CELLS AND PREPARATION METHODS

Abstract

A method of preparing cells for therapeutic use is provided, in which a population of progenitor cells is cultured in cell culture conditions that promote differentiation of the progenitor cells comprising the presence of: G-CSF, GM-CSF, IL-3 and TNF; to produce a population of granulopoietic cells. The granulopoietic cells produced may be useful in methods of treatment, including the treatment of cancer and infections, and to modulate inflammatory cell responses.

Description

"CELLS AND PREPARATION METHODS"

[0001] The present invention relates to methods of preparing cells for therapeutic use and to cell populations prepared by such methods. The invention also relates to granulopoietic cell populations and pharmaceutical compositions comprising such cell populations. The granulopoietic cell populations and pharmaceutical compositions may be suitable for use in modulating (e.g., amplifying) a therapeutic immune response, particularly in modulating non-granulocytic immune responses and / or in originating cells with cytocidal activity. The granulopoietic cell populations and pharmaceutical compositions may be useful in contexts such as the treatment of cancer or infection. The invention further relates to cell culture media.

[0002] Immunotherapies can work to amplify the native immune response of a cell or host to achieve a therapeutic goal. They are becoming increasingly important for use in a wide range of therapeutic settings.

[0003] Host therapeutic immune responses generally involve several types of immune cells and play a vital role in the body's fight against cancer, infections, and virtually all other diseases. However, a subject's native therapeutic immune response is not always sufficient to eradicate disease. For example, tumors can be adapted to be immunologically cold and can create an immunosuppressive tumor microenvironment (TME) that can render native antitumor therapeutic immune responses ineffective.

[0004] To successfully eradicate a tumor (e.g., cancer), a variety of different types of immune cells typically need to work together. However, in some cases, a subject's own immune cells may be defective, meaning there is a need for a variety of different types of immune cells from an alternative source. Currently, there are difficulties in manufacturing such cell combinations. Additionally or alternatively, such conventional cell combinations may have effects Petition 870250101699, dated 06 / 11 / 2025, page 7 / 275 2 / 243 adverse immunogenic effects.

[0005] Cell-based therapies can also make use of the cytocidal capacity of cells and their ability to kill cells, such as cancerous cells, infected cells, or cellular infectious agents, which play important roles in disease. Cells with cytocidal activity, or with the ability to generate such cytocidal cells, thus represent therapeutically important targets.

[0006] Consequently, there is a need for improved immunotherapies, particularly cell therapies, as well as methods of producing cells that can be used in said immunotherapies.

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

[0008] The inventors have identified conditions that can be used to generate large populations of granulopoietic cells that have highly desirable therapeutic capabilities. These cells are capable of modulating (such as amplifying) and, in appropriate modalities, serve to modulate or amplify the therapeutic immune response of non-granulocytic immune cells and also to give rise to cells with useful cytocidal activities. Such cell compositions and populations are therefore capable of modulating therapeutic immune responses (and can thus modulate, for example, amplify, a therapeutic host immune response) and / or generating cells with direct cytocidal activity after administration to a subject.

[0009] In a first aspect, the invention provides a method for preparing cells for therapeutic use, the method comprising: • To cultivate 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; Petition 870250101699, dated 06 / 11 / 2025, page 8 / 275 3 / 243

[0010] to produce a population of granulopoietic cells.

[0011] A method of the first aspect of the invention may optionally comprise a further step of purifying the produced granulopoietic cell population and / or formulating this cell population for medical use.

[0012] In a second aspect, the invention provides a population of granulopoietic cells prepared for therapeutic use by a method of the first aspect of the invention.

[0013] In a further aspect, the invention provides a cell culture medium for use in a method according to the first aspect of the invention, the medium comprising: G-CSF; GM-CSF; IL-3; and TNF. A cell culture medium according to this aspect of the invention may be referred to as a differentiation medium.

[0014] The granulopoietic cell populations produced by the methods of the invention may optionally be harvested once produced. For the purposes of this disclosure, cell harvesting may be taken to encompass cell suspension, cell isolation, or cell separation.

[0015] The granulopoietic cell populations produced by the methods of the invention can optionally be cryopreserved once produced. Granulocytes, such as neutrophils, are known not to respond well to cryopreservation, with low levels of viable cells remaining after a frozen cell population has been thawed. In contrast, the granulopoietic cells of the present invention are well adapted to cryopreservation, with high levels of viable cells being obtained after the freeze-thaw process. Consequently, the granulopoietic cell populations of the invention offer significant advantages, compared to mature granulocytic cells, in applications where it is desired to cryopreserve cells before their use for therapy.

[0016] The granulopoietic cell populations produced by the methods of the invention can optionally be formulated for medical use once Petition 870250101699, dated 06 / 11 / 2025, page 9 / 275 4 / 243 produced. The appropriate methods for formulating cell populations to be used therapeutically will be well known to those skilled in the art and can be used in formulating the granulopoietic cell populations of the invention, optionally to give rise to pharmaceutical compositions of the invention.

