Cells and preparation methods

Abstract

A method for preparing cells for therapeutic use is provided, comprising culturing a population of progenitor cells under cell culture conditions that promote progenitor cell differentiation, including the presence of G-CSF, GM-CSF, IL-3, and TNF, to generate a population of granulocyte-producing cells. The generated granulocyte-producing cells may be useful in therapeutic methods, including the treatment of cancer and infections, and may also be useful in modulating inflammatory cell responses.

Description

[Technical Field]

[0001] This invention relates to a method for preparing cells for therapeutic use and to populations of cells prepared by such a method. The invention also relates to populations of granulocyte-producing cells and to pharmaceutical compositions comprising such populations of cells. Populations of granulocyte-producing cells and pharmaceutical compositions may be suitable for use in modulating (e.g., amplifying) therapeutic immune responses, particularly in modulating non-granulocyte immune responses, and / or generating cells with cytodestructive activity. Populations of granulocyte-producing cells and pharmaceutical compositions can be used in situations such as the treatment of cancer or infection. The invention further relates to cell culture media.

[0002] Immunotherapy can work by amplifying the innate immune response of cells or the host to achieve therapeutic objectives. It is becoming increasingly important for use in a wide range of therapeutic situations.

[0003] The host therapeutic immune response often requires several types of immune cells and plays a crucial role in the body's fight against cancer, infections, and virtually all other diseases. However, the target's innate therapeutic immune response is not always sufficient to eradicate the disease. For example, tumors can adapt to an immunologically "cold" state and create an immunosuppressive tumor microenvironment (TME) that can neutralize the innate antitumor therapeutic immune response.

[0004] To successfully eradicate a tumor (e.g., cancer), various different types of immune cells usually need to work together. However, sometimes the target lacks its own immune cells, meaning that various different types of immune cells from alternative sources are needed. Currently, there are difficulties in producing such cell combinations. Furthermore, or conversely, such conventional cell combinations may have harmful immunogenic effects.

[0005] Cell-based therapies can also utilize the cellular destructive ability of cells and their ability to kill cells such as cancer cells, infected cells, or cellular infectious pathogens that play a crucial role in disease. Cells with cellular destructive activity, or cells capable of generating such destructive cells, are therefore important therapeutic targets.

[0006] Therefore, there is a need for improved immunotherapies, particularly cell therapies, and methods for generating cells that can be used in such immunotherapies.

[0007] The present invention addresses one or more of the above-mentioned problems.

[0008] The inventors have identified conditions that can be used to generate a large population of granulocyte-producing cells with highly desirable therapeutic activity. These cells can modulate (e.g., amplify) the therapeutic immune response of non-granulocyte immune cells, and in preferred embodiments, they can function to modulate or amplify the therapeutic immune response of non-granulocyte immune cells and can also produce cells with useful cytodestructive activity. Such compositions and populations of cells can therefore modulate (and thus modulate, e.g., amplify) the host therapeutic immune response and / or produce cells with direct cytodestructive activity after administration to a subject.

[0009] In a first aspect, the present invention relates to a method for preparing cells for therapeutic use, · G-CSF, GM-CSF, · IL-3 and TNF The present invention provides a method for generating a population of granulocyte-producing cells by culturing a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells, including the presence of a certain substance.

[0010] The method of the first aspect of the present invention may optionally further include the steps of purifying the population of granulocyte-producing cells that have been generated, and / or formulating the population of cells for medical use.

[0011] In a second aspect, the present invention provides a population of granulocyte-producing cells prepared for therapeutic use by the method of the first aspect of the present invention.

[0012] In a further embodiment, the present invention provides a cell culture medium for use in a method according to a first embodiment of the present invention, comprising G-CSF, GM-CSF, IL-3, and TNF. This cell culture medium according to this embodiment of the present invention may also be called a "differentiation medium."

[0013] The population of granulocyte-producing cells generated by the method of the present invention may be optionally recovered after generation. For the purposes of this disclosure, “recovery” of cells may be interpreted to include cell suspension, cell isolation, or cell separation.

[0014] The population of granulocyte-producing cells produced by the method of the present invention may optionally be cryopreserved after production. Granulocytes such as neutrophils do not respond well to cryopreservation, and it is known that the level of viable cells remaining after thawing of the frozen cell population is low. In contrast, the granulocyte-producing cells of the present invention are well-suited to cryopreservation, and a high level of viable cells is obtained after the freeze-thaw process. Therefore, the granulocyte-producing cell population of the present invention offers significant advantages compared to mature granulocyte cells in applications where it is desirable to cryopreserve cells before use for therapeutic purposes.

[0015] The population of granulocyte-producing cells produced by the method of the present invention may optionally be formulated for medical use after production. Suitable methods for formulating cell populations for therapeutic use are well known to those skilled in the art and can optionally be used to formulate the granulocyte-producing cell population of the present invention to produce the pharmaceutical composition of the present invention.

[0016] The characteristics of the population of granulocyte-producing cells generated, as well as the characteristics of the population of progenitor cells that can be used in such methods, are considered in more detail elsewhere in this specification.

[0017] Optionally, the cell culture conditions that promote the differentiation of progenitor cells may further include the presence of at least one cytokine selected from the group consisting of SCF and TPO. Preferably, the cell culture conditions include the presence of both SCF and TPO.

[0018] Similarly, the differentiation medium of the present invention may further include at least one cytokine selected from the group consisting of SCF and TPO. Preferably, such a cell culture medium includes both SCF and TPO.

[0019] In the following paragraphs, details of useful embodiments of the method of the first aspect of the present invention are described. These include useful embodiments of progenitor cells that can be used as starting materials, granulocyte-producing cells that can be generated by the method, and cell culture conditions that can be utilized. Except for any examples where otherwise noted, the considerations described regarding cell culture conditions that promote differentiation that can be used in the method of the first aspect of the present invention are also applicable as suitable embodiments of the differentiated cell culture medium of the present invention.

[0020] The cell culture conditions that promote differentiation, used in the method of the first aspect of the present invention, may include Iscove's Modified Dulbecco's Medium (IMDM) as the cell culture medium. Similarly, the cell culture medium of the present invention may also include IMDM. In either case, in a preferred embodiment, IMDM is in the form of a medium that includes high glucose, glutamine, HEPES, sodium pyruvate, and optionally may include phenol red.

[0021] The method of the first aspect of the present invention, or the differentiation medium of the present invention, utilizes cytokine granulocyte colony-stimulating factor (G-CSF) as an adjuvant.

[0022] Preferably, G-CSF is provided at a concentration of 0.013 μg / mL or higher. For example, G-CSF may be provided at concentrations of 0.016 μg / mL or higher, 0.02 μg / mL or higher, 0.03 μg / mL or higher, or 0.065 μg / mL or higher.

[0023] Preferably, G-CSF is provided at a concentration of 0.65 μg / mL or less. For example, G-CSF may be provided at concentrations of 0.52 μg / mL or less, 0.39 μg / mL or less, or 0.26 μg / mL or less.

[0024] Preferably, G-CSF is provided at concentrations of approximately 0.013 μg / mL to 0.65 μg / mL, 0.016 μg / mL to 0.52 μg / mL, 0.02 μg / mL to 0.39 μg / mL, 0.03 μg / mL to 0.26 μg / mL, or 0.065 μg / mL to 0.195 μg / mL. In a preferred embodiment, G-CSF is provided at a concentration of approximately 0.13 μg / mL. In fact, in a preferred embodiment, G-CSF is provided at a concentration of 0.13 μg / mL.

[0025] Examples of suitable forms of G-CSF that can be used in this way include products produced by Peprotech and GMP products produced by BioLegend, details of which are provided in Table 2.

[0026] The method according to the first aspect of the present invention, or the differentiation medium according to the present invention, utilizes cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) as an adjuvant.

[0027] Preferably, GM-CSF is provided at a concentration of 0.001 μg / mL or higher. For example, GM-CSF may be provided at concentrations of 0.00125 μg / mL or higher, 0.00167 μg / mL or higher, 0.0025 μg / mL or higher, or 0.005 μg / mL or higher.

[0028] Preferably, GM-CSF is provided at a concentration of 0.05 μg / mL or less. For example, GM-CSF may be provided at concentrations of 0.04 μg / mL or less, 0.03 μg / mL or less, or 0.02 μg / mL or less.

[0029] Preferably, GM-CSF is provided at concentrations of approximately 0.001 μg / mL to 0.05 μg / mL, 0.125 μg / mL to 0.04 μg / mL, 0.00167 μg / mL to 0.03 μg / mL, 0.0025 μg / mL to 0.02 μg / mL, or 0.005 μg / mL to 0.015 μg / mL. In a preferred embodiment, GM-CSF is provided at a concentration of approximately 0.01 μg / mL. In fact, in a preferred embodiment, GM-CSF is provided at a concentration of 0.01 μg / mL.

[0030] Examples of suitable forms of GM-CSF that can be used in this way include products produced by Peprotech and BioTechne, as well as GMP products produced by BioTechne, details of which are provided in Table 2.

[0031] The method according to the first aspect of the present invention, or the differentiation medium according to the present invention, utilizes the cytokine interleukin-3 (IL-3) as an adjuvant.

[0032] Preferably, IL-3 is provided at a concentration of 0.013 μg / mL or higher. For example, IL-3 may be provided at concentrations of 0.016 μg / mL or higher, 0.02 μg / mL or higher, 0.03 μg / mL or higher, or 0.065 μg / mL or higher.

[0033] Preferably, IL-3 is provided at a concentration of 0.65 μg / mL or less. For example, IL-3 may be provided at concentrations of 0.52 μg / mL or less, 0.39 μg / mL or less, or 0.26 μg / mL or less.

[0034] Preferably, IL-3 is provided at concentrations of approximately 0.013 μg / mL to 0.65 μg / mL, 0.016 μg / mL to 0.52 μg / mL, 0.02 μg / mL to 0.39 μg / mL, 0.03 μg / mL to 0.26 μg / mL, or 0.065 μg / mL to 0.195 μg / mL. In a preferred embodiment, IL-3 is provided at a concentration of approximately 0.13 μg / mL. In fact, in a preferred embodiment, IL-3 is provided at a concentration of 0.13 μg / mL.

[0035] Examples of suitable forms of IL-3 that can be used in this way include products manufactured by PeproTech and GMP products manufactured by PeproTech or BioTechne, details of which are provided in Table 2.

[0036] In preferred embodiments, GM-CSF and IL-3 are provided to cells for a period of 12 to 72 hours, preferably 48 hours, during the cell culture conditions. For example, GM-CSF and IL-3 may be provided to cells for the last 48 hours of the period the cells are in culture. GM-CSF and IL-3 may be provided to cells on days 4 and 5 of the cell culture conditions that promote the differentiation of progenitor cells. GM-CSF and IL-3 may be provided to cells on days 3 and 4 of the cell culture conditions that promote the differentiation of progenitor cells.

[0037] The method for obtaining granulocyte-producing cells utilizes the cytokine tumor necrosis factor (TNF) as an adjuvant. The terms TNF and TNF-alpha are used synonymously herein.

[0038] Preferably, TNF is provided at a concentration of 0.0001 μg / mL or higher. For example, TNF may be provided at concentrations of 0.000125 μg / mL or higher, 0.000167 μg / mL or higher, 0.00025 μg / mL or higher, or 0.0005 μg / mL or higher.

[0039] Preferably, TNF is provided at a concentration of 0.005 μg / mL or less. For example, TNF may be provided at a concentration of 0.004 μg / mL or less, 0.003 μg / mL or less, or 0.002 μg / mL or less.

[0040] Preferably, TNF is provided at a concentration of approximately 0.0001 μg / mL to 0.005 μg / mL, 0.000125 μg / mL to 0.004 μg / mL, 0.000167 μg / mL to 0.003 μg / mL, 0.00025 μg / mL to 0.002 μg / mL, or 0.0005 μg / mL to 0.0015 μg / mL. In a preferred embodiment, TNF is provided at a concentration of approximately 0.001 μg / mL. In fact, in a preferred embodiment, TNF is provided at a concentration of 0.001 μg / mL.

[0041] Examples of suitable forms of TNF that can be used in this way include products produced by PeproTech and GMP products produced by BioTechne, the details of which are described in Table 2.

[0042] In a preferred embodiment, TNF is provided to the cells for a period of 12 to 36 hours, preferably 24 hours, during the cell culture conditions. For example, TNF may be provided to the cells during the last 24 hours of the period during which the cells are in culture. TNF may be provided to the cells on the 4th to 5th day of the cell culture conditions that promote the differentiation of progenitor cells. TNF may be provided to the cells on the 5th day of the cell culture conditions that promote the differentiation of progenitor cells. TNF may be provided to the cells on the 4th day of the cell culture conditions that promote the differentiation of progenitor cells.

[0043] The method of the first aspect of the present invention, or the differentiation medium of the present invention, may optionally utilize cytokine stem cell factor (SCF) as an adjuvant.

[0044] Preferably, SCF is provided at a concentration of 0.013 μg / mL or more. For example, SCF may be provided at a concentration of 0.016 μg / mL or more, 0.02 μg / mL or more, 0.03 μg / mL or more, or 0.06 μg / mL or more.

[0045] Preferably, SCF is provided at a concentration of 0.65 μg / mL or less. For example, SCF may be provided at concentrations of 0.52 μg / mL or less, 0.39 μg / mL or less, or 0.26 μg / mL or less.

[0046] Preferably, SCF is provided at concentrations of approximately 0.013 μg / mL to 0.65 μg / mL, 0.016 μg / mL to 0.52 μg / mL, 0.02 μg / mL to 0.39 μg / mL, 0.03 μg / mL to 0.26 μg / mL, or 0.065 μg / mL to 0.195 μg / mL. In a preferred embodiment, SCF is provided at a concentration of approximately 0.13 μg / mL. In fact, in a preferred embodiment, SCF is provided at a concentration of 0.13 μg / mL.

[0047] Examples of suitable forms of SCF that can be used in this way include products produced by Peprotech and GMP products produced by Peprotech or BioTechne, details of which are provided in Table 2.

[0048] The method according to the first aspect of the present invention, or the differentiation medium according to the present invention, may optionally utilize the cytokine thrombopoietin (TPO) as an adjuvant.

[0049] Preferably, TPO is provided at a concentration of 0.013 μg / mL or higher. For example, TPO may be provided at concentrations of 0.016 μg / mL or higher, 0.02 μg / mL or higher, 0.03 μg / mL or higher, or 0.065 μg / mL or higher.

[0050] Preferably, TPO is provided at a concentration of 0.65 μg / mL or less. For example, TPO may be provided at concentrations of 0.52 μg / mL or less, 0.39 μg / mL or less, or 0.26 μg / mL or less.

[0051] Preferably, TPO is provided in concentrations of approximately 0.013 μg / mL to 0.65 μg / mL, 0.016 μg / mL to 0.52 μg / mL, 0.02 μg / mL to 0.39 μg / mL, 0.03 μg / mL to 0.26 μg / mL, or 0.065 μg / mL to 0.195 μg / mL. In a preferred embodiment, TPO is provided at a concentration of approximately 0.13 μg / mL. In fact, in a preferred embodiment, TPO is provided at a concentration of 0.13 μg / mL.

[0052] Examples of suitable forms of TPO that can be used in this way include products produced by Peprotech and GMP products produced by BioTechne or Peprotech, details of which are provided in Table 2.

[0053] In preferred embodiments, the cell culture conditions used when culturing a population of progenitor cells to generate granulocyte-producing cells further include the presence of at least one adjuvant selected from the group consisting of insulin transferrin selenium (ITS) and human serum albumin (HSA). In preferred embodiments, such cell culture conditions include the presence of both ITS and HSA. Preferably, both ITS and HSA are present in the differentiation medium of the present invention.

[0054] The method according to the first aspect of the present invention, or the differentiation medium of the present invention, may appropriately utilize insulin as an adjuvant at a concentration between about 0.1 g / L and about 5 g / L, for example, about 1.0 g / L. These methods and cell culture media may appropriately utilize transferrin as an adjuvant at a concentration between about 0.01 g / L and about 2.5 g / L, for example, about 0.55 g / L. Preferably, such methods and cell culture media may utilize selenium as an adjuvant at a concentration between about 0.0001 g / L and about 0.003 g / L, for example, about 0.00067 g / L.

[0055] The method according to the first aspect of the present invention, or the differentiation medium according to the present invention, may optionally utilize HSA as an auxiliary agent.

[0056] Preferably, HSA may be provided in a concentration between 0.1% and 5%. For example, HSA provided as an adjuvant may be provided in a concentration of approximately 1%.

[0057] Preferably, the cell culture conditions or differentiation medium used in the method of the present invention to promote the differentiation of progenitor cells may include GM-CSF, G-CSF, SCF, TPO, IL-3, TNF, ITS, and HSA. The cell culture medium may optionally include IMDM supplemented with Glutamax.

[0058] Therefore, in a preferred embodiment, the cell culture conditions or differentiation medium used in the method of the present invention to promote the differentiation of progenitor cells may include approximately 0.01 μg / mL of GM-CSF, approximately 0.13 μg / mL of G-CSF, approximately 0.13 μg / mL of SCF, approximately 0.13 μg / mL of TPO, approximately 0.13 μg / mL of IL-3, approximately 0.001 μg / mL of TNF, 1 × ITS, and approximately 1% HSA. The cell culture medium may optionally include IMDM supplemented with Glutamax.

[0059] The method of the present invention may include culturing a population of progenitor cells for any appropriate period of time under cell culture conditions that promote the differentiation of progenitor cells. For example, progenitor cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days under conditions that produce a population of granulocyte-producing cells. The method according to the first aspect of the present invention may include culturing a population of progenitor cells for a period of 1 to 7 days under cell culture conditions that promote the differentiation of progenitor cells. For example, such a method may include culturing cells for a period of 4 to 7 days under the relevant conditions. In a preferred embodiment, such a method may include culturing cells for approximately 1 day, or approximately 2 days, or approximately 3 days, or approximately 4 days, or approximately 5 days, or approximately 6 days, or approximately 7 days. Progenitor cells may be cultured for 1 to 10 days, 2 to 9 days, 3 to 8 days, 4 to 7 days, or 5 to 6 days under conditions that produce a population of granulocyte-producing cells. Preferably, the progenitor cells are cultured for 4, 5, or 6 days under conditions that produce a population of granulocyte-producing cells. In a preferred embodiment, the progenitor cells are cultured for 4 days under conditions that produce a population of granulocyte-producing cells. In a preferred embodiment, the progenitor cells are cultured for 5 days under conditions that produce a population of granulocyte-producing cells. In a preferred embodiment, the progenitor cells are cultured for 6 days under conditions that produce a population of granulocyte-producing cells.

[0060] In a preferred embodiment of the method of the present invention, the progenitor cells are 1 cm 2 Approximately 1 x 10 5 From 10x10 6 The cells may be cultured at an initial seeding density.

[0061] The method of the present invention may also include increasing the number of cells present in the culture so that the number of granulocyte-producing cells obtained by the method is greater than the number of progenitor cells present at the start of the method. In a preferred embodiment, the number of granulocyte-producing cells in the resulting population may increase by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 times compared to the number of progenitor cells present at the start of the method. The method described in the examples achieves a population of granulocyte-producing cells that is approximately 3.5 times larger than the initial population of progenitor cells.

[0062] In preferred embodiments, the method of the first aspect of the present invention is carried out with respect to a population of progenitor cells generated by in vitro proliferation of a population of stem cells. Thus, such a method of the present invention may further include the step of culturing a population of stem cells under cell culture conditions that generate a population of progenitor cells.

[0063] In a preferred embodiment, the method of the first aspect of the present invention further includes the step of culturing a population of stem cells under cell culture conditions that generate a population of progenitor cells, • The cell culture conditions for generating progenitor cells are: · SCF, • Flt-3 ligand, IL-3, · IL-6, and TPO This includes the presence of.

[0064] The number of progenitor cells generated by such methods may be significantly increased compared to the number of stem cells present at the start of the cell culture conditions. As just one example, such embodiments of the method of the present invention can achieve an increase in the number of progenitor cells of at least 50 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 300 times, or at least 350 times or more compared to the number of stem cells at the start of the cell culture conditions. The examples detail the protocol used by the inventors to achieve an increase of approximately 75 times in the number of progenitor cells compared to the initial stem cell population.

[0065] The present invention also provides a cell culture medium for use in the method of the present invention, comprising SCF, Flt-3 ligand, IL-3, IL-6, and TPO. The cell culture medium according to this aspect of the present invention may also be referred to as "expansion medium."

[0066] Therefore, the method for preparing cells for therapeutic use according to such embodiments of the present invention is a) · SCF, • Flt-3 ligand, IL-3, · IL-6, and TPO To generate a population of progenitor cells by culturing a population of stem cells under cell culture conditions that include the presence of, b) · G-CSF, GM-CSF, · IL-3 and TNF To culture a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells, including the presence of [specific element], to generate a population of granulocyte-producing cells, and optionally, c) To collect granulocyte-producing cells and It may include.

[0067] Representing the change in cell number from the initial population of stem cells to the population of granulocyte-generating cells generated, the total increase in cell number achieved by such a method of the present invention may be at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 300 times, at least 350 times, at least 400 times, at least 450 times, at least 500 times, at least 550 times, at least 600 times, at least 650 times, at least 700 times, at least 750 times, at least 800 times, at least 850 times, at least 900 times, at least 950 times, at least 1000 times, at least 1050 times, at least 1100 times, at least 1150 times, at least 1200 times, at least 1250 times, or at least 1300 times. Examples detail the protocols used by the inventors to achieve an increase of more than 250 times in the number of granulocyte-generating cells compared to the initial stem cell population.

[0068] The method according to such embodiments of the present invention may include a total incubation period of 10 to 25 days, for example, 11 to 20 days, for example, 12, 13, 14, 15, 6, 17, 18, or 19 days.

[0069] SCF may optionally be provided as an adjuvant in embodiments of the method of the present invention that include a step of generating a population of progenitor cells, and in the expanded medium of the present invention.

[0070] Preferably, SCF is provided at a concentration of 0.02 μg / mL or higher. For example, SCF may be provided at concentrations of 0.025 μg / mL or higher, 0.03 μg / mL or higher, 0.05 μg / mL or higher, or 0.1 μg / mL or higher.

[0071] Preferably, SCF is provided at a concentration of 1 μg / mL or less. For example, SCF may be provided at a concentration of 0.8 μg / mL or less, 0.6 μg / mL or less, or 0.4 μg / mL or less.

[0072] Preferably, SCF is provided at concentrations of approximately 0.02 μg / mL to 1 μg / mL, 0.025 μg / mL to 0.8 μg / mL, 0.03 μg / mL to 0.6 μg / mL, 0.05 μg / mL to 0.4 μg / mL, or 0.1 μg / mL to 0.3 μg / mL. In a preferred embodiment, SCF is provided at a concentration of approximately 0.2 μg / mL. In fact, in a preferred embodiment, SCF is provided at a concentration of 0.2 μg / mL.

[0073] The above-described form of SCF is also suitable for use in such embodiments.

[0074] Flt-3 ligand (F3L) may optionally be provided as an adjuvant in embodiments of the method of the present invention that include a step of generating a population of progenitor cells, and in the expanded medium of the present invention.

[0075] Preferably, F3L is provided at a concentration of 0.02 μg / mL or higher. For example, F3L may be provided at concentrations of 0.025 μg / mL or higher, 0.03 μg / mL or higher, 0.05 μg / mL or higher, or 0.1 μg / mL or higher.

[0076] Preferably, F3L is provided at a concentration of 1 μg / mL or less. For example, F3L may be provided at a concentration of 0.8 μg / mL or less, 0.6 μg / mL or less, or 0.4 μg / mL or less.

[0077] Preferably, F3L is provided at concentrations of approximately 0.02 μg / mL to 1 μg / mL, 0.025 μg / mL to 0.8 μg / mL, 0.03 μg / mL to 0.6 μg / mL, 0.05 μg / mL to 0.4 μg / mL, or 0.1 μg / mL to 0.3 μg / mL. In a preferred embodiment, F3L is provided at a concentration of approximately 0.2 μg / mL. In fact, in a preferred embodiment, F3L is provided at a concentration of 0.2 μg / mL.

[0078] Examples of suitable forms of F3L that can be used in this way include products produced by Peprotech and GMP products produced by Peprotech or BioTechne, details of which are provided in Table 2.

[0079] IL-3 may optionally be provided as an adjuvant in embodiments of the method of the present invention that include the step of generating a population of progenitor cells, and in the expanded medium of the present invention.

[0080] Preferably, IL-3 is provided at a concentration of 0.0015 μg / mL or higher. For example, IL-3 may be provided at concentrations of 0.0019 μg / mL or higher, 0.0025 μg / mL or higher, 0.00375 μg / mL or higher, or 0.0075 μg / mL or higher.

[0081] Preferably, IL-3 is provided at a concentration of 0.075 μg / mL or less. For example, IL-3 may be provided at concentrations of 0.06 μg / mL or less, 0.045 μg / mL or less, or 0.03 μg / mL or less.

[0082] Preferably, IL-3 is provided at concentrations of approximately 0.0015 μg / mL to 0.075 μg / mL, 0.0019 μg / mL to 0.06 μg / mL, 0.0025 μg / mL to 0.045 μg / mL, 0.00375 μg / mL to 0.03 μg / mL, or 0.0075 μg / mL to 0.0225 μg / mL. In a preferred embodiment, IL-3 is provided at a concentration of approximately 0.015 μg / mL. In fact, in a preferred embodiment, IL-3 is provided at a concentration of 0.015 μg / mL.

[0083] The above-described IL-3 configuration is suitable for use in such embodiments.

[0084] Interleukin-6 (IL-6) may optionally be provided as an adjuvant in embodiments of the method of the present invention that include a step of generating a population of progenitor cells, and in the expanded medium of the present invention.

[0085] Preferably, IL-6 is provided at a concentration of 0.0015 μg / mL or higher. For example, IL-6 may be provided at concentrations of 0.0019 μg / mL or higher, 0.0025 μg / mL or higher, 0.00375 μg / mL or higher, or 0.0075 μg / mL or higher.

[0086] Preferably, IL-6 is provided at a concentration of 0.075 μg / mL or less. For example, IL-6 may be provided at concentrations of 0.06 μg / mL or less, 0.045 μg / mL or less, or 0.03 μg / mL or less.

[0087] Preferably, IL-6 is provided at concentrations of approximately 0.0015 μg / mL to 0.075 μg / mL, 0.0019 μg / mL to 0.06 μg / mL, 0.0025 μg / mL to 0.045 μg / mL, 0.00375 μg / mL to 0.03 μg / mL, or 0.0075 μg / mL to 0.0225 μg / mL. In a preferred embodiment, IL-6 is provided at a concentration of approximately 0.015 μg / mL. In fact, in a preferred embodiment, IL-6 is provided at a concentration of 0.015 μg / mL.

[0088] Examples of suitable forms of IL-6 that can be used in this way include products manufactured by PeproTech and GMP products manufactured by PeproTech or BioTechne, details of which are provided in Table 2.

[0089] TPO may optionally be provided as an adjuvant in embodiments of the method of the present invention that include a step of generating a population of progenitor cells, and in the expanding medium of the present invention.

[0090] Preferably, TPO is provided at a concentration of 0.002 μg / mL or higher. For example, TPO may be provided at concentrations of 0.0025 μg / mL or higher, 0.003 μg / mL or higher, 0.005 μg / mL or higher, or 0.01 μg / mL or higher.

[0091] Preferably, TPO is provided at a concentration of 0.1 μg / mL or less. For example, TPO may be provided at a concentration of 0.08 μg / mL or less, 0.06 μg / mL or less, or 0.04 μg / mL or less.

[0092] Preferably, TPO is provided in concentrations of approximately 0.002 μg / mL to 0.1 μg / mL, 0.0025 μg / mL to 0.08 μg / mL, 0.003 μg / mL to 0.06 μg / mL, 0.005 μg / mL to 0.04 μg / mL, or 0.01 μg / mL to 0.03 μg / mL. In a preferred embodiment, TPO is provided at a concentration of approximately 0.02 μg / mL. In fact, in a preferred embodiment, TPO is provided at a concentration of 0.02 μg / mL.

[0093] The TPO forms described above are also suitable for use in these embodiments.

[0094] Preferably, the cell culture conditions or expansion medium used in the method of the present invention to promote the generation of progenitor cells may include SCF, and Flt-3 ligand, and IL-3, and IL-6, and TPO, and ITS, and HSA. The cell culture medium may optionally include IMDM to which Glutamax has been added.

[0095] Therefore, in a preferred embodiment, the cell culture conditions or expansion medium used in the method of the present invention to promote the generation of progenitor cells may include approximately 0.2 μg / mL of SCF, approximately 0.2 μg / mL of Flt-3 ligand, approximately 0.015 μg / mL of IL-3, approximately 0.015 μg / mL of IL-6, approximately 0.02 μg / mL of TPO, 1 × ITS, and approximately 1% HSA. The cell culture medium may optionally include IMDM with added Glutamax.

[0096] Examples of stem cells that can be used in such methods of the present invention as starting materials for the generation of progenitor cells (and ultimately granulocyte-producing cells) include, but are not limited to, hematopoietic stem cells (HSCs). Further details of suitable stem cells and sources of stem cells are provided elsewhere herein and include (but are not limited to) umbilical cord blood and mobilized blood.

[0097] In preferred embodiments, the cell culture conditions used when culturing stem cells to generate progenitor cells further include the presence of at least one adjuvant selected from the group consisting of ITS and HSA. In preferred embodiments, such cell culture conditions include the presence of both ITS and HSA. Preferably, both ITS and HSA are present in the expanded medium of the present invention.

[0098] ITS may be provided as an adjuvant in embodiments of the method of the present invention, which includes the step of generating a population of progenitor cells, and in the expanded medium of the present invention.

[0099] Such embodiments of the methods of the first aspect of the present invention, or the expanded culture medium of the present invention, may appropriately utilize insulin as an adjuvant at a concentration between about 0.1 g / L and about 5 g / L, for example, about 1.0 g / L. These methods and cell culture media may appropriately utilize transferrin as an adjuvant at a concentration between about 0.01 g / L and about 2.5 g / L, for example, about 0.55 g / L. Preferably, such methods and cell culture media may utilize selenium as an adjuvant at a concentration between about 0.0001 g / L and about 0.003 g / L, for example, about 0.00067 g / L.

[0100] HSA may be provided as an adjuvant in embodiments of the present invention that include a step of generating a population of progenitor cells, and in the expanded medium of the present invention.

[0101] Preferably, HSA may be provided in a concentration between 0.1% and 5%. For example, HSA provided as an adjuvant may be provided in a concentration of approximately 1%.

