it kills at least 80% or 90% of the cancer cells present.

[0135] In embodiments where the method includes using a 5:1 ratio of granulocytes to cancer cells, the granulocytes can be considered to kill cancer cells if they kill at least 30% of the cancer cells present. Preferably, where the method includes using a 5:1 ratio of granulocytes to cancer cells, the granulocytes can be considered to kill cancer cells if they kill at least 40% of the cancer cells present. In embodiments where the method includes using a 10:1 ratio of granulocytes to cancer cells, the granulocytes can be considered to kill cancer cells if they kill at least 45%, 50% or 60% (preferably at least 60%) of the cancer cells present. More preferably, where the method includes using a 10:1 ratio of granulocytes to cancer cells, the granulocytes can be considered to kill cancer cells if they kill at least 80% of the cancer cells present.

[0136] In some embodiments, the cancer cell line is a cervical cancer cell line (e.g., HeLa). In embodiments where the method includes using a 5:1 ratio of granulocytes to cervical cancer cells, the granulocytes can be considered to kill cancer cells if they kill at least 30% of the cervical cancer cells present. Preferably, when the method includes using a 5:1 ratio of granulocytes to cervical cancer cells, the granulocytes can be considered to kill cancer cells if they kill at least 40% of the cervical cancer cells present. In embodiments where the method includes using a 10:1 ratio of granulocytes to cervical cancer cells, the granulocytes can be considered to kill cancer cells if they kill at least 45%, 50% or 60% (preferably at least 60%) of the cervical cancer cells present. More preferably, when the method includes using a 10:1 ratio of granulocytes to cervical cancer cells, the granulocytes can be considered to kill cancer cells if they kill at least 80% of the cervical cancer cells present.

[0137] In some embodiments, the cancer cell line is a pancreatic cancer cell line (e.g., PANC-1). In embodiments where the method includes using a 5:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can be considered to kill the cancer cells if they kill at least 50% or 60% of the pancreatic cancer cells present. Preferably, when the method includes using a 5:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can be considered to kill the cancer cells if they kill at least 65% or 70% of the pancreatic cancer cells present. In embodiments where the method includes using a 10:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can be considered to kill the cancer cells if they kill at least 70% of the pancreatic cancer cells present. Preferably, when the method includes using a 10:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can be considered to kill the cancer cells if they kill at least 80% or 90% of the pancreatic cancer cells present.

[0138] Granulocytes that kill less than 5% of the cancer cells are preferably discarded.

[0139] The preceding paragraphs apply to each of the methods described herein, and the disclosure may be combined with any of the methods described herein.

[0140] A cell capable of killing a cancer cell ("likely to kill a cancer cell") can be defined as a cell having at least 70% or 75% CKA in a CKA assay herein (e.g., the CKA assay described above). Suitably, the cell may have at least 80% or 90% activity in a CKA assay herein (e.g., the CKA assay described above).

[0141] Cells that have the "ability to kill cancer cells" ("believed to kill cancer cells") are suitable for use in the treatment of cancer. Hematopoietic cells that can be differentiated into cells that have the "ability to kill cancer cells" are also believed to be suitable for use in the treatment of cancer.

[0142] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a. a density of at least 1.077 g / ml; and b. Ability to kill cancer cells The present invention provides an in vitro cell culture of hematopoietic cells which differentiate to form granulocytes, the cell culture being characterized by:

[0143] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a. density greater than 1.077 g / ml; and b. Ability to kill cancer cells The present invention provides an in vitro cell culture of hematopoietic cells which differentiate to form granulocytes, the cell culture being characterized by:

[0144] Granulocytes having a density of at least 1.077 g / ml may be obtainable by a method as described in Example 21. In one embodiment, such a method comprises: i. providing a sucrose solution adjusted to a density of 1.077 g / ml; ii. adding a composition comprising granulocytes; and iii. Centrifugation to sediment high density granulocytes (e.g., neutrophils) Includes.

[0145] Granulocytes having a density of at least 1.077 g / ml may be obtainable using commercially available kits such as the Histopaque®-1077 kit (commercially available from Sigma-Aldrich, catalog number 10771-100ML).

[0146] In some embodiments, granulocytes having a density of 1.077 g / ml are obtained by Ficoll-Paque separation. Typically, such granulocytes are found at the bottom of the 1.077 Ficoll-Paque medium after separation, while low-density granulocytes (e.g., having a density less than 1.077 g / ml) are found at the 1.077-plasma interface.

[0147] Thus, granulocytes according to (and for use in) the present invention may have a density of at least 1.077 g / ml, and granulocytes having a density less than 1.077 g / ml may be excluded from the present invention. In some embodiments, granulocytes according to the present invention have a density of at least 1.077 g / ml and a cell surface potential as described herein. Thus, all cell surface potential embodiments apply equally to said granulocytes.

[0148] Preferably, the granulocytes have a density greater than 1.077 g / ml.

[0149] In one embodiment, the granulocytes have a density of at least 1.078 g / ml. In one embodiment, the granulocytes have a density of at least 1.079 g / ml. In one embodiment, the granulocytes have a density of at least 1.080 g / ml. In one embodiment, the granulocytes have a density of at least 1.081 g / ml. In one embodiment, the granulocytes have a density of at least 1.082 g / ml. In one embodiment, the granulocytes have a density of at least 1.083 g / ml. Preferably, the granulocytes have a density of 1.082 g / ml or greater.

[0150] In one embodiment, the granulocytes have a density of less than 1.084 g / ml. In one embodiment, the granulocytes have a density of less than 1.083 g / ml. In one embodiment, the granulocytes have a density of less than 1.082 g / ml. In one embodiment, the granulocytes have a density of less than 1.081 g / ml. In one embodiment, the granulocytes have a density of less than 1.080 g / ml. In one embodiment, the granulocytes have a density of less than 1.079 g / ml. In one embodiment, the granulocytes have a density of less than 1.078 g / ml.

[0151] In one embodiment, the granulocytes have a density of 1.077 g / ml to 1.084 g / ml (e.g., a density greater than 1.077 g / ml but less than 1.084 g / ml). The granulocytes may have a density of 1.079 g / ml to 1.084 g / ml, e.g., a density of 1.080 g / ml to 1.084 g / ml. The granulocytes may have a density of 1.080 g / ml to 1.083 g / ml, e.g., a density of 1.080 g / ml to 1.082 g / ml.

[0152] In one embodiment, the hematopoietic cells have a density of at least 1.077 g / ml. Preferably, the hematopoietic cells have a density greater than 1.077 g / ml.

[0153] In one embodiment, the hematopoietic cells have a density of at least 1.078 g / ml. In one embodiment, the hematopoietic cells have a density of at least 1.079 g / ml. In one embodiment, the hematopoietic cells have a density of at least 1.080 g / ml. In one embodiment, the hematopoietic cells have a density of at least 1.081 g / ml. In one embodiment, the hematopoietic cells have a density of at least 1.082 g / ml. In one embodiment, the hematopoietic cells have a density of at least 1.083 g / ml.

[0154] In one embodiment, the hematopoietic cells have a density of less than 1.084 g / ml. In one embodiment, the hematopoietic cells have a density of less than 1.083 g / ml. In one embodiment, the hematopoietic cells have a density of less than 1.082 g / ml. In one embodiment, the hematopoietic cells have a density of less than 1.081 g / ml. In one embodiment, the hematopoietic cells have a density of less than 1.080 g / ml. In one embodiment, the hematopoietic cells have a density of less than 1.079 g / ml. In one embodiment, the hematopoietic cells have a density of less than 1.078 g / ml.

[0155] In one embodiment, the hematopoietic cells have a density of 1.077 g / ml to 1.084 g / ml (e.g., a density greater than 1.077 g / ml but less than 1.084 g / ml). The hematopoietic cells may have a density of 1.079 g / ml to 1.084 g / ml, e.g., a density of 1.080 g / ml to 1.084 g / ml. The hematopoietic cells may have a density of 1.080 g / ml to 1.083 g / ml, e.g., a density of 1.080 g / ml to 1.082 g / ml.

[0156] In one aspect, the invention provides a method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. Measuring the density of granulocytes available from the donor; and b. selecting hematopoietic cells from said donor if the measured density of granulocytes is at least 1.077 g / ml. The present invention provides a method comprising:

[0157] The present invention also provides a method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising the steps of: a. Measuring the density of hematopoietic cells; and b. Selecting hematopoietic cells that have a density less than 1.077 g / ml and / or that differentiate to form granulocytes with reduced ability to kill cancer cells, and that have a higher density than otherwise identical hematopoietic cells. Also provided is a method comprising:

[0158] In one aspect, a method is provided comprising differentiating an in vitro cell culture of hematopoietic cells, the hematopoietic cells being: a. a density of at least 1.077 g / ml; and b. Ability to kill cancer cells In one embodiment, a method is provided for differentiating a cell line comprising:

[0159] The present invention also provides an in vitro culture of granulocytes obtainable by said method.

[0160] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a. expression or activity of toll-like receptors; and / or the absence or inactivity of the programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2; and b. Ability to kill cancer cells The present invention provides an in vitro cell culture of hematopoietic cells which differentiate to form granulocytes, the cell culture being characterized by:

[0161] The presence or absence of said receptor can be determined using any technique known to those skilled in the art. For example, those skilled in the art can use labeled antibodies, optionally in combination with FACS, to detect the presence or absence of said receptor.

[0162] Receptor activity / inactivity can be determined using any known technique, for example, by detecting changes in gene expression that are associated with said activity / inactivity.

[0163] Preferably, the granulocytes express toll-like receptors; do not express programmed cell death 1 (PD-1) receptors (e.g., UniProt accession number Q15116); CD15 (e.g., UniProt accession number P07333); CD224 (e.g., UniProt accession number P19440); CXCR1 (e.g., UniProt accession number P25024); and / or CXCR2 (e.g., UniProt accession number P25025) (preferably do not express programmed cell death 1 (PD-1) receptors; CD115; CD224; CXCR1; and CXCR2).

[0164] Alternatively, or in addition, one or more of the PD-1 receptor; CD115; CD224; CXCR1; and CXCR2 may be expressed in an inactive form or inactivated after expression.

[0165] The toll-like receptor may be one or more of TLR1 (e.g., UniProt accession number Q15399), TLR2 (e.g., UniProt accession number O60603), TLR3 (e.g., UniProt accession number O15455), TLR4 (e.g., UniProt accession number O00206), TLR5 (e.g., UniProt accession number O60602), TLR6 (e.g., UniProt accession number Q9Y2C9), TLR7 (e.g., UniProt accession number Q9NYK1), TLR8 (e.g., UniProt accession number Q9NR97), TLR9 (e.g., UniProt accession number Q9NR96), TLR10 (e.g., UniProt accession number Q9BXR5), and / or TLR11 (e.g., UniProt accession number Q6R590). Preferably, the toll-like receptor is TLR4.

[0166] Without wishing to be bound by theory, the inventors believe that PD-L1 does not bind to its receptor PD-1 on granulocytes (e.g., neutrophils) with high CKA and / or granulocytes (e.g., neutrophils) with high CKA do not produce PD-L1; and / or Granulocytes (e.g., neutrophils) with high CKA have active toll-like receptors on their surface; and / or CD115 and CD224 markers are not expressed on granulocytes (e.g., neutrophils) with high CKA; CXCR1 and CXCR2 are receptors for granulocytes (e.g., neutrophils) with low CKA, i.e., granulocytes (e.g., neutrophils) with high CKA do not express CXCR1&CXCR2, or CXCR1 and CXCR2 are inhibited in said granulocytes (e.g., neutrophils). I think so.

[0167] In one embodiment, granulocytes having the above cell surface polypeptide expression profile also have a cell surface potential and / or density as described herein.

[0168] In one aspect, the invention provides a method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. detecting the expression or activity of toll-like receptors; programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2 on granulocytes obtainable from the donor; and b. selecting hematopoietic cells from said donor if toll-like receptors are expressed or active; and / or programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2 are not expressed or inactive. The present invention provides a method comprising:

[0169] In one aspect, a method is provided comprising differentiating an in vitro cell culture of hematopoietic cells, the hematopoietic cells being: a. expression or activity of toll-like receptors; and / or the absence or inactivity of the programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2; and b. Ability to kill cancer cells In one embodiment, a method is provided for differentiating a cell line comprising:

[0170] The present invention also provides an in vitro culture of granulocytes obtainable by said method.

[0171] Hematopoietic cells can be immortalized. The skilled artisan is familiar with immortalization techniques, including in particular the introduction of viral genes that deregulate the cell cycle (e.g., the E1 gene of adenovirus type 5) and the artificial expression of telomerase. Immortalization advantageously allows the preparation of cell lines that can be stably cultured in vitro. Thus, in one aspect, the present invention provides selected hematopoietic cells, as well as immortalized cell lines obtainable (e.g., obtained) from stable hematopoietic cell cultures. Advantageously, immortalized cell lines or stable hematopoietic cell cultures are obtainable (e.g., obtained) by the methods of the present invention.