[0017] The characteristics of the granulopoietic cell populations produced and also of the progenitor cell populations that can be used in such methods are considered in more detail in other parts of the descriptive report.

[0018] Optionally, cell culture conditions that promote progenitor cell differentiation may also include the presence of at least one cytokine selected from the group consisting of: SCF and TPO. Appropriately, cell culture conditions include the presence of SCF and TPO.

[0019] Similarly, a means of differentiation of the invention may further comprise at least one cytokine selected from the group consisting of: SCF and TPO. Suitably, such a cell culture medium comprises SCF and TPO.

[0020] The following paragraphs set forth details of useful embodiments of the methods of the first aspect of the invention. These include useful embodiments of the progenitor cells that can be used as starting material, the granulopoietic cells that can be produced by the methods, and the cell culture conditions that can be employed. Except for any examples where otherwise noted, the considerations set forth regarding the cell culture conditions that promote differentiation that can be used in the methods of the first aspect of the invention are also applicable as suitable embodiments of a differentiation cell culture medium of the invention.

[0021] The cell culture conditions that promote differentiation used in the methods of the first aspect of the invention may comprise Iscove-modified Dulbecco medium (IMDM) as a cell culture medium. Similarly, a cell culture medium of the invention may also comprise IMDM. In Petition 870250101699, dated 06 / 11 / 2025, page 10 / 275 5 / 243 In both cases, in a suitable embodiment, IMDM is a form of the medium comprising elevated glucose, glutamine, HEPES, sodium pyruvate and may optionally contain phenol red.

[0022] The methods of the first aspect of the invention, or a means of differentiating the invention, make use of granulocyte colony-stimulating factor cytokine (G-CSF) as a supplement.

[0023] Appropriately, G-CSF is supplied at a concentration of 0.013 μg / mL or more. For example, G-CSF may be supplied at a concentration of 0.016 μg / mL or more, 0.02 μg / mL or more, 0.03 μg / mL or more, or 0.065 μg / mL or more.

[0024] Appropriately, G-CSF is supplied at a concentration of 0.65 μg / mL or less. For example, G-CSF may be supplied at a concentration of 0.52 μg / mL or less, 0.39 μg / mL or less, or 0.26 μg / mL or less.

[0025] Suitably, G-CSF is supplied at a concentration 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 suitable embodiment, G-CSF is supplied at a concentration of approximately 0.13 μg / mL. In fact, in a suitable embodiment, G-CSF is supplied at a concentration of 0.13 μg / mL.

[0026] Examples of suitable forms of G-CSF that can be used in this way include the product produced by Peprotech and the GMP product produced by BioLegend, details of which are set out in Table 2.

[0027] The methods of the first aspect of the invention, or a means of differentiating the invention, make use of granulocyte-macrophage colony-stimulating factor cytokine (GM-CSF) as a supplement.

[0028] Appropriately, GM-CSF is supplied at a concentration of 0.001 μg / mL or more. For example, GM-CSF may be supplied at a concentration of 0.00125 μg / mL or more, 0.00167 μg / mL or more, 0.0025 μg / mL or more, or 0.005 μg / mL or more.

[0029] Appropriately, GM-CSF is supplied at a concentration of 0.05 Petition 870250101699, dated 06 / 11 / 2025, page 11 / 275 6 / 243 μg / mL or less. For example, GM-CSF can be supplied at a concentration of 0.04 μg / mL or less, 0.03 pg / mL or less, or less, or 0.02 pg / mL or less.

[0030] Suitablely, GM-CSF is supplied at a concentration of approximately 0.001 pg / mL to 0.05 pg / mL, 0.125 μg / mL to 0.04 μg / mL, 0.00167 μg / mL to 0.03 pg / mL, 0.0025 pg / mL to 0.02 pg / mL or 0.005 pg / mL to 0.015 pg / mL. In a suitable embodiment, GM-CSF is supplied at a concentration of approximately 0.01 pg / mL. In fact, in a suitable embodiment, GM-CSF is supplied at a concentration of 0.01 pg / mL.

[0031] Examples of suitable forms of GM-CSF that can be used in this way include the products produced by Peprotech and BioTechne and the GMP product produced by BioTechne, the details of which are set out in Table 2.

[0032] The methods of the first aspect of the invention, or a means of differentiating the invention, make use of the cytokine interleukin-3 (IL-3) as a supplement.

[0033] Appropriately, IL-3 is supplied at a concentration of 0.013 pg / mL or more. For example, IL-3 may be supplied at a concentration of 0.016 pg / mL or more, 0.02 pg / mL or more, 0.03 pg / mL or more, or 0.065 pg / mL or more.