[0102] In embodiments of the present invention's method for culturing stem cells to obtain progenitor cells, this may include increasing the number of cells present in the culture.

[0103] The method of the present invention may include culturing a population of stem cells for any appropriate period of time under cell culture conditions that generate a population of progenitor cells. For example, cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days under conditions that generate a population of progenitor cells. Preferably, cells are cultured for 8 or 9 days under conditions that generate a population of progenitor cells. Stem cells may be cultured for 1-15 days, 1-10 days, 2-14 days, 3-13 days, 4-12 days, 5-11 days, 6-10 days, 7-9 days, or 8-9 days under conditions that generate a population of progenitor cells. Preferably, stem cells such as HSCs are cultured for 8-9 days under conditions that generate a population of progenitor cells.

[0104] In a preferred embodiment of such a method of the present invention, stem cells are cultured for a period of 6 to 10 days under conditions that produce a population of progenitor cells. For example, such a method may include culturing the cells for a period of 7 to 8 days. In a preferred embodiment, such a method may include culturing the cells for approximately 6, or approximately 7, or approximately 8, or approximately 9, or approximately 10 days under cell culture conditions that produce a population of progenitor cells.

[0105] Therefore, the method of the present invention for preparing cells for therapeutic use is (a) A population of stem cells is cultured for 6 to 10 days, or preferably 8 days, under cell culture conditions for generating progenitor cells that include the presence of SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, thereby generating a population of progenitor cells. (b) To obtain a population of granulocyte-producing cells by culturing a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells. It may include.

[0106] A preferred method of the present invention for preparing cells for therapeutic use is: (a) A population of stem cells is cultured for 6 to 10 days, or preferably 8 days, under cell culture conditions for generating progenitor cells that include the presence of IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, thereby generating a population of progenitor cells. (b) A population of progenitor cells is cultured for 1 to 6 days, preferably 5 days, under cell culture conditions that promote the differentiation of progenitor cells, including IMDM, G-CSF, GM-CSF, IL-3, and TNF, to obtain a population of granulocyte-producing cells. It may include.

[0107] Such a method of the present invention for preparing cells for therapeutic use is (a) A population of stem cells is cultured for 6 to 10 days, or preferably 8 days, under cell culture conditions for generating progenitor cells that include the presence of IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, thereby generating a population of progenitor cells. (b) To obtain a population of granulocyte-producing cells by culturing a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells. It may include.

[0108] For example, the present invention's method for preparing cells for therapeutic use is: (a) A population of stem cells is cultured for 6 to 10 days, or preferably 8 days, under cell culture conditions for generating progenitor cells that include the presence of IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, thereby generating a population of progenitor cells. (b) Culture a population of progenitor cells for 1 to 6 days, or preferably 5 days, under cell culture conditions that promote the differentiation of progenitor cells including IMDM, SCF, TPO, GCSF, ITS, and HSA, to obtain a population of granulocyte-producing cells. It may include.

[0109] A properly supplemented cell culture medium may be replaced or supplemented at any appropriate time during the culture of stem cells under conditions for generating progenitor cells. For example, the cell culture medium may be supplemented on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, or 15th day of the culture of stem cells. Preferably, the cell culture medium is supplemented on the 1st and 6th days of the culture of stem cells. The cell culture medium may be replaced on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, or 15th day of the culture of stem cells. Preferably, the cell culture medium is replaced on the 4th day of the culture of stem cells.

[0110] Stem cells such as HSCs from which progenitor cells are generated may be seeded at any appropriate cell density. For example, the stem cells are 1×10 5 cells / mL to 1×10 6 cells / mL, 2.5×10 5 cells / mL to 1×10 6 cells / mL, 3×10 5 cells / mL to 8×10 5 cells / mL or 4×10 5 cells / mL to 6×10 5 cells / mL, preferably seeded at a density of 5×10 5 cells / mL. The stem cells are 1×10 5 cells / cm 2 ~1×10 6 cells / cm 2 、2.5×10 5 cells / cm 2 ~1×10 6 cells / cm 2 、3×10 5 cells / cm 2 ~8×10 5 cells / cm 2 または4×10 5 cells / cm 2 ~6×10 5 cells / cm 2 、好ましくは5×10 5 cells / cm 2They may be seeded at a density of 5 × 10. In a preferred embodiment, stem cells (e.g., HSCs) are arranged as follows: 5 cells / mL and 5 × 10 5 cells / cm 2 It is sown at this density.

[0111] Cells may be seeded in any suitable culture vessel. For example, cells may be seeded in a G-Rex 6M or G-Rex 10M culture vessel. Cells may be transferred to a new culture vessel at any appropriate time. Cells may be successively transferred to cell culture vessels with increasing surface area. Such transfers may be performed on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the culture for generating progenitor cells. For example, stem cells (e.g., HSCs) may be transferred from a smaller G-Rex to a G-Rex 100M on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the culture for generating progenitor cells. For example, stem cells (e.g., HSCs) may be transferred to a G-Rex 100M or a larger cell culture vessel, such as a G-Rex 500M, on day 4 of expansion. In a preferred embodiment, progenitor cells may be transferred to a new culture vessel on day 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of culture conditions that promote the differentiation of progenitor cells into granulocyte-producing cells.

[0112] According to such embodiments, a preferred method for preparing cells for therapeutic use is: (a) 5 × 10 5 cells / mL and 5 × 10 5 cells / cm 2 This involves seeding stem cells (e.g., HSCs), (b) To obtain a population of progenitor cells by culturing cells for 8 days in a cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, wherein the cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA is replenished on days 1 and 6 of the culture, and the cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA is replaced on day 4 of the culture, (c) A population of progenitor cells is cultured for 5-6 days in a cell culture medium containing IMDM, SCF, TPO, GCSF, ITS, and HSA to obtain a population of granulocyte-producing cells, wherein the cell culture medium containing IMDM, SCF, TPO, GCSF, ITS, and HSA is replenished on the third day of differentiation, and a population of progenitor cells is cultured to obtain a population of granulocyte-producing cells. It may include.

[0113] A preferred method for preparing cells for therapeutic use is: (a) 5 × 10 5 cells / mL and 5 × 10 5 cells / cm 2 This involves seeding stem cells (e.g., HSCs), (b) To obtain a population of progenitor cells by culturing cells for 8 days in a cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, wherein the cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA is replenished on days 1 and 6 of the culture, and the cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA is replaced on day 4 of the culture, (c) A population of progenitor cells is cultured for 5-6 days in a cell culture medium containing IMDM, SCF, TPO, G-CSF, ITS, and HSA to obtain a population of granulocyte-producing cells, wherein the cell culture medium containing IMDM, SCF, TPO, GCSF, ITS, HAS, GM-CSF, IL-3, and TNF is replenished on the third day of differentiation, and a population of progenitor cells is cultured to obtain a population of granulocyte-producing cells. It may include.

[0114] The inventors have also identified a method by which granulocyte-producing cells can be primed to amplify cellular properties that enhance their therapeutic utility. In particular, priming granulocyte-producing cells by such a method can amplify their cytotoxic activity in a manner that enhances their therapeutic utility.

[0115] Accordingly, a third aspect of the present invention provides a method for priming granulocyte-producing cells for therapeutic use, comprising culturing a population of granulocyte-producing cells in the presence of GM-CSF and optionally one or more cytokines selected from the group consisting of TNF, IFN-α, IFN-β, IL-15, and IL-18.

[0116] A method according to a first aspect of the present invention may also include the step of priming granulocyte-producing cells for therapeutic use by culturing a population of granulocyte-producing cells in the presence of GM-CSF and optionally one or more cytokines selected from the group consisting of TNF, IFN-α, IFN-β, IL-15, and IL-18.

[0117] A third or first embodiment of the present invention, comprising the step of priming granulocyte-producing cells, may optionally further comprise the steps of purifying the resulting population of primed granulocyte-producing cells and / or formulating the population of primed cells for medical use.

[0118] A fourth aspect of the present invention provides a population of primed granulocyte-producing cells that can be obtained by the method according to the third aspect of the present invention. The population of primed granulocyte-producing cells can be obtained by the method according to the third aspect of the present invention. The population of primed granulocyte-producing cells may be as defined elsewhere in this disclosure (for example, with respect to the biological activity of the primed cells or the expression of their specific markers).

[0119] GM-CSF may be used at concentrations of 1-1000 ng / mL, 2-500 ng / mL, 3-250 ng / mL, or 4-200 ng / mL in the cell culture conditions during the priming step. GM-CSF may also be used at concentrations of 5-150 ng / mL, for example, 10-130 ng / mL.

[0120] TNF may be used in the cell culture conditions of the priming step at concentrations of 0.001–10 ng / mL, 0.002–5 ng / mL, 0.003–2.5 ng / mL, or 0.004–2 ng / mL. TNF may also be used at concentrations of 0.005–1.5 ng / mL, for example, 0.01–1 ng / mL.

[0121] IFN-α may be used at concentrations of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, or 4-20 ng / mL in the cell culture conditions during the priming step. IFN-α may also be used at concentrations of 5-15 ng / mL, for example, 10 ng / mL.

[0122] IFN-β may be used at concentrations of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, or 4-20 ng / mL in the cell culture conditions during the priming step. IFN-β may also be used at concentrations of 5-15 ng / mL, for example, 10 ng / mL.

[0123] IL-15 may be used in the cell culture conditions of the priming step at concentrations of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, or 4-20 ng / mL. IL-15 may also be used at concentrations of 5-15 ng / mL, for example, 10 ng / mL.

[0124] IL-18 may be used in the cell culture conditions of the priming step at concentrations of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, or 4-20 ng / mL. IL-18 may also be used at concentrations of 5-15 ng / mL, for example, 10 ng / mL.

[0125] IL-3 may be used in the cell culture conditions of the priming step at concentrations of 1-1000 ng / mL, 2-500 ng / mL, 3-250 ng / mL, or 4-200 ng / mL. IL-3 may also be used at concentrations of 5-150 ng / mL, for example, 10-130 ng / mL.

[0126] In a preferred embodiment, priming includes culturing a population of granulocyte-producing cells in the presence of GM-CSF at a concentration of approximately 130 ng / mL, and optionally one or more cytokines selected from the group consisting of TNF at a concentration of approximately 0.01 to 1.0 ng / mL, IFN-α at a concentration of approximately 10 ng / mL, IFN-β at a concentration of approximately 10 ng / mL, IL-15 at a concentration of approximately 10 ng / mL, IL-18 at a concentration of approximately 10 ng / mL, and IL-3 at a concentration of approximately 130 ng / mL.

[0127] In preferred embodiments, the primed cells may be cultured in the presence of GM-CSF, G-CSF, SCF, TPO, and IL-15. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 10 ng / mL, G-CSF at a concentration of approximately 130 ng / mL, SCF at a concentration of approximately 130 ng / mL, TPO at a concentration of approximately 130 ng / mL, and IL-15 at a concentration of approximately 10 ng / mL.

[0128] In preferred embodiments, the primed cells may be cultured in the presence of GM-CSF, G-CSF, SCF, TPO, and TNF. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 100 ng / mL, G-CSF at a concentration of approximately 130 ng / mL, SCF at a concentration of approximately 130 ng / mL, TPO at a concentration of approximately 130 ng / mL, and TNF at a concentration of approximately 10 ng / mL.

[0129] In a preferred embodiment, the primed cells may be cultured in the presence of GM-CSF and IL-3. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL and IL-3 at a concentration of approximately 130 ng / mL.

[0130] In a preferred embodiment, the primed cells may be cultured in the presence of GM-CSF and IL-15. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL and IL-15 at a concentration of approximately 10 ng / mL.

[0131] In a preferred embodiment, the primed cells may be cultured in the presence of GM-CSF and IL-18. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL and IL-18 at a concentration of approximately 10 ng / mL.

[0132] In a preferred embodiment, the primed cells may be cultured in the presence of GM-CSF and IL-16. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL and IL-16 at a concentration of approximately 10 ng / mL.

[0133] In a preferred embodiment, the primed cells may be cultured in the presence of GM-CSF and TNF. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL and TNF at a concentration of approximately 1 ng / mL.

[0134] In preferred embodiments, the primed cells may be cultured in the presence of GM-CSF, G-CSF, SCF, TPO, and IFN-α. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL, G-CSF at a concentration of approximately 130 ng / mL, SCF at a concentration of approximately 130 ng / mL, TPO at a concentration of approximately 130 ng / mL, and IFN-α at a concentration of approximately 10 ng / mL.

[0135] The priming step can be continued for any appropriate period. For example, the priming step may be continued for 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, 72 hours, 78 hours, 84 hours, 90 hours, or 96 hours. The priming step may be continued for 1 to 96 hours, 2 to 90 hours, 3 to 84 hours, 6 to 78 hours, 12 to 72 hours, 18 to 54 hours, or 24 to 48 hours. Preferably, priming may include a culture incorporating the cytokines mentioned above for a period of 1, 2, or 3 days, for example, at the concentrations mentioned above. In particular, priming may include a culture incorporating the combination of priming cytokines mentioned for 2 days.

[0136] The priming step may be suitably incorporated at any appropriate stage of the method of the present invention. However, priming is typically performed during the period in which the progenitor cells are cultured under conditions that promote differentiation of the progenitor cells into granulocyte-producing cells. For example, priming may be initiated on day 1, day 2, day 3, day 4, or day 5 of culturing the progenitor cells under conditions that promote differentiation into granulocyte-producing cells.

[0137] Alternatively, in a preferred embodiment, the priming step may be performed after the granulocyte-producing cells have been generated and optionally after the granulocyte-producing cells have been collected. For example, priming may be performed before or after the cryopreservation of the population of granulocyte-producing cells according to the present invention.

[0138] As a mere example, if the priming step is performed over two days, priming may be performed on days 3 and 4 of culture conditions that promote differentiation of progenitor cells into granulocytes, on days 4 and 5 of such culture, or on days 5 and 6 of such culture. To avoid misunderstanding, any of the above priming protocols may be preferably performed on days 3 and 4, days 4 and 5, or days 5 and 6 of culture conditions that promote differentiation of progenitor cells into granulocytes.

[0139] The priming step developed by the present inventors does not appear to have a significant effect on the immunomodulatory capacity of the granulocyte population. Therefore, in embodiments where only the immunomodulatory activity of granulocytes is desired, it may be preferable to omit the priming step from the method of generating the granulocyte population.

[0140] A method according to the first aspect of the present invention results in the generation of a population of granulocyte-producing cells, which is the subject of the second aspect of the present invention. Similarly, a method according to the third aspect of the present invention relates to the priming of granulocyte-producing cells and results in a population of cells according to the fourth aspect of the present invention. Granulocyte-producing cells are also used in the pharmaceutical compositions, medical uses, and therapeutic methods of the present invention. Unless otherwise required by context, the following definitions are applicable to granulocyte-producing cells in each of these aspects of the present invention, or in any other context in which granulocyte-producing cells or populations of such cells are referred to.

[0141] Any of the cells or populations of cells disclosed herein may be derived from mammals such as humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Preferably, the cells or populations of cells are of human origin. Therefore, the cells may be human cells, or the populations of cells may be populations of human cells. In particular, the granulocyte-producing cells or populations of granulocyte-producing cells disclosed herein may be derived from mammals such as humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Preferably, the granulocyte-producing cells or populations of granulocyte-producing cells are of human origin. Therefore, the granulocyte-producing cells may be human granulocyte-producing cells. The populations of granulocyte-producing cells may be populations of human granulocyte-producing cells.

[0142] For a cell to be considered "granulocyte-generating" in the terminology of this invention, it must be able to produce granulocytes (e.g., neutrophils) or granulocyte precursor cells of the granulocyte lineage. Indeed, a suitable granulocyte-generating cell can produce such cells. To avoid misunderstanding, granulocytes themselves should be considered "granulocyte-generating" for the purposes of this invention, but in many embodiments, granulocyte-generating cells are not granulocytes themselves, but cells capable of producing granulocytes. Preferably, granulocyte-generating cells in the context of this invention may be interpreted as excluding other cell lineages, for example, monocyte lineages and / or lymphocyte lineages.

[0143] In the context of the present invention, a suitable population of granulocyte-producing cells may be defined by reference to the expression of their different markers. Those skilled in the art will be well aware of suitable methods by which cells can be characterized and / or isolated and, if necessary, enriched based on the expression of a particular profile of cell surface markers.

[0144] The following definitions, based on appropriate marker expression profiles, can be used alone or in combination to identify appropriate populations of granulocyte-producing cells.

[0145] Unless otherwise specified (for example, in lists that state "or" or "and / or"), any reference in this disclosure to cells that are positive or negative for the expression of certain markers should be interpreted as meaning that such cells must have the listed expression (either positive or negative) of each of the markers mentioned. Thus, for example, a reference to a cell or population of cells as "CD15+CD66b+" should be interpreted as meaning that the cell is positive for the expression of both CD15 and CD66b, and that the population of such cells includes cells that are CD15+ and cells that are CD66b+.

[0146] This disclosure includes definitions of cell populations or subpopulations with respect to the enumerated expression (either positive or negative) of several specific markers.

[0147] In a preferred embodiment, such a definition can be interpreted as requiring that the population or subpopulation of interest include cells that are positive or negative (if required by the definition) with respect to the enumerated markers. For example, in the case of a population defined as being positive for the expression of a first marker, negative for the expression of a second marker, and positive for the expression of a third marker, this requirement may be satisfied by a population of cells that include cells that are positive for the first marker, and at the same time also include cells that are negative for the second marker, and further include cells that are positive for the third marker. In such an embodiment, the population or subpopulation of cells may be heterogeneous with respect to cells having the enumerated expressions (whether positive or negative). Preferably, the cells that each show the required expression for each of the enumerated markers may constitute the largest group of cells in such a population or subpopulation. Preferably, the cells that each show the required expression for each of the enumerated markers may constitute the majority of cells in such a population or subpopulation. Preferably, cells that each exhibit the required expression for each of the enumerated markers constitute at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in such population or subpopulation.

[0148] In one embodiment, in a given population or subpopulation, each cell in that population or subpopulation may express at least two, three, four, or five of the enumerated markers.

[0149] In a preferred embodiment, such a definition can be interpreted as requiring that the population or subpopulation of interest consist of cells that are positive or negative (if required by the definition) with respect to the enumerated markers. In such an embodiment, the population or subpopulation of cells is homogeneous with respect to cells having the enumerated expression (whether positive or negative).

[0150] In a preferred embodiment, the population of granulocyte-producing cells includes cells that are "Lin-" (i.e., negative for a cocktail of common leukocyte markers, defined for this purpose as negative expression of each of CD3, CD16, CD19, CD20, CD14, and CD56). For example, a preferred population of granulocyte-producing cells may contain at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% Lin- cells. As an example, a preferred population of granulocyte-producing cells may contain at least 90% Lin- cells. A preferred population of granulocyte-producing cells may contain approximately 95-99% Lin- cells. Preferably, the population of granulocyte-producing cells may contain approximately 97% Lin- cells.

[0151] Alternatively, or furthermore, a preferred population of granulocyte-producing cells includes CD34+ cells. For example, such a population of granulocyte-producing cells may contain less than 50%, less than 45%, less than 40%, or less than 35% CD34+ cells. As an example, such a population of granulocyte-producing cells may contain less than 30% CD34+ cells. In such embodiments, the proportion of CD34+ cells may be between approximately 5% and 25%. Preferably, the population of granulocyte-producing cells contains approximately 14% CD34+ cells.

[0152] Alternatively, or furthermore, a preferred population of granulocyte-producing cells includes CD38+ cells. For example, such a population of granulocyte-producing cells may include at least 10%, at least 15%, or at least 20% CD38+ cells. In such embodiments, the proportion of CD38+ cells may be between approximately 10% and 80%, for example, between approximately 10% and 30%. Preferably, the population of granulocyte-producing cells includes approximately 12% CD38+ cells.

[0153] Alternatively, or furthermore, a preferred population of granulocyte-producing cells includes cells having a hematopoietic stem cell (HSC) phenotype (defined for this purpose as Lin-CD34+CD38-CD45RA-CD90+). For example, such a population of granulocyte-producing cells may include cells having less than 5%, less than 4%, less than 3%, or less than 2% of the HSC phenotype. As an example, such a population of granulocyte-producing cells may include cells having less than 1% of the HSC phenotype. A preferred population of granulocyte-producing cells may include cells having approximately 0.01–0.15% of the HSC phenotype. Preferably, the population of granulocyte-producing cells includes cells having approximately 0.04% of the HSC phenotype.

[0154] Alternatively, or furthermore, a preferred population of granulocyte-producing cells may include cells having less than 1% of the long-term reconstituted hematopoietic stem cell (LT-HSC) phenotype (defined for this purpose as Lin-CD34+CD38-CD45RA-CD90+CD49f+). For example, such a population of granulocyte-producing cells may include cells having less than 5%, less than 4%, less than 3%, or less than 2% of the LT-HSC phenotype. As an example, such a population of granulocyte-producing cells may include cells having less than 1% of the LT-HSC phenotype. A preferred population of granulocyte-producing cells may include cells having approximately 0.01–0.05% of the LT-HSC phenotype. Preferably, the population of granulocyte-producing cells includes cells having approximately 0.02% of the LT-HSC phenotype.

[0155] Alternatively, or further, a preferred population of granulocyte-producing cells may include cells having the lymphoid priming pluripotent progenitor (LMPP) phenotype (defined for this purpose as Lin-CD34+CD38-CD45RA+). For example, such a population of granulocyte-producing cells may include cells having less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, or less than 25% of the LMPP phenotype. As an example, such a population of granulocyte-producing cells may include cells having less than 20% of the LMPP phenotype. A preferred population of granulocyte-producing cells may include cells having approximately 2-15% of the LMPP phenotype. Preferably, the population of granulocyte-producing cells includes cells having approximately 5% of the LMPP phenotype.

[0156] Alternatively, or furthermore, a preferred population of granulocyte-producing cells includes cells having a pluripotent progenitor (MPP) phenotype (defined for this purpose as Lin-CD34+CD38-CD45RA-). For example, such a population of granulocyte-producing cells may include cells having less than 30%, less than 25%, less than 20%, or less than 15% of the MPP phenotype. As an example, such a population of granulocyte-producing cells may include cells having less than 10% of the MPP phenotype. A preferred population of granulocyte-producing cells may include cells having approximately 1-6% of the MPP phenotype. Preferably, the population of granulocyte-producing cells includes cells having approximately 2% of the MPP phenotype.

[0157] In a preferred embodiment, the population of granulocyte-producing cells may include more than 90% Lin- cells (e.g., approximately 97% Lin- cells), and / or less than 30% CD34+ cells (e.g., approximately 14% CD34+ cells), and / or more than 10% CD38+ cells (e.g., approximately 12% CD38+ cells), and / or less than 1% of cells having the above-defined HSC phenotype (e.g., approximately 0.04% of cells having the HSC phenotype), and / or less than 1% of cells having the above-defined LT-HSC phenotype (e.g., approximately 0.02% of cells having the LT-HSC phenotype), and / or less than 20% of cells having the above-defined LMPP phenotype (e.g., approximately 5% of cells having the LMPP phenotype), and / or less than 10% of cells having the above-defined MPP phenotype (e.g., approximately 2.5% of cells having the MPP phenotype).

[0158] In preferred embodiments, the population of granulocyte-producing cells may include more than 90% Lin- cells (e.g., approximately 97% Lin- cells), less than 30% CD34+ cells (e.g., approximately 14% CD34+ cells), more than 10% CD38+ cells (e.g., approximately 12% CD38+ cells), less than 1% of cells having the above-defined HSC phenotype (e.g., approximately 0.04% of cells having the HSC phenotype), less than 1% of cells having the above-defined LT-HSC phenotype (e.g., approximately 0.02% of cells having the LT-HSC phenotype), less than 20% of cells having the above-defined LMPP phenotype (e.g., approximately 5% of cells having the LMPP phenotype), and less than 10% of cells having the above-defined MPP phenotype (e.g., approximately 2.5% of cells having the MPP phenotype).

[0159] Alternatively, or furthermore, a preferred population of granulocyte-producing cells may include CD15- versus CD15+ cells in a ratio of approximately 1:1.

[0160] A suitable population of granulocyte-producing cells may contain approximately 25-75% or 35-60% CD15- cells. For example, a suitable population of granulocyte-producing cells may contain approximately 50% CD15- cells.

[0161] A suitable population of granulocyte-producing cells may contain approximately 30-70% or 40-65% CD15+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 50% CD15+ cells.

[0162] A preferred population of granulocyte-producing cells may contain approximately 5-25%, 5-20%, 7-18%, or 10-15% CD15+CD66b+ cells. For example, a preferred population of granulocyte-producing cells may contain approximately 12% CD15+CD66b+ cells.

[0163] A preferred population of granulocyte-producing cells may contain approximately less than 30% or less than 25% CD11b+ cells. For example, a preferred population of granulocyte-producing cells may contain approximately 10-25% or 15-25% CD11b+ cells, for example, approximately 19% CD11b+ cells.

[0164] A preferred population of granulocyte-producing cells may contain at least 30%, at least 35%, at least 40%, or at least 45% CD71+ cells. For example, a preferred population of granulocyte-producing cells may contain approximately 60% CD71+ cells.

[0165] A suitable population of granulocyte-producing cells may contain approximately 60-95% or 65-90% CD49d+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 75% CD49d+ cells.

[0166] A preferred population of granulocyte-producing cells may contain less than 5%, less than 4%, less than 3%, or less than 2% CD10+ cells. A preferred population of granulocyte-producing cells may contain approximately 0.03–2% CD10+ cells. For example, a preferred population of granulocyte-producing cells may contain approximately 0.5% CD10+ cells.

[0167] A suitable population of granulocyte-producing cells may contain approximately 1-120% or 2-15% CD177+ cells. A suitable population of granulocyte-producing cells may contain approximately 6% CD177+ cells.

[0168] A preferred population of granulocyte-producing cells may contain less than 20% or less than 15% CD62L+ cells. For example, a preferred population of granulocyte-producing cells may contain between approximately 2% and 15%, for example, approximately 8% CD62L+ cells.

[0169] A suitable population of granulocyte-producing cells may contain approximately 40-85% or 50-75% CD54+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 63% CD54+ cells.

[0170] A suitable population of granulocyte-producing cells may contain approximately 2-15% or approximately 5-10% CD63+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 7% CD63+ cells.

[0171] A suitable population of granulocyte-producing cells may contain approximately 70-90% or 75-85% CD18+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 80% CD18+ cells.

[0172] A suitable population of granulocyte-producing cells may contain approximately 35-55% HLA-DR+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 47% HLA-DR+ cells.

[0173] A suitable population of granulocyte-producing cells may contain approximately 6-8% CD115+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 5% CD115+ cells.

[0174] A suitable population of granulocyte-producing cells may contain approximately 5-30% CD40+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 15% CD40+ cells.

[0175] A suitable population of granulocyte-producing cells may contain approximately 5-30% CD64+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 15% CD64+ cells.

[0176] A suitable population of granulocyte-producing cells may contain approximately 20-55% CD32+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 40% CD32+ cells.

[0177] A suitable population of granulocyte-producing cells may contain approximately 4-9% CXCR2+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 6% CXCR2+ cells.

[0178] A suitable population of granulocyte-producing cells may contain approximately 0.04–1% CD16+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 0.25% CD16+ cells.

[0179] A suitable population of granulocyte-producing cells may contain approximately 2-15% CD14+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 8% CD14+ cells.

[0180] A suitable population of granulocyte-producing cells may contain approximately 0.5–4% CD68+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 1.5% CD68+ cells.

[0181] A suitable population of granulocyte-producing cells may contain approximately 2-18% CD206+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 10% CD206+ cells.

[0182] A fifth aspect of the present invention is: • Over 90% of Lin- cells (for example, approximately 97% of Lin- cells), • Less than 30% CD34+ cells (e.g., approximately 14% CD34+ cells), • Over 30% of CD38+ cells (for example, approximately 65% ​​of CD38+ cells), Cells with less than 1% HSC phenotype (for example, cells with approximately 0.04% HSC phenotype), • Cells with less than 1% LT-HSC phenotype (for example, cells with approximately 0.02% LT-HSC phenotype) • Cells with less than 20% LMPP phenotype (for example, cells with approximately 5% LMPP phenotype), and This provides an isolated population of granulocyte-producing cells containing cells with less than 10% MPP phenotype (e.g., cells with approximately 2.5% MPP phenotype).

[0183] A sixth aspect of the present invention is: • The first subgroup of cells that are CD15+CD64+CD18+CD49d+CD71+ • A second subgroup of cells that are CD15-CD11b+ / -CD18+CD49d+CD32+HLA-DR- This invention provides an isolated population of granulocyte-producing cells, including a third subpopulation of cells that are CD15-CD11b-HLA-DR+CD18+CD49d+ and CD71+.

[0184] The population of granulocyte-producing cells according to the sixth aspect of the present invention may further include a fourth subpopulation of cells that are CD15-CD11b+HLA-DR+.

[0185] It will be understood that if information on the markers expressed by these subpopulations of cells is obtained, one or more of these subpopulations can be easily isolated from the cell population of the sixth aspect of the present invention. This leads to further aspects of the present invention.

[0186] In a seventh aspect, the present invention provides a population of granulocyte-producing cells that are CD15+CD64+CD18+CD49d+CD71+. A preferred population of such cells (which may also constitute a first subpopulation of cells in relation to a sixth aspect of the present invention) may also be positive for one, two or more, or all of the markers selected from the group consisting of: CD177, CD11b, CD71, CD66b, HLA-DR, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14. The population of cells in the seventh aspect of the present invention may be negative for the markers CD16 and / or CD62L (in addition to the necessary or optional expression or absence of expression of the other markers described above). Preferably, the population or subpopulation of cells is heterogeneous with respect to the enumerated marker profile (which may preferably include any of the components referred to herein). Preferably, the population or subpopulation of cells according to this embodiment of the present invention is uniformly positive for CD15 and heterogeneous with respect to other markers in the enumerated marker profile (which may preferably include any of the components referred to herein). Preferably, the population or subpopulation of cells is uniform with respect to the enumerated marker profile (which may preferably include any of the components referred to herein).

[0187] A first subpopulation of cells present in a population of granulocyte-producing cells according to a sixth aspect of the present invention, or cells according to a seventh aspect of the present invention, express markers very similar to those expressed by committed neutrophil precursors. However, the cells disclosed according to this aspect of the present invention are CD64+, and may also be CD16- and / or CD62L-. This is in contrast to neutrophil precursor cells that are CD64-CD16+ and CD62L+, which are observed in circulation and during homeostasis. The expression of CD64 by CD15+CD64+CD18+CD49d+CD71+ cells therefore provides a useful means of distinguishing the cells disclosed herein from those naturally occurring, as well as the lack of CD16 and / or CD62L expression. Cells or populations of cells that are CD15+CD64+CD18+CD49d+CD71+ and also CD16- and / or CD62L- can be distinguished as being produced by the method according to the present invention, rather than being naturally occurring granulocyte-producing cells or populations of such cells.

