Cell that kills cancer

Abstract

To provide a therapy based on a cell suitable for treating cancer.SOLUTION: The present invention relates to an in vitro culture product of a hematopoietic cell that is differentiated so as to form a granulocyte having the capability of killing a cancer cell. The present invention also relates to the granulocyte, the hematopoietic cell, and a method for identifying a granulocyte, a composition and a kit including the same, and use thereof for treating cancer.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Cancer is a leading cause of morbidity and mortality worldwide, and cancer incidence in developed countries is increasing annually. The World Health Organization stated that in 2012 alone, there were approximately 14 million new cancer cases (8.2 million related 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, are being evaluated. However, this treatment is still viewed primarily as 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 remain cancer-free during 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 to directly transfer granulocytes (e.g., neutrophils) collected from donors into cancer patients. Current methods 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 (very rare) donors to obtain the required cell volume. Third, to avoid immune responses from repeated exposure, the same donor cannot be used for subsequent administrations, thus necessitating an increased pool of suitable donors. Fourth, donors cannot realistically be expected to be available on demand or willing to provide an endless supply 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 of 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 present inventors have surprisingly found that it is possible to select hematopoietic cells that can differentiate into granulocytes that have the ability to kill 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 and thus stored and / or propagated indefinitely. The present invention can provide stable cell lines capable of producing granulocytes from cancer cells. 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 demonstrates 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 be differentiated into granulocytes that also exhibit high cancer-killing activity.

[0011] Advantageously, such hematopoietic cells can be preserved 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 donors.

[0012] Furthermore, hematopoietic cell-derived granulocytes have been found to kill cancer cells more rapidly than donor-isolated granulocytes, and 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 pharmaceutical composition 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.The ability to kill cancer cells The present invention provides an in vitro cell culture of hematopoietic cells that 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 the 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, hematopoietic cells suitable for use in treating cancer are selected. 1. A method for selecting blood cells, 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 which 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 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 having a surface potential higher (e.g., more positive) than the surface potential of otherwise identical hematopoietic cells that differentiate to form granulocytes with 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 the subject with a cancer cell line; b. incubating the mixture; c. measuring the percentage of cancer cells killed in the 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 granulocytes from a subject kill less than 20% or 10% of cancer cells in a mixture, the subject is selected 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. Preferably, if granulocytes from a subject kill less than 5% or 1% of cancer cells in a mixture, the subject is selected 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.

[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. 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, obtaining hematopoietic cells from the sample derived from said donor. The present invention provides a method comprising:

[0023] The percentage of cancer cells killed in the control sample can be determined before performing the method of the invention or concurrently (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 the sample; and d. If the % of cancer cells killed in the test sample is higher than the % of cancer cells killed in the control sample, obtaining hematopoietic cells from the sample derived from said first donor. 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 contains granulocytes that kill at most 50%, 40%, 30%, 20%, or 10% of cancer cells in the methods described herein. Preferably, the control sample contains granulocytes that kill at most 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 the 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 when 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 the 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 the 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 the 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 the 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 10:1 ratio of granulocytes to cervical 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 cervical cancer cells in the mixture. More preferably, the method comprises using a 10:1 ratio of granulocytes to cervical cancer cells, and hematopoietic cells are obtained from the 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 includes using a 5:1 ratio of granulocytes to pancreatic cancer cells, and the hematopoietic cells are selected from the group consisting of granulocytes, granulocytes, and hematopoietic cells. Preferably, the method includes 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 10:1 ratio of granulocytes to pancreatic cancer cells, 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 10:1 ratio of granulocytes to pancreatic cancer cells, 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 aspect, there is provided an in vitro method for selecting granulocytes that selectively kill cancer cells, 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 the 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 which 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 before performing the method of the invention or concurrently (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 granulocytes obtainable from the same donor (e.g., control granulocytes) to form a control sample; c. incubating the sample; and d. Selecting the 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 includes 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 the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110 USA). available at

[0053] In one embodiment, there is provided a method for selecting granulocytes that selectively kill cancer cells, 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 (e.g., control granulocytes). To choose A method is provided which 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. measuring the percentage of pancreatic cancer cells killed in the mixture; and d. Selecting granulocytes that kill at least 5% of the 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 comprises using a 5:1 ratio of granulocytes to pancreatic cancer cells, wherein the granulocytes kill at least 50% or 60% of the pancreatic cancer cells in the mixture. The method involves 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 involves using a 10:1 ratio of granulocytes to pancreatic cancer cells, where the granulocytes kill at least 70% of the pancreatic cancer cells in the mixture. Preferably, the method involves using a 10:1 ratio of granulocytes to pancreatic cancer cells, 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 embodiment is a method for selecting suitable granulocytes for use in the treatment of cancer. An in vitro method comprising: a. mixing granulocytes with a plurality of different cancer cell lines to provide a plurality of mixtures; b. incubating the mixture; c. measuring the percentage of cancer cells killed in the 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 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, and 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, 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 from 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 include measuring and / or selecting granulocytes based on a surface potential (e.g., cell surface charge) as disclosed herein. The in vitro method may further include measuring and / or selecting granulocytes based on a cell density (e.g., of at least 1.077 g / ml) as disclosed herein. The in vitro method may further include measuring and / or selecting granulocytes based on the expression or activity of a toll-like receptor; and / or the absence or inactivity of the expression of programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2 on the granulocytes.

[0069] The in vitro method according to the above embodiment can be performed using a cancer killing activity (CKA) assay (e.g., , may be representative of claim 6).

[0070] As used herein, the term "mixing" means mixing one or more components together in any order, whether sequentially or simultaneously. By "mixing" is meant bringing a first component into contact with a second component (eg, granulocytes and 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, the granulocytes can be obtained from a donor, for example, a human donor. Alternatively, or in addition, the granulocytes can be obtained from a subject with a cancer of a different type / subset 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 with which the subject has been diagnosed.

[0073] Cancer cell lines for use in the methods of the present invention include 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. The cell line may be one or more selected from the following 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®; SW 1990, 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 HeLa cells.

[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 is carried out for 6 hours to 2 days, for example, 12 hours to 36 hours, for example, 16 hours to 36 days. The incubation step may be carried out for 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, 35°C to 42°C, preferably 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 (e.g., 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 device 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., after 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 using 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 methods 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 methods involve the use of a ratio of granulocytes to cancer cells of 5:1. More preferably, the methods involve the use of a ratio of granulocytes to cancer cells of 10:1.

[0082] Those skilled in the art will understand that when the methods of the present invention include 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, the samples are preferably equivalent. For example, the samples being compared may be of the same sample type (e.g., blood) and may have undergone 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, allowing for a suitable comparison to 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 cancer cells in the methods described herein. The granulocytes are capable of killing at least 10%, 20%, 30%, 40%, or 50% of cancer cells in the methods described herein. In one embodiment, the granulocytes are capable of killing at least 60% of cancer cells in the methods described herein. In one embodiment, the granulocytes are capable of killing at least 70% of cancer cells in the methods described herein. Preferably, the granulocytes are capable of killing at least 5% of cancer cells in the methods described herein. In this case, at least 80% or 90% of cancer cells can be killed.