[0034] Appropriately, IL-3 is supplied at a concentration of 0.65 pg / mL or less. For example, IL-3 may be supplied at a concentration of 0.52 pg / mL or less, 0.39 pg / mL or less, or 0.26 pg / mL or less.

[0035] Appropriately, IL-3 is delivered at a concentration of approximately 0.013 pg / mL to 0.65 pg / mL, 0.016 pg / mL to 0.52 pg / mL, 0.02 pg / mL to 0.39 pg / mL, 0.03 pg / mL to 0.26 pg / mL, or 0.065 pg / mL to 0.195 pg / mL. In an appropriate embodiment, IL-3 is delivered at a concentration of approximately 0.13 pg / mL. In fact, in an appropriate embodiment, IL-3 is delivered at a concentration of 0.13 pg / mL.

[0036] Examples of suitable forms of IL-3 that can be used in this way include the product produced by PeproTech and the GMP product produced Petition 870250101699, dated 06 / 11 / 2025, page 12 / 275 7 / 243 by PeproTech or BioTechne, whose details are set out in Table 2.

[0037] In a suitable embodiment, GM-CSF and IL-3 are delivered to cells for a period between 12 and 72 hours, ideally a 48-hour period under cell culture conditions. For example, GM-CSF and IL-3 may be delivered to cells during the final 48 hours of the period during which they are in culture. GM-CSF and IL-3 may be delivered to cells on the fourth and fifth days of cell culture conditions that promote progenitor cell differentiation. GM-CSF and IL-3 may be delivered to cells on the third and fourth days of cell culture conditions that promote progenitor cell differentiation.

[0038] The methods for obtaining a granulopoietic cell make use of the cytokine tumor necrosis factor (TNF) as a supplement. The terms TNF and TNF-alpha are used interchangeably in this document.

[0039] Appropriately, TNF is supplied at a concentration of 0.0001 μg / mL or more. For example, TNF may be supplied at a concentration of 0.000125 μg / mL or more, 0.000167 μg / mL or more, 0.00025 μg / mL or more, or 0.0005 μg / mL or more.

[0040] Appropriately, TNF is supplied at a concentration of 0.005 μg / mL or less. For example, TNF may be supplied at a concentration of 0.004 μg / mL or less, 0.003 μg / mL or less, or 0.002 μg / mL or less.

[0041] Suitably, TNF is supplied 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 suitable embodiment, TNF is supplied at a concentration of approximately 0.001 μg / mL. In fact, in a suitable embodiment, TNF is supplied at a concentration of 0.001 μg / mL.

[0042] Examples of suitable forms of TNF that can be used in this way include the product produced by PeproTech and the GMP product produced by BioTechne, details of which are set out in Table 2.

[0043] In a suitable embodiment, TNF is delivered to cells by a Petition 870250101699, dated 06 / 11 / 2025, page 13 / 275 8 / 243 period between 12 and 36 hours, appropriately a 24-hour period during cell culture conditions. For example, TNF can be delivered to cells during the final 24 hours of the period during which they are in culture. TNF can be delivered to cells on the fourth to fifth day of cell culture conditions that promote progenitor cell differentiation. TNF can be delivered to cells on the fifth day of cell culture conditions that promote progenitor cell differentiation. TNF can be delivered to cells on the fourth day of cell culture conditions that promote progenitor cell differentiation.

[0044] The methods of the first aspect of the invention, or a means of differentiating the invention, may optionally make use of cytokine stem cell factor (SCF) as a supplement.

[0045] Appropriately, SCF is supplied at a concentration of 0.013 μg / mL or more. For example, SCF may be supplied at a concentration of 0.016 μg / mL or more, 0.02 μg / mL or more, 0.03 μg / mL or more, or 0.065 μg / mL or more.

[0046] Appropriately, SCF is supplied at a concentration of 0.65 μg / mL or less. For example, SCF may be supplied at a concentration of 0.52 μg / mL or less, 0.39 μg / mL or less, or 0.26 μg / mL or less.

[0047] Appropriately, SCF is supplied at a concentration 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 suitable embodiment, SCF is supplied at a concentration of approximately 0.13 μg / mL. In fact, in a suitable embodiment, SCF is supplied at a concentration of 0.13 μg / mL.

[0048] Examples of suitable forms of SCF that can be used in this way include the product produced by Peprotech and the GMP product produced by PeproTech or BioTechne, the details of which are set out in Table 2.

[0049] The methods of the first aspect of the invention, or a means of differentiating the invention, may optionally make use of the cytokine thrombopoietin (TPO) as a supplement. Petition 870250101699, dated 06 / 11 / 2025, page 14 / 275 9 / 243

[0050] Appropriately, TPO is supplied at a concentration of 0.013 μg / mL or more. For example, TPO may be supplied at a concentration of 0.016 μg / mL or more, 0.02 μg / mL or more, 0.03 μg / mL or more, or 0.065 μg / mL or more.