[0188] The inventors have confirmed that cells from a first subpopulation of cells present in a population of granulocyte-producing cells according to the sixth aspect of the present invention, or cells according to the seventh aspect of the present invention, exhibit cytotoxic activity that makes the cells particularly effective for medical use. In fact, such cells appear to constitute the primary source of cytotoxic activity in a population of cells according to the sixth aspect of the present invention. Therefore, such cells may be particularly useful in clinical situations where it is necessary to kill cells (e.g., cancer cells, infected cells, or cellular infectious pathogens) to achieve a therapeutic effect.

[0189] A first subpopulation of cells present in a population of granulocyte-producing cells according to the sixth aspect of the present invention, or cells according to the seventh aspect of the present invention, may express 4-1BBL and / or OX40L. These markers are ligands for T cells and NK cells, and their expression by these cells can indicate that the cells have immunomodulatory activity. Similarly, a first subpopulation of cells present in a population of granulocyte-producing cells according to the sixth aspect of the present invention, or cells according to the seventh aspect of the present invention, may also express CD38 and / or CD40 and / or CD54, as well as costimulatory molecules associated with functional interactions with immune cells such as T cells. Therefore, such cells, or pharmaceutical compositions containing such cells, may be effective in biological or therapeutic applications that utilize the modulation of the activity of such nongranulocytic inflammatory cell types.

[0190] In addition to expressing markers indicating immunomodulatory capacity, this cell population also expresses molecules (particularly CD11b, CD18, Mac1, and CD32) that suggest they possess direct cytotoxic activity. This could make them suitable for applications where therapeutic elimination of cells, such as cancer cells or infected cells, is desired.

[0191] In the eighth aspect, the present invention provides a population of cells that are CD15-CD11b+ / -CD18+CD49d+CD32+HLA-DR-. A preferred population of such cells (which may also constitute a second subpopulation of cells in connection with the sixth aspect of the present invention) may also be positive for one, two or more, or all of the markers selected from the group consisting of: CD177, CD11b, CD71, CD66b, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14. Preferably, the population of cells, or subpopulation, is heterogeneous with respect to the enumerated marker profiles (which may preferably include any of the components referred to herein). Preferably, a population or subpopulation of cells according to this embodiment of the present invention is uniformly negative for CD15 and HLA-DR and heterogeneous for other markers in the enumerated marker profile (which may preferably include any of the components referred to herein). Preferably, a population or subpopulation of cells according to this embodiment is uniformly negative for CD15, HLA-DR, and CD11b and heterogeneous for other markers in the enumerated marker profile (which may preferably include any of the components referred to herein). Preferably, a population or subpopulation of cells according to this embodiment is uniformly positive for CD11b, uniformly negative for CD15 and HLA-DR, and heterogeneous for other markers in the enumerated marker profile (which may preferably include any of the components referred to herein). Preferably, a population or subpopulation of cells is uniform for the enumerated marker profile (which may preferably include any of the components referred to herein).

[0192] A second subpopulation of cells present in the population of granulocyte-producing cells according to the sixth aspect of the present invention, or cells according to the eighth aspect of the present invention, express markers such as Mac-1 (including CD11b and CD18) and CD32, consistent with a high capacity for cytotoxic activity. Therefore, these cells may also be useful in medical uses or therapeutic methods where direct cytotoxic activity, such as the killing of cancer cells or infected cells, is required. These cells may also express molecules such as 4-1BBL and / or OX40L, which indicate immunomodulatory potential and suitability for use in biological or therapeutic applications requiring such activity. Cells in this group may also express CXCR2, which can be elevated by exposure to IL-3 during methods according to the present invention, and is a marker that may contribute to increased chemotaxis (in response to drugs such as IL-8) and targeting of these cells to TMEs.

[0193] In a ninth aspect, the present invention provides a population of cells that are CD15-CD11b-HLA-DR+CD18+CD49d+ and CD71+. A preferred population of such cells (which may also constitute a third subpopulation of cells in connection with a sixth aspect of the present invention) may also be positive for one, two or more, or all of the markers selected from the group consisting of: CD177, CD71, CD66b, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14. Preferably, the population of cells, or subpopulation, is heterogeneous with respect to the enumerated marker profiles (which may preferably include any of the components referred to herein). Preferably, a population or subpopulation of cells according to this embodiment of the present invention is uniformly negative for CD15 and CD11b, uniformly positive for HLA-DR, and heterogeneous for other markers in the enumerated marker profile (which may preferably include any of the components referred to herein). Preferably, a population or subpopulation of cells is uniform in the enumerated marker profile (which may preferably include any of the components referred to herein).

[0194] A third subpopulation of cells present in the population of granulocyte-producing cells according to the sixth aspect of the present invention, or cells according to the ninth aspect of the present invention, express markers indicating relatively low levels of differentiation. Consistently, these cells may also be CD34+. Cells in this group may also express markers such as 4-1BBL and / or OX40L and / or CD40 and / or CD54, indicating their suitability for use in applications requiring immunomodulation of non-granulocyte immune cells. While cells in this group do not express markers indicating direct cytotoxic activity, they may possess the ability to further differentiate and express markers such as CD11b and CD15, which would confer such activity. Therefore, these cells can be utilized in medical uses or therapeutic methods in which in vivo signaling would induce such differentiation, resulting in the ability to kill harmful cell types.

[0195] In a tenth aspect, the present invention provides a population of granulocyte-producing cells that are CD15-CD11b+HLA-DR+. A preferred population of such cells (which may also constitute any fourth subpopulation of cells in connection with a sixth aspect of the present invention) may also be positive for one, two or more, or all of the following markers selected from the group: CD177, CD71, CD66b, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14. Preferably, the population or subpopulation of cells is heterogeneous with respect to the enumerated marker profiles (which may preferably include any of the components referred to herein). Preferably, a population or subpopulation of cells according to this embodiment of the present invention is uniformly CD15-negative, uniformly HLA-DR and CD11b-positive, and heterogeneous with respect to other markers in the enumerated marker profile (which may preferably include any of the components referred to herein). Preferably, a population or subpopulation of cells is uniform with respect to the enumerated marker profile (which may preferably include any of the components referred to herein).

[0196] These cells, according to the tenth aspect of the present invention, which may optionally be present in the population of granulocyte-producing cells according to the sixth aspect of the present invention, express markers similar to those expected to activate myeloid cells. These cells may further express markers such as CD14 and / or CD11b and / or CD206. They may be suitable for use in applications where either direct cytotoxicity or immunomodulatory activity is desired.

[0197] The granulocyte-producing cells of a population generated by the method of the present invention, or the granulocyte-producing cells present in the composition of the present invention, may be CD64+, CD16-, and / or CD62L-. For example, the granulocyte-producing cells may be CD64+. The granulocyte-producing cells may be CD64+ and CD16-. The granulocyte-producing cells may be CD64+ and CD62L-. The granulocyte-producing cells may be CD16- and CD62L-. Preferably, the granulocyte-producing cells are CD64+, CD16-, and CD62L-. The expression of CD64, as well as the absence of CD16 and CD62L expression, by the granulocyte-producing cells of the present invention are in contrast to neutrophils, which are CD64-CD16+ and CD62L+, observed in circulation and during homeostasis. The expression of CD64, therefore, provides a useful means of distinguishing the granulocyte-producing cells disclosed herein from those naturally occurring, as is the absence of CD16 and / or CD62L expression. Granulocytogenic cells that are CD64+, CD16-, and / or CD62L- can be distinguished as being produced by the method according to the present invention, rather than being naturally occurring granulocytogenic cells or populations of such cells.

[0198] Accordingly, in one embodiment, the present invention provides granulocyte-producing cells that are CD64+ granulocyte-producing cells, or a population of such cells. Preferably, CD64+ granulocyte-producing cells are CD64+ and CD16- granulocyte-producing cells. CD64+ granulocyte-producing cells may be CD64+ and CD62L- granulocyte-producing cells. CD64+ granulocyte-producing cells may be CD64+, CD16-, and CD62L- granulocyte-producing cells. A population of cells having each of these expression profiles is provided by this embodiment of the present invention.

[0199] In related embodiments, the present invention provides granulocyte-producing cells that are CD16-granulocyte-producing cells. CD16-granulocyte-producing cells may be CD16- and CD62L-granulocyte-producing cells.

[0200] In another related embodiment, the present invention provides granulocyte-producing cells that are CD62L-granulocyte-producing cells.

[0201] In the eleventh aspect, the present invention provides a pharmaceutical composition comprising a population of granulocyte-producing cells. Preferably, the population of granulocyte-producing cells may be a population according to the second aspect of the present invention, or the fourth to tenth aspects.

[0202] In a twelfth aspect, the present invention provides the use of a population of granulocyte-producing cells in the manufacture of a drug. Preferably, the population of granulocyte-producing cells may be a population according to the second aspect of the present invention, or the fourth to tenth aspects.

[0203] In the thirteenth aspect, the present invention provides a pharmaceutical composition according to the sixth aspect of the present invention, or a population of granulocyte-producing cells according to the second, fourth to tenth aspects of the present invention, for use as a drug.

[0204] In a fourteenth aspect, the present invention provides a method for treating a disease or disorder in a subject, the method comprising administering a pharmaceutical composition according to the sixth aspect of the present invention, or a population of granulocyte-producing cells according to the second, fourth to tenth aspects of the present invention, to a subject.

[0205] In one embodiment, the present invention provides a pharmaceutical composition for use in a method of treating a disease or disorder in a subject.

[0206] In one embodiment, the present invention provides a pharmaceutical composition for use in pharmaceuticals.

[0207] In one embodiment, the present invention provides a method for treating a disease or disorder in a subject, comprising administering a pharmaceutical composition of the present invention to the subject.

[0208] In one embodiment, the present invention provides a pharmaceutical composition for use in a method of treating cancer in a subject.

[0209] In one embodiment, the present invention provides a method for treating cancer in a subject, comprising administering the pharmaceutical composition of the present invention to the subject.

[0210] In one embodiment, the present invention provides the use of the pharmaceutical composition of the present invention in the manufacture of a drug for treating cancer in a subject.

[0211] In one embodiment, the present invention provides a pharmaceutical composition for use in a method of treating an infectious disease in a subject.

[0212] In one embodiment, the present invention provides a method for treating an infectious disease in a subject, comprising administering a pharmaceutical composition of the present invention to the subject.

[0213] In one embodiment, the present invention provides the use of the pharmaceutical composition of the present invention in the manufacture of a drug for treating an infectious disease in a subject.

[0214] In one embodiment, the present invention provides a pharmaceutical composition for use in amplifying a nongranulocyte therapeutic immune response.

[0215] In one embodiment, the present invention provides a method of treatment comprising amplifying a nongranulocyte therapeutic immune response, the method comprising providing a pharmaceutical composition of the present invention to a subject requiring such treatment.

[0216] In one embodiment, the present invention provides a pharmaceutical composition for use in the manufacture of a drug for use in amplifying a nongranulocyte therapeutic immune response.

[0217] The present invention is based on the inventors' development of a method for forming a population of granulocyte-producing cells that has highly beneficial properties in a wide range of therapeutic situations, at least to some extent. The method of the first aspect of the present invention results in the generation of a population of granulocyte-producing cells, which is the subject of the second aspect of the present invention. Similarly, the method of the third aspect of the present invention relates to the priming of granulocyte-producing cells and results in a population of cells according to the fourth aspect of the present invention. Granulocyte-producing cells are also used in the pharmaceutical compositions, medical uses, and therapeutic methods of the present invention.

[0218] Populations of granulocyte-producing cells described herein, for example, those that can be produced using the methods of the present invention, can amplify the therapeutic immune response of non-granulocyte immune cells. In preferred embodiments, populations of granulocyte-producing cells result in amplification of the immune response of non-granulocyte immune cells. Such populations of granulocyte-producing cells, or pharmaceutical compositions containing such populations, can be used to treat many conditions, including (but not limited to) cancer and infectious diseases. The populations and compositions can also be used to enhance immunotherapeutic treatments in many conditions, including (but not limited to) cancer and infectious diseases.

[0219] As described above, the population of granulocyte-producing cells may include cells expressing markers such as 4-1BBL and / or OX40L and / or CD40 and / or CD54, which are associated with interactions with non-granulocyte immune cells. Such cells, or pharmaceutical compositions containing such cells, can be used in medical uses or therapeutic methods requiring beneficial immunomodulatory activity.

[0220] Alternatively, or furthermore, a preferred population of granulocyte-producing cells may include cells expressing markers that demonstrate the ability to directly destroy cells, such as Mac-1 (or its components CD11b and CD18) or CD32. Such cells, or pharmaceutical compositions containing such cells, can be used in medical uses or therapeutic methods that require the killing of cells such as cancer cells or infected cells.

[0221] The method of the present invention utilizes a population of progenitor cells as a “starting material” from which granulocyte-generating cells are produced. As described above, some embodiments of the method of the present invention may also incorporate an optional step of culturing a population of stem cells to generate a population of progenitor cells.

[0222] In a manner similar to the populations of granulocyte-producing cells described above, progenitor cells and populations of progenitor cells can be usefully defined in the context of this disclosure by marker profiles and phenotypic expression. The following definitions based on appropriate marker expression profiles can be used alone or in combination to identify appropriate populations of progenitor cells. Unless otherwise specified in the context, they should be considered applicable to progenitor cells referred to in any embodiment of the present invention.

[0223] In a preferred embodiment, the progenitor cell population includes cells that are Lin- (as defined above). For example, a preferred population of progenitor cells may include at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% Lin- cells. As an example, a preferred population of progenitor cells may include at least 98% Lin- cells. A preferred population of progenitor cells may include approximately 98-99% Lin- cells. Preferably, the progenitor cell population includes approximately 99% Lin- cells.

[0224] Alternatively, or furthermore, a preferred population of progenitor cells includes CD34+ cells. For example, such a population of progenitor cells may contain approximately 5–90% or approximately 10–85% CD34+ cells. As an example, such a population of progenitor cells may contain approximately 15–80% CD34+ cells. In such embodiments, the proportion of CD34+ cells may be between approximately 20–70%. Preferably, the population of progenitor cells contains approximately 43% CD34+ cells.

[0225] Alternatively, or furthermore, a preferred population of progenitor cells includes CD38+ cells. For example, such a population of progenitor cells may contain CD38+ cells between approximately 10–65%, approximately 15–60%, or approximately 20–55%. As an example, such a population of progenitor cells may contain CD38+ cells between approximately 25–50%. In such embodiments, the proportion of CD38+ cells may be between approximately 30% and 41%. Preferably, the population of progenitor cells contains approximately 35% CD38+ cells.

[0226] Alternatively, or furthermore, a preferred population of progenitor cells includes cells having an HSC phenotype. For example, such a population of progenitor cells may include cells having less than 5%, less than 4%, less than 3%, or less than 2% of the HSC phenotype. As an example, such a population of progenitor cells may include cells having less than 1% of the HSC phenotype. A preferred population of progenitor cells may include cells having approximately 0.01–0.7% of the HSC phenotype. Preferably, the population of progenitor cells includes cells having approximately 0.3% of the HSC phenotype.

[0227] Alternatively, or furthermore, a preferred population of progenitor cells includes cells having the LT-HSC phenotype. For example, such a population of progenitor cells may include cells having less than 5%, less than 4%, less than 3%, or less than 2% of the LT-HSC phenotype. As an example, such a population of progenitor cells may include cells having less than 1% of the LT-HSC phenotype. A preferred population of progenitor cells may include cells having approximately 0.01–0.03% of the LT-HSC phenotype. Preferably, the population of progenitor cells includes cells having approximately 0.02% of the LT-HSC phenotype.

[0228] Alternatively, or furthermore, a preferred population of progenitor cells includes cells having the LMPP phenotype. For example, such a population of progenitor cells may include cells having less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, or less than 45% of the LMPP phenotype. As an example, such a population of progenitor cells may include cells having less than 40% of the LMPP phenotype. A preferred population of progenitor cells may include cells having approximately 5-30% of the LMPP phenotype. Preferably, the population of progenitor cells includes cells having approximately 13% of the LMPP phenotype.

[0229] Alternatively, or furthermore, a preferred population of progenitor cells includes cells having the MPP phenotype. For example, such a population of progenitor cells may include cells having less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, or less than 45% of the MPP phenotype. As an example, such a population of progenitor cells may include cells having less than 40% of the MPP phenotype. A preferred population of progenitor cells may include cells having approximately 1-35% of the MPP phenotype. Preferably, the population of progenitor cells includes cells having approximately 13% of the MPP phenotype.

[0230] In preferred embodiments, the progenitor cell population may include more than 98% Lin- cells (e.g., approximately 99% Lin- cells), and / or 15-18% CD34+ cells (e.g., approximately 43% CD34+ cells), and / or 25-50% CD38+ cells (e.g., approximately 35% CD38+ cells), and / or less than 1% of cells having the above-defined HSC phenotype (e.g., approximately 0.3% of cells having the HSC phenotype), and / or less than 1% of cells having the above-defined LT-HSC phenotype (e.g., approximately 0.02% of cells having the LT-HSC phenotype), and / or less than 40% of cells having the above-defined LMPP phenotype (e.g., approximately 13% of cells having the LMPP phenotype), and / or less than 40% of cells having the above-defined MPP phenotype (e.g., approximately 13% of cells having the MPP phenotype).

[0231] In preferred embodiments, the progenitor cell population may include more than 98% Lin- cells (e.g., approximately 99% Lin- cells), 15–18% CD34+ cells (e.g., approximately 43% CD34+ cells), 25–50% CD38+ cells (e.g., approximately 35% CD38+ cells), less than 1% of cells having the above-defined HSC phenotype (e.g., approximately 0.3% of cells having the HSC phenotype), less than 1% of cells having the above-defined LT-HSC phenotype (e.g., approximately 0.02% of cells having the LT-HSC phenotype), less than 40% of cells having the above-defined LMPP phenotype (e.g., approximately 13% of cells having the LMPP phenotype), and less than 40% of cells having the above-defined MPP phenotype (e.g., approximately 13% of cells having the MPP phenotype).

[0232] Alternatively, or furthermore, a preferred population of progenitor cells may include CD15- versus CD15+ cells in a ratio of approximately 2:1.

[0233] A suitable population of progenitor cells may contain approximately 60-95% CD15- cells. For example, a suitable population of progenitor cells may contain approximately 71% CD15- cells.

[0234] A suitable population of progenitor cells may contain approximately 10-50% CD15+ cells. For example, a suitable population of progenitor cells may contain approximately 35% CD15+ cells.

[0235] A preferred population of progenitor cells may contain approximately 0.02–1% CD15+CD66b+ cells. For example, a preferred population of progenitor cells may contain approximately 0.04–0.47% or 0.24% CD15+CD66b+ cells.

[0236] A preferred population of progenitor cells may contain less than 20% CD11b+ cells. For example, a preferred population of progenitor cells may contain approximately 2-6%, or approximately 3%, CD11b+ cells.

[0237] A suitable population of progenitor cells may contain approximately 25-60% CD71+ cells. For example, a suitable population of progenitor cells may contain approximately 33% CD71+ cells.

[0238] A suitable population of progenitor cells may contain approximately 90-100% CD49d+ cells. For example, a suitable population of progenitor cells may contain approximately 95% CD49d+ cells.

[0239] A suitable population of progenitor cells may contain approximately 0.01–1.5% CD10+ cells. For example, a suitable population of progenitor cells may contain approximately 0.5% CD10+ cells.

[0240] A suitable population of progenitor cells may contain approximately 0.25–3% CD177+ cells. For example, a suitable population of progenitor cells may contain approximately 1% CD177+ cells.

[0241] A preferred population of progenitor cells may contain approximately 20-60% or 40-60% CD62L+ cells. For example, a preferred population of progenitor cells may contain approximately 46% CD62L+ cells.

[0242] A suitable population of progenitor cells may contain approximately 1-17% CD54+ cells. For example, a suitable population of progenitor cells may contain approximately 6% CD54+ cells.

[0243] A suitable population of progenitor cells may contain approximately 2-20% CD63+ cells. For example, a suitable population of progenitor cells may contain approximately 5% CD63+ cells.

[0244] A suitable population of progenitor cells may contain approximately 70-90% CD18+ cells. For example, a suitable population of progenitor cells may contain approximately 87% CD18+ cells.

[0245] The population of granulocyte-producing cells or pharmaceutical compositions of the present invention can be used in a wide range of therapeutic applications as disclosed herein. In particular, the population of cells or pharmaceutical compositions can be used to increase the activation or recruitment of host immune cells, especially non-granulocyte immune cells, in a manner that enables amplification of the host's therapeutic immune response. This realization makes it possible to use such compositions to enhance immunotherapeutic treatments in many conditions, including (but not limited to) cancer treatment. By amplifying the host's immune response, the compositions, medical uses, and therapeutic methods of the present invention can make otherwise immunologically "cold" tumors "hot" and thus responsive to treatment.

[0246] The amplification that occurs with respect to the host therapeutic immune response is not solely attributable to an increase in the number of granulocytes and non-granulocyte immune cells as a result of administration of the compositions of the present invention. Instead, granulocyte-producing cells and compositions comprising said granulocyte-producing cells can significantly increase the activation of non-granulocyte immune cells, particularly T cells, e.g., CD8, CD4, and γδ T cells, monocytes, macrophages, dendritic cells (DCs), and NK cells. On the other hand, non-granulocyte immune cells can significantly increase the activation of granulocyte-producing cells. As will be discussed in more detail below and as shown in the examples, this can increase the expression of degranulation markers, costimulatory molecules, and cytokines by activation.

[0247] The inventors' remarkable discovery that a population of granulocyte-producing cells according to the present invention can amplify (preferably function to amplify) the therapeutic immune response of non-granulocyte immune cells also gives rise to further aspects and embodiments of the present invention. As used herein, “immune response” encompasses any response of immune cells to their environment. Immune cells are constantly responding to their environment, including in vitro, and therefore constantly producing immune responses, even during homeostasis. A “therapeutic immune response” can be an immune response that can contribute to the eradication of disease. Examples of therapeutic immune responses include increased activation of immune cells, increased expression of cell degranulation markers by immune cells, increased expression of costimulatory molecules by immune cells, or increased expression of cytokines by immune cells. Such therapeutic immune responses can occur in vitro or in vivo.

[0248] Therefore, the inventors have shown that, surprisingly, granulocyte-producing cells may (preferably) promote the proliferation and / or survival of non-granulocyte immune cells, including NK cells and T cells, thereby increasing the ex vivo proliferation of these cell types and improving their persistence in vivo. The inventors have also shown that granulocyte-producing cells may (preferably) increase the expression of costimulatory molecules, including 4-1BB and OX40, on non-granulocyte immune cells, such as NK cells and T cells, including γδT cells, thereby improving the therapeutic effect. The inventors have also discovered that, surprisingly, non-granulocyte immune cells may (preferably) increase the expression of costimulatory molecules, including CD54, on granulocyte-producing cells, thereby improving the therapeutic effect of granulocyte-producing cells.

[0249] Compositions comprising granulocyte-producing cells and non-granulocyte immune cells may therefore be useful for therapeutic purposes. Such compositions may contain cells having an amplified therapeutic immune response, which in turn may amplify the host therapeutic immune response after administration to a subject, for example.

[0250] Advantageously, such compositions can help in the successful eradication of tumors (e.g., cancer) by providing a combination of immune cells suitable for this purpose. This can be particularly advantageous when the subject's own immune cells may be deficient. Furthermore, the present invention can enable the production of such compositions without the conventional manufacturing difficulties and / or harmful immunogenic effects.

[0251] Accordingly, in one embodiment, the present invention provides a composition comprising a population of granulocyte-producing cells and non-granulocyte immune cells. Advantageously, such a composition can help in the successful eradication of tumors (e.g., cancer) by providing a combination of immune cells suitable for this purpose. This may be particularly advantageous when the subject's own immune cells may be deficient. Furthermore, the present invention can enable the production of such compositions without the conventional manufacturing difficulties and / or harmful immunogenic effects.

[0252] T cells containing αβT cell receptors (also called "αβT cells") are generally considered to be a central cell type involved in regulating the immune response. However, we have surprisingly shown that granulocyte-producing cells can amplify the therapeutic immune response of non-granulocyte immune cells in the absence of αβT cells.

[0253] Accordingly, in one embodiment, the present invention provides a composition comprising a population of granulocyte-producing cells and non-granulocyte immune cells, but not comprising αβT cells. For example, the composition may comprise a population of granulocyte-producing cells and terminally differentiated non-granulocyte immune cells, and the composition may not comprise αβT cells.

[0254] In one embodiment, the present invention provides a composition comprising granulocyte-producing cells and non-granulocyte immune cells, wherein the granulocyte-producing cells can (preferably) modulate the therapeutic immune response of the non-granulocyte immune cells.

[0255] In one embodiment, the present invention provides a composition comprising a population of granulocyte-producing cells and non-granulocyte immune cells, wherein the population of granulocyte-producing cells can amplify (preferably amplify) the therapeutic immune response of the non-granulocyte immune cells.

[0256] In one aspect, the present invention is (a) A composition according to the present invention, or (b) Populations of granulocyte-producing cells and non-granulocyte immune cells (e.g., terminally differentiated non-granulocyte immune cells), (c) Optionally, provide a kit that includes instructions for using it (for example, when treating cancer).

[0257] In one embodiment, the present invention provides a method for producing a composition (for example, the composition of the present invention), the method comprising culturing PBMCs in the presence of granulocyte-producing cells to form a composition, and optionally depleting αβT cells before, during, or after the culture.

[0258] In one embodiment, the present invention provides a method for producing a composition (for example, the composition of the present invention), the method comprising culturing αβT cell-depleted PBMCs under conditions that promote the differentiation of progenitor cells present in αβT cell-depleted PBMCs into granulocyte-producing cells, thereby forming the composition.

[0259] In one embodiment, the present invention provides a composition that can be obtained by the method of the present invention.

[0260] In one embodiment, the present invention provides compositions for use in methods of treating diseases or disorders in a subject.

[0261] In one embodiment, the present invention provides compositions for use in pharmaceuticals.

[0262] In one embodiment, the present invention provides a method for treating a disease or disorder in a subject, comprising administering a composition of the present invention to the subject.

[0263] In one embodiment, the present invention provides the use of the composition of the present invention in the manufacture of pharmaceuticals.

[0264] In one embodiment, the present invention provides a composition for use in a method of treating cancer in a subject.

[0265] In one embodiment, the present invention provides a method for treating cancer in a subject, comprising administering a composition of the present invention to the subject.

[0266] In one embodiment, the present invention provides the use of the composition of the present invention in the manufacture of a drug for treating cancer in a subject.

[0267] In one embodiment, the present invention provides a composition for use in a method of treating an infectious disease in a subject.

[0268] In one embodiment, the present invention provides a method for treating an infectious disease in a subject, comprising administering a composition of the present invention to the subject.

[0269] In one embodiment, the present invention provides the use of the composition of the present invention in the manufacture of a drug for treating an infectious disease in a subject.

[0270] In therapeutic applications, this composition can modulate (preferably amplify) the target therapeutic immune response, such as the nongranulocyte therapeutic immune response.

[0271] In one embodiment, the present invention provides compositions for use in modulating nongranulocyte therapeutic immune responses.

[0272] In one embodiment, the present invention provides a method of treatment comprising modulating a nongranulocyte therapeutic immune response, comprising providing a composition of the present invention to a subject requiring such treatment.

[0273] In one embodiment, the present invention provides compositions for use in amplifying nongranulocyte therapeutic immune responses.

[0274] In one embodiment, the present invention provides a method of treatment comprising amplifying a nongranulocyte therapeutic immune response, the method comprising providing a composition of the present invention to a subject requiring such treatment.

[0275] In one embodiment, the present invention provides compositions for use in the manufacture of agents for use in modulating nongranulocyte therapeutic immune responses.

[0276] In one embodiment, the present invention provides compositions for use in the manufacture of agents for use in amplifying nongranulocyte therapeutic immune responses.

[0277] The present invention is based on the inventors' discovery that, at least to some extent, granulocyte-producing cells described herein may be able to amplify (preferably amplify) the therapeutic immune response of non-granulocyte immune cells. Advantageously, this may make it possible to provide compositions that can be used to treat many conditions, including (but not limited to) cancer, by combining such granulocyte-producing cells with non-granulocyte immune cells. The compositions can also be used to enhance immunotherapeutic treatments in many conditions, including (but not limited to) cancer treatment.

[0278] Amplification of an immune response (e.g., a therapeutic immune response) may be demonstrated in vitro by one or more of the following: increased activation of immune cells, increased expression of degranulation markers by immune cells, increased expression of costimulatory molecules by immune cells, increased proliferation by immune cells, increased survival by immune cells, increased abundance of immune cells, increased expression of cytokines by immune cells, increased transport by immune cells, increased cytotoxicity by immune cells, and / or increased tumor cell death activity by immune cells.

[0279] The composition may also be used to increase the activation or recruitment of host immune cells, particularly nongranulocyte immune cells, in a manner that enables amplification of the host's therapeutic immune response. This realization may enable the use of such compositions to enhance immunotherapeutic treatments in many conditions, including (but not limited to) cancer. By amplifying the host's immune response, the compositions, medical uses, and therapeutic methods of the present invention may be able to make otherwise immunologically "cold" tumors "hot" and thus responsive to treatment.

[0280] In one embodiment, the amplification that occurs with respect to the host therapeutic immune response is not solely attributable to an increase in the number of granulocytes and non-granulocyte immune cells as a result of administration of the composition of the present invention. Instead, granulocyte-producing cells and compositions comprising said granulocyte-producing cells may significantly increase the activation of non-granulocyte immune cells, particularly T cells, e.g., γδT cells, monocytes, macrophages, and NK cells. Conversely, non-granulocyte immune cells may significantly increase the activation of granulocyte-producing cells. As will be discussed in more detail below and as shown in the examples, activated granulocyte-producing and non-granulocyte cells may increase the expression of degranulation markers, costimulatory molecules, and cytokines. It may also increase the proliferation and survival of activated non-granulocyte cells, leading to an increase in the accumulation of such cells. The inventors have also demonstrated that activated non-granulocyte immune cells may exhibit an increased degree of recruitment to TMEs and increased cell-destructive activity (particularly increased tumor cell-killing activity).