[0172] The term "stable" as used in reference to a hematopoietic cell culture or cell line means that the cell culture or cell line has been modified to be more suitable for in vitro cell culture than unmodified cells (i.e., cells obtained from a donor and directly subjected to in vitro cell culture). Thus, said "stable" cell culture or cell line can undergo a greater number of replications (preferably over a longer period of time) when compared to unmodified cells.

[0173] The hematopoietic cells are suitably obtainable (eg, obtained) from a donor, eg, a human donor.

[0174] As used herein, the term "donor" refers to a subject (preferably a human subject) from whom a biological fluid sample is obtained. Any suitable biological fluid sample from which hematopoietic cells or granulocytes can be obtained can be used in the present invention.

[0175] Thus, the term "sample" as used herein with reference to a donor-derived sample may be any sample that contains hematopoietic cells or from which hematopoietic cells are obtainable (e.g., where the hematopoietic cells are iPSCs, the sample may contain somatic cells).

[0176] In one embodiment, the biological fluid sample (or "sample") is a blood sample, such as a peripheral blood sample. As used herein, the term "blood" includes whole blood, serum, and plasma. Blood can be centrifuged to separate red blood cells, white blood cells, and plasma. After centrifugation, the mononuclear cell layer can be removed for use in the present invention.

[0177] Donors may be selected based on one or more of the following characteristics: sex, age, medical history, and / or blood type. In one embodiment, the donor may be selected if the donor is male. In another embodiment, the donor may be selected if the donor is between 18 and 25 years old (preferably between 18 and 24 years old). Preferably, the donor may be selected if the donor is male and between 18 and 25 years old (preferably between 18 and 24 years old). Without wishing to be bound by theory, it is believed that males in early adulthood are more likely to produce granulocytes (e.g., neutrophils) that have the ability to kill cancer cells.

[0178] Suitably, the granulocytes obtainable (e.g., obtained) from the donor may have a surface potential defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec and the ability to kill cancer cells. In another embodiment, the granulocytes obtainable (e.g., obtained) from the donor have a surface potential defined by an electrophoretic mobility of at least 1.0 μm.cm / volt.sec and the ability to kill cancer cells.

[0179] The present invention also relates to the measurement of cell surface potential. The surface potential of a cell can be determined using any suitable technique known in the art. In one embodiment, the surface potential is determined using electrophoresis. The electrophoresis technique can be performed by applying a voltage to cells contained within a suitable electrophoretic medium and measuring cell mobility. The following protocol can be used: i. applying a direct current of 200 V to cells (suspended in 10 mM Tris-HCl and 291 mM glucose buffer) contained in an electrophoresis chamber; and ii. Measure the time it takes for the cells (e.g., granulocytes or hematopoietic cells) to traverse a fixed length while applying a current of 3 mA (e.g., by use of a microscope, optionally connected to a CCD camera).

[0180] Electrophoretic mobility "μ" (expressed in units of μm.cm / volt.sec) was calculated using the following formula: μ=ugS / I (In the formula, "u" = electrophoretic velocity measured by step ii.; "g" = conductivity of the electrophoretic medium; "S" = cross-sectional area of ​​the electrophoresis chamber; and "I" = current) It can be calculated using the following formula:

[0181] In one embodiment, the electrophoretic mobility is i. adding hematopoietic cells or granulocytes suspended in 10 mM Tris-HCl and 291 mM glucose to the electrophoresis chamber; ii. Applying a direct current of 200V / 3mA; and iii. measuring the distance (mm) that the hematopoietic cells or granulocytes have traveled toward the electrode in a given time; and iv. Calculating the electrophoretic mobility using the above formula is determined by.

[0182] Electrophoresis may be carried out in a 0.9% (isotonic) NaCl solution, preferably using a constant current (e.g., 3 mA).

[0183] Electrophoretic mobility assays may be those described in "Cell Electrophoresis" edited by Johann Bauer (ISBN 0-8493-8918-6 published by CRC Press, Inc.), the teachings of which are incorporated herein in their entirety.

[0184] Without wishing to be bound by theory, it is believed that hematopoietic cells (e.g., hematopoietic stem cells expressing CD34) capable of differentiating into granulocytes (e.g., neutrophils) with a higher CKA are more positively charged and therefore will migrate further toward a negatively charged electrode at a given time than hematopoietic cells (e.g., hematopoietic stem cells expressing CD34) of similar size and weight / density that differentiate into granulocytes (e.g., neutrophils) with a lower CKA. Similarly, it is believed that granulocytes (e.g., neutrophils) with a higher CKA are more positively charged and therefore will migrate further toward a negatively charged electrode at a given time than granulocytes (e.g., neutrophils) of similar size and weight / density that may have a lower CKA.

[0185] Preferably, the cell surface potential is equal to the electrophoretic mobility and is proportional to the distance traveled by the cell in a given time during electrophoresis. Cells with a higher positive charge will move further toward the electrode during electrophoresis than less positively (or negatively) charged cells. Such cells will therefore have a higher value of cell surface potential than less positively (or negatively) charged cells. The cell surface potential can be defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec or at least 2.25 μm.cm / volt.sec. Advantageously, the cell surface potential can be defined by an electrophoretic mobility of at least 2.5 μm.cm / volt.sec or at least 2.75 μm.cm / volt.sec. Advantageously, the cell surface potential can be defined by an electrophoretic mobility of at least 3.0 μm.cm / volt.sec or at least 3.25 μm.cm / volt.sec. Preferably, the surface potential of the cell can be defined by an electrophoretic mobility of at least 3.5 μm.cm / volt.sec or at least 3.75 μm.cm / volt.sec. More preferably, the surface potential of the cell can be defined by an electrophoretic mobility of at least 4.0 μm.cm / volt.sec. The cell may be a hematopoietic cell or a granulocyte.

[0186] In one embodiment, the hematopoietic cells of the invention may have a surface potential defined by an electrophoretic mobility of at least 1.0 μm.cm / volt.sec or at least 1.25 μm.cm / volt.sec, such as at least 1.5 μm.cm / volt.sec or 1.75 μm.cm / volt.sec (preferably at least 2.0 μm.cm / volt.sec). In one embodiment, the hematopoietic cells may have a surface potential defined by an electrophoretic mobility of at least 2.25 μm.cm / volt.sec or at least 2.5 μm.cm / volt.sec. Suitably, the hematopoietic cells may have a surface potential defined by an electrophoretic mobility of at least 2.75 μm.cm / volt.sec or at least 3.0 μm.cm / volt.sec. Suitably, the hematopoietic cells may have a surface potential defined by an electrophoretic mobility of at least 3.25 μm.cm / volt.sec or at least 3.5 μm.cm / volt.sec. More preferably, the hematopoietic cells may have a surface potential defined by an electrophoretic mobility of at least 3.75 μm.cm / volt.sec or at least 4.0 μm.cm / volt.sec.

[0187] In one embodiment, the granulocytes of the invention may have a surface potential defined by an electrophoretic mobility of at least 1.0 μm.cm / volt.sec or at least 1.25 μm.cm / volt.sec, for example at least 1.5 μm.cm / volt.sec or at least 1.75 μm.cm / volt.sec (preferably at least 2.0 μm.cm / volt.sec). In one embodiment, the granulocytes may have a surface potential defined by an electrophoretic mobility of at least 2.25 μm.cm / volt.sec or at least 2.5 μm.cm / volt.sec. Suitably, the granulocytes may have a surface potential defined by an electrophoretic mobility of at least 2.75 μm.cm / volt.sec or at least 3.0 μm.cm / volt.sec. Preferably, the granulocytes may have a surface potential defined by an electrophoretic mobility of at least 3.25 μm.cm / volt.sec or at least 3.5 μm.cm / volt.sec, more preferably, the granulocytes may have a surface potential defined by an electrophoretic mobility of at least 3.75 μm.cm / volt.sec or at least 4.0 μm.cm / volt.sec.

[0188] The invention may include measuring the surface potential of granulocytes obtainable (e.g., obtained) from the donor. Hematopoietic cells are selected from the donor if the surface potential is defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec (or alternatively at least 1.0 μm.cm / volt.sec). Advantageously, this provides a simple and / or rapid and / or reliable screening method for the selection of hematopoietic cells suitable for use in the treatment of cancer. In some embodiments according to the method used, a rapid screening can be used to determine whether a donor is suitable without the need for more complicated assays (e.g., CKA assay, etc.). A functional assay (such as the CKA assay described herein) can then be performed to validate the screening, for example, by differentiating the hematopoietic cells selected from the donor into granulocytes and testing the granulocytes in the CKA assay. Cells that do not have the required cell surface potential and / or the required CKA can be discarded.

[0189] Additionally or alternatively, the surface potential of a hematopoietic cell can be measured and selected if it has a surface potential defined by an electrophoretic mobility of less than 2.0 μm.cm / volt.sec (preferably less than 2.5 μm.cm / volt.sec, more preferably less than 3.5 μm.cm / volt.sec or 4.0 μm.cm / volt.sec); and / or has a higher surface potential than an otherwise identical hematopoietic cell that differentiates to form granulocytes (e.g., neutrophils) that have a reduced ability to kill cancer cells. Advantageously, this allows for rapid screening of hematopoietic cells to determine whether the cells are suitable for use in the treatment of cancer. Thus, the invention encompasses hematopoietic cells that have a surface potential defined by an electrophoretic mobility of less than 2.0 μm.cm / volt.sec (preferably, less than 2.5 μm.cm / volt.sec, more preferably, less than 3.5 μm.cm / volt.sec or 4.0 μm.cm / volt.sec); and / or that differentiate to form granulocytes (e.g., neutrophils) that have a reduced ability to kill cancer cells, and that have a higher surface potential than otherwise identical hematopoietic cells.

[0190] In one embodiment, hematopoietic cells that do not meet the screening criteria (eg, do not have a higher surface potential and / or have a surface potential defined by electrophoretic mobility less than 2.0 μm.cm / volt.sec) are discarded.

[0191] Additionally, or alternatively, the surface potential of the hematopoietic cells can be measured and selected if they have a surface potential defined by an electrophoretic mobility of less than 1.0 μm.cm / volt.sec (preferably, less than 1.25 μm.cm / volt.sec, more preferably, less than 1.5 μm.cm / volt.sec or 1.75 μm.cm / volt.sec); and / or a higher surface potential than otherwise identical hematopoietic cells that differentiate to form granulocytes (e.g., neutrophils) that have a reduced ability to kill cancer cells. Thus, the invention encompasses hematopoietic cells that have a surface potential defined by an electrophoretic mobility of less than 1.0 μm.cm / volt.sec (preferably, less than 1.25 μm.cm / volt.sec, more preferably, less than 1.5 μm.cm / volt.sec or 1.75 μm.cm / volt.sec); and / or that differentiate to form granulocytes (e.g., neutrophils) that have a reduced ability to kill cancer cells, and that have a higher surface potential than otherwise identical hematopoietic cells.

[0192] In one embodiment, hematopoietic cells that do not meet the screening criteria (eg, do not have a higher surface potential and / or have a surface potential defined by electrophoretic mobility less than 1.0 μm.cm / volt.sec) are discarded.

[0193] The term "higher surface potential" means a more positive surface charge.

[0194] "Reduced ability to kill cancer cells" can be determined experimentally by testing two or more granulocytes (e.g., neutrophils) under the same experimental conditions and comparing the concentration / amount of cancer cells killed. "Reduced ability to kill cancer cells" can be determined using the methods described herein or CKA assay. In one embodiment, "reduced ability to kill cancer cells" means that the cells kill 10% or 20% fewer cancer cells than the cells of the invention (i.e., cells that have "cancer cell-killing ability" as defined herein). Preferably, "reduced ability to kill cancer cells" means that the cells kill 5% fewer cancer cells or fewer cancer cells than the cells of the invention.

[0195] Thus, according to this method, appropriate values ​​of hematopoietic cell surface potential (allowing for the selection of hematopoietic cells that can be differentiated into granulocytes suitable for use in the treatment of cancer) can be empirically determined based on correlations between the surface potentials of granulocytes obtainable therefrom and / or based on their ability to kill cancer cells.

[0196] In a related aspect, the invention provides a method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. Granulocytes (e.g., neutrophils) available from a donor i. a surface potential defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec (or at least 1.0 μm.cm / volt.sec); and ii. Ability to kill cancer cells measuring the surface potential of hematopoietic cells obtainable from said donor; and b. Using the measured surface potential in a method for selecting hematopoietic cells The present invention provides a method comprising:

[0197] A "method for selecting hematopoietic cells" may refer to selecting hematopoietic cells from the same donor from which the surface potential measurements were taken, or from a different donor.