[0051] Appropriately, TPO is supplied at a concentration of 0.65 μg / mL or less. For example, TPO may be supplied at a concentration of 0.52 μg / mL or less, 0.39 μg / mL or less, or 0.26 μg / mL or less.

[0052] Appropriately, TPO is delivered at a concentration 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 suitable embodiment, TPO is delivered at a concentration of approximately 0.13 μg / mL. In fact, in a suitable embodiment, TPO is delivered at a concentration of 0.13 μg / mL.

[0053] Examples of suitable forms of TPO that can be used in this way include the product produced by Peprotech and the GMP products produced by BioTechne or Peprotech, the details of which are set out in Table 2.

[0054] In a suitable embodiment, the cell culture conditions used in culturing the progenitor cell population to produce granulopoietic cells further comprise the presence of at least one supplement selected from the group consisting of: insulin transferrin selenium (ITS) and human serum albumin (HSA). In a suitable embodiment, such cell culture conditions comprise the presence of ITS and HSA. Suitablely, ITS and HSA are present in a differentiation medium of the invention.

[0055] The methods of the first aspect of the invention, or a means of differentiating the invention, can make appropriate use of insulin at a concentration between about 0.1 g / L and about 5 g / L, for example, at a concentration of approximately 1.0 g / L, as a supplement. These methods and cell culture media can make appropriate use of transferrin at a concentration between about 0.01 g / L and about 2.5 g / L, for example, at a concentration of approximately 0.55 g / L as a supplement. Appropriately, such methods and cell culture media can make appropriate use of selenium at a Petition 870250101699, dated 06 / 11 / 2025, p. 15 / 275 10 / 243 concentration between about 0.0001 g / L and about 0.003 g / L, for example, to a concentration of approximately 0.00067 g / L, as a supplement.

[0056] The methods of the first aspect of the invention, or a means of differentiating the invention, may optionally make use of HSA as a supplement.

[0057] Appropriately, HSA can be supplied at a concentration between 0.1% and 5%. For example, HSA supplied as a supplement can be supplied at a concentration of approximately 1%.

[0058] Suitably, the cell culture conditions that promote the differentiation of progenitor cells used in a method of the invention, or a differentiation medium of the invention, may comprise: GM-CSF; and G-CSF; and SCF; and TPO; and IL-3; and TNF; and ITS; and HSA. The cell culture medium may comprise IMDM, optionally with Glutamax supplementation.

[0059] Thus, in a suitable embodiment, the cell culture conditions that promote the differentiation of progenitor cells used in a method of the invention, or a differentiation medium of the invention, may comprise: GM-CSF at a concentration of approximately 0.01 pg / mL; and G-CSF at a concentration of approximately 0.13 pg / mL; and SCF at a concentration of approximately 0.13 pg / mL; and TPO at a concentration of approximately 0.13 pg / mL; and IL-3 at a concentration of approximately 0.13 pg / mL; and TNF at a concentration of approximately 0.001 pg / mL; and 1x ITS; and HSA at approximately 1%. The cell culture medium may comprise IMDM, optionally with Glutamax supplementation.

[0060] One method of the invention may comprise culturing a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells for any suitable period of time. For example, progenitor cells may be cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days under conditions to produce a population of granulopoietic cells. The methods according to the first aspect of the invention may comprise Petition 870250101699, dated 06 / 11 / 2025, page 16 / 275 11 / 243 Culture of progenitor cell populations under cell culture conditions that promote progenitor cell differentiation for a period of 1 to 7 days. For example, such methods may comprise culturing the cells under relevant conditions for a period of 4 to 7 days. In a suitable embodiment, such methods may comprise culturing the cells for approximately 1 day, or for approximately 2 days, or for approximately 3 days, or for approximately 4 days, or for approximately 5 days, or for approximately 6 days, or for approximately 7 days. Progenitor cells may be cultured for 1-10 days, 2-9 days, 3-8 days, 4-7 days, or 5-6 days under conditions to produce a population of granulopoietic cells. Suitablely, progenitor cells are cultured for 4, 5, or 6 days under conditions to produce a population of granulopoietic cells.In a suitable embodiment, progenitor cells are cultured for 4 days under conditions to produce a population of granulopoietic cells. In a suitable embodiment, progenitor cells are cultured for 5 days under conditions to produce a population of granulopoietic cells. In a suitable embodiment, progenitor cells are cultured for 6 days under conditions to produce a population of granulopoietic cells.

[0061] In a suitable embodiment of a method of the invention, the progenitor cells can be cultured at an initial seeding density between approximately 1x105 and 10x106 cells per cm2.

[0062] The methods of the invention may involve expanding the number of cells present in the culture, so that the number of granulopoietic cells produced by the method is greater than the number of progenitor cells present at the beginning of the method. In a suitable embodiment, the number of granulopoietic cells in the produced population may be increased, compared to the number of progenitor cells present at the beginning of the method, by at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, by Petition 870250101699, dated 06 / 11 / 2025, page 17 / 275 12 / 243 minus 13 times, at least 14 times, or at least 15 times. The methods established in the Examples achieve a population of granulopoietic cells that is approximately 3.5 times larger than the initial population of progenitor cells.