[0281] Surprisingly, the inventors have discovered that these effects can be achieved by using granulocyte-producing cells and / or non-granulocyte immune cells, as well as compositions containing the same type of cells, for subjects receiving a population or composition of granulocyte-producing cells therapeutically.

[0282] These properties suggest that granulocyte-producing cells, including compositions containing granulocytes and non-granulocyte immune cells, can be used therapeutically in the treatment of cancer, and that such treatments can also be used to enhance other cell-based immunotherapies.

[0283] Furthermore, the granulocyte-producing cells of the present invention, or granulocyte-producing cells used in accordance with the present invention, may be able to differentiate (preferably differentiate) into granulocytes that have the ability to kill cancer cells. Thus, the compositions and therapies according to the present invention may achieve a dual mode of action, amplifying the non-granulocyte immune response and generating granulocytes that can directly kill cancer cells.

[0284] The inventors have demonstrated that granulocyte-producing cells suitable for use in the compositions or medical uses of the present invention, or in the methods of the present invention, may amplify (preferably amplify) immune responses through several different mechanisms. In particular, granulocyte-producing cells can increase the activation of immune cells and increase the activity (such as cell transport and cell destructive activity) required for a successful therapeutic immune response.

[0285] Populations of granulocyte-producing cells suitable for use in these embodiments of the present invention can be characterized according to definitions presented elsewhere herein. Such populations of granulocyte-producing cells can be generated by methods for preparing cells for therapeutic uses as described herein.

[0286] Preferably, the composition includes a population of granulocyte-producing cells that can (preferably amplify) the therapeutic immune response of nongranulocyte immune cells. Thus, in one embodiment, a composition is provided comprising a population of granulocyte-producing cells and nongranulocyte immune cells (e.g., terminally differentiated nongranulocyte immune cells), wherein the population of granulocyte-producing cells can (preferably amplify) the therapeutic immune response of nongranulocyte immune cells.

[0287] The ability of a population of granulocyte-producing cells to amplify the therapeutic immune response of non-granulocyte immune cells can be determined by any appropriate means.

[0288] For example, the ability of a population of granulocyte-producing cells to amplify the therapeutic immune response of non-granulocyte immune cells can be determined by an in vitro assay. For example, the ability of a population of granulocyte-producing cells to amplify the therapeutic immune response of non-granulocyte immune cells can be determined by a method including the following: (a) Mixing PBMCs or αβT cell-depleted PBMCs in the presence of granulocyte-producing cells to form a mixture. (b) Incubate the mixture (c) After incubation, determine the therapeutic immune response of nongranulocyte immune cells present in the mixture, and (d) After incubation, compare the therapeutic immune response of nongranulocyte immune cells present in the mixture to a reference standard.

[0289] The reference standard may be any suitable control. For example, the reference standard may be the corresponding therapeutic immune response of nongranulocyte immune cells present in PBMCs or αβT cell-depleted PBMCs before mixing. The reference standard may be the corresponding therapeutic immune response of nongranulocyte immune cells present in the mixture before incubation. The reference standard may be the corresponding therapeutic immune response of nongranulocyte immune cells cultured in the absence of granulocyte-producing cells but otherwise subjected to the same conditions. The reference standard may be the corresponding therapeutic immune response of nongranulocyte immune cells cultured in the presence of fewer granulocyte-producing cells but otherwise subjected to the same conditions.

[0290] Such reference standards may be obtained using cells from the same donor or a different donor as those used in steps (a) to (c). Preferably, the reference standards can be obtained using cells from the same donor as those used in steps (a) to (c).

[0291] If the therapeutic immune response of non-granulocyte immune cells present in the mixture after incubation increases compared to a reference standard, the population of granulocyte-producing cells can be considered to amplify the therapeutic immune response of non-granulocyte immune cells. Therefore, if the therapeutic immune response of non-granulocyte immune cells present in the mixture after incubation increases compared to the corresponding therapeutic immune response of non-granulocyte immune cells present in PBMCs or αβT cell-depleted PBMCs before mixing, the population of granulocyte-producing cells can be considered to amplify the therapeutic immune response of non-granulocyte immune cells. Preferably, if the therapeutic immune response of non-granulocyte immune cells present in the mixture after incubation increases compared to the corresponding therapeutic immune response of non-granulocyte immune cells cultured in the absence of granulocyte-producing cells but otherwise subjected to the same conditions, the population of granulocyte-producing cells can be considered to amplify the therapeutic immune response of non-granulocyte immune cells. If the therapeutic immune response of nongranulocyte immune cells present in the mixture after incubation increases compared to the corresponding therapeutic immune response of nongranulocyte immune cells cultured in the presence of fewer granulocyte-producing cells but otherwise subjected to identical conditions, then the population of granulocyte-producing cells can be considered to amplify the therapeutic immune response of nongranulocyte immune cells.

[0292] Preferably, the ability of a population of granulocyte-producing cells to amplify the therapeutic immune response of non-granulocyte immune cells is determined by a method including: (a) Mixing PBMCs or αβT cell-depleted PBMCs in the presence of granulocyte-producing cells to form a mixture. (b) Incubate the mixture (c) After incubation, determine the therapeutic immune response of nongranulocyte immune cells present in the mixture, and (d) After incubation, compare the therapeutic immune response of nongranulocyte immune cells present in the mixture with the corresponding therapeutic immune response of nongranulocyte immune cells cultured in the absence of granulocyte-producing cells but otherwise subjected to the same conditions.

[0293] Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs can be mixed together in any appropriate ratio. Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs may be mixed together in a ratio of 100:1 to 0.01:1 for granulocyte-producing cells to PBMCs or αβT cell-depleted PBMCs. A population of granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs may be mixed together in a ratio of 100:1 to 0.01:1, 75:1 to 0.05:1, 50:1 to 0.1:1, 25:1 to 0.2:1, 10:1 to 0.25:1, 5:1 to 0.25:1, 3:1 to 0.25:1, or 2:1 to 0.5:1 for granulocyte-producing cells to PBMCs or αβT cell-depleted PBMCs. Preferably, granulocyte-producing cells and PBMCs are mixed together in a ratio of 3:1 to 0.25:1 for granulocyte-producing cells to PBMCs or αβT cell-depleted PBMCs.

[0294] Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs may be mixed together in a ratio of 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.1:1, 0.05:1, or 0.01:1 or less. Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs may be mixed together in a ratio of at least 0.01:1, 0.05:1, 0.1:1, 0.25:1, 0.5:1, 1:1, 2:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1. Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs may be mixed together in a ratio of 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.1:1, 0.05:1, or 0.01:1. Preferably, granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs are mixed together in a ratio of 2:1, 1:1, or 0.5:1. For example, granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs may be mixed together in a ratio of 2:1. Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs may be mixed together in a 1:1 ratio of granulocyte-producing cells to PBMCs or αβT cell-depleted PBMCs. Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs may be mixed together in a 0.5:1 ratio of granulocyte-producing cells to PBMCs or αβT cell-depleted PBMCs.

[0295] The mixture may be incubated for any appropriate time. For example, the mixture may be incubated for 1 to 240 hours. The mixture may be incubated for 1 to 240 hours, 2 to 220 hours, 4 to 200 hours, 8 to 180 hours, 12 to 160 hours, 16 to 140 hours, 20 to 120 hours, 24 to 100 hours, 24 to 96 hours, 48 ​​to 96 hours, or 48 to 72 hours. Preferably, the mixture is incubated for 48 to 96 hours.

[0296] The mixture may be incubated for 1, 2, 4, 8, 12, 16, 20, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 144, 168, 192, 216, or 240 hours. Preferably, the mixture is incubated for 72 hours.

[0297] Therefore, the ability of a population of granulocyte-producing cells to amplify the therapeutic immune response of non-granulocyte immune cells can be determined by a method including the following: (a) Mixing PBMCs or αβT cell-depleted PBMCs in the presence of granulocyte-producing cells to form a mixture containing granulocyte-producing cells in a 2:1, 1:1, or 0.5:1 ratio to PBMCs or αβT cell-depleted PBMCs. (b) Incubate the mixture for 72 hours. (c) After incubation, determine the therapeutic immune response of nongranulocyte immune cells present in the mixture, and (d) After incubation, compare the therapeutic immune response of nongranulocyte immune cells present in the mixture to a reference standard.

[0298] Preferably, the ability of a population of granulocyte-producing cells to amplify the therapeutic immune response of non-granulocyte immune cells may be determined by a method including: (a) Mixing PBMCs or αβT cell-depleted PBMCs in the presence of granulocyte-producing cells to form a mixture containing granulocyte-producing cells in a 2:1, 1:1, or 0.5:1 ratio to PBMCs or αβT cell-depleted PBMCs. (b) Incubate the mixture for 72 hours. (c) After incubation, determine the therapeutic immune response of nongranulocyte immune cells present in the mixture, and (d) After incubation, compare the therapeutic immune response of nongranulocyte immune cells present in the mixture with the corresponding therapeutic immune response of nongranulocyte immune cells cultured in the absence of granulocyte-producing cells but otherwise subjected to the same conditions.

[0299] The mixture may further contain a CD3 activator, such as OKT3.

[0300] The therapeutic immune response of nongranulocyte immune cells can be determined by any appropriate means. For example, the therapeutic immune response of nongranulocyte immune cells can be determined by measuring cell surface markers present on nongranulocyte immune cells using flow cytometry. The therapeutic immune response of nongranulocyte immune cells can be determined by measuring the levels of activation markers, degranulation markers, and / or costimulatory markers present on nongranulocyte immune cells using flow cytometry. The therapeutic immune response of nongranulocyte immune cells can be determined by measuring the proliferation and / or survival of nongranulocyte immune cells using flow cytometry.

[0301] Therefore, the therapeutic immune response of nongranulocyte immune cells can be determined by methods including the following: (a) Washing nongranulocyte immune cells, (b) Incubate the cells with a live / dead staining agent. (c) Wash the cells in flow cytometry buffer and surface stain the cells with an antibody to measure the number of nongranulocyte immune cells present, the level of activation markers present on the nongranulocyte immune cells, the level of degranulation markers, and / or the level of costimulatory markers. (d) Fixing the cells, and (e) Analyze the cells using a flow cytometer.

[0302] If the number of nongranulocyte immune cells present, the levels of activation markers present on nongranulocyte immune cells, the levels of degranulation markers, and / or co-stimulatory markers are increased compared to a reference standard, then the population of granulocyte-producing cells may be considered capable of amplifying the therapeutic immune response of nongranulocyte immune cells.

[0303] Preferably, the therapeutic immune response of nongranulocyte immune cells can be determined by a method including the following: (a) Washing nongranulocyte immune cells (for example, those present in the mixture after incubation, or in PBMCs or PBMCs depleted of αβT cells) in PBS. (b) Incubate the cells with a viability / death stain (Fixable Viability Dye eFluor 780, 1:50 dilution) and an FcγR block (Human TruStain FcX, 1:50 dilution) for 20 minutes. (c) Wash the cells in flow cytometry buffer and surface stain the cells with antibodies specific to CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (HCD56), CD107a (H4A3), 4-1BB (4B4-1) and / or (preferably, and) OX40 (Ber-ACT35), wherein the antibodies are used at a 1:50 dilution and staining is performed at 50 μl / sample. (d) Fix the cells using 100 μl of 1×BD CellFix, and (e) Analyze the cells using a flow cytometer (e.g., MACSQuant 16(Miltenyi)).

[0304] If the expression levels of CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (HCD56), CD107a (H4A3), 4-1BB (4B4-1), and / or (preferably, and) OX40 present on nongranulocyte immune cells are increased compared to a reference standard, the population of granulocyte-producing cells may be considered capable of amplifying the therapeutic immune response of nongranulocyte immune cells.

[0305] The data can be analyzed using any suitable software, preferably FlowLogic software. The stained cell population is preferably analyzed by gating with single living cells.

[0306] The therapeutic immune response of nongranulocyte immune cells can be determined by measuring the cytokines produced by these cells. For example, the therapeutic immune response of nongranulocyte immune cells can be determined by measuring the cytokines produced by these cells using ELISA.

[0307] Therefore, the therapeutic immune response of nongranulocyte immune cells can be determined by methods including the following: (a) Measuring the concentration of cytokines present in the cell culture supernatant of nongranulocyte immune cells using ELISA, and / or (b) Use LEGENDplex to measure the concentration of cytokines present in the cell culture supernatant of nongranulocyte immune cells.

[0308] If the concentration of cytokines present in the cell culture supernatant of nongranulocyte immune cells increases compared to a reference standard, the population of granulocyte-producing cells may be considered capable of amplifying the therapeutic immune response of nongranulocyte immune cells.

[0309] Preferably, the therapeutic immune response of nongranulocyte immune cells can be determined by a method including the following: (a) Measuring the concentration of secreted IFN-γ present in the cell culture supernatant of nongranulocyte immune cells (e.g., present in the mixture after incubation, or present in PBMCs or αβT cell-depleted PBMCs) using a quantitative sandwich ELISA (e.g., Abcam, ab174443) in accordance with the manufacturer's instructions, and / or (b) Measure the concentration of CXCL10 present in the cell culture supernatant of nongranulocyte immune cells (e.g., present in the mixture after incubation, or present in PBMCs or αβT cell-depleted PBMCs) using LEGENDplex (e.g., BioLegend, 740985) in accordance with the manufacturer's instructions.

[0310] If the concentrations of IFN-γ and / or CXCL10 (preferably and) present in the cell culture supernatant of nongranulocyte immune cells are increased compared to a reference standard, the population of granulocyte-producing cells may be considered capable of amplifying the therapeutic immune response of nongranulocyte immune cells.

[0311] The therapeutic immune response of nongranulocyte immune cells can be determined by measuring cell surface markers present on nongranulocyte immune cells and / or by measuring cytokines produced by nongranulocyte immune cells. Preferably, the therapeutic immune response of nongranulocyte immune cells is determined by a method including: (a)(i) Washing nongranulocyte immune cells (for example, those present in the mixture after incubation, or in PBMCs or PBMCs depleted of αβT cells) in PBS. (ii) Incubate the cells with a viability / death stain (Fixable Viability Dye eFluor 780, 1:50 dilution) and an FcγR block (Human TruStain FcX, 1:50 dilution) for 20 minutes. (iii) Wash the cells in flow cytometry buffer and surface stain the cells with antibodies specific to CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (HCD56), CD107a (H4A3), 4-1BB (4B4-1) and / or (preferably, and) OX40 (Ber-ACT35), wherein the antibodies are used at a 1:50 dilution and staining is performed at 50 μl / sample. (iv) Fix the cells using 100 μl of 1×BD CellFix, and (v) Analyzing the cells using a flow cytometer (e.g., MACSQuant 16(Miltenyi)), and / or (b)(i) Measuring the concentration of secreted IFN-γ present in the cell culture supernatant of nongranulocyte immune cells (e.g., present in the mixture after incubation, or present in PBMCs or αβT cell-depleted PBMCs) using a quantitative sandwich ELISA (e.g., Abcam, ab174443) in accordance with the manufacturer's instructions, and / or (ii) Measure the concentration of CXCL10 present in the cell culture supernatant of nongranulocyte immune cells (e.g., present in the mixture after incubation, or present in PBMCs or αβT cell-depleted PBMCs) using LEGENDplex (e.g., BioLegend, 740985) in accordance with the manufacturer's instructions.

[0312] The therapeutic immune response of nongranulocyte immune cells can be determined, for example, by measuring tumor death of nongranulocyte immune cells as determined by the method described herein. If the level of tumor death of nongranulocyte immune cells increases compared to a reference standard, the population of granulocyte-producing cells may be considered to be able to amplify the therapeutic immune response of nongranulocyte immune cells.

[0313] The ability of a population of granulocyte-producing cells to amplify the therapeutic immune response of non-granulocyte immune cells may be determined by a method including: (a) Mixing PBMCs or αβT cell-depleted PBMCs in the presence of granulocyte-producing cells to form a mixture containing granulocyte-producing cells in a 2:1, 1:1, or 0.5:1 ratio to PBMCs or αβT cell-depleted PBMCs. (b) Incubate the mixture for 72 hours. (c) Determining the therapeutic immune response of nongranulocyte immune cells present in the mixture after incubation by a method comprising the following: (i) Wash nongranulocyte immune cells (for example, those present in the mixture after incubation, or in PBMCs or PBMCs depleted of αβT cells) in PBS. (ii) Incubate the cells with a viability / death stain (Fixable Viability Dye eFluor 780, 1:50 dilution) and an FcγR block (Human TruStain FcX, 1:50 dilution) for 20 minutes. (iii) Wash the cells in flow cytometry buffer and surface stain the cells with antibodies specific to CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (HCD56), CD107a (H4A3), 4-1BB (4B4-1) and / or (preferably, and) OX40 (Ber-ACT35), wherein the antibodies are used at a 1:50 dilution and staining is performed at 50 μl / sample. (iv) Fix the cells using 100 μl of 1×BD CellFix, and (v) Analyzing the cells using a flow cytometer (e.g., MACSQuant 16(Miltenyi)) and / or determining the therapeutic immune response of nongranulocyte immune cells present in the mixture after incubation by a method including: (i) Using a quantitative sandwich ELISA (e.g., Abcam, ab174443) in accordance with the manufacturer's instructions, measure the concentration of secreted IFN-γ present in the cell culture supernatant of nongranulocyte immune cells (e.g., present in the mixture after incubation, or present in PBMCs or αβT cell-depleted PBMCs), and / or (ii) Using LEGENDplex (e.g., BioLegend, 740985) in accordance with the manufacturer's instructions, measure the concentration of CXCL10 present in the cell culture supernatant of nongranulocyte immune cells (e.g., present in the mixture after incubation, or present in PBMCs or αβT cell-depleted PBMCs), and (d) After incubation, compare the therapeutic immune response of nongranulocyte immune cells present in the mixture to a reference standard.

[0314] Preferably, the ability of a population of granulocyte-producing cells to amplify the therapeutic immune response of non-granulocyte immune cells may be determined by a method including: (a) Mixing PBMCs or αβT cell-depleted PBMCs in the presence of granulocyte-producing cells to form a mixture containing granulocyte-producing cells in a 2:1, 1:1, or 0.5:1 ratio to PBMCs or αβT cell-depleted PBMCs. (b) Incubate the mixture for 72 hours. (c) Determining the therapeutic immune response of nongranulocyte immune cells present in the mixture after incubation by a method comprising the following: (i) Wash nongranulocyte immune cells (for example, those present in the mixture after incubation, or in PBMCs or PBMCs depleted of αβT cells) in PBS. (ii) Incubate the cells with a viability / death stain (Fixable Viability Dye eFluor 780, 1:50 dilution) and an FcγR block (Human TruStain FcX, 1:50 dilution) for 20 minutes. (iii) Wash the cells in flow cytometry buffer and surface stain the cells with antibodies specific to CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (HCD56), CD107a (H4A3), 4-1BB (4B4-1) and / or (preferably, and) OX40 (Ber-ACT35), wherein the antibodies are used at a 1:50 dilution and staining is performed at 50 μl / sample. (iv) Fix the cells using 100 μl of 1×BD CellFix, and (v) Analyzing the cells using a flow cytometer (e.g., MACSQuant 16(Miltenyi)) and / or determining the therapeutic immune response of nongranulocyte immune cells present in the mixture after incubation by a method including: (i) Using a quantitative sandwich ELISA (e.g., Abcam, ab174443) in accordance with the manufacturer's instructions, measure the concentration of secreted IFN-γ present in the cell culture supernatant of nongranulocyte immune cells (e.g., present in the mixture after incubation, or present in PBMCs or αβT cell-depleted PBMCs), and / or (ii) Using LEGENDplex (e.g., BioLegend, 740985) in accordance with the manufacturer's instructions, measure the concentration of CXCL10 present in the cell culture supernatant of nongranulocyte immune cells (e.g., present in the mixture after incubation, or present in PBMCs or αβT cell-depleted PBMCs), and (d) After incubation, compare the therapeutic immune response of nongranulocyte immune cells present in the mixture with the corresponding therapeutic immune response of nongranulocyte immune cells cultured in the absence of granulocyte-producing cells but otherwise subjected to the same conditions.

[0315] The ability of a population of granulocytes to increase multiple therapeutic immune responses in non-granulocyte immune cells suggests that such populations are particularly suitable for inclusion in the compositions of the present invention. Therefore, the composition may include a population of granulocytes capable of amplifying (preferably amplifying) the levels of CD3, CD4, CD8, CD56, CD107a, 4-1BB, and OX40 in non-granulocyte immune cells compared to a reference standard. The composition may also include, for example, a population of granulocytes capable of amplifying (preferably amplifying) the levels of IFN-γ and CXCL10 in non-granulocyte immune cells compared to a reference standard.

[0316] The inventors have surprisingly discovered that a population of granulocyte-producing cells that can (preferably) amplify the therapeutic immune response of one type of nongranulocyte immune cell may also be able (preferably) amplify the therapeutic immune response of a different type of nongranulocyte immune cell. Therefore, if a population of granulocyte-producing cells can amplify the therapeutic immune response of NK cells and / or T cells, as determined, for example, using the method described herein, then the population of granulocyte-producing cells may be considered capable of amplifying the therapeutic immune response of nongranulocyte immune cells. Preferably, if a population of granulocyte-producing cells can amplify the therapeutic immune response of NK cells, as determined, for example, using the method described herein, then the population of granulocyte-producing cells may be considered capable of amplifying the therapeutic immune response of nongranulocyte immune cells.

[0317] The inventors have also shown that granulocyte-producing cells that can amplify (preferably amplify) a particular therapeutic immune response may also be able to amplify (preferably amplify) a different type of therapeutic immune response. For example, a population of granulocyte-producing cells that can increase (preferably increase) cell activation may also be able to increase (preferably increase) the expression of degranulation markers. Therefore, if a population of granulocyte-producing cells can increase NK cell activation, increase the expression of NK cell degranulation markers, increase the expression of NK cell costimulatory molecules, increase NK cell proliferation, increase NK cell survival, increase cytokine expression by NK cells, increase NK cell cytodestructive activity, and / or increase NK cell tumor cell killing activity, then the population of granulocyte-producing cells may be considered able to amplify the therapeutic immune response of non-granulocyte immune cells. Preferably, if a population of granulocyte-producing cells can increase (preferably increase) the levels of CD107a, 4-1BB and / or (preferably and) OX40 in NK cells, as determined, for example, using the method described herein, then the population of granulocyte-producing cells is considered to be able to amplify the therapeutic immune response of non-granulocyte immune cells.

[0318] Populations of granulocyte-producing cells suitable for use in various embodiments of the present invention may increase the activation of immune cells, such as non-granulocyte immune cells. In particular, populations of granulocyte-producing cells may increase (preferably increase) the activation of non-granulocyte immune cells present in the compositions of the present invention. Therefore, populations of granulocyte-producing cells may amplify the therapeutic immune response of non-granulocyte immune cells by increasing their activation.

[0319] Preferably, a population of granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocyte immune cells) such that the expression of one or more markers of degranulation by immune cells increases. Preferably, a population of granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocyte immune cells) such that the expression of one or more co-stimulatory molecules by immune cells increases. Preferably, a population of granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocyte immune cells) such that the proliferation of immune cells increases. Preferably, a population of granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocyte immune cells) such that the abundance of immune cells increases. Preferably, a population of granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocyte immune cells) such that the survival of immune cells increases. Preferably, a population of granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocyte immune cells) such that the expression of one or more cytokines by immune cells is increased. Preferably, a population of granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocyte immune cells) such that the transport of immune cells is increased. Preferably, a population of granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocyte immune cells) such that the cytodestructive activity of immune cells is increased.

[0320] As used herein, the term “one or more” may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, when “one or more” precedes a list, “one or more” may mean all the elements of the list. Similarly, as used herein, the term “at least one” may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, when “at least one” precedes a list, “at least one” may mean all the elements of the list.

[0321] Preferably, a population of granulocyte-producing cells suitable for use according to the present invention may increase the activation of immune cells (e.g., non-granulocyte immune cells) via "Signal 2" (co-stimulation). Alternatively, a population of granulocyte-producing cells suitable for use according to the present invention may further increase the activation of immune cells (e.g., non-granulocyte immune cells) via "Signal 3" (cytokine stimulation). A population of granulocyte-producing cells suitable for use according to the present invention may have the ability to increase the activation of immune cells (e.g., non-granulocyte immune cells) via both Signal 2 and Signal 3.

[0322] Signals 2 and 3 are both known to be important in generating an effective immune response against tumors and in overcoming the immunosuppressive effects of TME. Therefore, our data (shown in the examples) demonstrating that granulocyte-producing cells suitable for use according to the present invention may be able to provide these signals clearly demonstrate their suitability for use in amplifying potentially therapeutic immune responses related to cancer treatment.

[0323] Other useful methods for defining the relevant population of granulocyte-producing cells are listed below.

[0324] Granulocyte-producing cells suitable for use in the compositions, medical uses, and methods of the present invention may be defined with respect to their potency. The population of granulocyte-producing cells may include pluripotent cells. In preferred embodiments, the population of granulocyte-producing cells may include unipotent cells.

[0325] A suitable population of granulocyte-producing cells for use in various embodiments of the present invention may be defined with respect to their differentiation state within the granulocyte-producing pathway. In a preferred embodiment, the population of granulocyte-producing cells has a differentiation stage corresponding to the differentiation stage between myeloblasts and granulocytes. Preferably, the population of granulocyte-producing cells has a differentiation stage corresponding to the differentiation stage between myeloblasts and band neutrophils. For example, the population of granulocyte-producing cells may have a differentiation stage corresponding to the differentiation stage between myeloblasts and metamyelocytes. Preferably, the population of granulocyte-producing cells has a differentiation stage corresponding to the differentiation stage between myeloblasts and myelocytes. Preferably, the population of granulocyte-producing cells has a differentiation stage corresponding to the differentiation stage between myeloblasts and promyelocytes.

[0326] In a preferred embodiment, the population of granulocytes has a differentiation stage corresponding to myeloblasts. In a preferred embodiment, the population of granulocytes has a differentiation stage corresponding to promyelocytes. In a preferred embodiment, the population of granulocytes has a differentiation stage corresponding to myelocytes. In a preferred embodiment, the population of granulocytes has a differentiation stage corresponding to metamyelocytes. In a preferred embodiment, the population of granulocytes has a differentiation stage corresponding to band neutrophils.

[0327] In a preferred embodiment, the population of granulocyte-producing cells has a differentiation stage corresponding to that of granulocytes.

[0328] As described elsewhere in this specification, populations of granulocyte-producing cells suitable for use in various embodiments of the present invention may be derived from artificial stem cells such as iPSCs. Such populations of granulocyte-producing cells may not be identical to naturally occurring cells in the granulocyte-producing pathway, but it will be understood that they may share structural (e.g., marker expression) or functional (e.g., potency) characteristics with such naturally occurring cells. References in this disclosure to cells having a differentiation stage "corresponding" to the specified cell types should be interpreted accordingly.

[0329] Preferably, the population of granulocyte-producing cells is selected from a group that includes (or consists of) myeloblasts, promyelocytes, myelocytes, metamyelocytes, band neutrophils, and granulocytes. Preferably, the population of granulocyte-producing cells is selected from a group that includes (or consists of) myeloblasts, promyelocytes, myelocytes, metamyelocytes, and band neutrophils. Preferably, the population of granulocyte-producing cells is selected from a group that includes (or consists of) myeloblasts, promyelocytes, myelocytes, and metamyelocytes. Preferably, the population of granulocyte-producing cells is selected from a group that includes (or consists of) myeloblasts, promyelocytes, and myelocytes. Preferably, the population of granulocyte-producing cells is selected from a group that includes (or consists of) myeloblasts and promyelocytes.

[0330] In a preferred embodiment, the population of granulocyte-producing cells is myeloblasts. In a preferred embodiment, the population of granulocyte-producing cells is promyelocytes. In a preferred embodiment, the population of granulocyte-producing cells is myelocytes. In a preferred embodiment, the population of granulocyte-producing cells is metamyelocytes. In a preferred embodiment, the population of granulocyte-producing cells is band neutrophils. In a preferred embodiment, the population of granulocyte-producing cells is granulocytes.

[0331] Preferably, the population of granulocyte-producing cells may be committed to the neutrophil lineage. In such embodiments, the preferred population of granulocyte-producing cells may include, or consist of, cells selected from the group including (or comprising) neutrophil promyelocytes, neutrophils, neutrophil metamyelocytes, neutrophil band neutrophils, and neutrophils.

[0332] As further described elsewhere in this specification, populations of granulocyte-producing cells that can be used in various embodiments of the present invention may also be defined with respect to granulocytes that they can induce differentiation of. A preferred example of a population of granulocyte-producing cells may be able to produce granulocytes that have the ability to kill cancer cells and / or cells that have infected an infected organism. Alternatively, or further, a preferred population of granulocyte-producing cells may be able to produce granulocytes that have a desirable expression profile of molecules such as chemokines or costimulatory receptor ligands.

[0333] The inventors have surprisingly shown that populations of granulocyte-producing cells cultured in the presence of non-granulocyte-producing immune cells may have an amplified therapeutic immune response. Therefore, populations of granulocyte-producing cells suitable for use in the compositions, medical uses, and methods of the present invention may be characterized by having an amplified therapeutic immune response. For example, populations of granulocyte-producing cells suitable for use in the compositions, medical uses, and methods of the present invention may be characterized by one or more of the following, as determined by the method herein, increased activation, increased expression of degranulation markers, increased expression of costimulatory molecules, increased proliferation, increased survival, increased cytokine expression, increased cell destructive activity, or increased tumor cell killing activity, compared, for example, to the corresponding therapeutic immune response of non-granulocyte-producing immune cells cultured in the absence of granulocyte-producing cells as described herein, or to a reference standard. Preferably, the composition comprises a population of granulocyte-producing cells characterized by one or more of the following, as determined by the method herein, increased activation, increased expression of degranulation markers, increased expression of costimulatory molecules, increased proliferation, increased survival, increased cytokine expression, increased cell destructive activity, or increased tumor cell killing activity, compared, for example, to the corresponding therapeutic immune response of a population of granulocyte-producing cells cultured in the absence of non-granulocyte immune cells as described herein, or to a reference standard.