[0198] In one aspect, the present invention provides an in vitro method for obtaining hematopoietic cells suitable for use in the treatment of cancer, comprising: a. measuring the cell surface charge of granulocytes obtainable from a donor; and b. Obtaining hematopoietic cells from a sample derived from said donor, where said granulocytes have a higher positive cell surface charge when compared to control granulocytes. The present invention provides a method comprising:

[0199] The control granulocytes may have a negative, positive or neutral charge. Preferably, the control granulocytes have a positive charge. Preferably, the control granulocytes are granulocytes that do not kill cancer cells (e.g., do not kill at least 5% of cancer cells in the method described herein). The control granulocytes are preferably obtained from a donor different from the donor in step a.

[0200] In one embodiment, the method includes obtaining hematopoietic cells from a sample derived from the donor when the granulocytes have a cell surface charge that is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% more positively charged compared to control granulocytes.

[0201] In another aspect, the invention provides an in vitro method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. measuring the cell surface charge of hematopoietic cells obtainable from a donor; and b. selecting the hematopoietic cells as suitable for use in the treatment of cancer if the hematopoietic cells have a higher positive cell surface charge when compared to a control hematopoietic cell. The present invention provides a method comprising:

[0202] The cell surface charge of the control granulocytes or hematopoietic cells can be determined prior to carrying out the method of the invention or simultaneously (preferably simultaneously) with carrying out the method of the invention.

[0203] For example, in one embodiment, the method comprises: a. Measuring the cell surface charge of granulocytes or hematopoietic cells obtainable from a donor; b. Measuring the cell surface charge of control granulocytes or hematopoietic cells; and c. obtaining hematopoietic cells from a sample derived from the donor if the granulocytes have a higher positive cell surface charge compared to the control granulocytes; or selecting the hematopoietic cells as suitable for use in the treatment of cancer if the hematopoietic cells have a higher positive cell surface charge compared to the control hematopoietic cells. Includes.

[0204] In some embodiments, the method may involve the use of multiple different test samples that include granulocytes from additional donors (eg, a second, third, fourth donor, etc.).

[0205] The control hematopoietic cells may have a negative, positive or neutral charge. Preferably, the control hematopoietic cells have a positive charge. Preferably, the control hematopoietic cells are hematopoietic cells that do not differentiate into granulocytes that kill cancer cells (e.g., the hematopoietic cells do not differentiate into granulocytes that kill at least 5% of cancer cells in the methods described herein). The control hematopoietic cells are preferably obtained from a donor different from the donor in step a.

[0206] In one embodiment, the method includes obtaining hematopoietic cells from a sample derived from the donor when the hematopoietic cells have a cell surface charge that is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% more positively charged compared to control hematopoietic cells.

[0207] In one aspect, an in vitro method for selecting hematopoietic cells suitable for use in the treatment of cancer is provided, comprising: a. measuring the concentration of granulocytes having a positive cell surface charge in a sample obtainable from a donor; and b. Obtaining hematopoietic cells from a sample derived from a donor if the concentration of said granulocytes having a positive cell surface charge is greater than the concentration of granulocytes having a positive cell surface charge in an otherwise identical control sample derived from a different donor. A method is provided that includes:

[0208] The control sample referred to may be a sample derived from a donor that has granulocytes that do not kill cancer cells (e.g., granulocytes that do not kill at least 5% of cancer cells in the methods described herein).

[0209] In one embodiment, the method comprises obtaining hematopoietic cells from a sample derived from a donor when the concentration of granulocytes having a positive cell surface charge is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% higher than the concentration of granulocytes having a positive cell surface charge in an otherwise identical control sample derived from a different donor. Preferably, the concentration of granulocytes having a positive cell surface charge is at least 50% higher than the concentration of granulocytes having a positive cell surface charge in an otherwise identical control sample derived from a different donor.

[0210] In another aspect, there is provided an in vitro method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. measuring the concentration of hematopoietic cells having a positive cell surface charge in a sample obtainable from a donor; and b. selecting the hematopoietic cells as suitable for use in the treatment of cancer if the concentration of hematopoietic cells having a positive cell surface charge is greater than the concentration of hematopoietic cells having a positive cell surface charge in an otherwise identical control sample from a different donor. A method is provided that includes:

[0211] The concentration of granulocytes or hematopoietic cells having a positive cell surface charge in an otherwise identical control sample from a different donor can be determined prior to carrying out the method of the invention or simultaneously (preferably simultaneously) with carrying out the method of the invention.

[0212] For example, in one embodiment, the method comprises: a. Measuring the concentration of granulocytes or hematopoietic cells having a positive cell surface charge in a sample obtainable from a (first) donor; b. Measuring the concentration of granulocytes or hematopoietic cells with a positive cell surface charge in otherwise identical control samples from different donors; and c. obtaining hematopoietic cells from the sample from said (first) donor if the concentration of said granulocytes with a positive cell surface charge is greater than the concentration of granulocytes with a positive cell surface charge in an otherwise identical control sample; or selecting hematopoietic cells as suitable for use in the treatment of cancer if the concentration of said hematopoietic cells with a positive cell surface charge from said (first) donor is greater than the concentration of hematopoietic cells with a positive cell surface charge in an otherwise identical control sample. Includes.

[0213] In some embodiments, the method may involve the use of multiple different test samples that include granulocytes from additional donors (eg, a second, third, fourth donor, etc.).

[0214] The referenced control sample may be a sample derived from a donor having hematopoietic cells that do not differentiate into granulocytes that kill cancer cells (e.g., said hematopoietic cells do not differentiate into granulocytes that kill at least 5% of cancer cells in the methods described herein).

[0215] In one embodiment, the method comprises obtaining hematopoietic cells from a sample derived from a donor when the concentration of hematopoietic cells having a positive cell surface charge is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% greater than the concentration of hematopoietic cells having a positive cell surface charge in an otherwise identical control sample derived from a different donor. Preferably, the concentration of hematopoietic cells having a positive cell surface charge is at least 50% greater than the concentration of hematopoietic cells having a positive cell surface charge in an otherwise identical control sample derived from a different donor.

[0216] In one aspect, an in vitro method for selecting hematopoietic cells suitable for use in the treatment of cancer is provided, comprising: a. measuring the cell surface charge of granulocytes obtainable from a first donor; b. identifying obtainable granulocytes from said first donor that have a higher positive cell surface charge when compared to control granulocytes; c. determining the concentration of the granulocytes identified in step b.; d. comparing the concentration of granulocytes measured in step c. with a concentration of granulocytes obtainable from a second (or additional) donor, wherein the granulocytes from the second (or additional) donor have a higher positive cell surface charge when compared to control granulocytes; and e. Obtaining hematopoietic cells from a sample derived from said first donor if the comparison identifies a higher concentration of said granulocytes obtainable from said first donor as compared to the concentration of said granulocytes obtainable from said second (or further) donor. A method is provided that includes:

[0217] In one embodiment, the granulocytes obtainable from the first donor and the granulocytes obtainable from the second donor compared in step d. have equal cell surface charges.

[0218] The control granulocytes may have a negative, positive or neutral charge. Preferably, the control granulocytes have a positive charge. Preferably, the control granulocytes are granulocytes that do not kill cancer cells (e.g., do not kill at least 5% of cancer cells in the method described herein). The control granulocytes are preferably obtained from a donor different from the donor in step a.

[0219] In one embodiment, the granulocytes obtainable from the first donor have a cell surface charge that is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% more positively charged compared to control granulocytes.

[0220] In one embodiment, the comparison identifies a concentration of said granulocytes obtainable from said first donor that is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% higher than the concentration of said granulocytes obtainable from said second (or further) donor, preferably at least 50% higher.

[0221] In one aspect, an in vitro method for selecting hematopoietic cells suitable for use in the treatment of cancer is provided, comprising: a. measuring the cell surface charge of hematopoietic cells obtainable from a first donor; b. identifying hematopoietic cells obtainable from said first donor that have a higher positive cell surface charge when compared to control hematopoietic cells; c. determining the concentration of the hematopoietic cells identified in step b.; d. comparing the concentration of hematopoietic cells measured in step c. with a concentration of hematopoietic cells obtainable from a second (or additional) donor, wherein the hematopoietic cells derived from the second (or additional) donor have a higher positive cell surface charge when compared to control hematopoietic cells; and e. selecting hematopoietic cells obtainable from said first donor as suitable for use in treating cancer if the comparison identifies a higher concentration of said hematopoietic cells obtainable from said first donor as compared to the concentration of said hematopoietic cells obtainable from said second (or further) donor. A method is provided that includes:

[0222] In one embodiment, the hematopoietic cells obtainable from the first donor and the hematopoietic cells obtainable from the second donor compared in step d. have equal cell surface charges.

[0223] The cell surface charge of the control granulocytes or hematopoietic cells can be determined prior to carrying out the method of the invention or simultaneously (preferably simultaneously) with carrying out the method of the invention.

[0224] The control hematopoietic cells may have a negative, positive or neutral charge. Preferably, the control hematopoietic cells have a positive charge. Preferably, the control hematopoietic cells are hematopoietic cells that do not differentiate into granulocytes that kill cancer cells (e.g., the hematopoietic cells do not differentiate into granulocytes that kill at least 5% of cancer cells in the methods described herein). The control hematopoietic cells are preferably obtained from a donor different from the donor in step a.

[0225] In one embodiment, the hematopoietic cells obtainable from the first donor have a cell surface charge that is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% more positively charged compared to control hematopoietic cells.

[0226] In one embodiment, the comparison identifies a concentration of said hematopoietic cells obtainable from said first donor that is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% higher than the concentration of said hematopoietic cells obtainable from said second (or further) donor, preferably at least 50% higher.

[0227] In some embodiments, the additional donor may be a third, fourth, fifth or sixth donor.

[0228] In one embodiment, measuring the cell surface charge comprises contacting the granulocytes or hematopoietic cells with a positively charged means and a negatively charged means (e.g., nanoparticles), wherein preferential binding of the granulocytes or hematopoietic cells to the positively charged means indicates that the cell surface is negatively charged, and preferential binding of the granulocytes or hematopoietic cells to the negatively charged means indicates that the cell surface is positively charged.

[0229] In one embodiment, a granulocyte or hematopoietic cell has a positive cell surface charge if a negatively charged means can bind to it and a positively charged means cannot bind to it. In one embodiment, a granulocyte or hematopoietic cell has a negative cell surface charge if a positively charged means can bind to it and a negatively charged means cannot bind to it.

[0230] Such negatively and / or positively charged means can also be used to measure the concentration of granulocytes or hematopoietic cells in a sample.

[0231] The positively charged means may be a positively charged particle, nanoprobe or nanoparticle, or a cation exchange medium.

[0232] In one aspect, the invention includes isolating granulocytes or hematopoietic cells comprising a (higher) positive cell surface charge by means of said charge. For example, said cells can be isolated using negatively charged means such as negatively charged particles, nanoprobes or nanoparticles, or anion exchange media. Such techniques can be used to measure the cell surface charge of granulocytes or the concentration of granulocytes with a positive cell surface charge in said embodiments.

[0233] The cells can be isolated from negatively charged, neutrally charged, or weakly positively charged granulocytes or hematopoietic cells.

[0234] In one embodiment, the positively or negatively charged means (e.g., nanoparticles) may be detectable by fluorescence. In another embodiment, the positively or negatively charged means (e.g., nanoparticles) may be captured magnetically, thus allowing the isolation of cells that interact with said means.

[0235] Suitable nanoparticles are superparamagnetic iron(II,III) oxide (Fe 3 O 4) nanoparticles (NPs) were conjugated with (3-aminopropyl)triethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) and ammonium hydroxide (NH 4 Silicon dioxide (SiO OH) is deposited on the surface of the NPs upon reaction with 2 Fluorescein isothiocyanate (FITC) can be prepared by forming a thin layer of a SiO 2 Thus, the hydroxyl groups (SiO 2 Branched poly(ethyleneimine) (PEI) molecules can be used to bond SiO 2 in a non-covalent manner, exposing SiO 2 -OH groups and creating a negative surface charge. 2 In addition to covering the --OH groups, additional amine groups that carry a positive charge can also be exposed.

[0236] Thus, in one embodiment, the negatively charged nanoparticles are Fe 3 O 4 The nanoparticles were conjugated with APTES and then reacted with tetraethyl orthosilicate (TEOS) and ammonium hydroxide (NH 4 Upon reaction with OH, SiO 2 Forming a thin layer of the shell, and SiO 2 Embedding FITC in the shell, thus SiO 2 It is prepared by exposing --OH groups (creating a negative surface charge).

[0237] In another embodiment, positively charged nanoparticles are prepared by contacting negatively charged nanoparticles (as described herein) with PEI molecules (e.g., to expose additional amine groups that carry a positive charge).