[0085] In embodiments where the method involves using a 5:1 ratio of granulocytes to cancer cells, the granulocytes are capable of killing at least 30% of the cancer cells in the methods described herein. Preferably, when the method involves using a 5:1 ratio of granulocytes to cancer cells, the granulocytes are capable of killing at least 40% of the cancer cells in the methods described herein. In embodiments where the method involves using a 10:1 ratio of granulocytes to cancer cells, the granulocytes are capable of killing at least 45%, 50%, or 60% (preferably at least 60%) of the cancer cells in the methods described herein. More preferably, when the method involves using a 10:1 ratio of granulocytes to cancer cells, the granulocytes are capable of killing 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 the 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 the 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 the 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 the 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 involves using a 5:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can kill at least 50% or 60% of pancreatic cancer cells in the methods described herein. Preferably, when the method involves using a 5:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can kill at least 65% or 70% of pancreatic cancer cells in the methods described herein. In embodiments where the method involves using a 10:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can kill at least 70% of pancreatic cancer cells in the methods described herein. Preferably, when the method involves using a 10:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can kill at least 80% or 90% of pancreatic cancer cells in the methods described herein.

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

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

[0090] In one embodiment, the in vivo method for selecting suitable granulocytes for use in the treatment of cancer is In vitro methods include a. Mix granulocytes with cancer cell lines (preferably pancreatic cancer cell lines 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 target cell; b. Incubating the mixture(s) at 39°C for 24 hours; c. measuring the % of cancer cells killed in said mixture(s); 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 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 include simultaneously assaying the 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 granulocytes obtainable from a donor with multiple different cancer cell lines to provide multiple mixtures; b. incubating the mixture; c. measuring the percentage of cancer cells killed in the 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 cancer of the same type / subset 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 a toll-like receptor; and / or the absence or inactivity of the expression of programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2 on granulocytes differentiated from hematopoietic cells.

[0095] In one aspect, hematopoietic cells obtainable (eg, obtained) by the methods of the invention are provided.

[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 percentage 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 percentage within 10 hours of contact with cancer cells.

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

[0099] The term "half-maximal cancer killing%" used in this context refers to 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 half-maximal 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 methods of the present invention may include a combination of the techniques described herein to improve the selection of cells suitable for treating cancer. For example, if multiple hematopoietic cells or granulocytes are selected because they meet a specified density threshold, cell surface potential / electrophoretic mobility can be assessed to identify hematopoietic cells that produce granulocytes (e.g., neutrophils) with high CKA or to help 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 can be obtained from the donor.

[0102] The present invention also provides a differentiation method comprising differentiating an in vitro cell culture of hematopoietic cells of the present invention, or hematopoietic cells obtainable according to the method of the present 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.The ability to kill cancer cells The cells are enriched for granulocytes with

[0103] In one aspect, the present invention provides a method for the production of hematopoietic cells; and granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormones; serotonin, vitamin C , Vitamin D, Glutamine (Gln), Arachidonic acid, AGE-albumin, Interleukin Kinase, 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 included in the composition may be obtainable (e.g., obtained) by the methods 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 method for the treatment of granulocytes; granulocyte-macrophage colony-stimulating factor (GM-CSF) , granulocyte colony-stimulating factor (G-CSF), growth hormone; serotonin, vitamin C, vitamin Vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, T NF-alpha (e.g., UniProt accession number P01375), Flt-3 ligand, thrombopoiesis Pharmaceutical compositions are provided that include ethine (e.g., UniProt Accession No. P40225), fetal bovine serum (FBS), or a combination thereof. The granulocytes contained in the compositions are obtainable (e.g., obtained) by the methods 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 hormones, and and serotonin, and vitamin C, and vitamin D, and glutamine (Gln), and and arachidonic acid, and AGE-albumin, and interleukin, and TNF-alpha, and Flt-3 ligand, and thrombopoietin, and fetal bovine serum (FBS).

[0108] Interleukins include interleukin-3 (IL-3) (e.g., UniProt accession number P08700), interleukin-8 (IL-8) (e.g., UniProt accession number P10145), interleukin-4 (IL-4) (e.g., UniProt accession number P05112), interleukin-6 (IL-6) ( For example, the interleukin may be erythropoietin (E.g., UniProt Accession No. P05231), interleukin-18 (IL-18) (e.g., UniProt Accession No. Q14116), or a combination thereof. , interleukin-3 (IL-3), interleukin-8 (IL-8), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-18 (IL-18) It's okay to have it.

[0109] In one embodiment, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone, serotonin, AGE-albumin, interleukin (IL-1), TNF-alpha, Flt-3 ligand, or thrombopoietin are the main antagonists of human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and thrombopoietin. It may be growth hormone, serotonin, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, or thrombopoietin.

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

[0111] The present invention also relates to 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 related embodiments, hematopoietic cells, 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 are provided. Corresponding methods for treating cancer are also provided, comprising administering to a subject in need thereof an in vitro cell culture of hematopoietic cells, granulocytes, in vitro cell cultures of granulocytes, pharmaceutical compositions, or kits of the present invention.

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

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

[0114] As used herein, the term "hematopoietic cells" 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, hematopoietic cells are cells of the hematopoietic pathway or equivalent cells. In one embodiment, hematopoietic cells are induced pluripotent stem cells (iPSCs) or equivalent cells. In one embodiment, iPSCs are obtainable from the somatic cells of a donor. The 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, hematopoietic cells are obtained from a donor-derived sample, and the hematopoietic cells can be isolated from the sample. In another embodiment, the hematopoietic cells are obtained from a donor-derived sample, and the sample contains somatic cells, 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, for example, hematopoietic stem cells that express a cell surface receptor selected from 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 the cell surface polypeptide marker CD34. + , 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 cells are granulocyte progenitor cells. The granulocyte progenitor cells 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 performed as described 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 present invention provides a method for producing hematopoietic cells in a mammalian animal, comprising administering to the mammalian animal a therapeutic agent selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone; serotonin, vitamin C, vitamin B6, vitamin D, vitamin E, vitamin B12, vitamin B6, vitamin D2, vitamin B6, vitamin D3, vitamin B6, vitamin D4, vitamin B6, vitamin B12, vitamin B6, vitamin D5, vitamin B6, vitamin B12 ... 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 present invention provides a method for the production of hematopoietic cells in a mammalian animal, comprising administering to the mammalian animal a hematopoietic cell line ... Tonin, Vitamin C, Vitamin D, Glutamine (Gln), and Ala Quidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, and fetal bovine serum (FBS) and a method for differentiating the hematopoietic cells, the method comprising:

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

[0124] In one embodiment, differentiation of hematopoietic cells comprises culturing the hematopoietic cells with one or more support cells. Suitably, the support 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 support 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 may also Reactive oxygen species (O2 - ) may be produced.

[0127] The present invention encompasses granulocytes suitable for use in the treatment of cancer. Preferably, the 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, the 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, the granulocytes 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, the granulocytes 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. Granulocytes also have the ability to kill 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., have been killed), the cells exhibit the ability to kill cancer cells. In one embodiment, the ability to kill cancer cells is measured using the cancer killing activity (CKA) assay described herein. is determined.