[0334] A population of granulocytes exhibiting an amplified therapeutic immune response may be a population of granulocytes with increased activation. Therefore, the composition may include a population of granulocytes with increased activation. Increased activation of a population of granulocytes can be associated with increased expression of one or more markers selected from the group including (or consisting of) CD54, CD40, CD11b, and Mac1. Therefore, the composition of the present invention may include, for example, a population of granulocytes with increased expression of CD54, CD40, CD11b, and / or Mac1 compared to a population of granulocytes not cultured in the presence of non-granulocyte immune cells but otherwise subjected to the same conditions. The composition of the present invention may include, for example, a population of granulocytes with increased expression of CD40 compared to a population of granulocytes not cultured in the presence of non-granulocyte immune cells but otherwise subjected to the same conditions. The composition of the present invention may include, for example, a population of granulocyte-producing cells that have increased CD11b expression compared to a population of granulocyte-producing cells that have not been cultured in the presence of non-granulocyte-immune cells but have been subjected to otherwise identical conditions. The composition of the present invention may also include, for example, a population of granulocyte-producing cells that have increased Mac1 expression compared to a population of granulocyte-producing cells that have not been cultured in the presence of non-granulocyte-immune cells but have been subjected to otherwise identical conditions. Preferably, the composition of the present invention includes, for example, a population of granulocyte-producing cells that have increased CD54 expression compared to a population of granulocyte-producing cells that have not been cultured in the presence of non-granulocyte-immune cells but have been subjected to otherwise identical conditions.

[0335] The activation of such a population of granulocytes (determined, e.g., by CD54 expression) may increase by at least 5%. For example, the activation of a population of granulocytes may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The quantification of the increase in the activation of a population of granulocytes in such embodiments can be achieved by comparison with a suitable control, e.g., a population of granulocytes not cultured in the presence of non-granulocyte immune cells but otherwise subjected to identical conditions.

[0336] The compositions of the present invention may include, for example, a population of granulocytes that express increased CD54, a population of granulocytes that express increased CD40, a population of granulocytes that express increased CD11b, and / or a population of granulocytes that express increased Mac1, compared to a population of granulocytes that have not been cultured in the presence of non-granulocyte immune cells but have been subjected to otherwise identical conditions. In some embodiments, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% of the population of granulocytes in the composition express CD54, as determined, for example, by flow cytometry. Preferably, at least about 45% of the population of granulocytes in the composition express CD54, as determined, for example, by flow cytometry.

[0337] Populations of granulocyte-producing cells may be obtained from any suitable source. For example, populations of granulocyte-producing cells may be obtained from PBMC samples or umbilical cord blood samples. PBMC samples or umbilical cord blood samples may be obtained from donors (e.g., can be obtained). Preferably, populations of granulocyte-producing cells may be obtained from αβT cell-depleted PBMC samples (e.g., can be obtained). Populations of granulocyte-producing cells may be obtained from hematopoietic stem cells or stem cells such as iPSCs (e.g., can be differentiated in vitro).

[0338] As used herein, the term “possible to obtain” also includes the term “obtainable.” In one embodiment, “possible to obtain” means “obtainable.”

[0339] As used herein, the term “donor” refers to a subject (preferably a human subject) from which a sample can be obtained (e.g., can be obtained). Any suitable sample from which a population of granulocyte-producing cells and / or non-granulocyte immune cells can be obtained may be obtained from a donor. Donors may be selected based on one or more of the following characteristics: sex, age, medical history, and / or blood type. Donors may be selected if they are healthy. Donors may be selected if they do not have cancer and do not have infectious diseases. For example, donors may be selected if they do not have cancer. Donors may be selected if they do not have infectious diseases. Donors may be selected if they are male. Donors may be selected if they are between 18 and 55 years old, preferably between 18 and 35 years old (more preferably between 18 and 24 years old). Preferably, the donor may be selected if the donor is male between 18 and 55 years of age, preferably between 18 and 35 years of age (more preferably between 18 and 24 years of age). In another embodiment, the donor may be selected if the donor is female. The donor may be selected if the donor is over 40 years of age. Preferably, the donor may be selected if the donor is female over 40 years of age.

[0340] Populations of granulocyte-producing cells may be obtained from hematopoietic cells. As used herein, the term “hematopoietic cells” refers to cells that can (preferably differentiate) into granulocyte-producing cells or populations of granulocyte-producing cells. Thus, the term “hematopoietic cells” encompasses hematopoietic stem cells, as well as precursor cells (e.g., differentiated from hematopoietic stem cells), which can (preferably differentiate) into granulocyte-producing cells or populations of granulocyte-producing cells. Precursor cells may also be referred to herein as “granulocyte-producing precursor cells.” Preferred examples of such precursor cells are defined elsewhere herein in relation to the generation of populations of granulocyte-producing cells. Hematopoietic cells according to the present invention may relate to hematopoietic stem cells, granulocyte-producing precursor cells, or a combination thereof. 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. Stem cells may be obtained from umbilical cord blood.

[0341] In one embodiment, iPSCs can be obtained from donor somatic cells. The generation of iPSCs is a well-known technique in the art. See Yu et al (2007), Science, 318:1917-1920, the teachings of which are incorporated herein by reference. Thus, a population of granulocyte-producing cells may be obtained from induced pluripotent stem cells (iPSCs) or hematopoietic stem cells (HSCs). Preferably, a population of granulocyte-producing cells can be obtained from HSCs. A population of granulocyte-producing cells may be obtained by the methods for obtaining a population of granulocyte-producing cells described herein (for example).

[0342] Granulocyte-producing cells obtained by the above method (for example, obtained) can amplify (preferably amplify) the therapeutic immune response of non-granulocyte immune cells.

[0343] The population of granulocyte-producing cells present in the composition of the present invention may be a heterogeneous population of granulocyte-producing cells, i.e., a population of granulocyte-producing cells comprising multiple different types or subtypes of granulocyte-producing cells, or it may be a homogeneous population of granulocyte-producing cells, i.e., a population of granulocyte-producing cells comprising a single type of granulocyte-producing cell. Preferably, the population of granulocyte-producing cells is a heterogeneous population of granulocyte-producing cells.

[0344] As used herein, the term “non-granulocyte immune cells” refers to any cell of the immune system other than granulocytes (e.g., non-granulocytes). Therefore, non-granulocyte immune cells may be any immune cell other than neutrophils, eosinophils, or basophils.

[0345] Nongranulocyte immune cells may be dendritic cells, blood-derived myeloid cells, monocytes, macrophages, natural killer (NK) cells, B cells, or T cells, such as γδT cells. Preferably, nongranulocyte immune cells are dendritic cells, blood-derived myeloid cells, monocytes, macrophages, NK cells, B cells, or γδT cells. Particularly preferably, nongranulocyte immune cells are γδT cells (e.g., Vδ1 + or Vδ2 + These are γδT cells or NK cells.

[0346] Nongranulocyte immune cells suitable for use in the compositions, medical uses, and methods of the present invention may be characterized by having an amplified therapeutic immune response. For example, nongranulocyte immune cells suitable for use in the compositions, medical uses, and methods of the present invention may be characterized by one or more of the following, compared to the corresponding therapeutic immune response of nongranulocyte immune cells cultured in the absence of granulocyte-producing cells, or compared to a reference standard: increased activation, increased expression of degranulation markers, increased expression of costimulatory molecules, increased proliferation, increased survival, increased cytokine expression, increased cell destructive activity, or increased tumor cell killing activity. Preferably, the composition comprises nongranulocyte immune cells characterized by one or more of the following, compared to the corresponding therapeutic immune response of nongranulocyte immune cells cultured in the absence of a population of granulocyte-producing cells, or compared to a reference standard: increased activation, increased expression of degranulation markers, increased expression of costimulatory molecules, increased proliferation, increased survival, increased cytokine expression, increased cell destructive activity, or increased tumor cell killing activity.

[0347] For example, nongranulocyte immune cells may show increased expression of one or more markers selected from CD3, CD4, CD8, CD56, CD107a, 4-1BB, and OX40. Preferably, nongranulocyte immune cells show increased expression of one or more markers selected from CD107a, 4-1BB, and OX40. Nongranulocyte immune cells may show increased expression of CD107a. Nongranulocyte immune cells may show increased expression of 4-1BB. Nongranulocyte immune cells may show increased expression of OX40. Nongranulocyte immune cells may show increased expression of CXCL10. Nongranulocyte immune cells may secrete increased concentrations of CXCL10. Nongranulocyte immune cells may show increased expression of IFN-γ. Nongranulocyte immune cells may secrete increased concentrations of IFN-γ. Nongranulocyte immune cells may show increased proliferation. Nongranulocyte immune cells may show increased tumor-killing ability. This increase may be compared to non-granulocyte immune cells cultured in the absence of a population of granulocyte-producing cells, but otherwise subjected to identical conditions.

[0348] The inventors have surprisingly discovered that the population of granulocyte-producing cells described herein can amplify (preferably amplify) the therapeutic immune response of NK cells. For example, the inventors have shown that the population of granulocyte-producing cells described herein increases the proliferation of NK cells, thereby overcoming the problem of limited ex vivo expansion of NK cells. The inventors have shown that the granulocyte-producing cells described herein increase the survival of NK cells, thereby overcoming the problem of limited in vivo survival of NK cells. Furthermore, the inventors have shown that the population of granulocyte-producing cells described herein potently increases the expression of 4-1BB and OX40 on NK cells, thereby enhancing the cytotoxicity of NK cells.

[0349] Therefore, this composition may contain a population of granulocyte-producing cells and NK cells. The NK cells can be any suitable NK cells. NK cells are CD3 - , and CD56 + These could be NK cells. For example, NK cells are CD3 - CD56 dim , and / or CD16 + For example, CD3 - CD56 dim , and CD16 + These could be NK cells. NK cells are CD3 - CD56 bright , and / or CD16 - For example, CD3 - CD56 bright , and CD16 - These could be NK cells. NK cells are CD3 - CD56 + CD7 + CD127 - NKp46 + T-bet + , and / or Eomes + For example, CD3 - CD56 + CD7 + CD127 - NKp46+ T-bet + , and Eomes + It could be an NK cell. Although not bound by theory, CD56 dim , and CD16 + NK cells are mainly found in the blood, and CD56 bright , and CD16 - NK cells are thought to be primarily found in the lymphoid system. NK cells may be those that can be obtained by the method described in Oyer et al. Biol Blood Marrow Transplant 21(2015)632-639, which is incorporated in whole herein by reference.

[0350] Populations of granulocyte-producing cells suitable for use according to the present invention may increase NK cell activation. Therefore, the composition may contain increasedly activated NK cells. Increased NK cell activation may be associated with one or more of the following: increased expression of degranulation markers by NK cells (including, but not limited to, CD107a); increased expression of costimulatory molecules by NK cells (including, but not limited to, 4-1BB and / or OX40); increased expression of cytokines by NK cells (including, but not limited to, IFN-γ and / or TNF); increased NK cell transport; increased recruitment of NK cells to TMEs; increased cell destructive activity by NK cells (including, but not limited to, tumor cell death); increased NK cell proliferation; increased NK cell survival; and increased NK cell abundance. Changes in these properties associated with increased NK cell activation upon exposure to populations of granulocyte-producing cells suitable for use according to the present invention are shown in the examples. Further relevant considerations regarding these various properties are described elsewhere in this specification.

[0351] NK cell activation may increase by at least 5%. For example, NK cell activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the increase in NK cell activation in such embodiments can be achieved by comparison with a suitable control.

[0352] As used herein, the term “appropriate control” may refer to nongranulocyte immune cells that have not been cultured in the presence of a population of granulocyte-producing cells, but have otherwise been subjected to identical conditions.

[0353] In some embodiments, for example, as determined by flow cytometry, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the NK cells in the composition express 4-1BB. Preferably, for example, as determined by flow cytometry, at least about 10% of the NK cells in the composition express 4-1BB. In some embodiments, for example, as determined by flow cytometry, at least about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, or 35% of the NK cells in the composition express OX40. Preferably, for example, as determined by flow cytometry, at least about 5% of the NK cells in the composition express OX40. Particularly preferably, at least about 10% of the NK cells in the composition express 4-1BB and at least about 5% of the NK cells in the composition express OX40.

[0354] The inventors have surprisingly discovered that the population of granulocyte-producing cells described herein can amplify (preferably amplify) the therapeutic immune response of T cells. For example, the inventors have discovered that the population of granulocyte-producing cells described herein can amplify CD4 +and CD8 + It has been shown that the expression of 4-1BB and OX40 on T cells is increased, and the expression of 4-1BB and CD25 on γδT cells is increased, thereby improving the effector function of these cells. Therefore, the composition may include a population of granulocyte-producing cells and T cells. The T cells can be any suitable T cells. The T cells may be αβT cells or γδT cells. αβT cells are T cells that have an αβT cell receptor (TCR) on their cell surface. On the other hand, γδT cells are T cells that have a γδTCR on their cell surface. Preferably, the T cells are γδT cells. Particularly preferably, the γδT cells are Vδ1 or Vδ2 γδT cells. Preferably, the T cells are not αβT cells.

[0355] Populations of granulocyte-producing cells suitable for use according to the present invention may increase T cell activation. Therefore, the composition may contain increasedly activated T cells. Increased T cell activation may be associated with, but is not limited to, increased expression of degranulation markers by T cells (including, but not limited to, CD107a), increased expression of costimulatory molecules by T cells (including, but not limited to, 4-1BB and / or OX40), increased cytokine expression by T cells, increased T cell transport, increased recruitment of T cells to TMEs, increased cytodestructive activity by T cells (including, but not limited to, tumor cell death), increased T cell proliferation, increased T cell survival, and increased T cell abundance. Changes in these properties associated with increased T cell activation upon exposure to populations of granulocyte-producing cells suitable for use according to the present invention are shown in the examples. Further relevant considerations regarding these various properties are described elsewhere in this specification.

[0356] T cell activation may increase by at least 5%. For example, T cell activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The quantification of the increase in T cell activation in such embodiments can be achieved by comparison with a suitable control.

[0357] A population of granulocyte-producing cells suitable for use according to the present invention is CD8 + It may increase the activation of T cells. Therefore, this composition may increase the activation of CD8 + May contain T cells (CD8) + Increased T cell activation is CD8 + Increased expression of degranulation markers by T cells (including, but not limited to, CD107a), CD8 + Increased expression of co-stimulatory molecules by T cells (including, but not limited to, 4-1BB and / or OX40), and CD8 + This may be associated with one or more of the increased proliferation of T cells. Further relevant considerations regarding these various characteristics are described elsewhere in this specification.

[0358] Such CD8 + T cell activation may increase by at least 5%. For example, CD8 + T cell activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% or more. CD8 in such embodiments+ The quantification of the increase in T cell activation can utilize a comparison with a suitable control.

[0359] A population of granulopoietic cells suitable for use according to the present invention is CD4 + T cell activation may be increased. Thus, the composition may contain CD4 + T cells with increased activation. CD4 + The increase in CD4 + T cell activation may be associated with an increase in the expression of costimulatory molecules (including, but not limited to, 4-1BB and / or OX40) by CD4 + T cells, and / or an increase in the proliferation of CD4 + T cells. Further relevant considerations regarding these various properties are described elsewhere in this specification.

[0360] Such CD4 + T cell activation may increase by at least 5%. For example, CD4 + T cell activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The quantification of the increase in CD4 + T cell activation according to such embodiments can utilize a comparison with a suitable control.

[0361] A population of granulopoietic cells suitable for use according to the present invention may increase the activation of γδ T cells (e.g., Vδ1 + γδ T cells or Vδ2 + γδ T cells). Thus, the composition may contain γδ T cells (e.g., Vδ1 + γδ T cells or Vδ2 + γδ T cells) with increased activation. Vδ1 +Increased activation of γδT cells may be associated with increased expression of 4-1BB and / or CD25 on the cell surface. Vδ2 + Increased activation of γδT cells may be associated with increased expression of 4-1BB on the cell surface. Vδ1 + and Vδ2 + Increased activation of γδT cells was observed in Vδ1 and Vδ2 cells, respectively. + This may be associated with increased proliferation and / or survival of γδT cells.

[0362] Such γδT cell activation may increase by at least 5%. For example, γδT cell activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The quantification of the increase in γδT cell activation in such embodiments can be achieved by comparison with a suitable control.

[0363] In some embodiments, for example, Vδ1 in the composition is determined by flow cytometry. + At least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, or 5% of γδT cells express 4-1BB. Preferably, Vδ1 in the composition is expressed, for example, as determined by flow cytometry. + At least about 0.5% of γδT cells express 4-1BB. In some embodiments, for example, Vδ1 in the composition is determined by flow cytometry. + At least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% of γδT cells express CD25. Preferably, as determined by flow cytometry, for example, Vδ1 in the composition expresses CD25.+ At least about 1% of γδT cells express CD25. Preferably, Vδ1 in the composition is determined, for example, by flow cytometry. + At least about 0.5% of γδT cells express 4-1BB, and as determined, for example by flow cytometry, Vδ1 in the composition + At least about 1% of γδT cells express CD25. Particularly preferably, as determined by flow cytometry, Vδ1 in the composition is expressed. + At least about 5% of γδT cells express 4-1BB, and as determined, for example by flow cytometry, Vδ1 in the composition + At least about 30% of γδT cells express CD25.

[0364] In some embodiments, for example, Vδ2 in the composition is determined by flow cytometry. + At least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% of γδT cells express 4-1BB. Preferably, Vδ2 in the composition is expressed, for example, as determined by flow cytometry. + At least about 0.5% of γδT cells express 4-1BB. Particularly preferably, as determined by flow cytometry, Vδ2 in the composition is expressed. + At least about 10% of γδT cells express 4-1BB.

[0365] Dendritic cells can be any suitable dendritic cell. For example, dendritic cells can be classical or conventional dendritic cells (cDCs), plasmacytoid dendritic cells (pDCs), or monocyte-derived cells with dendritic cell-like characteristics (moDCs). cDCs can be type 1 cDCs (cDC1) or type 2 cDCs (cDC2). Although not bound by theory, cDC1 cells present exogenous antigens on MHC class I and naive CD8 + While inducing T cells to acquire cytotoxic T cell (CTL) effector function, cDC2 cells use antigen presentation on MHC class II to enable naive CD4 +It is thought that pDCs prime T cells. On the other hand, pDCs are thought to have a dedicated function of secreting type I interferon (IFN).

[0366] Therefore, dendritic cells are CD11c + HLA-DR + , and / or CD141 + For example, CD11c + HLA-DR + , and CD141 + These may be dendritic cells. This expression profile may be characteristic of cDC1 cells. Dendritic cells are CD11c + HLA-DR + CD141 + CLEC9A + , and / or CADM1 + For example, CD11c + HLA-DR + CD141 + CLEC9A + , and CADM1 + These can be dendritic cells (e.g., cDC1 cells). Dendritic cells are CD11c + HLA-DR + CD1c + , and / or CD11b + For example, CD11c + HLA-DR + CD1c + , and CD11b + These may be dendritic cells. This expression profile may be characteristic of cDC2 cells. Dendritic cells are CD11c + HLA-DR + CD1c + CD11b + FCER1A + CLEC10A + CD2 + CD172A + , and / or ILT1 + For example, CD11c + HLA-DR + CD1c + CD11b + FCER1A+ CLEC10A + CD2 + CD172A + , and ILT1 + These may be dendritic cells (e.g., cDC2 cells). Dendritic cells are HLA-DR + CD303 + , and / or CD123 + For example, HLA-DR + CD303 + , and CD123 + These may be dendritic cells. This expression profile may be characteristic of pDCs. Dendritic cells are HLA-DR + CD303 + CD123 + CD11c + (For example, CD11c int ), MHCII + (For example, MHC lo ), Bst2 + , and / or B220 + For example, HLA-DR + CD303 + CD123 + CD11c + (For example, CD11c int ), MHCII + (For example, MHC lo ), Bst2 + , and B220 + For example, HLA-DR + CD303 + CD123 + CD11c int MHC lo Bst2 + , and B220 + These can be dendritic cells (e.g., pDCs). Dendritic cells are CD11c + CD11b + CD1a + , and / or CD1c + For example, CD11c + CD11b + CD1a + , and CD1c +These may be dendritic cells. This expression profile may be characteristic of moDCs. Dendritic cells are CD11c + CD11b + CD1a + CD1c + CD206 + CD209 + , and / or CD172A + CD11c + CD11b + CD1a + CD1c + CD206 + CD209 + , and CD172A + These could be dendritic cells (e.g., moDCs).

[0367] Populations of granulocyte-producing cells suitable for use according to the present invention may increase dendritic cell activation. Therefore, the composition may contain dendritic cells with increased activation. Increased dendritic cell activation may be associated with increased expression of CD83, CD86, and / or CD80.

[0368] Such dendritic cell activation may increase by at least 5%. For example, dendritic cell activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The quantification of the increase in dendritic cell activation in such embodiments can be achieved by comparison with a suitable control.

[0369] Monocytes can be any suitable monocyte. For example, monocytes may be classical monocytes, intermediate monocytes, or non-classical monocytes. While not bound by theory, classical monocytes are considered the major monocyte population involved in phagocytic activity and have low production of inflammatory cytokines; intermediate monocytes produce inflammatory cytokines such as TNFα, IL-1β, and / or IL-6; and non-classical monocytes produce anti-inflammatory cytokines and constitutively produce IL-1RA.

[0370] Therefore, the monocyte is CD14 + CD16 + , or CD64 + It could be a monocyte. For example, a monocyte is CD14 + (For example, CD14 Hi ), CD64 + CD62L + TNFR1 + TNFR2 + (For example, TNFR2 Lo ), CD192 + (For example, CD192) Hi ), and / or CXCR1 + (For example, CXCR1 Lo ), for example, CD14 + (For example, CD14) Hi ), CD64 + CD62L + TNFR1 + TNFR2 + (For example, TNFR2 Lo ), CD192 + (For example, CD192) Hi ), and CXCR1 + (For example, CXCR1 Lo ), for example, CD14 Hi CD64 + CD62L + TNFR1 + TNFR2 Lo CD192 Hi , and CXCR1 Lo It may be a monocyte. This expression profile may be characteristic of classical monocytes. Monocytes are CD16 + CD14+ (For example, CD14) Hi ), CD64 + HLA-DR + (For example, HLA-DR Hi ), TNFR1 + (For example, TNFR1 Hi ), TNFR2 + CD192 + (For example, CD192) Lo ), CX3CR1 + (For example, CX3CR1 Hi ), and / or CD195 + For example, CD16 + CD14 + (For example, CD14) Hi ), CD64 + HLA-DR + (For example, HLA-DR Hi ), TNFR1 + (For example, TNFR1 Hi ), TNFR2 + CD192 + (For example, CD192) Lo ), CX3CR1 + (For example, CX3CR1 Hi ), and CD195 + For example, CD16 + CD14 Hi CD64 + HLA-DR Hi TNFR1 Hi TNFR2 + CD192 Lo , CX3CR1 Hi , and CD195 + It may be a monocyte. This expression profile may be characteristic of intermediate monocytes. Monocytes are CD14 + (For example, CD14) Lo ), CD16 + (For example, CD16) Hi ), TNFR1 + (For example, TNFR1 Lo ), and / or TNFR2 + (For example, TNFR2 Hi ), for example, CD14 + (For example, CD14) Lo), CD16 + (For example, CD16) Hi ), TNFR1 + (For example, TNFR1 Lo ), and TNFR2 + (For example, TNFR2 Hi ), for example, CD14 Lo CD16 Hi TNFR1 Lo , and TNFR2 Hi This could be a monocyte. This expression profile may be characteristic of non-classical monocytes.

[0371] A macrophage can be any suitable macrophage. For example, a macrophage may be a classically activated M1 macrophage or an alternatively activated M2 macrophage. While not bound by theory, M1 macrophages are thought to exhibit high antigen-presenting activity and high production of inflammatory cytokines, such as IL-1, IL-6, TNFα, nitric oxide, and reactive oxygen species (ROS). On the other hand, M2 macrophages are thought to exhibit low production of inflammatory cytokines such as IL-1, IL-6, and TNFα. Naturally, M1 and M2 macrophages may be further subdivided into further subclassifications.

[0372] Therefore, macrophages are CD11b + CD14 + CD15 + CD16 + , and / or CD68 + CD11b + CD14 + CD15 + CD16 + , and CD68 + It could be a macrophage. Macrophages are CD16 + CD32 + CD16 / CD32 + CD64 + CD68 + , CD80 + CD86 + CD369 +Mer + and / or MHC II + For example, CD16 + CD32 + CD16 / CD32 + CD64 + CD68 + , CD80 + CD86 + CD369 + Mer + , and MHC II + This may be a macrophage. This expression profile may be characteristic of M1 macrophages. M1 macrophages may be characterized by the secretion of IFNγ, IL-1α, IL-1β, IL-6, IL-12, IL-23 and / or TNFα, for example, IFNγ, IL-1α, IL-1β, IL-6, IL-12, IL-23 and TNFα. Macrophages are CD115 + CD163 + CD204 + CD206 + CD209 + FceR1 + , and / or VSIG4 + For example, CD115 + CD163 + CD204 + CD206 + CD209 + FceR1 + , and VSIG4 + This could be a macrophage. This expression profile may be characteristic of M2 macrophages. M2 macrophages may be characterized by the secretion of IDO, IL-10, and / or TGFβ, for example, IDO, IL-10, and TGFβ.

[0373] Populations of granulocyte-producing cells suitable for use according to the present invention may increase macrophage activation. Therefore, the composition may contain macrophages with increased activation. Increased macrophage activation may be associated with increased expression of CD86 and CD40, and / or enhanced secretion of TNFα.

[0374] Such macrophage activation may increase by at least 5%. For example, macrophage activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in macrophage activation in such embodiments can be quantified by comparison with a suitable control.

[0375] The B cell can be any suitable B cell. For example, the B cell can be any B cell containing a B cell receptor (BCR). The B cell can be a pro-B cell, pre-B cell, immature B cell, transitional B cell, naive B cell, B1 cell, memory B cell, or plasma cell. Preferably, the B cell is a transitional B cell, naive B cell, memory B cell, or plasma cell.

[0376] Therefore, B cells are CD19 + CD20 + CD34 + CD38 + , and / or CD45R + For example, CD19 + CD20 + CD34 + CD38 + , and CD45R + These could be B cells. This expression profile may be characteristic of pro-B cells. B cells are CD19 + CD20 + CD38 + CD40 + , and / or CD45R + For example, CD19 + CD20 + CD38 + CD40 + , and CD45R +These could be B cells. This expression profile may be characteristic of pre-B cells. B cells are CD19 + CD20 + CD40 + CD45R + , and / or IgM + CD19 + CD20 + CD40 + CD45R + , and IgM + These could be B cells. This expression profile may be characteristic of immature B cells. B cells are CD10 + CD19 + CD20 + CD24 hi , and / or CD28 hi For example, CD10 + CD19 + CD20 + CD24 hi , and CD28 hi It could be a B cell. B cells are CD10 + CD19 + CD20 + CD24 hi CD28 hi , BCL-2 lo , and / or CD27 - For example, CD10 + CD19 + CD20 + CD24 hi CD28 hi , BCL-2 lo , and CD27 - These could be B cells. This expression profile may be characteristic of transitional B cells. B cells are CD19 + CD20 + CD23 + CD40 + , and / or CD150 + For example, CD19 + CD20 + CD23 + CD40 + , and CD150 + It could be a B cell. B cells are CD19+ CD20 + CD23 + CD40 + CD150 + IgM + , and / or IgD + For example, CD19 + CD20 + CD23 + CD40 + CD150 + IgM + , and IgD + It could be a B cell. B cells are CD19 + CD20 + CD23 + CD40 + CD150 + IgM + IgD + , and / or CD38 lo For example, CD19 + CD20 + CD23 + CD40 + CD150 + IgM + IgD + , and CD38 lo These could be B cells. This expression profile may be characteristic of naive B cells. B cells are CD19 + CD20 + CD27 + , and / or IgM + For example, CD19 + CD20 + CD27 + , and IgM + It could be a B cell. B cells are CD19 + CD20 + CD27 + IgM + , and / or IgD lo For example, CD19 + CD20 + CD27 + IgM + , and IgD loThese could be B cells. This expression profile may be characteristic of B1 cells. B cells are CD19 + CD20 + CD27 + CD40 + , and / or CD150 - For example, CD19 + CD20 + CD27 + CD40 + , and CD150 - It could be a B cell. B cells are CD19 + CD20 + CD27 + CD40 + CD150 - IgA + , and / or IgG + For example, CD19 + CD20 + CD27 + CD40 + CD150 - IgA + , and IgG + It could be a B cell. B cells are CD19 + CD20 + CD27 + CD40 + CD150 - IgA + IgG + CD23 lo , and / or CD38 - For example, CD19 + CD20 + CD27 + CD40 + CD150 - IgA + IgG + CD23 lo , and CD38 - These could be B cells. This expression profile may be characteristic of memory B cells. B cells are CD9 hi CD27 hi CD38 hi CD40 + , and / or CD95 +For example, CD9 hi CD27 hi CD38 hi CD40 + , and CD95 + It could be a B cell. B cells are CD9 hi CD27 hi CD38 hi CD40 + CD95 + CXCR4 + , and / or CD138 + For example, CD9 hi CD27 hi CD38 hi CD40 + CD95 + CXCR4 + , and CD138 + It could be a B cell. B cells are CD9 hi CD27 hi CD38 hi CD40 + CD95 + CXCR4 + CD138 + CD19 lo , and / or CD20 - For example, CD9 hi CD27 hi CD38 hi CD40 + CD95 + CXCR4 + CD138 + CD19 lo , and CD20 - This could be a B cell. This expression profile may be characteristic of plasma cells.

[0377] The inventors have surprisingly discovered that the population of granulocyte-producing cells described herein can amplify (preferably amplify) the therapeutic immune response of blood-derived myeloid cells. For example, the inventors have shown that the granulocyte-producing cells described herein increase the viability and / or proliferation of blood-derived myeloid cells, thereby overcoming the problem of ex vivo expansion of blood-derived myeloid cells. Therefore, the composition may include a population of granulocyte-producing cells and blood-derived myeloid cells. The blood-derived myeloid cells can be any suitable blood-derived myeloid cells. The blood-derived myeloid cells are CD11b + CD15 + , and / or CD14 + For example, CD11b + CD15 + , and CD14 + These may be blood-derived bone marrow cells. Preferably, the blood-derived bone marrow cells are CD11 + These are blood-derived myeloid cells. Populations of granulocyte-producing cells suitable for use according to the present invention may increase the activation of blood-derived myeloid cells. Therefore, the composition may contain blood-derived myeloid cells with increased activation. Increased activation of blood-derived myeloid cells may be associated with increased CD11b expression.

[0378] The activation of blood-derived myeloid cells may increase by at least 5%. For example, the activation of blood-derived myeloid cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The quantification of the increase in macrophage activation by such embodiments can be achieved by comparison with a suitable control.

[0379] Nongranulocyte immune cells may be stem cells, precursor cells, or progenitor cells, such as dendritic cells, monocytes, macrophages, natural killer (NK) cells, B cells, or T cells, such as γδT cell stem cells, precursor cells, or progenitor cells. Nongranulocyte immune cells may be stem cells, precursor cells, or progenitor cells of any nongranulocyte immune cell described herein. Preferably, nongranulocyte immune cells are terminally differentiated immune cells.