[0238] In one embodiment, the negatively charged means (e.g., nanoparticles) may have a negative surface charge of at least -5mV, -10mV, -20mV, -30mV, or -40mV. Preferably, the negatively charged means (e.g., nanoparticles) may have a negative surface charge of at least -35mV.

[0239] In one embodiment, the positively charged means (e.g., nanoparticles) may have a positive surface charge of at least +5mV, +10mV, +20mV, +30mV, or +40mV. Preferably, the positively charged means (e.g., nanoparticles) may have a positive surface charge of at least +35mV.

[0240] The surface charge of the positively or negatively charged means (e.g., nanoparticles) may refer to the surface zeta potential of the positively or negatively charged means (e.g., nanoparticles). Surface zeta potential can be measured using a dynamic light scattering particle size analyzer (e.g., Zetasizer Nano-ZS90, Malvern, UK).

[0241] It is surprising that cell surface charge can be used to select granulocytes (which have high cancer killing activity) and related hematopoietic cells. For 60 years it has been supported that all mammalian cells are negatively charged and that this is consistent for the same cell type across individuals. While not wishing to be bound by theory, the inventors believe that the currently accepted theory that granulocytes are positive arises from limitations of the techniques used to analyze cell surface charge (e.g., missing soluble ions such as Na+, Ca+). The inventors believe that the positive charge on granulocytes can be provided by human neutrophil peptides (HNPs), the most abundant proteins in human neutrophils. HNPs contain 20-40 amino acids rich in arginine, lysine, and cysteine, making the peptides positively charged and are referred to as "cationic peptides." HNPs have an amphipathic folded rod-like structure with one side hydrophobic and the other hydrophilic and positively charged. The primary target of HNPs is believed to be the negatively charged lipid bilayer membrane on cancer cells. The activation mechanism of HNPs is the cleavage of a negatively charged leader peptide that neutralizes the positive charge in the peptide precursor. On target cells, mature amphipathic HNPs exert two main effector actions. First, HNPs form a barrel-like pore on the plasma membrane of the target cells. The hydrophobic side of the monomeric HNPs aligns against the hydrophobic portion of the target bilayer membrane, while the hydrophilic side of the HNPs forms a hydrophilic pore with other similarly aligned HNPs. This pore causes the swelling and rupture (cytolysis) of the target cancer cells. Second, once HNPs enter the cytoplasm of the target cells containing mitochondria, they can bind to and neutralize the negatively charged outer mitochondrial membrane, resulting in the loss of mitochondrial transmembrane potential, a well-known mechanism for inducing rapid apoptosis. The inventors therefore believe that granulocytes with a higher positive cell charge (eg, neutrophils) contain more HNPs and therefore have greater cancer killing activity.

[0242] In one embodiment, the granulocytes obtained by the method can be functionally assayed by a cancer killing assay or methods described herein.

[0243] The cells described herein may be part of a cell culture (e.g., an in vitro cell culture). The cell culture may include multiple different cell types (e.g., hematopoietic cells, or other than granulocytes).

[0244] In one embodiment, the cell culture is an in vitro cell culture of hematopoietic cells. The cell culture may be enriched for hematopoietic cells that differentiate to form granulocytes characterized by a surface potential defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec (or at least 1.0 μm.cm / volt.sec); and the ability to kill cancer cells. Alternatively, or in addition, the cell culture may be enriched for hematopoietic cells that differentiate to form granulocytes that have a density of at least 1.077 g / ml; and the ability to kill cancer cells. Alternatively, or in addition, the cell culture may be enriched for hematopoietic cells that differentiate to form granulocytes that have the expression or activity of toll-like receptors; and / or the absence or inactivity of expression of programmed cell death 1 (PD-1) receptors; CD115; CD224; CXCR1; and / or CXCR2; and the ability to kill cancer cells.

[0245] In one embodiment, the term "enriched for hematopoietic cells" means that the hematopoietic cells described herein (e.g., selected by the methods of the present invention) account for at least 70%, 75%, 80%, 85%, 90% or 95% of the total hematopoietic cells (preferably, total cells) contained in the cell culture.

[0246] In one embodiment, the term "enriched for hematopoietic cells" refers to (i) a surface potential defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec (or at least 1.0 μm.cm / volt.sec); or (ii) a density of at least 1.077 g / ml; or (iii) expression or activity of a toll-like receptor; and / or the absence or inactivity of the programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2; and This means that hematopoietic cells that differentiate to form granulocytes characterized by the ability to kill cancer cells account for at least 70%, 75%, 80%, 85%, 90% or 95% of all hematopoietic cells (preferably, all cells) contained in the cell culture.

[0247] In another embodiment, the cell culture is an in vitro cell culture of granulocytes. The granulocytes can be obtained by differentiating the hematopoietic cells of the present invention. The in vitro cell culture of granulocytes can be enriched for granulocytes having a surface potential defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec (or at least 1.0 μm.cm / volt.sec); and the ability to kill cancer cells. Alternatively, or in addition, the cell culture can be enriched for granulocytes having a density of at least 1.077 g / ml; and the ability to kill cancer cells. Alternatively, or in addition, the cell culture can be enriched for granulocytes having expression or activity of toll-like receptors; and / or the absence or inactivity of expression of programmed cell death 1 (PD-1) receptors; CD115; CD224; CXCR1; and / or CXCR2; and the ability to kill cancer cells.

[0248] In one embodiment, the term "enriched for granulocytes" means that the granulocytes described herein (e.g., selected by the methods of the present invention) account for at least 70%, 75%, 80%, 85%, 90% or 95% of the total granulocytes (preferably, total cells) contained in the cell culture.

[0249] In one embodiment, the term "enriched for granulocytes" refers to (i) a surface potential defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec (or at least 1.0 μm.cm / volt.sec); or (ii) a density of at least 1.077 g / ml; or (iii) expression or activity of a toll-like receptor; and / or the absence or inactivity of the programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2; and This means that the granulocytes capable of killing cancer cells account for at least 70%, 75%, 80%, 85%, 90% or 95% of the total granulocytes (preferably, the total cells) contained in the cell culture.

[0250] The cells, cell cultures or pharmaceutical compositions of the invention can be subjected to one or more further processing steps, such as cryogenic freezing. Further processing steps may include mixing the cells, cell cultures or pharmaceutical compositions with a storage medium, e.g., a cryopreservation medium.

[0251] The invention may further comprise depositing the cells, cell cultures, or pharmaceutical compositions of the invention in a cell bank, thus providing in a related aspect a cell bank comprising the cells, cell cultures, or pharmaceutical compositions. As used herein, the term "cell bank" refers to a storage facility that maintains cells under conditions favorable for cell survival. For example, cells can be stored in a metabolically dormant state (e.g., cryogenically frozen). Suitably, cells contained within a cell bank are classified for appropriate recovery (e.g., based on blood type, and / or human leukocyte antigen (HLA) type). In one embodiment, cells can be classified based on the type of cancer they (or cells differentiated from) kill. If the cell bank is a granulocyte cell bank, the cell bank can be supplemented using hematopoietic cells of the invention. In some embodiments, hematopoietic cells or granulocytes obtained from a donor can be stored and then administered to the donor (e.g., if the donor is diagnosed with cancer), thus constituting personalized medicine.

[0252] The cells or cell cultures of the present invention can be formulated in any suitable manner based on their downstream application (eg, storage in a cell bank, or use in therapy).

[0253] Thus, one aspect of the invention provides a cell bank comprising an in vitro cell culture of hematopoietic cells or an in vitro cell culture thereof, granulocytes or an in vitro cell culture thereof, or a pharmaceutical composition of the invention.

[0254] In one embodiment, the cells or cell cultures of the invention are formulated as a pharmaceutical composition comprising the cells or cell cultures of the invention and granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), growth hormone; serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, fetal bovine serum (FBS), or a combination thereof.

[0255] In one embodiment, the cells or cell cultures of the invention are formulated as a pharmaceutical composition comprising the cells or cell cultures of the invention and granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF), and growth hormone, and serotonin, and vitamin C, and vitamin D, and glutamine (Gln), and arachidonic acid, and AGE-albumin, and interleukin, and TNF-alpha, and Flt-3 ligand, and thrombopoietin, and fetal bovine serum (FBS).

[0256] In one embodiment, the cell, cell culture or pharmaceutical composition is formulated with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that can be administered to a subject (e.g., a patient) intravenously, intraarterially, intraperitoneally, intratumorally, intrathecally or a combination thereof (preferably intravenously) without causing harm to the subject. Thus, in one embodiment, the pharmaceutically acceptable carrier is an injectable carrier, such as a sterile saline solution.

[0257] The present invention provides cells, cell cultures, pharmaceutical compositions, and kits for use in medicine.For example, the present invention provides hematopoietic cells, in vitro cell cultures of hematopoietic cells, granulocytes, in vitro cell cultures of granulocytes, pharmaceutical compositions, or kits for use as medicine.The medicine is particularly useful in the treatment of cancer.

[0258] In one embodiment, the cancer is a solid tumor cancer. The term "solid tumor cancer" refers to an abnormal, malignant mass of tissue that does not contain cysts or liquid inclusions. Examples of solid tumor cancer include carcinomas, sarcomas, and lymphomas.

[0259] The solid tumor cancer may be a carcinoma. The carcinoma may be selected from one or more of adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, or a combination thereof. The carcinoma may also be selected from epithelial neoplasms, squamous cell neoplasms, squamous cell carcinoma, basal cell neoplasms, basal cell carcinoma, transitional cell carcinoma, adenocarcinoma (adenocarcinoma not otherwise specified (NOS), fibrosarcoma plastica, vipoma, cholangiocarcinoma, hepatocellular carcinoma NOS, adenoid cystic carcinoma, renal cell carcinoma, Grabitz tumor), adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic, mucinous and serous neoplasms, ductal, lobular and medullary neoplasms, acinar cell neoplasms, or mixed epithelial neoplasms.

[0260] Alternatively, the solid tumor cancer may be a sarcoma. The sarcoma may be selected from Askin's tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, or soft tissue sarcoma (such as alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans (DFSP), desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, malignant fibrous histiocytoma, undifferentiated pleomorphic sarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma).

[0261] Alternatively, the solid tumor may be a lymphoma, such as a B cell lymphoma, a T cell lymphoma, a NK cell lymphoma, or a Hodgkin's lymphoma.

[0262] In one embodiment, the in vitro cell culture of hematopoietic cells, hematopoietic cells, in vitro cell culture of granulocytes, granulocytes, pharmaceutical composition or kit of the invention is for use in the treatment of one or more of pancreatic cancer, liver cancer, esophageal cancer, gastric cancer, cervical cancer, ovarian cancer, lung cancer, bladder cancer, kidney cancer, brain tumor, prostate cancer, myeloma cancer, non-Hodgkin's lymphoma (NHL), laryngeal cancer, uterine cancer, or breast cancer.

[0263] Preferably, the in vitro cell culture of hematopoietic cells, hematopoietic cells, in vitro cell culture of granulocytes, granulocytes, pharmaceutical composition or kit of the present invention is for use in treating pancreatic cancer. The pancreatic cancer may be a pancreatic solid tumor cancer, such as pancreatic adenocarcinoma (e.g., pancreatic ductal adenocarcinoma).

[0264] References herein to "cancer cells" (e.g., in the context of "the ability to kill cancer cells") may refer to cancer cells of any of the above cancers. Suitably, the "cancer cells" may be solid tumor cancer cells, such as pancreatic cancer cells.

[0265] In one aspect, the present invention provides a method of treating cancer, comprising: a. Obtaining granulocytes or hematopoietic cells by the methods described herein; and b. Administering said granulocytes or hematopoietic cells to a subject. The present invention provides a method comprising:

[0266] In some embodiments, the hematopoietic cells can be differentiated into granulocytes prior to administration.

[0267] In one embodiment, the in vitro cell culture of hematopoietic cells, hematopoietic cells, in vitro cell culture of granulocytes, granulocytes, pharmaceutical composition or kit of the present invention is administered to a subject (e.g., a subject with cancer). Prior to administration, there may be a matching step between the agent (e.g., including the in vitro cell culture of hematopoietic cells, in vitro cell culture of granulocytes, or pharmaceutical composition of the present invention) and the subject to be treated. Matching may be based on data derived from a donor from which the hematopoietic cells or granulocytes are derived, and similar data obtained from the subject to be treated. Matching can be achieved based on blood type, human leukocyte antigen (HLA) type similarity, or a combination thereof.

[0268] A typical treatment regimen is 10 6 , 10 7 , 10 8 Or 10 9 cells, or up to 10 12 , 10 13 Or 10 14 In one embodiment, the treatment regimen may include administering at least 1x10 cells to the subject. 9 Preferably, the treatment regimen comprises administering to the subject a dose of at least 2x10 cells. 9 cells or at least 5x10 9 In one embodiment, the treatment regimen may include administering a dose of at least 1x10 cells to the subject. 10 cells or at least 5x10 10 The method may include administering a dose of at least 1x10 cells to the subject.11 Pieces or at least 2x10 11 In some embodiments, 1x10 cells may be administered to a subject. 9 ~3x10 11 Pieces or 1x10 10 ~3x10 11 Preferably, 5x10 cells are administered to the subject. 10 ~2.5x10 11 The cells are administered to the subject.