[0063] In a suitable embodiment, a method of the first aspect of the invention is practiced in relation to a population of progenitor cells that has been produced by in vitro expansion of a population of stem cells. Consequently, such a method of the invention may further comprise a step of cultivating a population of stem cells under cell culture conditions to produce the population of progenitor cells.

[0064] In a suitable embodiment, a method of the first aspect of the invention further comprises a step of cultivating a population of stem cells under cell culture conditions to produce the progenitor cell population: • where the cell culture conditions for the production of progenitor cells include the presence of • SCF, • Flt-3 ligand, • IL-3, • IL-6, and • TPO.

[0065] The number of progenitor cells produced by such a method can be markedly expanded compared to the number of stem cells present at the start of cell culture conditions. By way of example only, such an embodiment of a method of the invention can achieve an expansion of the number of progenitor cells that is 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 cell culture conditions. The Examples set forth details of a protocol that the inventors used to achieve an approximately 75-fold increase in the number of progenitor cells, in Petition 870250101699, dated 06 / 11 / 2025, page 18 / 275 13 / 243 comparison with the initial population of stem cells.

[0066] The invention also provides a cell culture medium, for use in a method of the invention, comprising SCF; Flt-3 ligand; IL-3; IL-6; and TPO. A cell culture medium according to this aspect of the invention may be referred to as an expansion medium.

[0067] Consequently, a method of preparing cells for therapeutic use according to such embodiments of the invention may comprise:

[0068] cultivate a population of stem cells under cell culture conditions to produce progenitor cells comprising the presence of: • SCF, • Flt-3 ligand, • IL-3, • IL-6, and • TPO;

[0069] to produce a population of progenitor cells; and

[0070] cultivate the population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells comprising the presence of: • G-CSF, • GM-CSF, • IL-3 and • TNF;

[0071] to produce a population of granulopoietic cells; and optionally

[0072] collect the granulopoietic cells.

[0073] The total increase in the number of cells achieved by such a method of the invention, representing the change in the number of cells from the initial population of stem cells to the population of granulopoietic cells produced, can 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, Petition 870250101699, dated 06 / 11 / 2025, page 19 / 275 14 / 243 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. The Examples set out details of a protocol that the inventors used to achieve a greater than 250-fold increase in the number of granulopoietic cells, compared to the initial population of stem cells.

[0074] A method according to such embodiments of the invention may involve a total time period in culture of between 10 and 25 days, for example, between 11 and 20 days, such as 12 days, 13 days, 14 days, 15 days, 6 days, 17 days, 18 days or 19 days.

[0075] The SCF may optionally be provided as a supplement in embodiments of the methods of the invention comprising a step of producing a population of progenitor cells as well as in an expansion medium of the invention.

[0076] Appropriately, SCF is supplied at a concentration of 0.02 μg / mL or more. For example, SCF may be supplied at a concentration of 0.025 μg / mL or more, 0.03 μg / mL or more, 0.05 μg / mL or more, or 0.1 μg / mL or more.

[0077] Appropriately, SCF is supplied at a concentration of 1 μg / mL or less. For example, SCF may be supplied at a concentration of 0.8 μg / mL or less, 0.6 μg / mL or less, or 0.4 μg / mL or less.

[0078] Suitablely, SCF is supplied at a concentration 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 suitable embodiment, SCF is supplied at a concentration of approximately 0.2 μg / mL. In fact, in a suitable embodiment, SCF is supplied at a concentration of 0.2 μg / mL.

[0079] The forms of SCF discussed above are also suitable for use in such modalities. Petition 870250101699, dated 06 / 11 / 2025, page 20 / 275 15 / 243

[0080] The Flt-3 (F3L) ligand may optionally be provided as a supplement in embodiments of the invention's methods comprising a step of producing a population of progenitor cells, as well as in an expansion medium of the invention.

[0081] Appropriately, F3L is supplied at a concentration of 0.02 μg / mL or more. For example, F3L may be supplied at a concentration of 0.025 μg / mL or more, 0.03 μg / mL or more, 0.05 μg / mL or more, or 0.1 μg / mL or more.

[0082] Appropriately, F3L is supplied at a concentration of 1 μg / mL or less. For example, F3L may be supplied at a concentration of 0.8 μg / mL or less, 0.6 μg / mL or less, or 0.4 μg / mL or less.

[0083] Suitablely, F3L is supplied at a concentration 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 suitable embodiment, F3L is supplied at a concentration of approximately 0.2 μg / mL. In fact, in a suitable embodiment, F3L is supplied at a concentration of 0.2 μg / mL.