[0380] Nongranulocyte immune cells can be obtained from any suitable source. For example, nongranulocyte immune cells can be obtained from umbilical cord blood samples, which can be obtained from donors (e.g., can be obtained). For example, nongranulocyte immune cells can be obtained from PBMC samples, which can be obtained from donors (e.g., can be obtained). Preferably, nongranulocyte immune cells can be obtained from αβT cell-depleted PBMC samples. Nongranulocyte immune cells can be obtained from αβT cell-depleted PBMC samples that can be obtained from G-CSF-mobilized blood. Nongranulocyte immune cells can be obtained from hematopoietic stem cells or stem cells such as iPSCs (e.g., they can then be differentiated in vitro).

[0381] While not bound by theory, it is believed that compositions containing multiple different types of nongranulocyte immune cells, when combined with the population of granulocyte-producing cells of the present invention, may have a synergistically amplified therapeutic immune response. Therefore, the composition may contain a population of granulocyte-producing cells and multiple different types of nongranulocyte immune cells. For example, the composition may contain at least two, at least three, at least four, at least five, or at least six different types of nongranulocyte immune cells.

[0382] This composition may contain several different types of nongranulocyte immune cells selected from dendritic cells, monocytes, macrophages, natural killer (NK) cells, B cells, and T cells (e.g., γδT cells). This composition may contain several different types of nongranulocyte immune cells selected from dendritic cells, monocytes, macrophages, natural killer (NK) cells, B cells, and γδT cells. This composition may contain several different types of nongranulocyte immune cells selected from monocytes, macrophages, NK cells, and γδT cells. Therefore, this composition may contain dendritic cells, monocytes, macrophages, NK cells, B cells, and T cells (e.g., γδT cells). This composition may contain dendritic cells, monocytes, macrophages, NK cells, B cells, and γδT cells. Preferably, this composition contains monocytes, macrophages, NK cells, and γδT cells. Particularly preferably, this composition contains NK cells and γδT cells. Particularly preferably, this composition contains NK cells and Vδ1 + γδT cells and / or Vδ2 + γδT cells, for example, NK cells, Vδ1 + γδT cells and Vδ2 + Includes γδT cells.

[0383] Multiple different types of nongranulocyte immune cells may be obtained from any suitable source. For example, multiple different types of nongranulocyte immune cells may be obtained from the same donor or different donors. Preferably, multiple different types of nongranulocyte immune cells may be obtained from the same donor. Multiple different types of nongranulocyte immune cells may be obtained from a single source or different sources. Preferably, multiple different types of nongranulocyte immune cells may be obtained from a single source. For example, multiple different types of nongranulocyte immune cells may be obtained from a sample of PBMCs. Preferably, multiple different types of nongranulocyte immune cells can be obtained from a sample of αβT cell-depleted PBMCs. Multiple different types of nongranulocyte immune cells may be obtained from iPSCs or a population of iPSCs.

[0384] Populations of granulocyte-producing cells and non-granulocyte immune cells may be obtained from the same donor or different donors. Preferably, populations of granulocyte-producing cells and non-granulocyte immune cells may be obtained from the same donor. For example, populations of granulocyte-producing cells and non-granulocyte immune cells may be obtained from a healthy donor. Preferably, populations of granulocyte-producing cells and non-granulocyte immune cells may be obtained from a donor that does not have cancer. Obtaining cells from a single donor may be particularly advantageous because it allows for the extraction of the entire innate immune component of that donor. Therefore, donors having particularly beneficial innate immune cells (e.g., innate immune cells that are highly cytotoxic to disease stimuli such as cancer cells, or innate immune cells that are particularly good at mobilizing other immune cells to diseased tissue) may be selected, and those innate immune cells may be included in the composition of the present invention. The cells in these compositions are expected to have synergistically improved properties (e.g., synergistically improved cytotoxicity and / or synergistically improved recruitment) compared to compositions containing only a single cell type, which at least partially arises from the synergistic effects between the different cell types present in the composition. In particular, as shown herein, the granulocyte-generating cells of the present invention have shown a particularly remarkable tendency to synergistically improve the activation, cytotoxicity, and / or recruitment of nongranulocyte immune cells.

[0385] Populations of granulocyte-producing cells and non-granulocyte immune cells may be obtained from the same or different sources. For example, populations of granulocyte-producing cells may be obtained from isolated hematopoietic stem cell samples, and non-granulocyte immune cells may be obtained from isolated PBMC samples. Preferably, populations of granulocyte-producing cells and non-granulocyte immune cells may be obtained from the same source. For example, populations of granulocyte-producing cells and non-granulocyte immune cells may be obtained from iPSCs or populations of iPSCs. Preferably, populations of granulocyte-producing cells and non-granulocyte immune cells may be obtained from isolated PBMCs, for example, samples of PBMCs derived from recruited blood. Particularly preferably, populations of granulocyte-producing cells and non-granulocyte immune cells may be obtained from αβT cell-depleted PBMCs, for example, samples of αβT cell-depleted PBMCs derived from recruited blood. Therefore, in a preferred embodiment, the population of granulocyte-producing cells and non-granulocyte immune cells can be obtained from a sample of alpha-beta T cell-depleted PBMCs derived from recruited blood, which can be obtained from a single donor. Advantageously, this makes it possible to prepare the composition using cells from a single source, thereby providing an extremely rational and efficient method for preparing the composition of the present invention.

[0386] As used herein, the term “mobilized blood” refers to blood circulating in a body treated with a mobilizing agent such as plerixafor and / or G-CSF. The term “mobilizing agent” refers to CD34 from the bone marrow into the bloodstream. + This refers to drugs that help mobilize hematopoietic stem cells and / or progenitor cells. Therefore, mobilized blood has a higher CD34 count compared to unmobilized blood. + The concentration of hematopoietic stem cells and / or precursor cells is high. Mobilized blood can be collected via leukocyte removal, allowing for the collection of PBMCs containing nongranulocyte immune cells as well as hematopoietic stem cells and / or precursor cells.

[0387] The inventors have discovered that granulocyte-producing cells can amplify (preferably amplify) the therapeutic immune response of non-granulocyte immune cells when they are present in different ratios. Therefore, populations of granulocyte-producing cells and non-granulocyte immune cells may be present in any appropriate ratio in the composition. Populations of granulocyte-producing cells and non-granulocyte immune cells may be present in a ratio of granulocyte-producing cells to non-granulocyte immune cells from 100:1 to 0.01:1. Populations of granulocyte-producing cells and non-granulocyte immune cells may be present in a ratio of granulocyte-producing cells to non-granulocyte immune cells from 100:1 to 0.01:1, 75:1 to 0.05:1, 50:1 to 0.1:1, 25:1 to 0.2:1, 10:1 to 0.25:1, 5:1 to 0.25:1, 3:1 to 0.25:1, or 2:1 to 0.5:1. Preferably, the population of granulocyte-producing cells and non-granulocyte cells are present in a ratio of 3:1 to 0.25:1 between granulocyte-producing cells and non-granulocyte immune cells.

[0388] The populations of granulocyte-producing cells and non-granulocyte immune cells may be present in a ratio of 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.1:1, 0.05:1, or 0.01:1 or less of granulocyte-producing cells to non-granulocyte immune cells. The populations of granulocyte-producing cells and non-granulocyte immune cells may be present in a ratio of at least 0.01:1, 0.05:1, 0.1:1, 0.25:1, 0.5:1, 1:1, 2:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1 of granulocyte-producing cells to non-granulocyte immune cells. The population of granulocyte-producing cells and non-granulocyte immune cells may exist in a ratio of 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.1:1, 0.05:1, or 0.01:1. Preferably, the population of granulocyte-producing cells and non-granulocyte immune cells may exist in a ratio of 2:1, 1:1, or 0.5:1. For example, the population of granulocyte-producing cells and non-granulocyte immune cells may exist in a ratio of 2:1. The population of granulocyte-producing cells and non-granulocyte immune cells may also exist in a ratio of 1:1. The population of granulocyte-producing cells and non-granulocyte immune cells may be present in a ratio of 0.5:1 between granulocyte-producing cells and non-granulocyte immune cells.

[0389] The compositions of the present invention may be suitable for homogeneous administration. Therefore, the compositions may be substantially or completely free of any components that cause graft-versus-host disease, such as αβT cells. Thus, in one embodiment, the composition is αβT cell-free. The term "αβT cell-free" means that the composition is free of or substantially free of αβT cells. As used in this context, the term "substantially absent" may also mean that less than 10% of the cells in the composition may be αβT cells, less than 5% of the cells in the composition may be αβT cells, less than 4% of the cells in the composition may be αβT cells, less than 3% of the cells in the composition may be αβT cells, less than 2% of the cells in the composition may be αβT cells, less than 1% of the cells in the composition may be αβT cells, less than 0.1% of the cells in the composition may be αβT cells, less than 0.01% of the cells in the composition may be αβT cells, or less than 0.0001% of the cells in the composition may be αβT cells. The term "substantially absent" as used in this context means that the composition is at most about 1 × 10 9 αβ T cells / kg of recipient, maximum approximately 1 × 10⁶ 8 αβ T cells / kg of recipient, maximum approximately 1 × 10⁶ 7 αβ T cells / kg of recipient, maximum approximately 1 × 10⁶ 6 αβ T cells / kg of the subject to be treated, preferably up to approximately 1 × 10⁶ 5 This may mean that the composition contains approximately 1 × 10 kg of αβ T cells / subjects being treated. The term "substantially none" as used in this context means that the composition contains approximately 1 × 10 1 ~1 × 10 9 αβ T cells / kg of treated subjects, approximately 1 × 10 2 ~1 × 10 8 αβ T cells / kg of treated subjects, approximately 1 × 10 3 ~1 × 10 7 αβ T cells / kg of treated subjects, approximately 1 × 10 4 ~1 × 10 6 αβ T cells / kg of the subject to be treated, preferably about 1 × 10⁻⁶ 4 ~1 × 10 5This may mean that the composition contains αβ T cells / kg of the subject being treated. The term "substantially none" as used in this context means that the composition contains up to approximately 7 × 10 10 αβ T cells, up to approximately 7 × 10⁶ 9 αβ T cells, up to approximately 7 × 10⁶ 8 αβ T cells, up to approximately 7 × 10⁶ 7 αβ T cells, preferably up to approximately 7 × 10⁶ 6 This may mean that the composition contains αβ T cells. The term "substantially absent" as used in this context means that the composition contains approximately 7 × 10 2 ~7×10 10 αβ T cells / kg of treated subjects, approximately 7 x 10 3 ~7×10 9 αβ T cells / kg of treated subjects, approximately 7 x 10 4 ~7×10 8 αβ T cells / kg of treated subjects, approximately 7 x 10 5 ~7×10 7 αβ T cells / kg of the subject to be treated, preferably about 7 × 10 5 ~7×10 6 This may mean that the composition contains αβT cells. Particularly preferably, the composition does not contain αβT cells.

[0390] As used herein, “subject” or “patient” may be a mammal, such as a human or other mammal. Preferably, “subject” means a human subject. Preferably, “patient” means a human patient.

[0391] In one embodiment, a composition is provided comprising one or more granulocytes differentiated from a population of granulocyte-producing cells that can (preferably amplify) the therapeutic immune response of nongranulocyte cells, and nongranulocyte cells.

[0392] In various embodiments, the composition may be a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier, excipient, auxiliary agent and / or salt.

[0393] In various embodiments, the composition (e.g., a pharmaceutical composition) can be obtained by the method of preparing the composition disclosed herein.

[0394] In one embodiment, a method for preparing the composition of the present invention is provided, comprising culturing non-granulocyte immune cells in the presence of a population of granulocyte-producing cells of the present invention. Preferably, the population of granulocyte-producing cells can amplify (preferably amplify) the therapeutic immune response of the non-granulocyte immune cells.

[0395] As described above, the granulocyte-producing cells of the present invention can, surprisingly, amplify (preferably amplify) the therapeutic immune response of different types of non-granulocyte immune cells. Therefore, the method may include culturing NK cells in the presence of a population of granulocyte-producing cells to form the composition. The method may also include culturing T cells (e.g., γδT cells) in the presence of a population of granulocyte-producing cells to form the composition. Preferably, the method includes culturing NK cells and T cells (e.g., γδT cells) in the presence of a population of granulocyte-producing cells to form the composition. Particularly preferably, the method includes culturing NK cells and γδT cells (e.g., Vδ1 + γδT cells or Vδ2 + The method includes culturing γδT cells to form the composition. Populations of nongranulocyte immune cells and granulocyte-producing cells may be cultured in the absence of αβT cells.

[0396] The inventors have shown that, surprisingly, certain cytokines can synergistically amplify the therapeutic immune response of nongranulocyte immune cells. In particular, the inventors hypothesize that cytokines that signal via a common gamma chain, or via interleukin-2 receptor subunit gamma (IL-2RG), may be particularly useful in amplifying the therapeutic immune response of nongranulocyte immune cells cultured in the presence of a population of granulocyte-producing cells. Such cytokines include IL-15, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Therefore, the method may include culturing a population of granulocyte-producing cells and nongranulocyte immune cells in the presence of cytokines that signal via IL-2RG. The method may also include culturing a population of granulocyte-producing cells and nongranulocyte immune cells in the presence of one or more cytokines selected from IL-15, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Preferably, this method involves culturing a population of granulocyte-producing cells and non-granulocyte immune cells in the presence of IL-15.

[0397] A population of granulocyte-producing cells and non-granulocyte-producing cells may be cultured together in any appropriate ratio. A population of granulocyte-producing cells and non-granulocyte-producing immune cells may be cultured together in a granulocyte-to-non-granulocyte-to-immune cell ratio of 100:1 to 0.01:1. A population of granulocyte-producing cells and non-granulocyte-producing immune cells may be cultured together in a granulocyte-to-non-granulocyte-to-immune cell ratio of 100:1 to 0.01:1, 75:1 to 0.05:1, 50:1 to 0.1:1, 25:1 to 0.2:1, 10:1 to 0.25:1, 5:1 to 0.25:1, 3:1 to 0.25:1, or 2:1 to 0.5:1. Preferably, a population of granulocyte-producing cells and non-granulocyte-producing cells may be cultured together in a granulocyte-to-non-granulocyte-to-immune cell ratio of 3:1 to 0.25:1.

[0398] A population of granulocyte-producing cells and non-granulocyte immune cells may be cultured together in a ratio of 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.1:1, 0.05:1, or 0.01:1 or less of granulocyte-producing cells to non-granulocyte immune cells. Granulocyte-producing cells and non-granulocyte immune cells may be cultured together in a ratio of at least 0.01:1, 0.05:1, 0.1:1, 0.25:1, 0.5:1, 1:1, 2:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1 of granulocyte-producing cells to non-granulocyte immune cells. A population of granulocyte-producing cells and non-granulocyte immune cells may be cultured together in a ratio of 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.1:1, 0.05:1, or 0.01:1. Preferably, the population of granulocyte-producing cells and non-granulocyte immune cells are cultured together in a ratio of 2:1, 1:1, or 0.5:1. For example, a population of granulocyte-producing cells and non-granulocyte immune cells may be cultured together in a ratio of 2:1. A population of granulocyte-producing cells and non-granulocyte immune cells may be cultured together in a ratio of 1:1. A population of granulocyte-producing cells and non-granulocyte immune cells may be cultured together in a ratio of 0.5:1 between granulocyte-producing cells and non-granulocyte immune cells.

[0399] A suitable source for non-granulocyte immune cells may be PBMCs. Therefore, this method may include culturing PBMCs in the presence of granulocyte-producing cells. This method (a) Isolating PBMCs from samples obtainable from donors, (b) Culturing the PBMC in the presence of granulocyte-producing cells to form the composition. It may include.

[0400] To improve the suitability of compositions for allogeneic administration, components that may cause graft-versus-host disease may be removed from the composition. This method may include removing components that cause graft-versus-host disease from the composition. For example, this method may include a step of depleting αβT cells from the composition. Therefore, this method may include culturing PBMCs in the presence of granulocyte-producing cells and depleting αβT cells from the PBMCs. Preferably, this method may include culturing αβT cell-depleted PBMCs in the presence of granulocyte-producing cells.

[0401] This method, (a) Isolating PBMCs from samples obtainable from donors, (b) Depleting αβT cells from isolated PBMCs, (c) Culturing αβT cell-depleted PBMCs in the presence of granulocyte-producing cells, thereby forming the composition. It may include.

[0402] αβT cells can be depleted from PBMCs using any appropriate means. For example, the step of depleting αβT cells from PBMCs is: (a) Incubating PBMCs in the presence of biotin-conjugated anti-TCR αβ antibody and anti-biotin macrobeads so that αβ T cells present in the PBMCs bind to the biotin-conjugated anti-TCR αβ antibody, (b) For example, separating antibody-bound αβT cells from PBMCs using magnetically activated cell sorting (MACS) It may include.

[0403] The step of depleting αβT cells from isolated PBMCs is, (a) Incubate PBMCs at room temperature for 15 minutes in the presence of biotin-conjugated anti-TCR αβ antibody (Clone BW242 / 412, 1:50 dilution) so that αβ T cells present in the PBMCs bind to the biotin-conjugated anti-TCR αβ antibody, (b) Wash the antibody-bound cells in MACS buffer, (c) Centrifuge antibody-bound cells at 300 × g for 5 minutes, (d) Centrifuged cells are placed in antibiotin microbeads (20 μl / 1 × 10 7 MACS buffer containing cells (80 μl / 1 × 10⁻⁶) 7 Resuspending in cells, (e) Incubate the resuspended cells at 4°C for 15 minutes so that the antibody-bound cells bind to the antibiotin microbeads. (f) Wash the microbead-bound cells in MACS buffer and centrifuge the microbead-bound cells at 300 × g for 5 minutes. (g) Max. 1.25×10 8 The microbead-conjugated cells were resuspended in 500 μl of MACS buffer, and then added to an LD column placed within the magnetic field of a MACS MultiStand (Miltenyi Biotec). It may include, Here, unlabeled cells (αβTCR - The cells pass through the column and are collected.

[0404] As used herein, terms such as “αβT cell depleted PBMC” refer to a population of PBMCs that are substantially or completely devoid of αβT cells. Therefore, in one embodiment, the term “αβT cell depleted PBMC” means a sample of PBMCs that do not contain αβT cells. The term “sample of αβT cell-free PBMCs” in this context means that the PBMCs do not contain or substantially contain αβT cells. As used in this context, “substantially depleted” may refer to a sample of PBMCs in which less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of the PBMCs are αβT cells. As used in this context, “substantially depleted” means that the PBMCs are at most approximately 1 × 10⁻⁶ 9 αβ T cells / kg of recipient, maximum approximately 1 × 10⁶ 8 αβ T cells / kg of recipient, maximum approximately 1 × 10⁶7 αβ T cells / kg of recipient, maximum approximately 1 × 10⁶ 6 αβ T cells / kg of the subject to be treated, preferably up to approximately 1 × 10⁶ 5 This may mean that the αβ T cells / kg of the target being treated contain approximately 1 × 10⁻⁶ αβ T cells. The term "substantially none" as used in this context means that the PBMCs contain approximately 1 × 10⁻⁶ αβ T cells / kg of the target being treated. 1 ~1 × 10 9 αβ T cells / kg of treated subjects, approximately 1 × 10 2 ~1 × 10 8 αβ T cells / kg of treated subjects, approximately 1 × 10 3 ~1 × 10 7 αβ T cells / kg of treated subjects, approximately 1 × 10 4 ~1 × 10 6 αβ T cells / kg of the subject to be treated, preferably about 1 × 10⁻⁶ 4 ~1 × 10 5 This may mean that the αβ T cells / kg of the target being treated include. The term "substantially none" as used in this context means that the PBMCs are up to approximately 7 × 10 10 αβ T cells, up to approximately 7 × 10⁶ 9 αβ T cells, up to approximately 7 × 10⁶ 8 αβ T cells, up to approximately 7 × 10⁶ 7 αβ T cells, preferably up to approximately 7 × 10⁶ 6 This may mean that the PBMC contains αβT cells. The term "substantially absent" as used in this context means that the PBMC contains approximately 7 × 10⁶ cells. 2 ~7×10 10 αβ T cells / kg of treated subjects, approximately 7 x 10 3 ~7×10 9 αβ T cells / kg of treated subjects, approximately 7 x 10 4 ~7×10 8 αβ T cells / kg of treated subjects, approximately 7 x 10 5 ~7×10 7 αβ T cells / kg of the subject to be treated, preferably about 7 × 10 5 ~7×10 6 This may mean that the αβT cells are present. Particularly preferably, the PBMC does not contain αβT cells.

[0405] αβT cells may be depleted at any appropriate time, for example, before administration to the subject. For example, PBMCs may be cultured in the presence of granulocyte-producing cells before or after the step of depleting αβT cells from isolated PBMCs.

[0406] Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) may be cultured together in any appropriate ratio. Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) may be cultured together in a ratio of granulocyte-to-PBMC ratio from 100:1 to 0.01:1. A population of granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) may be cultured together in a ratio of granulocyte-to-PBMC or αβT cell-depleted PBMCs of 100:1 to 0.01:1, 75:1 to 0.05:1, 50:1 to 0.1:1, 25:1 to 0.2:1, 10:1 to 0.25:1, 5:1 to 0.25:1, 3:1 to 0.25:1, or 2:1 to 0.5:1. Preferably, granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) are cultured together in a ratio of granulocyte-to-PBMC or αβT cell-depleted PBMCs of 3:1 to 0.25:1.

[0407] Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) may be cultured together in ratios of 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.1:1, 0.05:1, or 0.01:1 or less of granulocyte-producing cells to PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs). Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs may be cultured together in a ratio of at least 0.01:1, 0.05:1, 0.1:1, 0.25:1, 0.5:1, 1:1, 2:1, 3:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1 for granulocyte-producing cells to PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs). Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) may be cultured together in a ratio of 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.1:1, 0.05:1, or 0.01:1. Preferably, granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) are cultured together in a ratio of 2:1, 1:1, or 0.5:1. For example, granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) may be cultured together in a ratio of 2:1 granulocyte-producing cells to PBMCs or αβT cell-depleted PBMCs. Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) may be cultured together in a ratio of 1:1 granulocyte-producing cells to PBMCs or αβT cell-depleted PBMCs. Granulocyte-producing cells and PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) may be cultured together in a ratio of 0.5:1 granulocyte-producing cells to PBMCs or αβT cell-depleted PBMCs.

[0408] PBMCs and αβT cell-depleted PBMCs may contain granulocyte-producing cells. Although not bound by theory, it is thought that PBMCs obtainable from recruited blood, such as G-CSF recruited blood, contain a higher concentration of hematopoietic cells capable of differentiating into granulocyte-producing cells (preferably, they differentiate). Preferably, PBMCs or αβT cell-depleted PBMCs can be obtained from recruited blood, such as G-CSF recruited blood. This method may include obtaining PBMCs or αβT cell-depleted PBMCs from recruited blood, such as G-CSF recruited blood.

[0409] This method may include increasing the number of granulocyte-producing cells present in PBMCs or αβT cell-depleted PBMCs. This method may also include increasing the concentration of granulocyte-producing cells present in PBMCs or αβT cell-depleted PBMCs. The number or concentration of granulocyte-producing cells present in PBMCs or αβT cell-depleted PBMCs may be increased by any appropriate means. Therefore, the step of culturing PBMCs or αβT cell-depleted PBMCs in the presence of granulocyte-producing cells may include culturing PBMCs or αβT cell-depleted PBMCs under conditions suitable for the expansion and / or differentiation of hematopoietic cells present in PBMCs or αβT cell-depleted PBMCs.

[0410] Particularly preferred, a method for preparing the composition of the present invention comprises culturing αβT cell-depleted PBMCs (e.g., which can be obtained from recruited blood, e.g., a sample of G-CSF recruited blood) under conditions that promote the differentiation of progenitor cells present in αβT cell-depleted PBMCs into granulocyte-producing cells, thereby forming the composition. Advantageously, this makes it possible to prepare the composition using cells from a single source, thereby providing an extremely rational and efficient method for preparing the composition of the present invention.

[0411] This method may also include culturing αβT cell-depleted PBMCs (which can be obtained, for example, from recruited blood, for example, from G-CSF recruited blood samples) under conditions that generate progenitor cells from stem cells present in αβT cell-depleted PBMCs.

[0412] This method may optionally include depleting αβT cells from PBMCs (for example, which can be obtained from mobilized blood samples, such as G-CSF mobilized blood samples).

[0413] Therefore, this method, (a) Depleting αβT cells from PBMCs (for example, which can be obtained from mobilized blood samples, for example, G-CSF mobilized blood samples), (b) Culture the αβT cell-depleted PBMC under conditions that promote the differentiation of progenitor cells present in the αβT cell-depleted PBMC into granulocyte-producing cells, thereby forming the composition. It may include.

[0414] This method, (a) Depleting αβT cells from PBMCs (for example, which can be obtained from mobilized blood samples, for example, G-CSF mobilized blood samples), (b) Culturing αβT cell-depleted PBMCs under conditions that generate progenitor cells from stem cells present in αβT cell-depleted PBMCs, (c) Culture the progenitor cells present in αβT cell-depleted PBMCs under conditions that promote the differentiation of these progenitor cells into granulocyte-producing cells, thereby forming the composition. It may include.

[0415] The conditions that promote the differentiation of progenitor cells present in PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) into granulocyte-producing cells may be any suitable conditions. For example, the conditions that promote the differentiation of progenitor cells present in PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) into granulocyte-producing cells may be the conditions described herein that are suitable for obtaining a population of granulocyte-producing cells.

[0416] Similarly, the conditions for generating progenitor cells from stem cells present in PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) may be any suitable conditions. For example, the conditions for generating progenitor cells from stem cells present in PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) may be the conditions described herein used in a method for obtaining a population of granulocyte-producing cells, which includes the step of culturing stem cells under culture conditions that generate progenitor cells.

[0417] This method may further include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) under conditions suitable for maintaining NK cells. This method may further include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) under conditions suitable for maintaining γδT cells. Preferably, this method includes culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) under conditions suitable for maintaining NK cells and γδT cells.

[0418] Therefore, this method, (a) Culturing αβT cell-depleted PBMCs under conditions that promote the differentiation of progenitor cells present in αβT cell-depleted PBMCs into granulocyte-producing cells, (b) Culture the αβT cell-depleted PBMC under conditions suitable for maintaining NK cells and γδT cells present in the αβT cell-depleted PBMC, thereby forming the composition. It may include.

[0419] This method, (a) Culturing αβT cell-depleted PBMCs under conditions suitable for generating progenitor cells from stem cells present in αβT cell-depleted PBMCs, and under conditions suitable for maintaining NK cells and γδT cells present in αβT cell-depleted PBMCs, (b) Culture the progenitor cells present in αβT cell-depleted PBMCs under conditions that promote the differentiation of these progenitor cells into granulocyte-producing cells, thereby forming the composition. It may include.

[0420] Conditions suitable for maintaining NK cells and γδT cells may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21. Conditions suitable for maintaining NK cells and γδT cells may also include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 at concentrations of 1-20 ng / mL, 5-15 ng / mL, or 7.5-12.5 ng / mL. Conditions suitable for maintaining NK cells and γδT cells may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21, present at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL. Preferably, one or more cytokines are present at a concentration of 10 ng / mL during the maintenance phase. Therefore, this method may also include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21, present at a concentration of 10 ng / mL. This method may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for an appropriate amount of time in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 10 ng / mL). For example, this method may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 10 ng / mL).This method may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 10 ng / mL). This method may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 10 ng / mL). This method may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for 1 to 10 days, 2 to 10 days, 3 to 10 days, 4 to 10 days, 5 to 9 days, 6 to 9 days, or 7 to 9 days in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 10 ng / mL). Preferably, this method includes culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for 7 to 9 days in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 10 ng / mL).

[0421] This method may further include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) under conditions suitable for NK cell activation. This method may further include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) under conditions suitable for γδT cell activation. Preferably, this method includes culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) under conditions suitable for NK cell and γδT cell activation.

[0422] Therefore, this method, (a) Culturing αβT cell-depleted PBMCs under conditions that promote the differentiation of progenitor cells present in αβT cell-depleted PBMCs into granulocyte-producing cells, (b) Culturing αβT cell-depleted PBMCs under conditions suitable for activating NK cells and γδT cells present in αβT cell-depleted PBMCs, thereby forming the composition. It may include.

[0423] Conditions suitable for the differentiation of hematopoietic cells present in αβT cell-depleted PBMCs may also be suitable for the activation of NK cells and γδT cells present in αβT cell-depleted PBMCs.

[0424] This method, (a) Culturing αβT cell-depleted PBMCs under conditions that generate progenitor cells from stem cells present in αβT cell-depleted PBMCs, and culturing αβT cell-depleted PBMCs under conditions suitable for maintaining NK cells and γδT cells present in αβT cell-depleted PBMCs, (b) Culture the progenitor cells present in the αβT cell-depleted PBMC under conditions that promote the differentiation of the progenitor cells present in the αβT cell-depleted PBMC into granulocyte-producing cells, and culture the αβT cell-depleted PBMC under conditions suitable for the activation of NK cells and γδT cells present in the αβT cell-depleted PBMC, thereby forming the composition. It may include.

[0425] Conditions suitable for activating NK cells and γδT cells may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21. Conditions suitable for activating NK cells and γδT cells may also include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 at concentrations of 20-200 ng / mL, 50-150 ng / mL, or 75-125 ng / mL. Conditions suitable for activating NK cells and γδT cells may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21, present at concentrations of 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL. Preferably, one or more cytokines are present at a concentration of 100 ng / mL during the activation phase. Therefore, this method may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 at a concentration of 100 ng / mL. This method may also include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for any appropriate time at the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 100 ng / mL). For example, this method may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for at least 1, 2, 3, 4, 5, or 6 days in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 100 ng / mL).This method may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for up to 1, 2, 3, 4, 5, or 6 days in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 100 ng / mL). This method may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for up to 1, 2, 3, 4, 5, or 6 days in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 100 ng / mL). This method may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for 1 to 6 days, 2 to 6 days, 3 to 6 days, or 4 to 6 days in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 100 ng / mL). Preferably, this method includes culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) for 4 to 6 days in the presence of one or more cytokines selected from IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21 (e.g., at a concentration of 100 ng / mL). Conditions suitable for the activation of NK cells and γδT cells may further include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of T cell receptor activators, such as OKT3 activators or T cell receptor antibodies such as anti-CD3 antibodies. Although not bound by theory, it is thought that culturing PBMCs or αβT cell-depleted PBMCs in the presence of T cell receptors can synergistically enhance the expansion and activation of γδT cells. Therefore, this method may include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of T cell receptor activators, such as OKT3 activators or T cell receptor antibodies such as anti-CD3 antibodies.