[0269] The subject for treatment can be administered once, twice, three times, four times, five times, or six times per week. Alternatively, the subject can be administered daily (e.g., once or twice a day). In other embodiments, the subject can be administered once a week or once every two weeks. Preferably, administration is weekly. Those skilled in the art will understand that dosage can be adjusted based on the needs of the subject and the efficacy of the drug. For example, if the efficacy of the drug is high, the dosage can be reduced.

[0270] In one embodiment, a subject for treatment has at least 2x10 9 cells or at least 2x10 10 The cells are administered weekly (e.g., once a week). Preferably, at least 1x10 cells are administered to a subject for treatment. 11 Pieces or at least 2x10 11 The cells can be administered weekly.

[0271] Treatment period can be changed based on the response of the subject to treatment and / or the type and / or severity of cancer.For example, the subject for treatment can be administered for at least 1 or 2 weeks.Preferably, the subject for treatment can be administered for at least 3 or 4 weeks.In one embodiment, the subject for treatment is administered for at least 5 or 6 weeks, preferably at least 7 or 8 weeks.

[0272] In one embodiment, subjects for treatment receive at least 2x10 9Preferably, subjects for treatment receive at least 2x10 cells, where the cells are administered once a week. 9 cells (preferably at least 2x10 10 pcs or 2x10 11 cells), where the cells are administered once a week.

[0273] Administration may be by any suitable technique or route, including, but not limited to, intravenous injection, intraarterial injection, intraperitoneal injection, injection into a tumor resection cavity, intrathecal injection, or a combination thereof. Preferably, the agent may be administered intravenously.

[0274] White blood cell growth factors can be administered together with the agents of the present invention. The administration can be sequential or simultaneous (preferably simultaneous). Suitable white blood cell growth factors can include granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), growth hormone; serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, fetal bovine serum (FBS), or combinations thereof. Suitably, the leukocyte growth factors may include granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), growth hormone, serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukins, TNF-alpha, Flt-3 ligand, thrombopoietin, and fetal bovine serum (FBS). Specific examples of the above include, but are not limited to, LEUKINE® brand sargramostim, NEUPOGEN® brand filgrastim, and NEULAST A® brand 5PEG-filgrastim.

[0275] When the agent is a hematopoietic cell (e.g., hematopoietic cell culture), the agent can be administered (e.g., sequentially or simultaneously, preferably simultaneously) with granulocyte-colony stimulating factor; and growth hormone; and serotonin; and interleukin. In one embodiment, granulocyte progenitor cells (e.g., granulocyte progenitor cell culture) are administered (e.g., sequentially or simultaneously, preferably simultaneously) with granulocyte-colony stimulating factor; and growth hormone; and serotonin; and interleukin.

[0276] The present invention also provides a kit comprising an in vitro cell culture of hematopoietic cells, granulocytes, an in vitro cell culture of granulocytes, or a pharmaceutical composition of the present invention; and instructions for its use in medicine. Suitably, the instructions may be for its use in treating cancer as described in any one of the above embodiments. In some embodiments, the instructions also detail a suitable dosing regimen (e.g., as described in the above embodiments). In one embodiment, the instructions are for the use of the kit in treating cancer, preferably pancreatic cancer.

[0277] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th ed., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0278] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numerical ranges are inclusive of the numbers defining the range.

[0279] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0280] Definitions of other terms may occur throughout this specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and as such may vary. It should also be understood that the terms used herein are for the purpose of describing only specific embodiments, since the scope of the present disclosure will be limited only by the appended claims.

[0281] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, rounded to two decimal places of the unit of the lower limit, is also specifically disclosed, unless the text clearly indicates otherwise. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either limit is included, neither limit is included, or both limits are included in the smaller range is also encompassed within the disclosure, and any limit in a stated range may be explicitly excluded. When a stated range includes one or both limits, ranges excluding either or both of these included limits are also included in the disclosure.

[0282] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "hematopoietic cells" includes a plurality of such candidate agents, reference to "hematopoietic cells" includes reference to one or more hematopoietic cells and equivalents thereof known to those skilled in the art, and so forth.

[0283] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and their inclusion herein should not be construed as an admission that such publications are prior art to the claims appended hereto.

[0284] The invention will now be described, by way of example only, with reference to the following figures and examples.

[0285] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0286] [Figure 1] Figure 1 shows the cytotoxicity results (MTT assay) of donor-derived neutrophils (DDNs) from different donors and at various ratios of effector to target cells. The difference in CKA levels between donors is maintained at higher effector:target cell ratios. Effector: DDNs; Target: HeLa cells. [Diagram 2] Figure 2 shows the cytotoxicity results of three CD34+ stem cell-derived neutrophil populations from different donors and at various ratios of effector to target cells. The results show that stem cell-derived neutrophils from different donors have differential CKA. Effector: SCDN; Target: HeLa cells and PANC-1 (pancreatic cancer). [Diagram 3]Figure 3 shows the cytotoxicity results (xCELLigence assay) of donor-derived neutrophils (DDNs) from different donors and at various ratios of effector to target cells. The difference in CKA levels between donors is maintained at higher effector:target cell ratios. Effector: DDNs; Target: HeLa cells. [Figure 4] Figure 4 shows the cytotoxicity results of fresh donor-derived neutrophils against different cancer cell types and at various ratios of effector to target cells. The results show that DDNs derived from different donors have differential CKA, with higher CKA against pancreatic cancer cells. Effector: DDN; Target: HeLa cells (cervical cancer) and PANC-1 cells (pancreatic cancer). [Diagram 5] Figure 5 shows the selective cytotoxicity of donor-derived neutrophils for cancer cell types compared to non-cancer cells and various ratios of effector to target cells. The results show that DDNs that kill cancer cells have minimal effects on non-cancer cells, confirming the selectivity. Effector: DDN; Target: HeLa cells (cervical cancer) and PANC-1 cells (pancreatic cancer) and MCF-12F (non-cancer cells, normal breast epithelium). [Figure 6] Figure 6 shows the cytotoxicity results of CD34+ stem cell-derived neutrophil populations (derived from umbilical cord blood stem cells) derived from five different cultures and at various ratios of effector to target cells. The results were generated using the MTT assay and show that ex vivo generated neutrophils have differential CKA. Effector: SCDN; Target: HeLa cells. [Figure 7] Figure 7 shows the cytotoxicity results of three CD34+ stem cell-derived neutrophil populations against different cancer cell types and at various ratios of effector to target cells. The results show that SCDNs derived from different donors have differential CKA, with higher CKA against pancreatic cancer cells. Effector: SCDN; Target: HeLa cells (cervical cancer) and PANC-1 cells (pancreatic cancer). [Figure 8]Figure 8 shows the cytotoxicity results of three CD34+ stem cell-derived neutrophil populations from different donors and at various ratios of effector to target cells. The results show that stem cell-derived neutrophils from different donors have differential CKA. Effector: SCDN; Target: HeLa cells (cervical cancer) and PANC-1 cells (pancreatic cancer). [Figure 9] Figure 9 shows the cytotoxicity results of three CD34+ stem cell-derived neutrophil populations derived from different donors (LC267, LC268, LC269) and at various ratios of effector to target cells. The results show that stem cell-derived neutrophils derived from different donors have selective cytotoxicity. Effector: SCDN; Target: HeLa cells (cervical cancer), PANC-1 cells (pancreatic cancer) and MCF-12F cells (non-cancerous cells, normal breast epithelium). [Figure 10] Figure 10 shows the cytotoxicity results of three CD34+ stem cell-derived neutrophil populations derived from different donors (LC252, LC253, LC254) and at various ratios of effector to target cells. The results show that stem cell-derived neutrophils derived from different donors have selective cytotoxicity. Effector: SCDN; Target: HeLa cells (cervical cancer), PANC-1 cells (pancreatic cancer) and MCF-12F cells (non-cancerous cells, normal breast epithelium). [Figure 11] Figure 11 shows the cytotoxicity results of three CD34+ stem cell-derived neutrophil cultures, together with the cytotoxicity results of donor-derived neutrophils from the same donor and at various ratios of effector to target cells.SCDN and DDN from the same donor have similar CKA levels.The similar CKA relationship between DDN and SCDN is maintained at various ratios of effector to target cells for donor LC253.Effector: SCDN and DDN; target: HeLa cells (cervical cancer). [Figure 12]Figure 12 shows the cytotoxicity results of three CD34+ stem cell-derived neutrophil cultures, together with the cytotoxicity results of fresh donor-derived neutrophils from the same donor and at various ratios of effector to target cells. SCDN and DDN have similar CKA levels. The same CKA relationship between DDN and SCDN is maintained at various ratios of effector to target cells for donor LC253. Effector: SCDN and DDN; target: HeLa cells (cervical cancer). [Figure 13] Figure 13 shows a comparison of cytotoxicity between natural and stem cell-derived neutrophils over time. Higher CKA (donor LC269) is maintained between DDN and SCDN. Effector: SCDN and DDN (three different SCDN and DDN cultures); Target: HeLa cells (cervical cancer). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0287] Example 1 Donor recruitment Donors are preselected based on the probability of having neutrophils that exhibit high levels of cancer-killing activity (CKA) in the CKA assay described in Example 2. The preselection criteria are: · You do not have a serious medical or psychiatric condition that would affect your ability to provide consent or sample collection; ·No personal or family history of cancer that is targeted for therapy; · No history of chemotherapy or radiotherapy in the 3 months prior to the sample collection date; ·Be between 18 and 24 years of age; Optionally, being male (without wishing to be bound by theory, it is believed that neutrophils from males exhibit the highest levels of CKA when tested in a CKA assay); and Optionally, blood type O or Rh- Includes.

[0288] White blood cells (WBCs) are collected by drawing approximately 18 ml of human blood from a donor. The blood is divided into three BD Vacutainer™ CPT tubes and centrifuged at 175xg 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 420xg for 5 minutes at 23°C and washed with 10 ml of Dulbecco's modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA) + 10% fetal bovine serum (FBS) (Sigma. St. Louis, MO). Cells are counted and 1.6x10 6 Resuspend in medium to a final concentration of cells / ml.

[0289] Example 2 Testing CKA of extracted granulocytes in the CKA assay Cells are cultured in DMEM + 10% FBS in T25 flasks until 80% confluency. Cell lines are cultured in T75cm 2 Cell culture flasks were incubated at 37°C, 8% CO in DMEM supplemented with the following components: 10% volume / volume FBS, penicillin (Sigma. St. Louis, MO), streptomycin (Sigma. St. Louis, MO), and L-glutamine supplement (Sigma. St. Louis, MO). 2and propagate and maintain it. Cultured pancreatic cancer cells (e.g., commercially available from American Type Culture Collection - United Kingdom (UK), Guernsey, Ireland, Jersey and Liechtenstein, LGC Standards, Queens Road, Teddington, Middlesex TW11 0LY, UK; 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™) are split and passaged in culture flasks until they reach 70% surface confluency.

[0290] Cells are trypsinized, harvested and counted using Trypan Blue. Assay plates (24 wells) contain 8x10 cells per well in 24 well flat bottom plates. 4 Seed 1000 pancreatic cancer cells (e.g., pancreatic ductal adenocarcinoma cells). Keep the plate in a 5% CO 2 The cells are incubated at 37° C. for 24 hours in 5% CO. The cells are labeled with 2.5 μM CellTracker™ Green for 45 minutes. Fresh medium is added to the cells and they are incubated at 37° C. for 24 hours in 5% CO. 2 Return to incubator.

[0291] 500 μl of MN cell suspension (8x10 5 Perform the CKA assay by adding 100 ng / ml of granulocytes (100 ng / ml) to each well in which pancreatic cancer cells have been grown for 24 h. Mix the cells and incubate for 24 h at 39 °C, 5% CO. 2After 24 hours of incubation, cells are harvested by trypsinization and centrifugation. Cells are resuspended in 100 μl of cold phosphate buffered saline (PBS) and then 125 μl of 0.4% Trypan Blue is added. Cells are counted under a microscope (using phase contrast and fluorescence microscopes).

[0292] Granulocytes (e.g., neutrophils) capable of killing at least 70% or at least 80% of cancer cells in this assay (i.e., having at least 70% or 80% CKA, respectively) are believed to be particularly suitable for use in treating cancer.