[0084] Examples of suitable F3L forms that can be used in this way include the product produced by Peprotech and the GMP product produced by PeproTech or BioTechne, details of which are set out in Table 2.

[0085] IL-3 may optionally be provided as a supplement in embodiments of the invention's methods comprising a step of producing a population of progenitor cells, as well as in an expansion medium of the invention.

[0086] Appropriately, IL-3 is supplied at a concentration of 0.0015 μg / mL or more. For example, IL-3 may be supplied at a concentration of 0.0019 μg / mL or more, 0.0025 μg / mL or more, 0.00375 μg / mL or more, or 0.0075 μg / mL or more.

[0087] Appropriately, IL-3 is supplied at a concentration of 0.075 μg / mL or less. For example, IL-3 may be supplied at a concentration of 0.06 μg / mL or less, 0.045 μg / mL or less, or 0.03 μg / mL or less. Petition 870250101699, dated 06 / 11 / 2025, page 21 / 275 16 / 243

[0088] Appropriately, IL-3 is delivered at a concentration 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 suitable embodiment, IL-3 is delivered at a concentration of approximately 0.015 μg / mL. In fact, in a suitable embodiment, IL-3 is delivered at a concentration of 0.015 μg / mL.

[0089] The IL-3 forms discussed above are suitable for use in such modalities.

[0090] Interleukin 6 (IL-6) may optionally be provided as a supplement in embodiments of the invention's methods comprising a step of producing a population of progenitor cells, as well as in an expansion medium of the invention.

[0091] Appropriately, IL-6 is supplied at a concentration of 0.0015 μg / mL or more. For example, IL-6 may be supplied at a concentration of 0.0019 μg / mL or more, 0.0025 μg / mL or more, 0.00375 μg / mL or more, or 0.0075 μg / mL or more.

[0092] Appropriately, IL-6 is supplied at a concentration of 0.075 μg / mL or less. For example, IL-6 may be supplied at a concentration of 0.06 μg / mL or less, 0.045 μg / mL or less, or 0.03 μg / mL or less.

[0093] Appropriately, IL-6 is delivered at a concentration 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 suitable embodiment, IL-6 is delivered at a concentration of approximately 0.015 μg / mL. In fact, in a suitable embodiment, IL-6 is delivered at a concentration of 0.015 μg / mL.

[0094] Examples of suitable forms of IL-6 that can be used in this way include the product produced by PeproTech and the GMP product produced by PeproTech or BioTechne, details of which are set out in Table 2.

[0095] TPO may optionally be provided as a supplement in embodiments of the invention methods comprising a step of producing a Petition 870250101699, dated 06 / 11 / 2025, page 22 / 275 17 / 243 progenitor cell population, as well as in an expansion medium of the invention.

[0096] Appropriately, TPO is supplied at a concentration of 0.002 μg / mL or more. For example, TPO may be supplied at a concentration of 0.0025 μg / mL or more, 0.003 μg / mL or more, 0.005 pg / mL or more, or 0.01 μg / mL or more.

[0097] Appropriately, TPO is supplied at a concentration of 0.1 pg / mL or less. For example, TPO may be supplied at a concentration of 0.08 pg / mL or less, 0.06 pg / mL or less, or 0.04 pg / mL or less.

[0098] Appropriately, TPO is delivered at a concentration of approximately 0.002 pg / mL to 0.1 pg / mL, 0.0025 pg / mL to 0.08 pg / mL, 0.003 pg / mL to 0.06 pg / mL, 0.005 pg / mL to 0.04 pg / mL, or 0.01 pg / mL to 0.03 pg / mL. In a suitable embodiment, TPO is delivered at a concentration of approximately 0.02 pg / mL. In fact, in a suitable embodiment, TPO is delivered at a concentration of 0.02 pg / mL.

[0099] The TPO forms discussed above are also suitable for use in these modalities.

[0100] Appropriately, the cell culture conditions that promote the production of progenitor cells used in a method of the invention, or a means of expansion of the invention, may comprise: SCF; and Flt-3 Ligand; and IL-3; and IL-6; and TPO; and ITS; and HSA. The cell culture medium may comprise IMDM, optionally with Glutamax supplementation.

[0101] Thus, in a suitable embodiment, the cell culture conditions that promote the production of progenitor cells used in a method of the invention, or a means of expanding the invention, may comprise: SCF at a concentration of approximately 0.2 pg / mL; and Flt-3 Ligand at a concentration of approximately 0.2 pg / mL; and IL-3 at a concentration of approximately 0.015 pg / mL; and IL-6 at a concentration of approximately 0.015 pg / mL; and TPO at a concentration of approximately 0.02 pg / mL; and 1x ITS; and HSA at approximately 1%. The cell culture medium may comprise IMDM, optionally with Glutamax supplementation. Petition 870250101699, dated 06 / 11 / 2025, page 23 / 275 18 / 243

[0102] Stem cells that can be employed in such methods of the invention, as a starting material for the production of progenitor cells (and ultimately granulopoietic cells) include, but are not limited to, hematopoietic stem cells (HSCs). Further details of suitable stem cells and stem cell sources are provided elsewhere in this descriptive report and (without limitation) include umbilical cord blood and mobilized blood.