[0130] In one embodiment, the CKA assay comprises: a. contacting a cancer cell line with granulocytes to form a test sample (preferably at a granulocyte to cancer cell ratio of 10:1); b. incubating the test sample; and c. Measuring the percent 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 were mixed and cultured 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 to 24 hours (e.g., at 30 to 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 A compound 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 involves 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, when the method involves 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 involves 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, when the method involves 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 involves 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 involves 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 involves 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 involves 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 involves using a 5:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can be considered to kill cancer cells if they kill at least 50% or 60% of the pancreatic cancer cells present. Preferably, when the method involves using a 5:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can be considered to kill cancer cells if they kill at least 65% or 70% of the pancreatic cancer cells present. In embodiments where the method involves using a 10:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can be considered to kill cancer cells if they kill at least 70% of the pancreatic cancer cells present. Preferably, when the method involves using a 10:1 ratio of granulocytes to pancreatic cancer cells, the granulocytes can be considered to kill 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] Cells that have the ability to kill cancer cells ("believed to kill cancer cells") are those that are at least 70% or more potent in a CKA assay herein (e.g., the CKA assay described above). Preferably, the cells have a CKA of at least 8% or 75%. It may have 0% or 90% activity.

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

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

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

[0144] Granulocytes having a density of at least 1.077 g / ml may be obtainable by the method 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. Centrifuging 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 have a density of 1.077 g / ml after separation. They are found at the bottom of the Ficoll-Paque medium, while low density granulocytes (eg, 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 such 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. 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 present invention provides a method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. Measuring the density of granulocytes obtainable 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: a. Measuring hematopoietic cell density; and b. Selecting hematopoietic cells that have a density less than 1.077 g / ml and / or that differentiate to form granulocytes with a 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, there is provided a method comprising differentiating an in vitro cell culture of hematopoietic cells, wherein the hematopoietic cells are: a. a density of at least 1.077 g / ml; and b.The ability to kill cancer cells In one embodiment, a method is provided for differentiating the cells to form granulocytes characterized by:

[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 pharmaceutical composition comprising: a. Expression or activity of toll-like receptors; and / or absence or inactivity of programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2; and b.The ability to kill cancer cells The present invention provides an in vitro cell culture of hematopoietic cells that differentiate to form granulocytes, characterized by:

[0161] The presence or absence of the 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 the receptor.

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

[0163] Preferably, the granulocytes express toll-like receptors; do not express programmed cell death 1 (PD-1) receptor (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 the programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and CXCR2).

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

[0165] Toll-like receptors include TLR1 (e.g., UniProt accession number Q15399), TLR2 (e.g., For example, TLR1 (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). 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 present 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 the 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, there is provided a method comprising differentiating an in vitro cell culture of hematopoietic cells, wherein the hematopoietic cells are: a. Expression or activity of toll-like receptors; and / or absence or inactivity of programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2; and b.The ability to kill cancer cells In one embodiment, a method is provided for differentiating the cells to form granulocytes characterized by:

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

[0171] Hematopoietic cells can be immortalized. Those skilled in the art are familiar with immortalization techniques, including, inter alia, 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. Preferably, 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, the "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 preferably 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., if 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" encompasses 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: gender, age, medical history, and / or blood type. In one embodiment, the donor may be selected if he / she is male. In another embodiment, the donor may be selected if he / she is 18-25 years old (preferably 18-24 years old). Preferably, the donor may be selected if he / she is male and aged 18-25 years old (preferably 18-24 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 measuring 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 in 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. While applying a current of 3 mA, measure the time it takes for the cells (e.g., granulocytes or hematopoietic cells) to traverse a fixed length (e.g., optionally monitored by a CCD camera). (by using a connected microscope).

[0180] Electrophoretic mobility "μ" (expressed in units 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:

[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 electrophoretic mobility using the above formula is determined by.

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

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

[0184] Without wishing to be bound by theory, it is believed that hematopoietic cells (e.g., hematopoietic stem cells expressing CD34) that can differentiate into granulocytes (e.g., neutrophils) with a higher CKA are more positively charged and therefore will migrate further toward a negatively charged electrode in 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 in 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 migrate further toward the electrode during electrophoresis than less positively (or negatively) charged cells. Such cells will therefore have a higher 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. Preferably, 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. Preferably, 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, 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, e.g., 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, 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, hematopoietic cells 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. 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. 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 present 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, e.g., 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. Preferably, 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 at least 3.75 μm.cm / volt.sec or at least 4.0 μm.cm / volt.sec.

[0188] The invention may involve measuring the surface potential of granulocytes obtainable (e.g., obtained) from a 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, depending on the method used, rapid screening can be used to screen for more complex assays (e.g., CKA). A functional assay (such as the CKA assay described herein) can then be performed to determine whether a donor is suitable, for example. For example, the screen can be validated by differentiating selected hematopoietic cells from the donor into granulocytes and testing the granulocytes in a CKA assay. Cells that do not have the required cell surface potential and / or the required CKA may be discarded.

[0189] Additionally or alternatively, the surface potential of hematopoietic cells can be measured and selected if they 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 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. Advantageously, this allows for rapid screening of hematopoietic cells to determine whether they are suitable for use in the treatment of cancer. Thus, the present 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 an electrophoretic mobility of less than 2.0 μm.cm / volt.sec) are discarded.

[0191] Additionally or alternatively, the surface potential of 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 have 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 present 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 an electrophoretic mobility of 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 killed cancer cells. "Reduced ability to kill cancer cells" can be determined using the methods described herein or a 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 present invention (i.e., cells having "cancer cell-killing ability" as defined herein). Preferably, "reduced ability to kill cancer cells" means that the cells kill 5% or fewer cancer cells than the cells of the present 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 cancer treatment) 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 the 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 the donor; and b. Using the measured surface potential in a method for selecting hematopoietic cells The present invention provides a method comprising:

[0197] The "method for selecting hematopoietic cells" may be performed using the same donor from which the surface potential measurements were obtained. Alternatively, it may refer to selecting hematopoietic cells from different donors.

[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 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 present 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 treating cancer if the hematopoietic cells have a higher positive cell surface charge when compared to control hematopoietic cells. The present invention provides a method comprising:

[0202] The cell surface charge of the control granulocytes or hematopoietic cells can be determined before carrying out the method of the invention or at the same time (preferably simultaneously) as 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 the sample 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 treating 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 method 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 comprises the step of: increasing the hematopoietic cells by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 27 and obtaining said hematopoietic cells from a sample derived from said donor if said hematopoietic cells have a cell surface charge that is 00%, 200%, or 300% more positive.

[0207] In one 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 granulocytes with 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 referenced control sample may be a sample derived 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).

[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% greater 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% greater 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 embodiment, methods for selecting hematopoietic cells suitable for use in the treatment of cancer are provided. An in vitro method comprising: a. Measuring the concentration of hematopoietic cells with a positive cell surface charge in a sample obtainable from a donor; and b. selecting the hematopoietic cells as suitable for use in treating 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 with a positive cell surface charge in an otherwise identical control sample from a different donor can be determined before performing the method of the invention or simultaneously (preferably simultaneously) with performing the method of the invention.