[0426] Preferably, the method includes culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of IL-15. The method may further include culturing PBMCs or αβT cell-depleted PBMCs (e.g., αβT cell-depleted PBMCs) in the presence of T cell receptor activators, such as OKT3 activators, or T cell receptor antibodies, such as anti-CD3 antibodies.

[0427] This method may include culturing non-granulocyte immune cells differentiated from iPSCs (e.g., iPSC-derived γδT cells and / or iPSC-derived NK cells) in the presence of a population of granulocyte-producing cells to form a composition. This method may include culturing non-granulocyte immune cells in the presence of a population of granulocyte-producing cells differentiated from iPSCs to form a composition. This method may include culturing non-granulocyte immune cells differentiated from iPSCs (e.g., iPSC-derived γδT cells and / or iPSC-derived NK cells) in the presence of a population of granulocyte-producing cells differentiated from iPSCs to form a composition. This method may include culturing iPSC-derived αβT cells, iPSC-derived γδT cells, iPSC-derived NK cells, or a combination thereof in the presence of iPSC-derived granulocyte-producing cells to form a composition. For example, this method may include culturing iPSC-derived NK cells in the presence of iPSC-derived granulocyte-producing cells to form a composition. This method may include culturing iPSC-derived γδT cells in the presence of iPSC-derived granulocyte-producing cells to form a composition. This method may also include culturing iPSC-derived γδT cells and iPSC-derived NK cells in the presence of iPSC-derived granulocyte-producing cells to form a composition.

[0428] This method may include differentiating iPSCs into a population of granulocyte-producing cells, for example, a population of granulocyte-producing cells as defined herein, and culturing non-granulocyte immune cells in the presence of iPSC-derived granulocyte-producing cells. This method may also include differentiating iPSCs into non-granulocyte immune cells, for example, γδT cells and / or NK cells, and culturing iPSC-derived non-granulocyte immune cells in the presence of a population of granulocyte-producing cells. This method may also include differentiating iPSCs into a population of granulocyte-producing cells, differentiating iPSCs into non-granulocyte immune cells, for example, γδT cells and / or NK cells, and culturing iPSC-derived non-granulocyte immune cells in the presence of iPSC-derived granulocyte-producing cells.

[0429] Therefore, this method, (a) Differentiation of iPSCs into iPSC-derived granulocyte-producing cells, (b) Differentiation of iPSCs into iPSC-derived γδT cells, (c) Differentiation of iPSCs into iPSC-derived NK cells, The composition is formed by co-culturing iPSC-derived granulocyte-producing cells, iPSC-derived γδT cells, and iPSC-derived NK cells. It may include.

[0430] iPSCs may be obtained from any suitable donor. For example, iPSCs may be obtained from a donor that produces granulocytes capable of killing cancer cells, as defined using the assay described herein.

[0431] iPSCs may be obtained from any suitable source. For example, iPSCs may be obtained from somatic cells, such as αβT cells or γδT cells. iPSCs may be obtained from stem cells. In embodiments of this method that include differentiating iPSCs into γδT cells, iPSCs may be obtained from γδT cells. In embodiments of this method that include differentiating iPSCs into αβT cells, iPSCs may be obtained from αβT cells.

[0432] Therefore, this method, (a) Differentiation of iPSCs into iPSC-derived granulocyte-producing cells, (b) Differentiating iPSCs obtained from γδT cells into iPSC-derived γδT cells, (c) Differentiation of iPSCs into iPSC-derived NK cells, The composition is formed by co-culturing iPSC-derived granulocyte-producing cells, iPSC-derived αβT cells, and iPSC-derived NK cells. It may include.

[0433] This method, (a) Differentiation of iPSCs obtained from γδT cells into iPSC-derived granulocyte-producing cells, (b) Differentiating iPSCs obtained from γδT cells into iPSC-derived γδT cells, (c) Differentiation of iPSCs obtained from γδT cells into iPSC-derived NK cells, The composition is formed by co-culturing iPSC-derived granulocyte-producing cells, iPSC-derived αβT cells, and iPSC-derived NK cells. It may include.

[0434] Cell culture additives may enhance the amplification of therapeutic immune responses. Therefore, populations of granulocyte-producing cells and non-granulocyte immune cells may be cultured in the presence of any suitable cell culture additive, such as growth factors, cytokines, or chemokines. For example, populations of granulocyte-producing cells and non-granulocyte immune cells may be cultured in the presence of 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, serum (e.g., fetal bovine serum [FBS]), retinoic acid, lipopolysaccharide (LPS), IFN-gamma, IFN-beta, or a combination thereof. Populations of granulocyte-producing cells and non-granulocyte immune cells may also be cultured in the presence of IFN-gamma and GM-CSF. Populations of granulocyte-producing cells and non-granulocyte immune cells may be cultured in the presence of TNF-alpha. Populations of granulocyte-producing cells and non-granulocyte immune cells may be cultured in the presence of 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, and fetal bovine serum (FBS). Populations of granulocyte-producing cells and non-granulocyte immune cells may be cultured in the presence of 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), retinoic acid, lipopolysaccharide (LPS), IFN-gamma, and IFN-beta. Populations of granulocyte-producing cells and non-granulocyte immune cells may be cultured in the presence of an anti-CD3 agonist, such as an anti-OKT3 antibody. Populations of granulocyte-producing cells and non-granulocyte immune cells may be cultured in the presence of an anti-OKT3 antibody.

[0435] Populations of granulocyte-producing cells and non-granulocyte immune cells that can be used in various embodiments of the present invention may be provided in the form of enriched populations of such granulocyte-producing cells and non-granulocyte immune cells. In one embodiment, the present invention provides a pharmaceutical composition comprising enriched populations of granulocyte-producing cells and non-granulocyte immune cells.

[0436] As a mere example, such an enriched population may be a population of cells in which granulocyte-producing cells and non-granulocyte immune cells constitute at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, or at least 1% of the total cell population. Such an enriched population may further be a population of cells in which granulocyte-producing cells constitute at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% of the total cell population. In fact, a concentrated population may be a population of cells in which granulocyte-producing cells constitute at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or substantially 100% of the total cell population present.

[0437] The granulocyte-producing cells and non-granulocyte immune cells of such enriched populations may be as defined in any preferred embodiments described elsewhere in this specification. For example, the granulocyte-producing cells of the enriched population may be CD62L - It is possible.

[0438] In one aspect of the present invention, a pharmaceutical composition is provided that includes an enriched population of granulocyte-producing cells and non-granulocyte immune cells. The enriched population of granulocyte-producing cells and non-granulocyte immune cells incorporated into the pharmaceutical composition of the present invention may be as considered above.

[0439] Preferably, the granulocyte-producing cells present in the pharmaceutical composition of the present invention are CD62L - This is possible. Preferably, the granulocyte-producing cells present in the pharmaceutical composition of the present invention are CD16 - This is possible. Preferably, the granulocyte-producing cells present in the pharmaceutical composition of the present invention are CD10 - This is possible. Preferably, the granulocyte-producing cells present in the pharmaceutical composition of the present invention are CD16 - CD10 - and CD62L - It is possible.

[0440] The pharmaceutical composition may be formulated in any conventional manner for its intended route of administration. For example, the pharmaceutical composition may be formulated for administration by injection or infusion.

[0441] Preferably, the composition of the present invention (e.g., a pharmaceutical composition) may contain granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-stimulating factor (G-CSF), growth hormone, serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, serum (e.g., fetal bovine serum [FBS]), retinoic acid, lipopolysaccharide (LPS), IFN-gamma, IFN-beta, or a combination thereof. Preferably, the composition (e.g., a pharmaceutical composition) contains IFN-gamma and GM-CSF. Preferably, the composition (e.g., a pharmaceutical composition) contains TNF-alpha. Particularly preferably, the composition (for example, a pharmaceutical composition) comprises 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, and fetal bovine serum (FBS). Preferably, the composition (for example, a pharmaceutical composition) contains 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), retinoic acid, lipopolysaccharide (LPS), IFN-gamma, and IFN-beta.

[0442] In the context of the present invention, a host therapeutic immune response should preferably be interpreted as an immune response that contributes to or achieves a desired therapeutic outcome. In a preferred embodiment, the host therapeutic immune response may be an immune response that leads (directly or indirectly) to the death of cancer cells, thereby enabling the treatment of cancer. In a preferred embodiment, the host therapeutic immune response may be an immune response that leads (directly or indirectly) to the death of infected cells or cellular infectious pathogens, thereby enabling the treatment of an infection.

[0443] A host therapeutic immune response may involve the action of any cell in the immune system. A “non-granulocyte immune response” may involve the action of any cell in the immune system other than granulocytes. A non-granulocyte immune response can be beneficially amplified by any suitable population of granulocyte-producing cells according to the present invention, or by any suitable pharmaceutical composition containing such a population of granulocyte-producing cells. A suitable composition may utilize a population of granulocyte-producing cells that does not contain non-granulocyte immune cells. Similarly, a composition containing a population of granulocyte-producing cells and / or non-granulocyte immune cells can amplify a host therapeutic immune response, for example, after administration to a subject. As merely an example, a host therapeutic immune response that can be amplified by a composition of the present invention (e.g., a pharmaceutical composition), medical use, or method of treatment may include T cells (CD8 + T cells, CD4 + This may involve the action of one or more cell types selected from the group including (or consisting of) T cells, NK cells, αβT cells, γδT cells, peripheral blood T cells, and tumor-infiltrating T cells (including, but not limited to, NK cells, monocytes, macrophages, dendritic cells (DCs), and B cells.

[0444] Amplification of an immune response (e.g., a host therapeutic immune response) may be demonstrated in vitro by one or more of the following: increased activation of immune cells involved in the immune response; increased expression of degranulation markers by immune cells involved in the immune response; increased expression of costimulatory molecules by immune cells involved in the immune response; increased proliferation by immune cells involved in the immune response; increased survival by immune cells involved in the immune response; increased abundance of immune cells involved in the immune response; increased expression of cytokines by immune cells involved in the immune response; increased transport by immune cells involved in the immune response; increased recruitment of immune cells involved in the immune response to TMEs; increased cytotoxicity by immune cells involved in the immune response; or increased tumor cell death activity by immune cells involved in the immune response.

[0445] Alternatively, or furthermore, amplification of the host therapeutic immune response may be evaluated in relation to the outcomes achieved by the therapeutic immune response.

[0446] For example, in the case of a host therapeutic immune response used in the treatment of cancer, amplification of the immune response can be demonstrated by an increase in the efficacy of the cancer treatment. Such an increase in efficacy can be demonstrated by symptom reduction, increased patient survival rate and / or survival time, reduced tumor burden, prevention or delay of recurrence, reduced severity of recurrence, reduced number of recurrences, reduced number of metastases, and / or prevention or delay of metastases.

[0447] In the case of host therapeutic immune responses used to treat infectious diseases, amplification of the immune response can be demonstrated by an increase in the efficacy of treating the infectious disease. Such an increase can be demonstrated by symptom reduction, increased patient survival rate and / or survival time, reduced infectious burden, and / or a shorter time to elimination of infection.

[0448] For the purposes of this disclosure, references to “host” cells (e.g., host immune cells) or “host” immune responses may be interpreted as referring to cells or immune responses of a subject that are being treated, or are thought to be being treated, with a population or composition of granulocyte-producing cells according to any of the various aspects of the Invention. Unless otherwise specified in the context, all references to immune cells or immune responses relating to the various aspects and embodiments of the Invention should be interpreted as applicable to host immune cells or host immune responses.

[0449] Populations or compositions of granulocyte-producing cells suitable for use in various embodiments of the present invention may be able to increase (preferably increase) the activation of host immune cells. Therefore, such cells may be able to amplify (preferably amplify) the host therapeutic immune response by increasing the activation of host immune cells. It will be understood that these are the activated immune cells primarily involved in providing the desired activity in the therapeutic immune response. Thus, the ability of medical uses and therapeutic methods to increase the activation of host immune cells would be beneficial in almost all situations where a therapeutic immune response is required. In particular, the amplification of the therapeutic immune response by increasing the activation of host immune cells may be advantageous in the treatment of cancer or infectious diseases, but not limited to these cases.

[0450] Populations of granulocyte-producing cells suitable for use according to the present invention may exhibit some or all of the above characteristics.

[0451] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or a composition of the present invention containing a population of granulocyte-producing cells [e.g., a pharmaceutical composition]) is sufficient to increase the activation of immune cells, such as host immune cells. The degree of increase, the host immune cells involved, and appropriate indicators of the increase in activation may be as considered in the preceding paragraph and / or in the following paragraph.

[0452] A population or composition of granulocyte-producing cells suitable for use according to the present invention may increase host T cell activation. Therefore, such a population or composition of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing host T cell activation.

[0453] CD8 + and CD4 + It will be understood that increased activation of host T cells, such as CD8 cells, significantly contributes to the desired activity in the therapeutic immune response. + While cytotoxic T cells such as T cells are known to have direct cell-destroying activity, CD4 + Helper T cells, such as T cells, are known to assist in regulating the immune response by further stimulating other immune cells. Therefore, the use of populations or compositions of granulocyte-producing cells to increase host T cell activation may be beneficial in a wide range of situations where a therapeutic immune response is required. In particular, the amplification of the host therapeutic immune response by increasing host T cell activation may be advantageous in the treatment of cancer or infectious diseases, though not limited to these applications.

[0454] Host T cells that may experience increased activation are CD8 + T cells, CD4 + The group may be selected from (or consist of) T cells, NK T cells, αβ T cells, γδ T cells, peripheral blood T cells, and tumor-infiltrating T cells.

[0455] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the activation of host T cells. The degree of increase, and appropriate indicators of the increase in activation, may be as considered in the preceding paragraph and / or in the following paragraph.

[0456] A population or composition of granulocyte-producing cells suitable for use according to the present invention is a host CD8 +It may increase T cell activation. Therefore, such a population or composition of granulocyte-producing cells may increase the host CD8 + In some cases, increasing T cell activation can amplify (preferably amplify) the host therapeutic immune response.

[0457] A population or composition of granulocyte-producing cells suitable for use according to the present invention is a host CD4 + Host CD4 cells such as T cells + It may increase T cell activation. Therefore, such a population or composition of granulocyte-producing cells may increase the host CD4 + In some cases, increasing T cell activation can amplify (preferably amplify) the host therapeutic immune response.

[0458] A population or composition of granulocyte-producing cells suitable for use according to the present invention may increase the activation of host NK T cells. Therefore, such a population or composition of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the activation of host NK T cells.

[0459] Increased NK T cell activation may be associated with one or more of the following: increased expression of degranulation markers by NK T cells (including, but not limited to, CD107a), increased expression of costimulatory molecules by NK T cells (including, but not limited to, 4-1BB and / or OX40), and increased NK T cell survival. Further relevant considerations regarding these various properties are described elsewhere in this specification.

[0460] The host NK T cells whose activation is increased may be peripheral blood NK T cells or tumor-infiltrating NK T cells.

[0461] Such NK T cell activation may increase by at least 5%. For example, NK T cell activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in NK T cell activation in such embodiments can be quantified by comparison with a suitable control.

[0462] Populations or compositions of granulocyte-producing cells suitable for use according to the present invention may increase the activation of host γδT cells. Therefore, such populations or compositions of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the activation of host γδT cells.

[0463] Populations or compositions of granulocyte-producing cells suitable for use according to the present invention may increase the activation of host NK cells. Therefore, such populations or compositions of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the activation of host NK cells.

[0464] Those skilled in the art will understand that NK cells play a crucial role in providing the activity necessary to achieve a therapeutic immune response. NK cells exhibit strong cytolytic activity against physiologically stressed cells, such as tumor cells and virus-infected cells. Therefore, the use of populations of granulocyte-producing cells to increase NK cell activation would be beneficial in a wide range of situations where a therapeutic immune response is required. In particular, the amplification of a therapeutic immune response by increasing NK cell activation may be advantageous in the treatment of cancer or infectious diseases, though not limited to these cases.

[0465] The host NK cells whose activation is increased may be peripheral blood NK cells or tumor-infiltrating NK cells.

[0466] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the activation of host NK cells. The degree of increase, and appropriate indicators of the increase in activation, may be as considered in the preceding paragraph and / or in the following paragraph.

[0467] Populations or compositions of granulocyte-producing cells suitable for use according to the present invention may increase the activation of host monocytes or macrophages. Therefore, such populations or compositions of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the activation of host monocytes or macrophages.

[0468] Populations or compositions of granulocyte-producing cells suitable for use according to the present invention may increase the activation of host PBMCs. Therefore, such populations or compositions of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the activation of host PBMCs.

[0469] It will be understood that PBMCs play a crucial role in providing cells that contribute to any effective therapeutic immune response. PBMCs can be interpreted as any peripheral blood cells with a single round nucleus, such as T cells and NK cells. These cells possess a variety of functions that are important in bringing about an immune response, including the cytotoxic activity or activation of further immune cells. Therefore, the use of a population or composition of granulocyte-producing cells to increase the activation of host PBMCs would be beneficial in almost any situation where an effective immune response is required. In particular, amplification of a therapeutic immune response by increasing the activation of host PBMCs may be advantageous in the treatment of cancer or infectious diseases, though not limited to these cases.

[0470] Host PBMCs whose activation is increased include peripheral blood T cells (e.g., peripheral blood CD8 cells). + T cells, peripheral blood CD4 + Examples include, but are not limited to, those selected from the group including (or consisting of) T cells, peripheral blood NK T cells, peripheral blood αβ T cells, or peripheral blood γδ T cells, and peripheral blood NK cells.

[0471] Increased activation of host PBMCs can be demonstrated by any suitable marker of activation. For example, increased PBMC activation can be demonstrated by increased expression of cytokines (e.g., IFN-γ, and / or TNF). The ability of host PBMCs exposed to a population of granulocyte-producing cells suitable for use according to the present invention to increase cytokine expression is shown in the examples. Further relevant considerations regarding these various properties are described elsewhere in this specification.

[0472] Host PBMC activation may increase by at least 5%. For example, PBMC activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in host PBMC activation in such embodiments can be quantified by comparison with a suitable control.

[0473] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the activation of host PBMCs. The degree of increase, and appropriate indicators of the increase in activation, may be as considered in the preceding paragraph and / or in the following paragraph.

[0474] Populations or compositions of granulocyte-producing cells suitable for use according to the present invention may increase the activation of host TILs. Therefore, such populations or compositions of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the activation of host TILs.

[0475] For the purposes of this invention, host TILs can be interpreted as encompassing all lymphocyte cell populations that have infiltrated tumor tissue. Considering this, it will be recognized that TILs play a crucial role in exerting a therapeutic immune response against tumor cells. TILs can exert specific cytotoxic antitumor activity (e.g., against CD8 cells that have entered the tumor). + (Cells), activation of other immune cells (e.g., CD4 cells in tumors) + Antitumor responses can be promoted by (cellular) activation. Therefore, amplifying the therapeutic immune response by increasing host TIL activation can play a very advantageous role in cancer treatment.

[0476] In particular, the inventors have determined that a population of granulocyte-producing cells suitable for use according to the present invention may increase the activation of tumor-infiltrating T cells and / or NK cells. Such granulocyte-producing cells are tumor-infiltrating CD8 cells, as shown in the examples. + T cells and / or CD4 + This may increase the activation of T cells.

[0477] Increased activation of host TILs, such as increased activation of tumor-infiltrating T cells or tumor-infiltrating NK cells, can be demonstrated by any suitable marker of activation. For example, increased TIL activation can be demonstrated by increased expression of degranulation markers (e.g., CD107a, perforin, or granzyme). Alternatively, increased TIL activation can be demonstrated by increased expression of costimulatory molecules (e.g., 4-1BB, OX40, CD27, CD28, ICOS, HVEM, LIGHT, CD40L, DR3, GITR, CD30, TIM1, CD2, or CD226). The ability of TILs exposed to a population of granulocyte-producing cells suitable for use according to the present invention to increase the expression of degranulation markers or costimulatory molecules is demonstrated in the examples. Further relevant considerations regarding these various properties are described elsewhere in this specification.

[0478] TIL activation may increase by at least 5%. For example, TIL activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in TIL activation in such embodiments can be quantified by comparison with a suitable control.

[0479] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the activation of host TILs. The degree of increase, and appropriate indicators of the increase in activation, may be as considered in the preceding paragraph and / or as considered in the following paragraph.

[0480] Populations of granulocyte-producing cells suitable for use according to the present invention may increase the expression of degranulation markers by immune cells. In particular, granulocyte-producing cells may increase (preferably increase) the expression of degranulation markers by non-granulocyte immune cells present in the composition of the present invention. Populations or compositions of granulocyte-producing cells may increase (preferably increase) the expression of degranulation markers by host immune cells. Therefore, such populations or compositions of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the expression of degranulation markers by host immune cells.

[0481] Degranulation supports therapeutic immune activity, CD8 + This is a crucial process in the cell-destructive activity of immune cells such as T cells or NK cells. Therefore, it would be understood that an increase in the expression of degranulation markers such as CD107 provides an indicator that the therapeutic immune activity of such cells has increased, and that the therapeutic immune response has been amplified accordingly.

[0482] In preferred embodiments, the degranulation markers whose expression is increased by host immune cells are selected from the group comprising (or consisting of) CD107a, perforin, and granzymes. Preferably, the expression of two or more of these degranulation markers may be increased. For example, the expression of at least two such degranulation markers may be increased. In particular, the expression of CD107a by host immune cells may be increased.

[0483] Increased expression of degranulation markers can be evaluated by any appropriate method and quantified as needed.

[0484] In a preferred embodiment, the expression of the degranulation marker increases by at least 5%. For example, the expression of the degranulation marker may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in the expression of the degranulation marker in such embodiments can be quantified by comparison with a suitable control.

[0485] The expression of degranulation markers may increase in nongranulocyte immune cells present in the composition of the present invention, or in host immune cells selected from the group including (or comprising) T cells and NK cells. If the expression of degranulation markers is increased in T cells, such T cells may be CD8 + The group may be selected from (or consist of) T cells, NK T cells, αβ T cells, and γδ T cells.

[0486] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the expression of one or more degranulation markers by immune cells, such as host immune cells. The degranulation markers, the degree of increase, and the host immune cells involved may be as considered in the preceding paragraph.

[0487] Populations of granulocyte-producing cells suitable for use according to the present invention may increase the expression of co-stimulatory molecules by immune cells. In particular, granulocyte-producing cells may increase (preferably increase) the expression of co-stimulatory molecules by non-granulocyte immune cells present in the composition of the present invention. Populations or compositions of granulocyte-producing cells may increase (preferably increase) the expression of co-stimulatory molecules by host immune cells. Therefore, such populations or compositions of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the expression of co-stimulatory molecules by host immune cells.

[0488] Co-stimulatory molecules act to amplify or suppress the activation signals provided to T cells that trigger T cell differentiation. T cell differentiation is a crucial process in therapeutic immune responses, leading to the generation of cytotoxic T cells or helper T cells. Therefore, increasing the expression of co-stimulatory molecules can amplify therapeutic immune responses by inducing functional differentiation of T cells. The use of granulocyte-producing cell populations to increase the expression of co-stimulatory molecules would be beneficial in a wide range of situations where a therapeutically effective immune response is required. In particular, the amplification of therapeutic immune responses by increasing the activation of co-stimulatory molecules may be advantageous in the treatment of cancer or infectious diseases, though not limited to these applications.

[0489] In preferred embodiments, the co-stimulatory molecules whose expression is increased by non-granulocyte immune cells and / or host immune cells are selected from the group including (or consisting of) 4-1BB, OX40, CD27, CD28, ICOS, HVEM, LIGHT, CD40L, DR3, GITR, CD30, TIM1, CD2, and CD226. Preferably, the expression of two or more of these co-stimulatory molecules may be increased. For example, the expression of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 such co-stimulatory molecules may be increased. In particular, the expression of both 4-1BB and OX40 by non-granulocyte immune cells and / or host immune cells may be increased.

[0490] The expression of co-stimulatory molecules can be evaluated by any appropriate method and quantified as needed.

[0491] In a preferred embodiment, the expression of the co-stimulatory molecule increases by at least 5%. For example, the expression of the co-stimulatory molecule may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in the expression of the co-stimulatory molecule in such embodiments can be quantified by comparison with a suitable control.

[0492] The expression of costimulatory molecules may increase in nongranulocyte immune cells and / or host immune cells selected from a group including (or consisting of) T cells and NK cells. If the expression of costimulatory molecules increases in T cells, such T cells may be CD8 + T cells, CD4 +The group may be selected from (or consist of) T cells, NK T cells, αβ T cells, γδ T cells, peripheral blood T cells, and tumor-infiltrating T cells.

[0493] Preferably, for use according to the present invention, the therapeutically effective amount of such granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the expression of one or more co-stimulatory molecules by immune cells, such as non-granulocyte immune cells or host immune cells. The co-stimulatory molecules, the degree of increase, and the host immune cells involved may be as considered in the preceding paragraph.

[0494] Populations of granulocyte-producing cells suitable for use according to the present invention may increase cytokine expression by immune cells. In particular, granulocyte-producing cells may increase (preferably increase) cytokine expression by non-granulocyte immune cells present in the composition of the present invention. Populations or compositions of granulocyte-producing cells may increase (preferably increase) cytokine expression by host immune cells. Therefore, such populations or compositions of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the expression of co-stimulatory molecules by host immune cells.

[0495] Cytokines are crucial chemical messengers in the immune response. They signal for cell activation (inducing immune cells), differentiation of immune cells such as T cells, and proliferation of immune cells such as NK cells. The use of granulocyte-producing cell populations to increase cytokine activation would be beneficial in almost any situation where a therapeutic immune response is required. In particular, the amplification of a therapeutic immune response by increasing cytokine activation could be advantageous in the treatment of cancer or infectious diseases, though not limited to these applications.

[0496] For the purposes of this invention, cytokines should be interpreted as encompassing chemokines, interferons, interleukins, lymphokines, and TNF.

[0497] In preferred embodiments, cytokines whose expression is increased by nongranulocyte immune cells and / or host immune cells are selected from the group including (or consisting of) IFN-γ and TNF. Preferably, the expression of two or more of these co-stimulatory molecules may be increased. In particular, the expression of IFN-γ by nongranulocyte immune cells and / or host immune cells may be increased.

[0498] Increased cytokine expression can be evaluated by any appropriate method and quantified as needed.

[0499] In a preferred embodiment, cytokine expression increases by at least 5%. For example, cytokine expression may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of cytokine expression in such embodiments can be achieved by comparison with a suitable control.

[0500] Cytokine expression may be increased in host immune cells selected from a group including (or consisting of) PBMCs and TILs. The ability of a population of granulocyte-producing cells suitable for use according to the present invention to increase cytokine (e.g., IFN-γ) expression by PBMCs and TILs is shown in the examples.

[0501] Preferably, for use according to the present invention, a therapeutically effective amount of such a population of granulocyte-producing cells (or a composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the expression of one or more cytokines by immune cells such as nongranulocyte immune cells and / or host immune cells. The cytokines, the degree of increase, and the host immune cells involved may be as considered in the preceding paragraph.

[0502] Populations of granulocyte-producing cells suitable for use according to the present invention may be able to increase immune cell transport. In particular, populations of this type of granulocyte-producing cell may be able to increase (preferably increase) the transport of host immune cells. Therefore, such populations of granulocyte-producing cells may be able to amplify (preferably amplify) the host therapeutic immune response by increasing the transport of host immune cells.

[0503] The transport of immune cells plays a crucial role in their ability to access sites where they need to exert therapeutic activity, such as tumors or infection sites. Therefore, it will be understood that the ability of a population or composition of granulocyte-producing cells suitable for use according to the present invention to increase immune cell transport provides a clear advantage in promoting an effective therapeutic immune response.

[0504] With respect to PBMCs, and especially with respect to host PBMCs, an increase in cell transport may be observed. As mentioned elsewhere, the inventors have found that a population of granulocyte-producing cells suitable for use according to the present invention is CXCR3 + We demonstrated that it may be possible to generate granulocytes expressing CXCL10, which is known to act as a chemotactic factor for immune cells. Therefore, the medical use and therapeutic method of the present invention involves generating a population of cells expressing CXCL10, thereby enabling CXCR3 + T cells and CXCR3 + It may be particularly beneficial in increasing NK cell transport.

[0505] Increased immune cell transport can be assessed by any appropriate method and quantified as needed.

[0506] In a preferred embodiment, the transport of immune cells increases by at least 5%. For example, the transport of immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the transport of immune cells in such embodiments can be achieved by comparison with a suitable control.

[0507] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the transport of immune cells, such as host immune cells. The degree of increased transport and the host immune cells involved may be as considered in the preceding paragraph.

[0508] In particular, increased transport of immune cells may lead to increased recruitment of immune cells to TMEs.

[0509] As described above, the inventors noted that exposure to a population of granulocyte-producing cells suitable for use according to the present invention increases immune cell transport. In particular, the inventors noted that a population or composition of granulocyte-producing cells suitable for use according to the present invention may increase the recruitment of immune cells to the TME. As demonstrated in the examples, such a population or composition of granulocyte-producing cells may increase (preferably increase) the recruitment of host immune cells to the TME. Therefore, such a population or composition of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the recruitment of host immune cells to the TME.

[0510] It is well known that immune cells have a low tendency to enter tumor-mediated membranous cells (TMEs). Many immune cells have shown little ability to enter tumors, and TMEs possess immunosuppressive properties. Therefore, the ability of a population or composition of granulocyte-producing cells suitable for use according to the present invention to increase the recruitment of immune cells, such as host immune cells, into TMEs offers significant advantages in tumor treatment. By increasing the number of immune cells present within the tumor, the antitumor activity of cells that exert a therapeutic immune response can be dramatically increased.

[0511] With respect to PBMCs, and particularly with respect to host PBMCs, an increase in immune cell recruitment to TMEs may be observed. The ability of a population or composition of granulocyte-producing cells suitable for therapeutic use of the present invention to increase such recruitment to TMEs is demonstrated in the examples.

[0512] In the examples, the inventors also demonstrate that populations and compositions of granulocyte-producing cells suitable for use according to the present invention may differentiate to produce granulocytes expressing CXCL10. CXCL10 is CXCR3 + T cells and CXCR3 + CXCR3 may contain NK cells. + It is a chemotactic factor for immune cells. Therefore, a population and composition of granulocyte-producing cells suitable for use according to the present invention is CXCR3 to TME. + T cells and CXCR3 + This can be particularly beneficial in establishing a population of granulocyte progeny cells that can (preferably increase) the recruitment of NK cells.