[0293] Example 3 Examination of the surface potential of hematopoietic cells and neutrophils Electrophoresis is used to investigate changes in surface potential in hematopoietic cells (e.g., hematopoietic stem and / or progenitor cells) and neutrophils by measuring electrophoretic mobility. Suspended cells are collected from cultures by mechanical detachment and collection from the culture substrate. The collected cells are redistributed in an electrophoresis buffer solution containing 10 mM Tris-HCl and 291 mM glucose and introduced into a square glass electrophoresis chamber. 200 V DC is applied across the electrophoresis chamber. The electrophoretic velocity, u, of the cells is measured by recording the time required for the cells to pass through a fixed length at 3 mA under a microscope equipped with a CCD camera. The electrophoretic mobility, μ, is calculated by μ=ugS / I, where g is the conductivity of the medium, S is the cross-sectional area of ​​the electrophoresis chamber, and I is the current. For each condition, typically at least nine readings are taken to calculate the electrophoretic mobility of the cells.

[0294] Example 4 Extraction of hematopoietic stem cells from peripheral blood Upon giving consent, the donor is given granulocyte-colony stimulating factor (G-CSF) and / or granulocyte-macrophage colony stimulating factor (GM-CSF), e.g., Neupogen® (commercially available from Amgen Inc. USA), to aid in the recovery of peripheral hematopoietic stem cells while minimizing possible discomfort to the donor. To identify long-term persisting pluripotent stem cells, cell surface polypeptide markers are used. Suitably, the markers are CD34 + , CD59 + , Thy1 + , CD38 low / - , C-kit - / low , and lin - may include:

[0295] Example 5 Hematopoietic cell expansion and differentiation Hematopoietic cells (e.g., hematopoietic stem cells) are stimulated using a supernatant growth factor suspension to generate more stem cells or differentiate into progenitor 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 Mar;6(3):296-313.

[0296] The protocol has four main steps: Hematopoietic cell culture and expansion; Short-term expansion of multipotent myeloid progenitor cells with high doses of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), 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, fetal bovine serum (FBS), or combinations thereof; and ·Directed differentiation of myeloid progenitor cells into neutrophils, eosinophils, dendritic cells (DCs), Langerhans cells (LCs), macrophages and osteoclasts It consists of:

[0297] Example 6 Preparation of cell banks Hematopoietic stem cells, granulocyte progenitor cells and granulocytes obtainable therefrom are cryogenically frozen and stored in appropriate cell banks.

[0298] Example 7 Use in patients for the treatment of solid tumors Stored hematopoietic cells (e.g., hematopoietic stem cells or granulocyte progenitor cells obtainable therefrom), and granulocytes (e.g., neutrophils) differentiated therefrom, are matched to cancer patients based on the patient's cancer type, blood type (ABO, rh, and HLA), and / or genetics. Patients may also be matched based on human leukocyte antigen (HLA) similarity.

[0299] Patients are treated with: ·IV infusion of hematopoietic cells (including hematopoietic stem cells and granulocyte progenitor cells) into the patient along with granulocyte-colony stimulating factor, human growth hormone, serotonin, and interleukins; or IV infusion of stimulated granulocyte progenitor cells (which may be derived from hematopoietic stem cells) into a patient, which cells are believed to spontaneously differentiate into granulocytes (e.g., neutrophils) with high CKA in an in vivo CKA assay; or Direct IV infusion of granulocytes (e.g., neutrophils) with high CKA in the CKA assay that have been differentiated from hematopoietic cells (e.g., hematopoietic stem cells).

[0300] Typically, cells are dosed at 2x10 11 A cell dose of 100 mg / kg / day will be infused once a week for 8 weeks. The progress of therapy will be monitored and dosing will be adapted accordingly.

[0301] Example 8 Treatment of Patients with Pancreatic Cancer Mary is diagnosed with metastatic pancreatic ductal adenocarcinoma (PDAC) at age 69. Surgery is no longer an option (unresectable), gemcitabine is insufficient to prevent disease progression, and Abraxane or Forfirinox are not appropriate per her oncologist's recommendation due to side effects that would prevent her from enjoying what time she has left with her family. Mary's prognosis is 3-6 months of survival, making it unlikely that she will live long enough to see her expected soon-to-be-born grandchild.

[0302] Mary is encouraged to try Leukocyte Infusion Therapy (LIFT). To assess the potential suitability of the therapy, the hospital draws 20 ml of Mary's blood and sends it for analysis using a cancer-killing activity assay, which identifies the pancreatic cancer-killing activity of her granulocytes as less than 5%. Such a low reading indicates the inadequacy of her own innate immune system to fight off her cancer, which would kill her if the efficacy of her body's granulocytes was not improved.

[0303] Mary's patient notes and assay results are used to find a suitable cancer-killing granulocyte match. Mary's blood type is Type A. Mary's profile is processed using the cell database for the cell bank to identify suitable granulocytes (which show 70-90% cancer-killing activity (CKA) in the cancer-killing activity assay of Example 2 before cryogenic freezing). Cryogenic freezing of the granulocytes helps preserve the CKA, so the cells can be sent directly to the hospital (The Royal Marsden) without further testing. Mary is scheduled to visit that week for her first procedure. The hospital will store the cells appropriately. Mary will receive 2x10 cells with CKA on December 13th under strict supervision. 9 Mary receives her first infusion of 2x10 granulocytes. Mary is advised to return to the hospital in 3 days, where she undergoes an ultrasound scan, which shows significant tumor lysis and no signs of tumor lysis syndrome. The medical team determines that the granulocyte dose is 2x10 11It is decided to gradually increase it over three successive treatment sessions until it reaches

[0304] An ultrasound will be performed on January 17th; completed one week into the four week course of four treatments, observing complete tumor destruction and conversion to scar tissue, with successful healing occurring. 20ml of Mary's blood will be taken i) in parallel with the biopsy to assess the presence of metastatic cancer cells in Mary's blood (to confirm complete disappearance of the cancer); and ii) to test the cancer killing activity of Mary's granulocytes (to indicate risk of remission). Mary will have regular check-ups, monthly at first, then every six months.

[0305] Two years later, a new tumor is discovered in Mary's pancreas. Her doctors treat the tumor with radiation therapy and administer a single high dose of LIFT to ensure that any cancer cells that may be present in the blood are destroyed. Mary enjoys the life she and her family have regained and she has the opportunity to see growth.

[0306] After therapy, hematopoietic cells (e.g., hematopoietic stem cells) from the cell bank are stimulated to produce more granulocytes (with the desired CKA when tested using the assay of Example 2) to replenish the stock, thus ensuring a sufficient stock of granulocytes needed for similar patient situations.

[0307] Example 9 MTT "CKA Assay" MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow tetrazole that is positively charged and easily penetrates living eukaryotic cells. Live cells exhibiting active metabolism convert MTT to a purple formazan product (1(E,Z)-5-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan) by the NAD(P)H-dependent oxidoreductase mitochondrial enzyme, which exhibits a maximum absorbance near 570 nm. When cells die, they lose the ability to convert MTT to formazan, and thus the formation of color serves as a useful and convenient marker of only live cells. A solubilizing solution is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified by measuring it at a wavelength of 570 nm by a spectrophotometer. The absorbance at a reference wavelength of 690 nm is subtracted from the absorbance at 570 nm wavelength. Thus, the MTT assay was used to measure how many viable cells remain as a method to determine cancer killing activity (CKA), i.e., cytotoxicity against cancer cells.

[0308] Methods for preparing HeLa target cells Day 1: 1) HeLa cells (a robust type of cervical cancer cell line) were cultured and harvested when they reached logarithmic growth phase. 2) 10,000 HeLa cells (target cells) were added to each well of a 96-well flat-bottom plate in a final volume of 100 μL. 3) Target cells were left to adhere overnight, after which leukocytes (effector cells) were added, and all experimental conditions were set up in triplicate.

[0309] Day 2: 4) Effector cells were added to target cells at various ratios (eg, 1:1, 5:1, 10:1, 50:1 effector to target cell ratios). 5) The cells were left to stand and incubated at 37°C for 16 to 24 hours. 6) Target cells alone and target cells in the presence of Triton X were also plated in triplicate as controls for 0% and 100% cytotoxicity, respectively.

[0310] After the desired incubation time: 1) Wells were washed twice with PBS to remove effector cells and dead target cells. 2) MTT solution was prepared by dissolving the kit solution 10x with culture medium, i.e., per 100 wells, took 1000 μL (1 mL) of MTT stock (provided in the kit) and added 9000 μL (9 mL) of culture medium (RPMI-1640). 3) 100 μl of the MTT solution prepared in step 2 was added per well. 4) This was incubated for 4 hours. 5) The MTT solution was removed from all wells and 100 μl / well of solvent was added (provided in the kit). 6) Formazan crystals were dissolved by pipetting if necessary and plates were read at 570 and 690 nm. The background absorbance measured at 690 nm was subtracted from the absorbance measured at 570 nm.

[0311] Evidence for variable CKA in donor neutrophils 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 MTT assay described above at effector to target cell ratios of 1:1 and 5:1.

[0312] Figure 1 shows the cytotoxicity percentage recorded by MTT for different donors. There are differences between donors at ratios of 1:1 and 5:1. In conclusion, the MTT assay is able to demonstrate differences in CKA between neutrophils derived from different donors.

[0313] Example 10 Demonstration of CKA in stem cell-derived neutrophils Cultivation of neutrophils derived from CD34+ stem cells We cultured neutrophils derived from umbilical cord blood-derived stem cells expressing the CD34 protein using the protocol described by Timmins NE, 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.

[0314] The resulting cultures were tested for neutrophil content using CD11b+ and CD15 markers by fluorescence-activated cell sorting (FACS). We also assessed the production of reactive oxygen species (ROS), more specifically, superoxide anion (O), by use of the nitroblue tetrazolium (NBT) assay (commercially available kit and protocol from Sigma-Aldrich, catalog number 840W-1KT). 2 - ) production was also measured.

[0315] Because differences in ROS activity were found based on the age of stem cell-derived neutrophils (data not shown), we counted three stem cell batches on the same day for consistency / comparability. The results of FACS-derived counts of the percentage of CD11b+ and CD15+ cells are listed in Table 1.

[0316] [Table 1]

[0317] Demonstration of CKA in CD34+-derived neutrophils Stem cell-derived neutrophils (batches 008A, 709A and 915) were used as effector cells in a 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 Figure 2, which shows that CD34+ stem cell-derived neutrophils show cytotoxicity in the CKA assay of HeLa cells, and that the results differ between different donors. Batch 008A shows consistently lower cytotoxicity than batches 915 and 709A at effector to target ratios up to 10:1, despite being prepared at the same time as the other batches and cultured in the same way.

[0318] These results demonstrate that stem cells derived from different donors a) can be differentiated in vitro to produce neutrophils that exhibit cancer-killing capacity, and b) that this cancer-killing activity varies depending on the source donor.

[0319] This result supports the fact that innate immune system cancer-killing activity (CKA) varies from individual to individual, and that the same innate variation in CKA seen in neutrophils taken directly from donors by leukocyte cone is also seen in donor stem cells. By selecting donors who demonstrate high innate immune system cancer-killing activity and using their hematopoietic cells (i.e., hematopoietic stem cells) for ex vivo expansion and differentiation, a cell bank containing leukocytes with high cancer-killing activity for use in cancer treatment can be created.

[0320] Example 11 xCELLigence "CKA Assay" An ACEA Biosciences xCELLigence RTCA DP Analyzer system® was used, following the manufacturer's instructions. The xCELLigence System is a real-time cell analyzer that can continuously and dynamically monitor changes in cell phenotype without the use of labels by measuring electrical impedance. The system measures impedance using interdigitating gold microelectrodes integrated into the bottom of each well of a tissue culture E-Plate. The impedance measurements are displayed as Cell Index (CI) values ​​and provide quantitative information about the biological state of the cells, including viability. Impedance-based monitoring of cell viability correlates with cell number and MTT-based readouts. The dynamic aspect of the impedance-based cell viability measurements provides the necessary temporal information when neutrophils are used to induce cytotoxic effects. Notably, the xCELLigence System can also pinpoint the optimal time point when neutrophils achieve their maximal effect (if such data is desired), as indicated by the lowest CI value, in cytotoxicity and cell death assays. Typically, 6,000 cancer cells (HeLa or PANC-1) or healthy, non-cancerous cells (MCF-12F) are placed in the bottom of a 16-well plate (the system can read up to three plates simultaneously). For the first few hours after cells are added to the wells, the impedance increases rapidly. This is caused by cells falling out of suspension and depositing on the electrode, forming focal adhesions. If the initial number of cells added is low and there is empty space at the bottom of the well, the cells will proliferate, causing a gradual but steady increase in CI. When the cells reach confluence, the CI value reaches a plateau, reflecting the fact that the electrode surface area accessible to the bulk medium no longer changes. At this point, called the "normalization point", neutrophils are added (typically with a varying effector:target ratio). The percentage of cell lysis is determined by a simple formula: percentage of cell lysis = ((cell index) エフェクターなし -Cell index エフェクターあり ) / cell index エフェクターなし ) x 100.