[0103] In a suitable embodiment, the cell culture conditions used in culturing stem cells to produce progenitor cells further comprise the presence of at least one supplement selected from the group consisting of: ITS and HSA. In a suitable embodiment, such cell culture conditions comprise the presence of ITS and HSA. Suitablely, ITS and HSA are present in an expansion medium of the invention.

[0104] ITS can be provided as a supplement in embodiments of the invention's methods comprising a step of producing a population of progenitor cells, as well as in an expansion medium of the invention.

[0105] Such embodiments of the methods of the first aspect of the invention, or a means of expanding the invention, may make appropriate use of insulin at a concentration between about 0.1 g / L and about 5 g / L, for example, at a concentration of approximately 1.0 g / L, as a supplement. These methods and cell culture media may make appropriate use of transferrin at a concentration between about 0.01 g / L and about 2.5 g / L, for example, at a concentration of approximately 0.55 g / L, as a supplement. Appropriately, such methods and cell culture media may make appropriate use of selenium at a concentration between about 0.0001 g / L and about 0.003 g / L, for example, at a concentration of approximately 0.00067 g / L, as a supplement.

[0106] HSA can be provided as a supplement in embodiments of the invention's methods comprising a step of producing a population of progenitor cells, as well as in a means of expanding the invention.

[0107] Appropriately, HSA can be supplied at a concentration between 0.1% and 5%. For example, HSA supplied as a supplement can be supplied Petition 870250101699, dated 06 / 11 / 2025, page 24 / 275 19 / 243 at a concentration of approximately 1%.

[0108] In embodiments of the invention's methods in which stem cells are cultured to produce progenitor cells, this may involve expanding the number of cells present in the culture.

[0109] One method of the invention may comprise culturing a population of stem cells under cell culture conditions to produce a population of progenitor cells for any suitable period of time. For example, the cells may be cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 15 days under conditions to produce a population of progenitor cells. Preferably, the cells are cultured for 8 or 9 days under conditions to produce a population of progenitor cells. The 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 to produce a population of progenitor cells. Preferably, stem cells, such as HSCs, are cultured for 8-9 days under conditions to produce a population of progenitor cells.

[0110] In suitable embodiments of such methods of the invention, stem cells are cultured under conditions to produce a population of progenitor cells for a period of 6 to 10 days. For example, such methods may comprise culturing the cells for a period of 7 to 8 days. In one suitable embodiment, such methods may comprise culturing the cells under cell culture conditions to produce a population of progenitor cells for approximately 6 days, or for approximately 7 days, or for approximately 8 days, or for approximately 9 days, or for approximately 10 days.

[0111] Consequently, a method of the invention for preparing cells for therapeutic use may comprise: (a) cultivate a population of stem cells under cell culture conditions to produce progenitor cells comprising the presence of SCF, FLT-3, TPO, IL-3, IL-6, ITS and HSA for 6-10 days, or Petition 870250101699, dated 06 / 11 / 2025, page 25 / 275 20 / 243 preferably 8 days, to produce a population of progenitor cells; and (b) cultivate the population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells to obtain a population of granulopoietic cells.

[0112] A method of the invention for preparing cells for therapeutic use may comprise: (a) Cultivate a population of stem cells under cell culture conditions to produce progenitor cells comprising the presence of IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS and HSA for 6-10 days, or preferably 8 days, to produce a population of progenitor cells; and (b) Cultivate the population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells comprising IMDM, G-CSF, GM-CSF, IL-3 and TNF for 1-6 days, or preferably 5 days, to obtain a population of granulopoietic cells.

[0113] Such a method of the invention for preparing cells for therapeutic use may comprise: (a) Cultivate a population of stem cells under cell culture conditions to produce progenitor cells comprising the presence of IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS and HSA for 6-10 days, or preferably 8 days, to produce a population of progenitor cells; and (b) Cultivate the population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells to obtain a population of granulopoietic cells.

[0114] For example, a method of the invention for preparing cells for therapeutic use may comprise: (a) cultivate a population of stem cells under culture conditions Petition 870250101699, dated 06 / 11 / 2025, page 26 / 275 21 / 243 cell culture to produce progenitor cells comprising the presence of IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS and HSA for 6-10 days, or preferably 8 days, to produce a population of progenitor cells; and (b) cultivate the population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells comprising IMDM, SCF, TPO, GCSF, ITS and HSA for 1-6 days, or preferably 5 days, to obtain a population of granulopoietic cells.