[0513] The increase in immune cell recruitment to TME can be assessed by any appropriate method and quantified as needed.

[0514] In preferred embodiments, the recruitment of immune cells to TMEs increases by at least 5%. For example, the recruitment of immune cells to TMEs may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the recruitment of immune cells to TMEs in such embodiments can be achieved by comparison with a suitable control.

[0515] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the recruitment of immune cells, such as host immune cells, to the TME. The degree of increase in the recruitment of immune cells to the TME, and the host immune cells involved, may be as considered in the preceding paragraph.

[0516] Populations of granulocyte-producing cells suitable for use according to the present invention may increase the cytodestructive activity of immune cells. In particular, granulocyte-producing cells may increase (preferably increase) the cytodestructive activity of non-granulocyte immune cells present in the compositions of the present invention. Populations or compositions of granulocyte-producing cells may increase (preferably increase) the cytodestructive activity of host immune cells. Therefore, such populations or compositions of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the cytodestructive activity of host immune cells.

[0517] The catastrophic killing of infected cells, cancer cells, or other diseased cells is a crucial mechanism by which many immune cells exert their therapeutic activity. Therefore, the ability of granulocyte-producing cell populations and compositions suitable for use according to the present invention to increase the cellular destructive activity of immune cells will be seen as advantageous in increasing the effectiveness of therapeutic immune responses that can be used to treat many conditions, including cancer and infectious diseases.

[0518] Regarding PBMCs, particularly host PBMCs, an increase in the cell-destroying activity of immune cells may be observed.

[0519] The increase in the cell-destructive activity of immune cells can be evaluated by any appropriate method and quantified as necessary.

[0520] In a preferred embodiment, the cytotoxic activity of immune cells increases by at least 5%. For example, the cytotoxic activity of immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the cytotoxic activity of immune cells in such embodiments can be achieved by comparison with a suitable control.

[0521] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the cytodestructive activity of immune cells, such as host immune cells. The degree of increase in the cytodestructive activity of immune cells and the host immune cells involved may be as considered in the preceding paragraph.

[0522] In particular, increased cellular destructiveness of immune cells may lead to increased tumor cell death activity of immune cells, especially host immune cells.

[0523] Populations of granulocyte-producing cells suitable for use according to the present invention may be able to increase the tumor cell-killing activity of immune cells. Immune cells whose tumor cell-killing activity is increased may be in vitro cells or in vivo cells, for example, immune cells present in the subject to which a suitable population of granulocyte-producing cells is provided. In particular, granulocyte-producing cells may be able to increase (preferably increase) the tumor cell-killing activity of non-granulocyte immune cells present in the suitable compositions of the present invention. Populations or compositions of granulocyte-producing cells may be able to increase (preferably increase) the tumor cell-killing activity of host immune cells. Therefore, such populations or compositions of granulocyte-producing cells may be able to amplify (preferably amplify) the host therapeutic immune response by increasing the tumor cell-killing activity of host immune cells.

[0524] The use of immune cells to target and kill cancer cells forms the basis of most anti-cancer immunotherapies. Therefore, it will be readily apparent that the ability of populations and compositions of granulocyte-producing cells suitable for use according to the present invention to increase the tumor cell-killing activity of immune cells, such as host immune cells, provides a clear and desirable advantage in anti-cancer therapy.

[0525] With regard to PBMCs, and particularly with respect to host PBMCs, an increase in the tumor cell killing activity of immune cells may be observed. Such an increase is shown in the results provided in the examples.

[0526] The increased tumor cell death activity of immune cells can be evaluated by any appropriate method and quantified as needed.

[0527] In preferred embodiments, the tumor cell killing activity of immune cells increases by at least 5%. For example, the tumor cell killing activity of immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the tumor cell killing activity of immune cells in such embodiments can be achieved by comparison with a suitable control.

[0528] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the tumor cell-killing activity of immune cells, such as host immune cells. The degree of increase in the tumor cell-killing activity of immune cells and the host immune cells involved may be as considered in the preceding paragraph.

[0529] Populations of granulocyte-producing cells suitable for use according to the present invention may increase the proliferation of immune cells. In particular, granulocyte-producing cells may increase (preferably increase) the proliferation of non-granulocyte immune cells present in the compositions of the present invention. Populations or compositions of granulocyte-producing cells may increase (preferably increase) the proliferation of host immune cells. Therefore, such populations or compositions of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the proliferation of host immune cells.

[0530] Immune cell-based therapies rely on the development of appropriate immune cells in therapeutically effective quantities to deliver the necessary therapeutic immune response (e.g., in the treatment of cancer or infection). Therefore, the ability of granulocyte-producing cell populations and compositions suitable for use according to the present invention to increase the proliferation of immune cells, such as host immune cells, will be extremely beneficial in achieving this. For example, by increasing the proliferation of immune cells, granulocyte-producing cells and compositions suitable for use according to the present invention may amplify (preferably amplify) immune responses that would otherwise not reach a therapeutic threshold, or they may shorten the time required to generate therapeutically effective quantities of immune cells.

[0531] In preferred embodiments, the proliferation of T cells, such as host T cells, may increase. Preferred T cells include αβT cells and CD8 cells. + T cells, CD4 + The group may be selected from (or consist of) T cells, NK T cells, and γδ T cells. In particular, the proliferation of αβ T cells may increase, as demonstrated by the data shown in the examples. For example, αβ T cells are CD4 + It may also be a T cell, or CD8 + These may also be T cells.

[0532] Increased proliferation of immune cells can be assessed by any appropriate method and quantified as necessary.

[0533] Preferably, the proliferation of host immune cells may increase by at least 5%. For example, the proliferation of host immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The quantification of the increase in host immune cell proliferation in such embodiments can be achieved by comparison with a suitable control.

[0534] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the proliferation of immune cells, such as host immune cells. The degree of increase in immune cell proliferation and the host immune cells involved may be as considered in the preceding paragraph.

[0535] Populations of granulocyte-producing cells suitable for use according to the present invention may increase the survival of immune cells. In particular, granulocyte-producing cells may increase (preferably increase) the survival of non-granulocyte immune cells present in the compositions of the present invention. Populations or compositions of granulocyte-producing cells may increase (preferably increase) the survival of host immune cells. Therefore, such populations or compositions of granulocyte-producing cells may amplify (preferably amplify) the host therapeutic immune response by increasing the survival of host immune cells.

[0536] It is well known that immune cells have a limited lifespan and undergo rapid metabolic turnover in the body. This is exacerbated in situations such as TME, where immunosuppressive conditions further shorten the lifespan of immune cells entering tumors. Our discovery that granulocyte-producing cells and compositions suitable for therapeutic use of the present invention can increase the survival of immune cells, therefore, indicates that therapies utilizing such granulocyte-producing cells can offer advantages in extending the period during which immune cells can produce an effective therapeutic immune response. This may be particularly beneficial in the treatment of conditions such as cancer, where an immunosuppressive environment shortens the lifespan of immune cells in other ways.

[0537] In preferred embodiments, the survival of T cells (e.g., NK T cells) or NK cells may be increased. For example, the survival of host T cells (e.g., NK T cells) or NK cells may be increased. The examples show data demonstrating the ability of the useful granulocyte-producing cells and compositions according to the present invention to increase the survival of NK T cells and NK cells.

[0538] The increase in immune cell survival can be assessed by any appropriate method and quantified as needed.

[0539] Preferably, the survival of host immune cells may increase by at least 5%. For example, the survival of host immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The quantification of the increase in host immune cell survival by such embodiments can be achieved by comparison with a suitable control.

[0540] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the survival of immune cells, such as host immune cells. The degree of increase in immune cell survival and the host immune cells involved may be as considered in the preceding paragraph.

[0541] Populations of granulocyte-producing cells suitable for use according to the present invention may be able to increase the abundance of immune cells. In particular, populations or compositions of this type of granulocyte-producing cells may be able to increase (preferably increase) the abundance of host immune cells. Therefore, such populations or compositions of granulocyte-producing cells may be able to amplify (preferably amplify) the host therapeutic immune response by increasing the abundance of host immune cells.

[0542] While we do not wish to be bound by any hypothesis, the increased abundance of immune cells observed when exposing populations and compositions of granulocyte-producing cells suitable for use according to the present invention to such cells may result from a combination of increased proliferation and increased survival of immune cells, which are discussed in more detail above. Whatever it may be, it will provide a practical benefit with respect to the medical use and methods of the present invention. By increasing the abundance of immune cells that can participate in a therapeutic immune response, the medical use and therapeutic methods of the present invention have the ability to amplify such a therapeutic immune response in terms of both degree and duration. This clearly provides a benefit in many therapeutic situations.

[0543] The increase in the abundance of immune cells can be assessed by any appropriate method and quantified as needed.

[0544] Preferably, the abundance of host immune cells may increase by at least 5%. For example, the abundance of host immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in the abundance of host immune cells in such embodiments can be quantified by comparison with a suitable control.

[0545] In preferred embodiments, the abundance of T cells, such as host T cells, may increase. T cells whose abundance may increase include αβT cells and CD8 cells. + T cells, CD4 + The group may be selected from (or consist of) T cells, NK T cells, and γδ T cells. In particular, the abundance of host αβ T cells may increase, as further shown in the examples. αβ T cells are CD4 + It may also be a T cell, or CD8 + These may also be T cells.

[0546] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or the composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the abundance of immune cells, such as host immune cells. The degree of increase in the abundance of immune cells, and the host immune cells involved, may be as considered in the preceding paragraph.

[0547] Many of the properties of the cells and compositions suitable for use in the medical use and methods of the present invention indicate that these cells and compositions are well-suited for use in combination with other cell therapies, particularly for use with further cellular immunotherapy.

[0548] The ability of the cells and compositions of the present invention to increase the proliferation, abundance, and survival of immune cells suggests that therapies using the cells and compositions of the present invention may be particularly advantageous when used in combination with other cell therapies. These may be therapies using the host's own cells or therapies using cells of the same species. By providing therapies according to the present invention, cells involved in further cell therapies may be induced to proliferate, survive longer, and increase in abundance and accumulation. This may improve the effectiveness of such therapies.

[0549] As described above, the inventors have identified the ability of granulocyte-producing cell populations to provide “Signal 2” (co-stimulation) and “Signal 3” (cytokine simulation) to other immune cells, such as those constituting part of further cellular immunotherapy. The provision of these signals is important in generating an effective immune response against tumors and overcoming the immunosuppressive effects of TME. This property of granulocyte-producing cell populations suggests that they may be used in combination with further cellular immunotherapy, thereby potentially improving the proliferation, survival, and accumulation of cells involved in such further cellular therapy.

[0550] Our discovery that a population of granulocyte-producing cells can generate granulocytes that secrete chemokines such as CXCL10 also suggests potential utility in combination with further cellular immunotherapy. Chemokines play a crucial role in the migration, positioning, and release of immune cells during therapeutic immune responses. The ability of a population of granulocyte-producing cells to produce chemokine-secreting granulocyte progeny suggests that the use of such a population in combination with further cellular immunotherapy may generate granulocytes that can favorably enhance the activity of the cells in the further therapy.

[0551] The inventors also confirmed that granulocytes generated during the differentiation of a population of granulocyte-producing cells suitable for use in various embodiments of the present invention express ligands for costimulatory molecules such as 4-1BBL and OX40L. The interaction between these ligands and their receptors plays a crucial role in regulating T cell activation and the generation of effector T cell responses. Therefore, the expression of such receptors by the offspring of a population of granulocyte-producing cells suggests that the use of this population in combination with further cellular immunotherapy may enable the granulocyte-producing cell population to generate granulocytes that thus positively influence T cell responses.

[0552] Preferably, for use according to the present invention, the therapeutically effective amount of such a population of granulocyte-producing cells (or a composition of the present invention [e.g., a pharmaceutical composition]) is sufficient to increase the proliferation, survival, and / or abundance of immune cells associated with the further cellular immunotherapy when combined with further cell therapy. The degree of increase, the immune cells involved, and appropriate indicators of the increased activation may be as considered elsewhere herein.

[0553] Those skilled in the art will recognize many examples of cellular immunotherapies that can be beneficially used in combination with therapies using populations or compositions of granulocyte-producing cells according to the present invention. These include, but are not limited to, NK cell therapy, chimeric antigen receptor (CAR)-based therapies (including CAR-T cell therapy, e.g., CAR-γδ T cell therapy and CAR-NK cell therapy), TIL therapy, and engineered T cell receptor (TCR) therapy.

[0554] Medical uses, methods of treatment, and compositions (e.g., pharmaceutical compositions) may include populations of granulocyte-producing cells for use in treating a target by amplifying a nongranulocyte-mediated therapeutic immune response.

[0555] As used herein, the terms “to treat,” “to treat,” or “to treat” include preventive measures (e.g., to prevent the onset of a disease) and corrective measures (treatment of a subject already suffering from a disease). Preferably, as used herein, “to treat,” or “to treat” means corrective measures.

[0556] As used herein, the terms “to treat, to treat” or “to treat, to treat” may refer to both the disorder and / or its symptoms.

[0557] A population of granulocyte-producing cells as part of the composition of the present invention (for example, a pharmaceutical composition) may be administered to a subject in a therapeutically effective or prophylactically effective dose.

[0558] ...

Claims

1. A method for preparing cells for therapeutic use, G-CSF, GM-CSF, IL-3 and TNF A method comprising culturing a population of granulocytes in cell culture conditions that promote the differentiation of progenitor cells and generate a population of granulocyte-producing cells, including the presence of a certain element.

2. The method according to claim 1, wherein the cell culture conditions that promote the differentiation of the progenitor cells further comprise at least one adjuvant selected from the group consisting of SCF, TPO, ITS, and HSA.

3. The cell culture conditions that promote the differentiation of the aforementioned progenitor cells are: GM-CSF at a concentration of approximately 0.01 μg / mL, and - G-CSF at a concentration of approximately 0.13 μg / mL, and - SCF with a concentration of approximately 0.13 μg / mL, and - TPO with a concentration of approximately 0.13 μg / mL, and IL-3 at a concentration of approximately 0.13 μg / mL, - TNF at a concentration of approximately 0.001 μg / mL, and ITS, and Approximately 1% HSA The method according to claim 2, including the method described in claim 2.

4. The method according to any one of claims 1 to 3, wherein the progenitor cells are cultured for 4 to 6 days under conditions that produce a population of granulocyte-producing cells.

5. The method according to claim 4, wherein the progenitor cells are cultured for 4 or 5 days under conditions that produce a population of granulocyte-producing cells.

6. The method according to any of the prior claims, wherein GM-CSF and IL-3 are provided to the cells during the last 48 hours of the period in which the cells are in culture.

7. The method according to any of the prior claims, wherein TNF is provided to the cells during the last 24 hours of the period in which the cells are in culture.

8. The method further includes the step of culturing a population of stem cells under cell culture conditions that generate a population of progenitor cells, The cell culture conditions for generating progenitor cells are: SCF, • Flt-3 ligand, IL-3, IL-6, and TPO A method according to any of the prior claims, including the presence of [a specific element].

9. The cell culture conditions for generating progenitor cells are: - SCF with a concentration of approximately 0.2 μg / mL, and • Flt-3 ligand at a concentration of approximately 0.2 μg / mL, and IL-3 at a concentration of approximately 0.015 μg / mL, IL-6 at a concentration of approximately 0.015 μg / mL, - TPO with a concentration of approximately 0.02 μg / mL, and ITS, and Approximately 1% HSA The method according to claim 8, including the method described in claim 8.

10. The method according to claim 8 or 9, wherein the stem cells are cultured for 8 to 9 days under conditions that generate a population of progenitor cells.

11. The method according to claim 10, wherein the stem cells are cultured for eight days under conditions that generate a population of progenitor cells.

12. The method according to any one of claims 8 to 11, wherein the stem cells are HSCs.

13. The method according to any of the prior claims, further comprising the step of purifying the population of granulocyte-producing cells that have been generated.

14. The method according to any of the prior claims, further comprising the step of formulating a population of granulocyte-producing cells for medical use.

15. The method according to any of the prior claims, further comprising priming granulocyte-producing cells for therapeutic use by a method comprising culturing a population of granulocyte-producing cells in the presence of GM-CSF and, optionally, one or more cytokines selected from the group consisting of TNF, IFN-α, IFN-β, IL-15, and IL-18.

16. The method according to claim 15, further comprising the steps of purifying a population of primed granulocyte-producing cells that have been generated, and / or formulating the population of cells for medical use.

17. A cell culture medium for use in the method according to any one of claims 1 to 16, comprising G-CSF at a concentration of approximately 0.13 μg / mL, SCF at a concentration of approximately 0.13 μg / mL, TPO at a concentration of approximately 0.13 μg / mL, ITS, and approximately 1% HSA.

18. The cell culture medium according to claim 17, further comprising GM-CSF at a concentration of approximately 0.01 μg / mL and IL-3 at a concentration of approximately 0.13 μg / mL.

19. The cell culture medium according to claim 17 or claim 18, further comprising TNF at a concentration of approximately 0.001 μg / mL.

20. A cell culture medium for use in the method according to any one of claims 8 to 19, comprising SCF at a concentration of approximately 0.2 μg / mL, Flt-3 ligand at a concentration of approximately 0.2 μg / mL, IL-3 at a concentration of approximately 0.015 μg / mL, IL-6 at a concentration of approximately 0.015 μg / mL, TPO at a concentration of approximately 0.02 μg / mL, ITS, and approximately 1% HSA.

21. A population of granulocyte-producing cells prepared for therapeutic use by the method described in any one of claims 1 to 14.

22. A population of granulocyte-producing cells prepared for therapeutic use by the method of any one of claims 1 to 20.

23. CD15 ＋ 64 ＋ CD18 ＋ CD49d ＋ CD71 ＋ The first subgroup of cells, ・CD15 － CD11b ＋／－ CD18 ＋ CD49d ＋ CD32 ＋ HLA - DR － a second subpopulation of cells that are, and CD15 － CD11b － HLA-DR ＋ CD18 ＋ CD49d ＋ and CD71 ＋ The third subgroup of cells is A population of granulocyte-producing cells according to claim 21 or claim 22.

24. A population of isolated granulocyte-producing cells, CD15 ＋ 64 ＋ CD18 ＋ CD49d ＋ CD71 ＋ The first subgroup of cells, CD15 － CD11b ＋／－ CD18 ＋ CD49d ＋ CD32 ＋ HLA-DR － The second subgroup of cells, and CD15 － CD11b － HLA-DR ＋ CD18 ＋ CD49d ＋ and CD71 ＋ The third subgroup of cells A population of isolated granulocyte-producing cells, including [specific cells / organisms].

25. CD15 － CD11b ＋ HLA-DR ＋ The fourth subgroup of cells A population of granulocyte-producing cells according to claim 23 or claim 24, further comprising:

26. CD15 ＋ 64 ＋ CD18 ＋ CD49d ＋ CD71 ＋ A group of granulocyte-producing cells.

27. 64 ＋ and / or CD16 － and / or CD62L － A population of granulocyte-producing cells according to any one of claims 23 to 26.

28. A population of granulocyte-producing cells according to any one of claims 23 to 27, wherein the first and / or second subpopulation of cells is also positive for one, two or more, or all of the markers selected from the group consisting of CD177, CD11b, CD71, CD66b, HLA-DR, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14.

29. The population of granulocyte-producing cells according to any one of claims 23 to 28, wherein the third subpopulation of cells is also positive for one, two or more, or all of the markers selected from the group consisting of CD177, CD71, CD66b, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14.

30. 64 ＋ Granulocyte-producing cells, or a group of such cells.

31. 64 ＋ and CD16 － A granulocyte-producing cell according to claim 30, or a population of such cells.

32. 64 ＋ and CD62L － A granulocyte-producing cell, as described in claim 30 or claim 31, or a population of such cells.

33. 64 ＋ CD16 － and CD62L － A granulocyte-producing cell according to any one of claims 30 to 32, or a population of such cells.

34. CD16 － Granulocyte-producing cells, or a group of such cells.

35. CD16 － and CD62L － A granulocyte-producing cell as described in claim 34, or a population of such cells.

36. CD62L － Granulocyte-producing cells, or a population of such cells.

37. A population of isolated granulocyte-producing cells, Over 90% Lin － Cells (for example, approximately 97% Lin － cell), CD34 less than 30% ＋ Cells (for example, approximately 14% CD34) ＋ cell), Over 30% of CD38 ＋ Cells (for example, approximately 65% ​​CD38) ＋ cell), - Cells with less than 1% HSC phenotype (for example, cells with approximately 0.04% HSC phenotype), - Cells with less than 1% LT-HSC phenotype (for example, cells with approximately 0.02% LT-HSC phenotype) - Cells with less than 20% LMPP phenotype (for example, cells with approximately 5% LMPP phenotype), and - Cells with less than 10% MPP phenotype (for example, cells with approximately 2.5% MPP phenotype) A population of isolated granulocyte-producing cells, including [specific cells / organisms].

38. A pharmaceutical composition comprising a population of granulocyte-producing cells according to any one of claims 23 to 37.

39. A pharmaceutical composition according to claim 38, or a population of granulocyte-producing cells according to any one of claims 23 to 37, for use as a pharmaceutical.

40. A pharmaceutical composition or population of granulocyte-producing cells according to claim 39, for use in the treatment of cancer.

41. A pharmaceutical composition or population of granulocyte-producing cells according to claim 40, for use in treating one or more of the following: 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 lymphoma (NHL), laryngeal cancer, uterine cancer, or breast cancer.

42. A pharmaceutical composition or population of granulocyte-producing cells according to claim 41, for use in treating pancreatic cancer.

43. A pharmaceutical composition or population of granulocyte-producing cells according to claim 39, for use in the treatment of infectious diseases.

44. A pharmaceutical composition or population of granulocyte-producing cells according to claim 39, for use in promoting a non-granulocyte immune response.

45. Use of a population of granulocyte-producing cells according to any one of claims 23 to 37 in the manufacture of a pharmaceutical product.

46. A method for treating a disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition according to the sixth aspect of the present invention, or a population of granulocyte-producing cells according to the second, fourth to tenth aspects of the present invention.

47. A method for treating cancer in a subject, comprising administering the pharmaceutical composition of the present invention to the subject.

48. Use of the pharmaceutical composition of the present invention in the manufacture of a pharmaceutical for treating cancer in a target.

49. A method for treating an infectious disease in a subject, comprising administering the pharmaceutical composition of the present invention to the subject.

50. Use of the pharmaceutical composition of the present invention in the manufacture of drugs for treating infectious diseases in a subject.

51. The pharmaceutical composition of the present invention for use in amplifying nongranulocyte therapeutic immune responses.

52. A method of treatment comprising amplifying a nongranulocyte therapeutic immune response, comprising providing the pharmaceutical composition of the present invention to a subject requiring such treatment.

53. The present invention relates to a pharmaceutical composition for use in the manufacture of a pharmaceutical for use in amplifying a nongranulocyte therapeutic immune response.

54. A composition comprising a population of granulocyte-producing cells and non-granulocyte immune cells as defined herein.

55. A composition comprising a population of granulocyte-producing cells and non-granulocyte immune cells as defined herein, but without αβT cells.

56. A composition comprising granulocyte-producing cells and non-granulocyte immune cells as defined herein, wherein the granulocyte-producing cells can (preferably) modulate the therapeutic immune response of the non-granulocyte immune cells.

57. A composition comprising a population of granulocyte-producing cells and non-granulocyte immune cells as defined herein, wherein the population of granulocyte-producing cells can amplify (preferably amplify) the therapeutic immune response of the non-granulocyte immune cells.

58. (a) A composition according to the present invention, or (b) Populations of granulocyte-producing cells and non-granulocyte immune cells as defined herein (e.g., terminally differentiated non-granulocyte immune cells), as well as (c) Instructions for using it (for example, when treating cancer), at your discretion. A kit that includes this.

59. A method for producing a composition (for example, the composition of the present invention), comprising culturing PBMCs in the presence of granulocyte-producing cells as defined herein to form the composition, and optionally depleting αβT cells before, during, or after the culture.

60. A method for producing a composition (for example, the composition of the present invention), comprising culturing the αβT cell-depleted PBMC under conditions that promote the differentiation of progenitor cells present in the αβT cell-depleted PBMC into granulocyte-producing cells, by a method defined herein, thereby forming the composition.

61. A method for treating a disease or disorder in a subject, comprising administering a composition of the present invention to the subject.

62. The present invention provides a composition for use in modulating nongranulocyte therapeutic immune responses.

63. The present invention provides a composition for use in amplifying nongranulocyte-mediated therapeutic immune responses.

64. A method of treatment comprising modulating a nongranulocyte therapeutic immune response, comprising providing the composition of the present invention to a subject requiring such treatment.

65. A method of treatment comprising amplifying a nongranulocyte therapeutic immune response, comprising providing the composition of the present invention to a subject requiring such treatment.

66. The present invention provides a composition for use in the manufacture of pharmaceuticals for use in modulating nongranulocyte therapeutic immune responses.

67. The present invention provides a composition for use in the manufacture of pharmaceuticals for use in amplifying nongranulocyte therapeutic immune responses.

68. A method for preparing the composition of the present invention, comprising culturing nongranulocyte immune cells in the presence of a population of granulocyte-producing cells of the present invention.

69. A composition comprising a population of granulocyte-producing cells and non-granulocyte immune cells (e.g., terminally differentiated non-granulocyte immune cells) as defined herein, wherein the population of granulocyte-producing cells can amplify (preferably amplify) the therapeutic immune response of the non-granulocyte immune cells.

70. A composition comprising one or more granulocytes differentiated from a population of granulocyte-producing cells as defined herein, and nongranulocytes, which can (preferably amplify) the therapeutic immune response of nongranulocytes.

71. A method of treatment comprising modulating a nongranulocyte therapeutic immune response, comprising providing the composition of the present invention to a subject requiring such treatment.

72. A method of treatment comprising amplifying a nongranulocyte therapeutic immune response, comprising providing the composition of the present invention to a subject requiring such treatment.

73. A method for promoting the therapeutic activity of nongranulocyte immune cells, comprising incubating nongranulocyte immune cells with a population of granulocyte-producing cells as defined herein.

74. A method for increasing the viability of immune cells in culture, comprising culturing the immune cells in the presence of a feeder layer of granulocyte-producing cells as defined herein.

75. A method for increasing the proliferation of immune cells in culture, comprising culturing the immune cells in the presence of a feeder layer of granulocyte-producing cells as defined herein.

76. A method for selecting an appropriate treatment regimen for a patient, wherein the method is: This includes determining whether the patient has impaired nongranulocyte immune response, If the patient is identified as having impaired nongranulocyte immune response, treatment with a population of granulocyte-producing cells as defined herein is selected as the appropriate treatment. A method wherein, if the patient is identified as having no impairment in the non-granulocyte immune response, treatment with a therapy other than that of a population of granulocyte-producing cells as defined herein is selected.

77. A method for selecting an appropriate treatment regimen for a patient, wherein the method is: - Includes incubating the patient's nongranulocyte immune cells with a population of granulocyte-producing cells as defined herein, If the activation of the non-granulocyte immune cells in the patient increases in response to the incubation, then treatment with a population of granulocyte-producing cells as defined herein is selected as an appropriate treatment. A method wherein, if the activation of the non-granulocyte immune cells of the patient increases in response to the incubation, treatment with a therapy other than that of a population of granulocyte-producing cells as defined herein is selected.

78. A method for determining whether a population of granulocyte-producing cells as defined herein is suitable for use in the treatment of cancer by beneficially modulating the tumor microenvironment, - To evaluate whether the population of granulocyte-producing cells as defined herein, or cells derived from the population of granulocyte-producing cells, can express inflammatory cytokines, and / or - To evaluate whether the population of granulocyte-producing cells as defined herein, or cells derived from the population of granulocyte-producing cells, can stimulate the expression of inflammatory cytokines by non-granulocyte immune cells, Based on this evaluation, we will determine whether the population of granulocyte-producing cells as defined herein is suitable for use in the treatment of cancer by beneficially modulating the tumor microenvironment. Methods that include...

79. A method for determining whether a population of granulocyte-producing cells as defined herein is suitable for use in the treatment of cancer by increasing the recruitment of immune cells to a tumor and / or activation of immune cells, - To evaluate whether the population of granulocyte-producing cells as defined herein, or cells derived from the population of granulocyte-producing cells, can express chemokines associated with promoting cell transport, and / or - To evaluate whether the population of granulocyte-producing cells as defined herein, or cells derived from the population of granulocyte-producing cells, can stimulate the expression of degranulation markers by non-granulocyte immune cells, Based on this evaluation, we will determine whether the population of granulocyte-producing cells as defined herein is suitable for use in the treatment of cancer by increasing the recruitment of immune cells to tumors and / or immune cell activation. Methods that include...

80. A method for determining whether a population of granulocyte-producing cells as defined herein is suitable for use in the treatment of cancer by directly promoting the death of cancer cells, - Incubating the population of granulocyte-producing cells as defined herein, or cells derived from the population of granulocyte-producing cells, together with cells of a cancer cell line, - To evaluate whether the population of granulocyte-producing cells as defined herein, or cells derived from the population of granulocyte-producing cells, can increase the death of the cancer cell line to a greater extent than the death of non-cancer cells, Based on this evaluation, we will determine whether the population of granulocyte-producing cells as defined herein is suitable for use in the treatment of cancer by directly promoting the death of cancer cells. Methods that include...

81. A method for determining whether a population of granulocyte-producing cells as defined herein is suitable for use in treating an infection by directly promoting the death of cellular infectious pathogens or infected cells, • Incubating a population of granulocyte-producing cells as defined herein, or cells derived from such a population of granulocyte-producing cells, together with a sample of a cellular infectious pathogen or infected cells, - To evaluate whether the population of granulocyte-producing cells as defined herein, or cells derived from the population of granulocyte-producing cells, can increase the death of the cellular infectious pathogen or the infected cells, Based on this assessment, we will determine whether the population of granulocyte-producing cells defined herein is suitable for use in treating infections by directly promoting the death of cellular infectious pathogens or infected cells. Methods that include...

82. A method for determining whether a population of granulocyte-producing cells as defined herein is suitable for use in therapy by amplifying a therapeutic immune response, • Incubating the population of granulocyte-producing cells as defined herein, or cells derived from the population of granulocyte-producing cells, together with immune cells, - To evaluate whether the population of granulocyte-producing cells as defined herein, or the cells derived from the population of granulocyte-producing cells, can increase the activation of the immune cells, Based on this evaluation, we will determine whether the population of granulocyte-producing cells as defined herein is suitable for use in therapy by amplifying the therapeutic immune response. Methods that include...

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