[0212] For example, in one embodiment, the method comprises: a. Measuring the concentration of granulocytes or hematopoietic cells with 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 an otherwise identical control sample from a different donor; and c. obtaining hematopoietic cells from the sample from the (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 treating cancer if the concentration of said hematopoietic cells with a positive cell surface charge from the (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., the 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, there is provided an in vitro method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. Measuring the cell surface charge of granulocytes obtainable from a first donor; b. identifying obtainable granulocytes from said first donor having a higher positive cell surface charge when compared to control granulocytes; c. measuring the concentration of the granulocytes identified in step b.; d. comparing the concentration of granulocytes measured in step c. with the 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 the first donor if the comparison identifies a higher concentration of the granulocytes obtainable from the first donor compared to the concentration of the granulocytes obtainable from the second (or additional) donor. A method is provided which 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, there is provided 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 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. measuring the concentration of the hematopoietic cells identified in step b.; d. comparing the concentration of hematopoietic cells measured in step c. with the concentration of hematopoietic cells obtainable from a second (or additional) donor, wherein the hematopoietic cells 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 the first donor as suitable for use in treating cancer if the comparison identifies a higher concentration of hematopoietic cells obtainable from the first donor as compared to the concentration of hematopoietic cells obtainable from the second (or additional) donor. A method is provided which 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 before carrying out the method of the invention or at the same time (preferably simultaneously) as 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 method 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, hi another embodiment, the positively or negatively charged means (e.g., nanoparticles) may be captured magnetically, thus allowing for the isolation of cells that interact with said means.

[0235] Suitable nanoparticles are superparamagnetic iron(II, III) oxide (Fe3O4) nanoparticles (NPs), (3 -aminopropyl)triethoxysilane (APTES) to form tetraethoxysilane. NP upon reaction with tetrafluoroethylene orthosilicate (TEOS) and ammonium hydroxide (NH4OH) It can be prepared by forming a thin layer of silicon dioxide (SiO2) shell on the surface of Fluorescein isothiocyanate (FITC) is embedded in the SiO2 shell, thus This exposes hydroxyl groups (SiO2-OH) attached to silicon, creating a negative surface charge. Branched poly(ethyleneimine) (PEI) molecules can be used to bind the polymer in a non-covalent manner. The SiO2-OH groups are not only covered with SiO2 but also expose additional amine groups that carry positive charges. It can also be done.

[0236] Thus, in one embodiment, negatively charged nanoparticles are prepared by conjugating Fe3O4 nanoparticles with APTES to form a thin layer of SiO2 shell on the nanoparticle surface upon reaction with tetraethyl orthosilicate (TEOS) and ammonium hydroxide (NH4OH), and embedding FITC in the SiO2 shell, thus exposing the SiO2-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 -5 mV, -10 mV, -20 mV, -30 mV, or -40 mV. Preferably, the negatively charged means (e.g., nanoparticles) may have a negative surface charge of at least -35 mV.

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

[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). The 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 in the techniques used to analyze cell surface charge (e.g., the loss of soluble ions such as Na+ and Ca+). The inventors propose that the positive charge on granulocytes is provided by human neutrophil peptide (HNP), the most abundant protein in human neutrophils. We believe that HNPs can be used to treat cancer cells. HNPs contain 20–40 amino acids rich in arginine, lysine, and cysteine, making the peptides positively charged and called "cationic peptides." HNPs have an amphiphilic, folded, rod-like structure with one side hydrophobic and the other hydrophilic and positively charged. The primary target of HNPs is thought to be the negatively charged lipid bilayer membrane on cancer cells. The activation mechanism of HNPs is the cleavage of the negatively charged leader peptide, which neutralizes the positive charge in the peptide precursor. On target cells, mature amphiphilic HNPs exert two main effector actions. First, HNPs form barrel-like pores on the plasma membrane of target cells. The hydrophobic side of the monomeric HNPs aligns with the hydrophobic portion of the target bilayer membrane, while the hydrophilic side of the HNPs forms hydrophilic pores with other similarly aligned HNPs. This pore causes the target cancer cells to swell and burst (cytolysis). Second, once HNPs enter the cytoplasm of 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. We therefore believe that granulocytes (e.g., neutrophils) with a higher positive cellular charge 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 the methods described herein.

[0243] The cells described herein can be part of a cell culture (e.g., an in vitro cell culture). The cell culture can include multiple different cell types (e.g., hematopoietic cells, or cells 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 with 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 with the expression or activity of toll-like receptors; and / or the absence or inactivity of the 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" refers to the hematopoietic cells described herein. This means that the cells (e.g., selected by the methods of the present invention) account for at least 70%, 75%, 80%, 85%, 90%, or 95% of all hematopoietic cells (preferably, all 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 toll-like receptors; and / or the absence or inactivity of 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 are obtainable by differentiating the hematopoietic cells of the present invention. The in vitro cell culture may 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 may 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 may be enriched for granulocytes having expression or activity of a toll-like receptor; and / or absent or inactive expression of programmed cell death 1 (PD-1) receptor; 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 toll-like receptors; and / or the absence or inactivity of 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, all 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 deep-freezing, which may include mixing the cells, cell cultures or pharmaceutical compositions with a preservation 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, and thus in a related aspect, 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 may be stored in a metabolically dormant state (e.g., cryogenically frozen). Preferably, cells contained within the cell bank are classified (e.g., based on blood type and / or human leukocyte antigen (HLA) type) for proper recovery. In one embodiment, cells are stored in a cell bank that has been isolated from (or isolated from) the cell bank. The cell bank can be classified based on the type of cancer that the hematopoietic cells of the present invention kill. If the cell bank is a granulocyte cell bank, the cell bank can be replenished using the hematopoietic cells of the present 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 present 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 present invention.

[0254] In one embodiment, the cells or cell cultures of the invention are administered in combination with 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. It is formulated as follows.

[0255] In one embodiment, the cells or cell cultures of the invention are prepared by combining the cells or cell cultures of the invention 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 B6. Vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, and and interleukin, and TNF-alpha, and Flt-3 ligand, and thrombopoietin, and fetal bovine serum (FBS).

[0256] In one embodiment, the cells, cell cultures, or pharmaceutical compositions are 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 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 medicines.The medicines are 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 fluid inclusions. Examples of solid tumor cancers include carcinomas, sarcomas, and lymphomas.

[0259] The solid tumor cancer may be a carcinoma, such as adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, adenosquamous carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, and the like. The carcinoma may be selected from one or more of epithelial neoplasms, squamous cell neoplasms, squamous cell carcinoma, basal cell neoplasms, basal cell carcinoma, transitional cell carcinoma, adenocarcinoma (adenocarcinoma not otherwise specified (NOS), fibrositis plastica, vipoma, cholangiocarcinoma, hepatocellular carcinoma NOS, adenoid cystic carcinoma, renal cell carcinoma, Grawitz 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, such as Askin's tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, or soft tissue sarcomas (alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberances (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, an 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 present invention is for treating cancers including pancreatic cancer, liver cancer, esophageal cancer, gastric cancer, cervical cancer, ovarian cancer, lung cancer, bladder cancer, kidney cancer, brain cancer, prostate cancer, myeloma, non-Hodgkin's lymphoma (NHL), laryngeal cancer, uterine cancer, or is for use in the treatment of one or more of breast cancers.