[0321] Typically, assays were run for up to 70 hours and used to generate the results presented in Examples 12 to 20. The results presented in Examples 12 to 19 are the maximum % cell lysis achieved during the assay for each cell type.

[0322] The ratios shown in Figures 3-13 are effector (e.g., neutrophil) to target (e.g., cancer cell) ratios. Typically, neutrophil to cancer cell ratios of 5:1 or 10:1 were used.

[0323] Example 12 Evidence for variable CKA in donor-derived neutrophils Figure 3 shows the maximum cytotoxicity percentage recorded by the xCELLigence assay for different donors. The assay also shows differences between donors at 1:1 and 5:1 ratios (neutrophil to HeLa cell ratios). In conclusion, the xCELLigence assay can also demonstrate differences in CKA between neutrophils from different donors, which were consistent across various ratios of granulocytes to cancer cells.

[0324] The assay was carried out for up to 40 hours.

[0325] Example 13 Demonstration of CKA of donor-derived neutrophils on different cancer cell types Neutrophils isolated from five different donors were tested for CKA against both HeLa cells (cervical cancer) and PANC-1 cells (pancreatic cancer).

[0326] Figure 4 shows the maximum cytotoxicity percentage recorded by CKA assay (xCELLigence assay) for each cancer cell type and for different donors. The cytotoxicity percentage for pancreatic cancer cells was higher, which was surprising given that pancreatic cancer is typically one of the most difficult cancers to treat. Again, donor-derived neutrophils (DDN) from different donors were shown to have differential CKA.

[0327] Example 14 Demonstration of selectivity of donor-derived neutrophil CKA for cancer cells Neutrophils isolated from five different donors were tested for CKA against both HeLa cells (cervical cancer), PANC-1 cells (pancreatic cancer) as well as non-cancerous MCF-12F cells (normal breast epithelial cells).

[0328] Figure 5 shows the maximum cytotoxicity percentage recorded by CKA assay (xCELLigence assay) for neutrophils from each donor against HeLa and PANC-1 cancer cell lines and MCF-12F non-cancer cell line. Advantageously, DDN was highly selective for cancer cells and showed minimal effect on non-cancer cells.

[0329] Example 15 Cultivation of neutrophils derived from CD34+ stem cells Further results of culturing neutrophils derived from umbilical cord blood-derived stem cells are presented in Table 2, which shows that neutrophils can be generated from CD34+ hematopoietic stem cells isolated from umbilical cord blood. CD34+ is a hematopoietic stem cell marker. CD11b and CD15 are mature neutrophil markers.

[0330] [Table 2]

[0331] Example 16 Demonstration of CKA in CD34+ stem cell-derived neutrophils (SCDN) Results obtained by xCELLigence assay using an additional population of CD34+ stem cell-derived neutrophils (Figure 6) were consistent with those obtained by the MTT assay described above. SCDNs (generated ex vivo) were again shown to have differential CKA, with culture 5 showing low CKA neutrophils and culture 1 showing high CKA neutrophils.

[0332] Example 17 Demonstration of CKA of stem cell-derived neutrophils on different cancer cell types CD34+ stem cell-derived neutrophils isolated from three different donors were tested for CKA against both HeLa cells (cervical cancer) and PANC-1 cells (pancreatic cancer).

[0333] Figure 7 shows the maximum cytotoxicity percentage recorded by CKA assay (xCELLigence assay) for each cancer cell type and for donors LC267, LC268 and LC269. Similar to the results obtained for DDN (see Example 14), the cytotoxicity percentage for pancreatic cancer is higher than that observed for HeLa cells (at both effector to target cell ratios of 5:1 and 10:1). SCDN from different donors are also shown to have differential CKA. This assay is performed for up to 45 hours.

[0334] Similar results were obtained for SCDNs derived from donors LC252, LC253 and LC254 (Figure 8).

[0335] Example 18 Demonstration of selectivity of CKA of stem cell-derived neutrophils for cancer cells Neutrophils derived from CD34+ stem cells from three different donors were tested for CKA against both HeLa cells (cervical cancer), PANC-1 cells (pancreatic cancer) as well as non-cancerous MCF-12F cells (normal breast epithelial cells).

[0336] Figure 9 shows the maximum cytotoxicity percentage recorded by CKA assay (xCELLigence assay - performed for up to 45 hours) for donors LC267, LC268 and LC269 against each cancer and non-cancer cell type. Advantageously, SCDNs are highly selective for cancer cells and show minimal effects on non-cancer cells. Similar to Figure 3 showing DDNs from the same donor, SCDNs from donor LC269 have the highest CKA, LC268 is second, and LC267 shows the lowest CKA. Thus, it can be concluded that CKA is a genetically defined rather than an epigenetically defined trait.

[0337] Similar results were obtained for the SCDNs of donors LC252, LC253 and LC254 (Figure 10).

[0338] Example 19 Evidence that neutrophil CKA is genetically encoded Neutrophils isolated from three different donors (DDNs) as well as SCDNs derived from CD34+ stem cells of the same donors were tested for CKA.

[0339] Figure 11 shows the maximum cytotoxicity percentage recorded by CKA assay (xCELLigence assay - performed for up to 50 hours) on HeLa cells for donors LC252, LC253 and LC254 for DDN and SCDN. Surprisingly, SCDN showed high CKA similar to that of DDN from the same donor, again indicating that CKA is encoded at the genetic level. As in Figure 10, donor LC253 provided neutrophils (and SCDN) with the highest CKA, while donors LC252 and LC254 provided neutrophils (and SCDN) with lower CKA.

[0340] This indicates that donors found to have neutrophils with high CKA (e.g., DDN) can also be used as a source of CD34+ stem cells that can be differentiated into neutrophils with similarly high CKA (e.g., SCDN).

[0341] Similar results were obtained for SCDN from donors LC267, LC268 and LC269 (FIG. 12 - run for up to 45 hours).

[0342] Example 20 Evidence that SCDN kills cancer cells more quickly than DDN The CKA of SCDN and DDN of donors LC267, LC268, and LC269 was determined for periods up to 45 hours. The assay was performed according to Example 11.

[0343] Surprisingly, the results (Figure 13) show that SCDN kills cancer cells more rapidly than DDN from the same donor. SCDN from donor LC269 showed particularly rapid cancer killing efficacy, killing about 50% of cancer cells in about 18 hours (compared to 35% for DDN), with half-maximal killing by SCDN occurring within 10 hours (compared to negligible killing at this time for DDN).

[0344] Example 21 Isolation of high density neutrophils 10 ml of heparinized (20 U / ml) human blood is mixed with an equal volume of 3% dextran T500 in saline and incubated at room temperature for 30 min to allow sedimentation of red blood cells. A 50 ml conical polypropylene tube is prepared with 10 ml of sucrose 1.077 g / ml and the leukocyte-rich supernatant is slowly layered on top of the 1.077 g / ml sucrose layer before centrifugation at 400 x g for 30 min at room temperature without using the brake. High density neutrophils (HDN) appear in the pellet. Low density neutrophils (LDN) are co-purified with monocytes and lymphocytes at the interface between the 1.077 g / ml sucrose layer and plasma.

[0345] HDN can be tested in the CKA assay described herein. Hematopoietic cells are suitably obtained from a donor with HDN.

[0346] Example 22 Differentiation of induced pluripotent stem cells (iPSCs) into neutrophils with high CKA Identify donors that contain neutrophils with high CKA. Isolate somatic cells (e.g., fibroblasts) from donors and use them to establish the culture of iPSCs. Differentiate iPSCs into mature neutrophils, for example, using the protocol described by Sweeney CL, Merling RK, Choi U, Priel DB, Kuhns DB, Wang H and Malech HL, Generation of functionally mature neutrophils from induced pluripotent stem cells. Neutrophil Methods and Protocols, Methods in Molecular Biology. 2014;1124:189-206, and Sweeney et al. (2016), Stem Cells, 34(6), 1513-1526 (the teachings of which are incorporated herein by reference).

[0347] The resulting mature neutrophils are shown to have CKA levels similar to those of DDNs and SCDNs derived from HSCs from the same donor (as tested by both MTT and xCELLigence assays).

[0348] The mature neutrophils are then infused into the donor from which the iPSCs were originally derived, which does not elicit any immune response.

[0349] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain preferred embodiments, it should be understood that the claimed invention 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 the art of biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

[0350] section 1. a. a surface potential defined by an electrophoretic mobility of at least 1.0 μm.cm / volt.sec; and b. Ability to kill cancer cells An in vitro cell culture of hematopoietic cells that differentiate to form granulocytes characterized by:

[0351] 2. a. a density of at least 1.077 g / ml; and b. Ability to kill cancer cells An in vitro cell culture of hematopoietic cells that differentiate to form granulocytes characterized by:

[0352] 3. a. Expression or activity of toll-like receptors; and / or absent or inactive expression of programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2; and b. Ability to kill cancer cells An in vitro cell culture of hematopoietic cells that differentiate to form granulocytes characterized by:

[0353] 4. Hematopoietic cells a. a density of at least 1.077 g / ml; and / or b. Expression or activity of toll-like receptors; and / or the absence or inactivity of expression of programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2 2. The in vitro cell culture of clause 1, further characterized in that the cells differentiate to form granulocytes.

[0354] 5. The in vitro cell culture of paragraph 3 or 4, wherein the granulocytes are characterized by expression or activity of toll-like receptors; and absent or inactive expression of programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and CXCR2.

[0355] 6. The in vitro cell culture of any one of paragraphs 1-5, wherein the cell culture is enriched for hematopoietic cells.

[0356] 7. The in vitro cell culture of any one of clauses 1-6, wherein at least 70% of the cells in the in vitro cell culture are hematopoietic cells.

[0357] 8. The in vitro cell culture of any one of clauses 1-7, wherein the hematopoietic cells are obtainable from a donor, preferably a human donor.

[0358] 9. The in vitro cell culture of paragraph 8, wherein the donor is a male donor.

[0359] 10. The in vitro cell culture of paragraph 8 or 9, wherein the donor is aged between 18 and 25 years.

[0360] 11. The in vitro cell culture of any one of clauses 1-10, wherein the hematopoietic cells have a surface potential defined by an electrophoretic mobility of less than 1.0 μm.cm / volt.sec, and / or have a higher surface potential than otherwise identical hematopoietic cells that have differentiated to form granulocytes that have a reduced ability to kill cancer cells when compared to characteristic b as defined in clauses 1, 2 or 3.

[0361] 12. The in vitro cell culture of any one of clauses 1-11, wherein the hematopoietic cells have a surface potential defined by an electrophoretic mobility of at least 1.0 μm.cm / volt.sec or at least 2.0 μm.cm / volt.sec or at least 2.5 μm.cm / volt.sec or at least 3.0 μm.cm / volt.sec.

[0362] 13. The in vitro cell culture of any one of clauses 1-12, wherein the granulocytes have a surface potential defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec or at least 2.5 μm.cm / volt.sec or at least 3.0 μm.cm / volt.sec.

[0363] 14. The in vitro cell culture of any one of clauses 1-13, wherein the granulocytes are neutrophils.

[0364] 15. A method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. Measuring the surface potential of granulocytes obtainable from a donor; and b. selecting hematopoietic cells from said donor if the measured surface potential is defined by an electrophoretic mobility of at least 1.0 μm.cm / volt.sec. The method includes:

[0365] 16. A method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. Measuring the density of granulocytes available from the donor; and b. selecting hematopoietic cells from said donor if the measured density of granulocytes is at least 1.077 g / ml. The method includes:

[0366] 17. A method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. detecting the expression or activity of toll-like receptors; programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2 on granulocytes obtainable from the donor; and b. selecting hematopoietic cells from said donor if toll-like receptors are expressed or active; and / or programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2 are not expressed or inactive. The method includes:

[0367] 18. A method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. Measuring the surface potential of hematopoietic cells; and b. selecting hematopoietic cells that have a surface potential defined by an electrophoretic mobility of less than 1.0 μm.cm / volt.sec and / or have a higher surface potential than otherwise identical hematopoietic cells that differentiate to form granulocytes with reduced ability to kill cancer cells; The method includes:

[0368] 19. A method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. Measuring the density of hematopoietic cells; and b. Selecting hematopoietic cells that have a density less than 1.077 g / ml and / or that differentiate to form granulocytes with reduced ability to kill cancer cells, and that have a higher density than otherwise identical hematopoietic cells. The method includes:

[0369] 20. The method of any one of clauses 15-19, wherein the hematopoietic cells have a surface potential defined by an electrophoretic mobility of at least 1.0 μm.cm / volt.sec.

[0370] 21. The method of any one of clauses 15-20, wherein the hematopoietic cells have a surface potential defined by an electrophoretic mobility of at least 2.0 μm.cm / volt.sec or at least 2.5 μm.cm / volt.sec or at least 3.0 μm.cm / volt.sec.

[0371] 22. The method of any one of clauses 15-21, wherein the surface potential is determined by electrophoresis.

[0372] 23. The method of any one of clauses 15 to 22, further comprising discarding hematopoietic cells that are not selected in step b of any one of clauses 15 to 19.

[0373] 24. The method of any one of clauses 15-23, wherein the hematopoietic cells are hematopoietic stem cells.

[0374] 25. The method of any one of clauses 15-24, wherein the hematopoietic cells are granulocyte progenitor cells, such as common myeloid progenitor cells, myeloblasts, N. promyelocytes, N. myelocytes, N. metamyelocytes, N. neutrophil band cells, or combinations thereof.

[0375] 26. The method of any one of clauses 15 to 25, wherein the granulocytes are neutrophils.

[0376] 27. The method of any one of clauses 15-26, further comprising differentiating the hematopoietic cells into granulocytes.

[0377] 28. The method of any one of clauses 16-27, wherein the hematopoietic cells are obtainable from a donor, preferably a human donor.

[0378] 29. The method of any one of clauses 15 to 28, wherein the donor is a male donor.

[0379] 30. The method of any one of paragraphs 15-29, wherein the donor is aged between 18 and 25 years.

[0380] 31. An in vitro method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. mixing granulocytes obtainable from a donor with a cell line to form a mixture; b. incubating the mixture; c. determining the percentage of cancer cells killed in said mixture; and d. Selecting hematopoietic cells from said donor if said granulocytes kill at least 70% of the cancer cells in the mixture. The method includes:

[0381] 32. Use of the surface potential of hematopoietic cells to select cells that can be differentiated into granulocytes suitable for treating cancer, the surface potential being greater than that of otherwise identical hematopoietic cells that have a surface potential defined by an electrophoretic mobility of less than 1.0 μm.cm / volt.sec and / or that differentiate to form granulocytes with reduced ability to kill cancer cells.

[0382] 33. The use of clause 32, wherein the hematopoietic cells have a surface potential defined by an electrophoretic mobility of at least 1.0 μm.cm / volt.sec, or at least 2.0 μm.cm / volt.sec, or at least 2.5 μm.cm / volt.sec, or at least 3.0 μm.cm / volt.sec.

[0383] 34. An in vitro method for selecting granulocytes suitable for use in the treatment of pancreatic cancer, comprising: a. mixing granulocytes with a pancreatic cancer cell line to form a mixture; b. incubating the mixture; c. determining the percentage of pancreatic cancer cells killed in said mixture; and D. Selecting granulocytes that kill at least 70% of the pancreatic cancer cells in the mixture. The method includes:

[0384] 35. The in vitro method of paragraph 34, wherein the pancreatic cancer cell line is a pancreatic ductal adenocarcinoma cell line.

[0385] 36. An in vitro method for selecting granulocytes suitable for use in the treatment of cancer, comprising: a. mixing granulocytes with a plurality of different cancer cell lines to provide a plurality of mixtures; b. incubating the mixture; c. determining the percentage of cancer cells killed in said mixture; and d. If the granulocytes kill at least 70% of the cancer cells in the mixture, selecting the granulocytes as suitable for use in treating the same type / subset of cancer as the cancer cell line. The method includes:

[0386] 37. The in vitro method of any one of paragraphs 34-36, further comprising discarding granulocytes that kill less than 70% of the cancer cells in the mixture.

[0387] 38. The in vitro method according to any one of paragraphs 34 to 37, wherein the granulocytes are obtainable from a donor, preferably a human donor.

[0388] 39. The in vitro method according to any one of clauses 34 to 38, wherein the granulocytes are obtainable from a subject having a different type / subset of cancer than the cancer cell line used in the method.

[0389] 40. The in vitro method of any one of clauses 34-39, wherein the cancer cell line is one or more selected from a pancreatic cancer cell line, a liver cancer cell line, an esophageal cancer cell line, a gastric cancer cell line, a cervical cancer cell line, an ovarian cancer cell line, a lung cancer cell line, a bladder cancer cell line, a kidney cancer cell line, a brain tumor cell line, a prostate cancer cell line, a myeloma cancer cell line, a non-Hodgkin's lymphoma (NHL) cell line, a laryngeal cancer cell line, a uterine cancer cell line, or a breast cancer cell line.

[0390] 41. The in vitro method of any one of paragraphs 38 to 40, further comprising obtaining hematopoietic cells from a donor from which selected granulocytes are obtainable.

[0391] 42. A granulocyte obtainable by the method according to any one of paragraphs 34 to 41.

[0392] 43. A method comprising differentiating an in vitro cell culture of hematopoietic cells according to any one of clauses 1 to 14, or of hematopoietic cells obtainable according to the method according to any one of clauses 15 to 31, into granulocytes.

[0393] 44. An in vitro cell culture of granulocytes obtainable by the method according to paragraph 43, comprising: a. a surface potential defined by an electrophoretic mobility of at least 1.0 μm.cm / volt.sec; and b. Ability to kill cancer cells A cell culture enriched for granulocytes having

[0394] 45. An in vitro cell culture of granulocytes obtainable by the method according to paragraph 43, comprising: a. a density of at least 1.077 g / ml; and b. Ability to kill cancer cells A cell culture enriched for granulocytes having

[0395] 46. ​​An in vitro cell culture of granulocytes obtainable by the method according to paragraph 43, comprising: a. expression or activity of toll-like receptors; and / or the absence or inactivity of the programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2; and b. Ability to kill cancer cells A cell culture enriched for granulocytes having

[0396] 47. a. hematopoietic cells or granulocytes; and b. Granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone; serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, fetal bovine serum (FBS), or a combination thereof. 23. A pharmaceutical composition comprising:

[0397] 48. The in vitro cell culture of hematopoietic cells according to any one of clauses 1 to 14, or the in vitro cell culture of granulocytes according to clause 42, or the in vitro cell culture of granulocytes according to clauses 44 to 46, or the pharmaceutical composition according to clause 47, for use in the treatment of cancer.

[0398] 49. Use of an in vitro cell culture of hematopoietic cells according to any one of clauses 1 to 14, or a granulocyte according to clause 42, or an in vitro cell culture of granulocytes according to any one of clauses 44 to 46, or a pharmaceutical composition according to clause 47, in the manufacture of a medicament for the treatment of cancer.

[0399] 50. A method for treating cancer comprising administering to a subject in need thereof an in vitro cell culture of hematopoietic cells according to any one of clauses 1-14, or a granulocyte according to clause 42, or an in vitro cell culture of granulocytes according to any one of clauses 44-46, or a pharmaceutical composition according to clause 47.

[0400] 51. An in vitro cell culture of hematopoietic cells according to any one of clauses 1 to 14, or a granulocyte according to clause 42, or an in vitro cell culture of granulocytes according to any one of clauses 44 to 46, or a pharmaceutical composition according to clause 47 for use, or a use or method according to any one of clauses 48 to 50, wherein the cancer is a solid tumor cancer.

[0401] 52. The in vitro cell culture of hematopoietic cells according to any one of clauses 1-14, or the granulocytes according to clause 42, or the in vitro cell culture of granulocytes according to any one of clauses 44-46, or the pharmaceutical composition according to clause 47 for use, the use or method according to any one of clauses 48-50, wherein the cancer is one or more of pancreatic cancer, liver cancer, esophageal cancer, gastric cancer, cervical cancer, ovarian cancer, lung cancer, bladder cancer, kidney cancer, brain tumor, prostate cancer, myeloma cancer, non-Hodgkin's lymphoma (NHL), laryngeal cancer, uterine cancer, or breast cancer.

[0402] 53. An in vitro method for selecting a subject for treatment with an in vitro cell culture of hematopoietic cells according to any one of clauses 1 to 14, or a granulocyte according to clause 42, or an in vitro cell culture of granulocytes according to any one of clauses 44 to 46, or a pharmaceutical composition according to clause 47, comprising: a. mixing granulocytes from said subject with a cancer cell line; b. incubating the mixture; c. determining the percentage of cancer cells killed in said mixture; and d. if granulocytes from the subject kill less than 70% of the cancer cells in the mixture, selecting said subject for treatment with the in vitro cell culture of hematopoietic cells of any one of clauses 1-14, or the granulocytes of clause 42, or the in vitro cell culture of granulocytes of any one of clauses 44-46, or the pharmaceutical composition of clause 47. The method includes:

[0403] 54. The in vitro method of clause 53, wherein the subject is selected for treatment if granulocytes from the subject kill less than 50% or less than 25% (preferably less than 10% or 5%) of the cancer cells in the mixture.

[0404] 55. A cell bank comprising an in vitro cell culture of hematopoietic cells according to any one of clauses 1 to 14, or a granulocyte according to clause 42, or an in vitro cell culture of granulocytes according to any one of clauses 44 to 46, or a pharmaceutical composition according to clause 47.

[0405] 56. a. an in vitro cell culture of a hematopoietic cell according to any one of clauses 1 to 14, or a granulocyte according to clause 42, or an in vitro cell culture of a granulocyte according to any one of clauses 44 to 46, or a pharmaceutical composition according to clause 47; and b. Instructions for its use in medicine Kit including:

[0406] 57. The kit of clause 56, wherein the instructions are for its use in treating cancer, preferably pancreatic cancer.

Claims

1. An in vitro method for obtaining hematopoietic cells that can be differentiated into granulocytes suitable for use in the treatment of cancer, comprising: the hematopoietic cells are hematopoietic stem cells, granulocytic progenitor cells, or induced pluripotent stem cells obtained from the donor's somatic cells; a. mixing granulocytes obtainable from a donor with a cancer cell line to form a mixture; b. incubating the mixture; c. determining the percentage of cancer cells killed in said mixture; and d. If the granulocytes kill at least 40% of the cancer cells in the mixture, obtaining hematopoietic cells from the donor-derived sample. Including, The method according to claim 1, wherein the mixture ratio of granulocytes to cancer cells (granulocytes:cancer cells) is 10:

1.

2. An in vitro method for obtaining hematopoietic cells capable of being differentiated into granulocytes suitable for use in the treatment of cancer, comprising selecting hematopoietic cells from a sample derived from a donor, the hematopoietic cells are hematopoietic stem cells, granulocytic progenitor cells, or induced pluripotent stem cells obtained from the donor's somatic cells; granulocytes obtained from said donor have the ability to kill at least 40% of cancer cells in a cancer killing assay; The cancer killing assay comprises: a. mixing granulocytes with a cancer cell line to form a mixture; b. incubating the mixture; c. determining the percentage of cancer cells killed in said mixture; Including, The method according to claim 1, wherein the mixture ratio of granulocytes to cancer cells (granulocytes:cancer cells) is 10:

1.

3. The in vitro method according to claim 1 or 2, wherein the hematopoietic cells are hematopoietic stem cells.

4. 3. The in vitro method of claim 1 or 2, wherein the hematopoietic cells are granulocyte progenitor cells such as common myeloid progenitor cells, myeloblasts, N. promyelocytes, N. myelocytes, N. metamyelocytes, N. bands, or combinations thereof.

5. 3. The in vitro method according to claim 1 or 2, wherein the hematopoietic cells are induced pluripotent stem cells obtained from somatic cells of the donor.

6. The in vitro method according to any one of claims 1 to 5, wherein the donor is a human donor.

7. The in vitro method according to any one of claims 1 to 6, wherein the donor is a male donor.

8. An in vitro method according to any one of claims 1 to 7, wherein the donor is aged between 18 and 25 years.

9. 9. The in vitro method of any one of claims 1 to 8, wherein the cancer cell line is one or more selected from pancreatic cancer cell lines, liver cancer cell lines, esophageal cancer cell lines, gastric cancer cell lines, cervical cancer cell lines, ovarian cancer cell lines, lung cancer cell lines, bladder cancer cell lines, kidney cancer cell lines, brain cancer cell lines, prostate cancer cell lines, myeloma cancer cell lines, non-Hodgkin's lymphoma (NHL) cell lines, laryngeal cancer cell lines, uterine cancer cell lines, or breast cancer cell lines.

10. The in vitro method according to any one of claims 1 to 9, wherein the cancer cell line is a pancreatic cancer cell line.

11. The in vitro method according to any one of claims 1 to 10, wherein the cancer cell line is a pancreatic ductal adenocarcinoma cell line.

12. The in vitro method of claim 11, wherein the cancer cell line is the PANC-1 cell line.

13. The in vitro method according to any one of claims 1 to 12, wherein the granulocytes are neutrophils.

14. The in vitro method according to any one of claims 1 to 13, further comprising obtaining hematopoietic cells from a sample derived from said donor from which selected granulocytes are obtainable.

15. The in vitro method of any one of claims 1 to 14, further comprising differentiating the hematopoietic cells into granulocytes.

Images