[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, which 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 cancers listed above. 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 the 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, an 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 step of matching 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) with 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 A dose of cells is administered to a subject. Preferably, the treatment regimen comprises at least 2x10 9 cells or at least 5x 10 9 In one embodiment, the treatment regimen may include administering a dose of cells to the subject. Men should be at least 1x1010 cells or at least 5x10 10 The method may include administering a dose of at least 1 x 10 cells to a subject. 11 pieces or at least 2x10 11 In some embodiments, 1 x 10 cells may be administered to a subject. 9 ~3x10 11 Also is 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 one, two, three, four, five, or six times per week. Alternatively, the subject may be administered daily (e.g., once or twice daily). In this embodiment, it can be administered to the subject once a week or once every two weeks.Preferably, it is administered every week.Those skilled in the art will understand that dosage can be adjusted based on the need of the subject and the efficacy of the drug.For example, if the efficacy of the drug is high, dosage can be reduced.

[0270] In one embodiment, subjects for treatment receive at least 2x10 9 cells or at least Also 2x10 10 Preferably, at least 1 x 10 cells are administered weekly (e.g., once a week). 11 pieces or at least 2x10 11 The cells can be administered weekly.

[0271] Treatment period can be varied 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 9 Preferably, subjects for treatment receive at least 2x10 cells, where the cells are administered once a week. 9 cells (preferably at least 2 x10 10 pcs or 2x10 11 cells), wherein 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] A leukocyte growth factor can be administered together with the agent of the present invention. The administration can be sequential or simultaneous (preferably simultaneous). Suitable leukocyte growth factors include granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone (GH), and erythrocyte leukocyte-associated factor (YAG). 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. Growth factors include granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone, serotonin, vitamin C, and and vitamin D, glutamine (Gln), arachidonic acid, and AGE-albumin Min, interleukin, TNF-alpha, and Flt-3 ligand, and thrombopoietin, and fetal bovine serum (FBS). Examples 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., a hematopoietic cell culture), the agent can be administered together with (e.g., sequentially or simultaneously, preferably simultaneously) granulocyte colony-stimulating factor, growth hormone, serotonin, and interleukin. In one embodiment, granulocyte progenitor cells (e.g., a granulocyte progenitor cell culture) are administered together with (e.g., sequentially or simultaneously, preferably simultaneously) granulocyte colony-stimulating factor, growth hormone, 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 a cancer described in any one of the above embodiments. In some embodiments, the instructions also detail an appropriate 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, NY York (1994), and Hale & Marham, THE HARPER The COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provides those of 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] Other term definitions may appear throughout this specification. Before describing exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the particular embodiments described, and as such may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only, since the scope of the present disclosure will be limited only by the appended claims.

[0281] Where a range of values is provided, unless the context clearly indicates otherwise, it is understood that each intervening value between the upper and lower limit of that range, rounded to two decimal places of the unit of the lower limit, is also specifically disclosed. 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 independently be included in or excluded from the range, and each range where either limit, neither limit, or both limits are included in the smaller range is also encompassed within the disclosure, and any limit in a stated range is expressly excluded. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0282] It should 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, a reference to "hematopoietic cells" includes a plurality of such candidate agents, a reference to "hematopoietic cells" includes a 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 explanation 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 effector to target cell ratios. The difference in CKA levels between donors is maintained at higher effector:target cell ratios. Effector:DDNs; target:HeLa cells. [Figure 2] Figure 2 shows the cytotoxicity results of three CD34+ stem cell-derived neutrophil populations derived from different donors and at various effector to target cell ratios. The results show that stem cell-derived neutrophils derived from different donors have differential CKA. Effector: SCDN; Target: HeLa cells and PANC-1 (pancreatic cancer). [Figure 3] Figure 3 shows the cytotoxicity results (xCELLigence assay) of donor-derived neutrophils (DDNs) from different donors and at various effector to target cell ratios. 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 of fresh donor-derived neutrophils against different cancer cell types and at various effector to target cell ratios. The results show that DDNs 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). [Figure 5] Figure 5 shows the selective cytotoxicity of donor-derived neutrophils against cancer cell types compared with non-cancer cells and various effector to target cell ratios. The results demonstrate that DDNs kill cancer cells with minimal effect on non-cancer cells, confirming selectivity. Effector: DDN; Target: HeLa cells (cervical cancer), PANC-1 cells (pancreatic cancer), and MCF-12F cells (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) from five different cultures and at various ratios of effector to target cells. The results were generated using the MTT assay and demonstrate that ex vivo-generated neutrophils have differential CKA. Effector: SCDN; Target: HeLa cells. [Figure 7] Figure 7 shows the cytotoxicity of three CD34+ stem cell-derived neutrophil populations against different cancer cell types and at various effector to target cell ratios. 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 derived from different donors and at various effector to target cell ratios. The results show that stem cell-derived neutrophils derived 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 effector to target cell ratios. The results demonstrate 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 effector to target cell ratios. The results demonstrate 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-cancer cells, normal breast epithelium). [Figure 11] Figure 11 shows the cytotoxicity results of three CD34+ stem cell-derived neutrophil cultures, along with the cytotoxicity results of donor-derived neutrophils from the same donor and at various effector to target cell ratios. SCDN and DDN from the same donor have similar CKA levels. The similar CKA relationship between DDN and SCDN was maintained for donor LC253 at various effector to target cell ratios. 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, along with the cytotoxicity results of fresh donor-derived neutrophils from the same donor at various effector to target cell ratios. SCDN and DDN have similar CKA levels. The same CKA relationship between DDN and SCDN was maintained for donor LC253 at various effector to target cell ratios. Effector: SCDN and DDN; Target: HeLa cells (cervical cancer). [Figure 13] Figure 13 shows a comparison of the cytotoxicity of natural neutrophils and stem cell-derived neutrophils over time. Higher CKA (donor LC269) is maintained between DDN and SCDN. Effectors: SCDN and DDN (three different SCDN and DDN cultures); Target: HeLa cells (cervical cancer). DETAILED DESCRIPTION OF THE INVENTION [Example]

[0287] Example 1 Donor recruitment Donors are pre-selected 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 pre-selection criteria are: ·Not have a serious medical or psychiatric condition that would affect providing 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 date of sample collection; ·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 withdrawing approximately 18 ml of human blood from a donor. The blood is divided into three BD Vacutainer™ CPT tubes and centrifuged at 175 x g for 35 minutes at 23°C. The mononuclear cell (MN) layer is collected and transferred to a 15 ml conical tube. The MN cells are centrifuged at 420 x g 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). The cells are counted and collected at 1.6 x 10 6 cells / m Resuspend in medium to a final concentration of 1 ml.

[0289] Example 2 Testing CKA of extracted granulocytes in the CKA assay Cells are cultured in DMEM + 10% FBS in T25 flasks to 80% confluence. Cell lines are cultured in T75cm 2 In a cell culture flask, add the following components: 10% volume / volume in DMEM supplemented with FBS, penicillin (Sigma. St. Louis, MO), streptomycin (Sigma. St. Louis, MO), and L-glutamine supplement (Sigma. St. Louis, MO). Grow and maintain at 37°C and 8% CO2. 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 (trademark) The target cells are split and passaged in culture flasks until they reach 70% surface confluence.

[0290] Cells are trypsinized, harvested and counted using Trypan Blue. Assay plates (24 wells) contain 8x10 cells per well in a 24 well flat bottom plate. 4 pieces Pancreatic cancer cells (e.g., pancreatic ductal adenocarcinoma cells) are seeded on the plate. The plate is incubated at 37°C in 5% CO2. Incubate for 24 hours. Label the cells with 2.5 μM CellTracker™ Green for 45 minutes. Add fresh medium to the cells and incubate them in CO2 ink. Return to the lab.

[0291] 500 μl of MN cell suspension (8x10 5 granulocytes) and pancreatic cancer cells were grown for 24 hours. The CKA assay is performed by adding 1000 mg of 10 ... After centrifugation, cells are harvested by trypsinization and centrifuged. Cells are resuspended in 100 μl of cold phosphate-buffered saline (PBS) and then resuspended in 125 μl of 0.4% Trypan B. Lue 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 considered particularly suitable for use in the treatment of cancer.

[0293] Example 3 Testing 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 culture by mechanical detachment and collection from the culture substrate. Collected cells are then transferred to electrophoretic media containing 10 mM Tris-HCl and 291 mM glucose. The cells are redistributed in a running buffer solution and introduced into a square glass electrophoresis chamber. A DC voltage of 200 V 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=μgS / 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 will receive granulocyte-colony stimulating factor (G-CSF) and erythropoietic stem cells to aid in the recovery of peripheral hematopoietic stem cells while minimizing potential discomfort to the donor. and / or granulocyte-macrophage colony-stimulating factor (GM-CSF), e.g., Neupogen® (commercially available from Amgen Inc. USA). To identify long-term persisting pluripotent stem cells, cell surface polypeptide markers are used. Preferably, the marker is CD34 + , CD59 + , Thy1 + , CD38 low / - , C-kit - / low ,oh Yobi 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 consists of four main steps: ·Culture and expansion of hematopoietic cells; 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 a combination thereof. Short-term expansion of progenitor cells; 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 The stored hematopoietic cells (e.g., hematopoietic stem cells or granulocyte progenitor cells obtainable therefrom) and the granulocytes differentiated therefrom (e.g., neutrophils) are analyzed based on the patient's cancer type, blood type (ABO), and other factors. match cancer patients based on their ancestry, rhesus macaques, and HLA), and / or genetics; and Patients may 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) along with granulocyte-colony stimulating factor, human growth hormone, serotonin, and interleukins to the patient; or IV infusion into a patient of stimulated granulocyte progenitor cells (obtainable from hematopoietic stem cells), which, without wishing to be bound by theory, 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 a CKA assay that have been differentiated from hematopoietic cells (e.g., hematopoietic stem cells).

[0300] Typically, cells are dosed at 2x1011 A cell dose of 100 mg / kg / day will be infused weekly 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 the time she has left with her family. Mary's prognosis is 3 to 6 months of survival, making it unlikely she will live long enough to see her soon-to-be-born grandchild.

[0302] Mary is advised to try leukocyte infusion therapy (LIFT). To assess the potential suitability of the therapy, the hospital draws 20 ml of blood from Mary 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 were 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 that, prior to cryogenic freezing, exhibit 70-90% cancer-killing activity (CKA) in the cancer-killing activity assay of Example 2. Deep freezing of granulocytes helps preserve the CKA, so the cells can be sent directly to the hospital (The Royal Marsden) without further testing. Mary is She will visit later that week for her first procedure. The hospital will store the cells properly. Mary will undergo a 2x10 procedure with CKA on December 13th under strict supervision. 9The first round of 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 recommends that the granulocyte dose be 2x10 11 It is decided to gradually increase it over three consecutive 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, indicating successful healing. 20 ml of Mary's blood will be taken i) in parallel with the biopsy to assess the presence of metastatic cancer cells in her 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 checkups, initially monthly, 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 watch her grow.

[0306] After therapy, hematopoietic cells (e.g., hematopoietic stem cells) from the cell bank are stimulated to produce more granulocytes (having the desired CKA when tested using the assay of Example 2); Stocks are replenished, thus ensuring sufficient stocks of granulocytes are available 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 readily penetrates living eukaryotic cells. Viable cells exhibiting active metabolism convert MTT to a purple formazan product (1(E,Z)-5-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan) via the NAD(P)H-dependent mitochondrial oxidoreductase enzyme, which exhibits an absorbance maximum near 570 nm. When cells die, they lose the ability to convert MTT to formazan, and thus color formation serves as a useful and convenient marker for viable cells only. 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 spectrophotometrically at a wavelength of 570 nm. The absorbance at a reference wavelength of 690 nm is subtracted from the absorbance at 570 nm wavelength. Thus, the MTT assay can be used to determine cancer killing activity (CKA), i.e., The number of viable cells remaining was measured as a method to determine 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) After the target cells were allowed to adhere overnight, leukocytes (effector cells) were added, and all experimental conditions were set in triplicate.

[0309] Day 2: 4) Effector cells were added to target cells at various ratios (e.g., 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 diluting the kit solution 10 times with culture medium, i.e., per 100 wells, 1000 μL (1 mL) of MTT stock (provided in the kit) was taken and 9000 μL (9 mL) of culture medium (RPMI-1640) was added. 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 (provided in the kit) was added. 6) Formazan crystals were dissolved by pipetting if necessary and the 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 can demonstrate differences in CKA between neutrophils derived from different donors.

[0313] Example 10 Demonstration of CKA in stem cell-derived neutrophils Culturing neutrophils derived from CD34+ stem cells We cultured neutrophils derived from umbilical cord blood-derived stem cells expressing 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, by use of a nitroblue tetrazolium (NBT) assay (kit and protocol commercially available from Sigma-Aldrich, catalog number 840W-1KT). contains superoxide anion (O2 - ) 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 (batch 008A, 709A and 915) were cultured in a C-cell culture medium using HeLa target cells. These cells were used as effector cells in the KA MTT assay (see Example 9). The effector to target cell ratio was based on CD11b+ / CD15+. The results are summarized in Figure 2, which shows that CD34+ stem cell-derived neutrophils exhibit cytotoxicity in the CKA assay of HeLa cells, and that the results vary between different donors. Batch 008A consistently exhibits lower cytotoxicity than batches 915 and 709A at effector to target ratios up to 10:1, despite being prepared simultaneously with the other batches and cultured identically.

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

[0319] These results suggest that the cancer-killing activity (CKA) of the innate immune system differs between individuals, and that the leukocyte colony This supports the fact that the same innate variations in CKA seen in neutrophils directly collected from donors by immunofluorescence are also found in donor stem cells. By selecting donors with demonstrated high cancer-killing activity of the innate immune system and using their hematopoietic cells (i.e., hematopoietic stem cells) for ex vivo expansion and differentiation, it is possible to create a cell bank containing leukocytes with high cancer-killing activity for use in cancer treatment.

[0320] Example 11 xCELLigence "CKA Assay" The 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. This system measures impedance using interdigitating gold microelectrodes integrated into the bottom of each well of a tissue culture E-Plate. Impedance measurements are displayed as Cell Index (CI) values and provide quantitative information about the biological state of cells, including viability. Impedance-based monitoring of cell viability correlates with cell number and MTT-based readouts. The dynamic aspect of impedance-based cell viability measurements provides the temporal information necessary when using neutrophils to induce cytotoxic effects. Notably, the xCELLigence System also detects the maximal effect of neutrophils, as indicated by the lowest CI values, in cytotoxicity and cell death assays. It can also pinpoint the optimal time point for achieving this (if such data is desired). 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 accommodate up to 3 (The plates can be read simultaneously.) For the first few hours after cells are added to the wells, there is a rapid increase in impedance. This is caused by cells falling out of suspension, depositing on the electrode, and 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 at varying effector:target ratios). The percentage of cytolysis is determined by the simple formula: Percentage of Cytolysis = ((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 were used to generate the results presented in Examples 12 through 20. The results presented in Examples 12 through 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 was also able to demonstrate differences in CKA between neutrophils from different donors, and that this was 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 percentage cytotoxicity recorded by the CKA assay (xCELLigence assay) for each cancer cell type and for different donors. The percentage of cytotoxicity against 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 (DDNs) 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 results for HeLa and PANC-1 cancer cell lines and MCF-12F non-cancer cell line. CKA assay (xCELLigence assay) on neutrophils from each donor The maximum percentage of cytotoxicity recorded is shown. Advantageously, DDN was highly selective for cancer cells and showed minimal effect on non-cancer cells.

[0329] Example 15 Culturing 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 the 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 a differential CKA. Culture 5 showed low CKA neutrophils and culture 1 showed 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 cell proliferation rate recorded by the CKA assay (xCELLigence assay) for each cancer cell type and for donors LC267, LC268, and LC269. The percentage of cytotoxicity is shown. Similar to the results obtained with DDN (see Example 14), the percentage of cytotoxicity against pancreatic cancer was higher than that observed with HeLa cells (at both effector to target cell ratios of 5:1 and 10:1). SCDNs derived from different donors were also shown to have differential CKA. The assay was performed for up to 45 hours.

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

[0335] Example 18 Demonstration of CKA selectivity of stem cell-derived neutrophils against 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), and non-cancerous MCF-12F cells (normal breast epithelial cells).

[0336] Figure 9 shows the CKA assay (xCELLigence assay - up to 45 hours) for donors LC267, LC268, and LC269 against each cancer and non-cancer cell type. Figure 1 shows the maximum percentage of cytotoxicity recorded by SCDNs (performed over a period of 10 days). Advantageously, SCDNs were highly selective for cancer cells and showed minimal effects on non-cancerous cells. Similar to Figure 3, which shows SCDNs from the same donor, SCDNs from donor LC269 had the highest CKA, LC268 was second, and LC267 had 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 (FIG. 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 CKA assay (xCELLigence assay - up to 50 hours) of HeLa cells from donors LC252, LC253, and LC254 for DDN and SCDN. Figure 1 shows the maximum percentage of cytotoxicity recorded by the neutrophils (performed separately). Surprisingly, SCDNs exhibited high CKA similar to that of DDNs from the same donor, again indicating that CKA is encoded at the gene level. As in Figure 10, donor LC253 provided neutrophils (and SCDNs) with the highest CKA, whereas donors LC252 and LC254 provided neutrophils (and SCDNs) 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 approximately 50% of cancer cells in approximately 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 Ten 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 minutes to sediment red blood cells. A 50 ml conical polypropylene tube is prepared with 10 ml of 1.077 g / ml sucrose. The leukocyte-rich supernatant is slowly layered on top of the 1.077 g / ml sucrose layer and then centrifuged at 400 x g for 30 minutes at room temperature without using the brake. High-density neutrophils (HDN) appear in the pellet. Low-density neutrophils (LDN) appear in the 1.077 g / ml sucrose. They co-purify with monocytes and lymphocytes at the interface between the sucrose layer and the 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. Somatic cells (such as fibroblasts) are isolated from donors and used to establish iPSC cultures. iPSC is differentiated 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, without eliciting 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 specific 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 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.The 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.The 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 absence or inactivity of programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2; and b.The 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 absence or inactivity 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, wherein 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 absence or inactivity of 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 paragraphs 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 paragraphs 1 to 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 differentiate 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 paragraphs 1 to 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 paragraphs 1 to 13, wherein the granulocytes are neutrophils.

[0364] 15. A method for selecting hematopoietic cells suitable for use in the treatment of cancer. hand, a. Measuring the surface potential of granulocytes obtainable from the 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. A method comprising:

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

[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 the 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. A method comprising:

[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 with a surface potential defined by an electrophoretic mobility of less than 1.0 μm.cm / volt.sec and / or with a higher surface potential than otherwise identical hematopoietic cells that differentiate to form granulocytes with a reduced ability to kill cancer cells. A method comprising:

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

[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 according to any one of paragraphs 15 to 21, wherein the surface potential is determined by electrophoresis. How to do it.

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

[0373] 24. The method of any one of paragraphs 15 to 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 progenitors such as common myeloid progenitors, myeloblasts, N. promyelocytes, N. myelocytes, N. metamyelocytes, N. neutrophil band cells, or combinations thereof.

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

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

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

[0378] 29. The method of any one of paragraphs 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. measuring the percentage of cancer cells killed in the mixture; and d. Selecting hematopoietic cells from said donor if said granulocytes kill at least 70% of the cancer cells in the mixture. A method comprising:

[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 higher 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 paragraph 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 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. measuring the percentage of pancreatic cancer cells killed in the mixture; and d. Selecting granulocytes that kill at least 70% of the pancreatic cancer cells in the mixture. A method comprising:

[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 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. measuring the percentage of cancer cells killed in the 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 cancer of the same type / subset as the cancer cell line. A method comprising:

[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 of 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 of 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. Cancer cell lines include 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, and non-Hodgkin's lymphoma (NHL) cancer cell lines. 40. The in vitro method of any one of clauses 34 to 39, wherein the cell line is one or more selected from a leukemia 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 38-40, further comprising obtaining hematopoietic cells from a donor from which selected granulocytes are available.

[0391] 42. Granulocytes 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 paragraphs 1 to 14, or hematopoietic cells obtainable according to the method according to any one of paragraphs 15 to 31, into granulocytes.

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

[0394] 45. An in vitro cell culture of granulocytes obtainable by the method described in paragraph 43, comprising: a. a density of at least 1.077 g / ml; and b.The ability to kill cancer cells Cell cultures enriched for granulocytes with

[0395] 46. An in vitro cell culture of granulocytes obtainable by the method described in paragraph 43, comprising: a. Expression or activity of toll-like receptors; and / or absence or inactivity of programmed cell death 1 (PD-1) receptor; CD115; CD224; CXCR1; and / or CXCR2; and b.The ability to kill cancer cells Cell cultures enriched for granulocytes with

[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 A pharmaceutical composition comprising:

[0397] 48. 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 clauses 44 to 46, or a 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 for use according to clause 47, or a use or method according to any one of clauses 48 to 50, wherein the cancer is a solid tumor cancer.

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

[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 the subject with a cancer cell line; b. incubating the mixture; c. measuring the percentage of cancer cells killed in the mixture; and d. if granulocytes from the subject kill less than 70% of the cancer cells in the mixture, selecting the 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. A method comprising:

[0403] 54. The in vitro method of paragraph 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 paragraphs 1 to 14, or a granulocyte according to paragraph 42, or an in vitro cell culture of granulocytes according to any one of paragraphs 44 to 46, or a pharmaceutical composition according to paragraph 47.

[0405] 56. a. 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; and b. Instructions for its use in medicine Kit including:

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

Claims

1. 1. 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 the test sample; and b. 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, obtaining hematopoietic cells from the sample derived from said donor. A method comprising:

2. 1. 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 the test sample; and d. If the granulocytes kill at least 5% of the cancer cells in the test sample, obtaining hematopoietic cells from the donor-derived sample. A method comprising:

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. neutrophil band cells, or combinations thereof.

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

6. 1. 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. measuring the percentage of pancreatic cancer cells killed in the mixture; and d. Selecting granulocytes that kill at least 5% of the pancreatic cancer cells in the mixture. A method comprising:

7. An in vitro method for selecting granulocytes that selectively kill cancer cells, comprising: a. contacting a cancer cell line with granulocytes obtainable from a donor to form a test sample, and incubating the test sample; and b. Selecting the granulocytes as selective for cancer cells if the percentage of cancer cells killed in the test sample is higher than the percentage of non-cancerous cells killed in a control sample containing a non-cancerous cell line and granulocytes obtainable from the same donor. A method comprising:

8. 1. An in vitro method for selecting 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 compared to control granulocytes. A method comprising:

9. 1. An in vitro method for selecting hematopoietic cells suitable for use in the treatment of cancer, comprising: a. Measuring the concentration of granulocytes with 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 comprising:

10. 1. An in vitro method for selecting hematopoietic cells suitable for use in the treatment of cancer, 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. Measuring the concentration of granulocytes identified in step b.; d. comparing the concentration of granulocytes measured in step c. with the 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. If the comparison identifies a higher concentration of said granulocytes obtainable from said first donor compared to the concentration of said granulocytes obtainable from said second (or additional) donor, obtaining hematopoietic cells from a sample derived from said first donor. A method comprising:

11. An in vitro method according to any one of claims 8 to 10, wherein granulocytes are contacted by negatively charged nanoprobes or nanoparticles, preferably said granulocytes being isolated after said contacting.

12. Cancer cell lines include 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, and non-Hodgkin's lymphoma (NHL) cell lines.

12. The in vitro method of claim 1, wherein the cell line is one or more selected from the group consisting of a laryngeal cancer cell line, a uterine cancer cell line, and a breast cancer cell line.

13. The in vitro method according to any one of claims 1 to 12, wherein the cancer cell line is a pancreatic cancer cell line.

14. The in vitro method according to any one of claims 1 to 13, wherein the cancer cell line is a pancreatic ductal adenocarcinoma cell line, preferably the cancer cell line is the PANC-1 cell line.

15. The in vitro method according to any one of claims 1 to 14, wherein the granulocytes are neutrophils.

16. 16. The in vitro method of any one of claims 6 or 7 or 12-15, further comprising discarding granulocytes that kill less than 5% of the cancer cells in the mixture.

17. 17. The in vitro method of any one of claims 6 or 7 or 12-16, further comprising obtaining hematopoietic cells from a sample derived from said donor from which selected granulocytes are obtainable.

18. Preferably, hematopoietic cells or in vitro cell cultures thereof obtainable by the method of any one of claims 1 to 5, 8 to 15 or 17, wherein the hematopoietic cells have a positively charged cell surface.

19. Preferably, a granulocyte or an in vitro cell culture thereof obtained by the method according to any one of claims 6 or 7 or 12 to 17, or differentiated from a hematopoietic cell or an in vitro cell culture thereof according to claim 18, wherein the granulocyte has a positively charged cell surface.

20. An in vitro cell culture of hematopoietic cells, the hematopoietic cells comprising: a. a surface potential defined by an electrophoretic mobility of at least 1.0 μm.cm / volt.sec and / or a density greater than 1.077 g / ml; and b. Ability to kill cancer cells A cell culture that differentiates to form granulocytes characterized by:

21. a. a hematopoietic cell or an in vitro cell culture thereof according to claim 18, a granulocyte or an in vitro cell culture thereof according to claim 19, or an in vitro cell culture of a hematopoietic cell according to claim 20; 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 10. A pharmaceutical composition comprising:

22. a) a hematopoietic cell or an in vitro cell culture thereof according to claim 18, a granulocyte or an in vitro cell culture thereof according to claim 19, an in vitro cell culture of a hematopoietic cell according to claim 20, or a pharmaceutical composition according to claim 21; and b. Instructions for its use in medicine Kit including:

23. 23. A hematopoietic cell or an in vitro cell culture thereof according to claim 18, a granulocyte or an in vitro cell culture thereof according to claim 19, an in vitro cell culture of hematopoietic cells according to claim 20, a pharmaceutical composition according to claim 21, or a kit according to claim 22, for use in the treatment of cancer.

24. Use of hematopoietic cells or in vitro cell cultures thereof described in claim 18, granulocytes or in vitro cell cultures thereof described in claim 19, an in vitro cell culture of hematopoietic cells described in claim 20, a pharmaceutical composition described in claim 21, or a kit described in claim 22 in the manufacture of a medicament for the treatment of cancer.

25. A method for the treatment of cancer, comprising administering to a subject in need thereof the hematopoietic cells or in vitro cell culture thereof described in claim 18, the granulocytes or in vitro cell culture thereof described in claim 19, the in vitro cell culture of hematopoietic cells described in claim 20, or the pharmaceutical composition described in claim 21.

26. The hematopoietic cells or in vitro cell cultures thereof, granulocytes or in vitro cell cultures thereof, pharmaceutical composition, or kit for use, use, or method according to any one of claims 18 to 25, wherein the cancer is a solid tumor cancer.

27. Cancers include pancreatic cancer, liver cancer, esophageal cancer, stomach cancer, cervical cancer, ovarian cancer, lung cancer, bladder cancer, kidney cancer, brain tumors, prostate cancer, myeloma, non-Hodgkin's lymphoma (NHL), and throat cancer. The hematopoietic cells or in vitro cell cultures thereof, granulocytes or in vitro cell cultures thereof, or pharmaceutical composition, use or method for use, according to any one of claims 18 to 26, wherein the hematopoietic cells or in vitro cell cultures thereof are one or more of head cancer, uterine cancer, or breast cancer.

28. A cell bank comprising hematopoietic cells or in vitro cell cultures thereof according to claim 18, granulocytes or in vitro cell cultures thereof according to claim 19, an in vitro cell culture of hematopoietic cells according to claim 20, or a pharmaceutical composition according to claim 21.

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