[0115] The appropriately supplemented cell culture medium may be replaced or replenished at any appropriate time during stem cell culture under conditions to produce progenitor cells. For example, the cell culture medium may be replenished on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, or day 15 of stem cell culture. Appropriately, the cell culture medium is replenished on day 1 and day 6 of stem cell culture. The cell culture medium may be replaced on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, or day 15 of stem cell culture. Preferably, the cell culture medium is replaced on day 4 of stem cell culture.

[0116] Stem cells, such as HSCs, from which progenitor cells are to be produced, can be seeded at any suitable cell density. For example, stem cells can be seeded at a density of 1x10⁵ cells / mL - 1x10⁶ cells / mL, 2.5x10⁵ cells / mL - 1x10⁶ cells / mL, 3x10⁵ cells / mL - 8x10⁵ cells / mL or 4x10⁵ cells / mL - 6x10⁵ cells / mL, preferably 5x10⁵ cells / mL. Stem cells can be seeded at a density of 1x10⁵ cells / cm²-1x10⁶ cells / cm², 2.5x10⁵ cells / cm²-1x10⁶ cells / cm², 3x10⁵ cells / cm²-8x10⁵ cells / cm² or 4x10⁵ cells / cm²-6x10⁵ cells / cm², preferably 5x10⁵ cells / cm². In a suitable embodiment, stem cells (such as HSCs) are seeded at a density of 5x10⁵ cells / mL and 5x10⁵ cells / cm². Petition 870250101699, dated 06 / 11 / 2025, page 27 / 275 22 / 243

[0117] Cells can be seeded into any suitable culture vessel. For example, cells can be seeded into a G-Rex 6M or G-Rex 10M culture vessel. Cells can be transferred to a new culture vessel at any suitable time. Cells can be transferred sequentially to cell culture vessels of increasing surface area. Such transfers can occur on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, or day 15 of the culture to produce progenitor cells. For example, stem cells (such as HSCs) can be transferred from a smaller G-Rex to a 100M G-Rex on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, or day 15 of culture to produce progenitor cells.For example, stem cells (such as HSCs) can 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 suitable embodiment, progenitor cells can be transferred to a new culture vessel on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, or day 10 of culture conditions that promote the differentiation of progenitor cells into granulopoietic cells.

[0118] According to such modalities, a method of preparing cells for therapeutic use may comprise: (a) seeding stem cells (such as HSCs) at 5x105 cells / mL and 5x105 cells / cm2; (b) cultivate the cells in cell culture medium comprising IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS and HSA for 8 days to obtain a population of progenitor cells, wherein the cell culture medium comprising IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS and HSA is replenished on day 1 and day 6 of such culture, and wherein the cell culture medium comprising IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS and HSA is replaced on day 4 of such culture; (c) cultivate the progenitor cell population in a cell culture medium comprising IMDM, SCF, TPO, GCSF, ITS and HSA for 5-6 Petition 870250101699, dated 06 / 11 / 2025, page 28 / 275 23 / 243 days to obtain a population of granulopoietic cells, in which the cell culture medium comprising IMDM, SCF, TPO, GCSF, ITS and HSA is replenished on day 3 of differentiation.

[0119] A suitable method for preparing cells for therapeutic use may include: (a) seeding stem cells (such as HSCs) at 5x105 cells / mL and 5x105 cells / cm2; (b) cultivate the cells in cell culture medium comprising IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS and HSA for 8 days to obtain a population of progenitor cells, wherein the cell culture medium comprising IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS and HSA is replenished on day 1 and day 6 of such culture, and wherein the cell culture medium comprising IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS and HSA is replaced on day 4 of such culture; (c) Cultivate the progenitor cell population in a cell culture medium comprising IMDM, SCF, TPO, G-CSF, ITS, and HSA for 5-6 days to obtain a granulopoietic cell population, wherein the cell culture medium comprising IMDM, SCF, TPO, G-CSF, ITS, HSA, GM-CSF, IL-3, and TNF is replenished on day 3 of differentiation.

[0120] The inventors also identified methods by which granulopoietic cells can be primed in order to amplify the properties of the cells that increase their therapeutic usefulness. In particular, priming granulopoietic cells by such methods can amplify their cytocidal activity in a way that can increase their therapeutic usefulness.

[0121] Consequently, the third aspect of the invention provides a method for priming granulopoietic cells for therapeutic use, the method comprising culturing a population of granulopoietic 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.

[0122] A method according to the first aspect of the invention may also Petition 870250101699, dated 06 / 11 / 2025, page 29 / 275 24 / 243 comprise a priming step of granulopoietic cells for therapeutic use, by a method comprising culturing the granulopoietic cell population 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.

[0123] A method of the third or first aspect of the invention comprising a priming step of granulopoietic cells may optionally comprise a further step of purifying the primed granulopoietic cell population produced and / or formulating this primed cell population for medical use.

[0124] The fourth aspect of the invention provides a population of primed granulopoietic cells that can be obtained by a method according to the third aspect of the invention. The population of primed granulopoietic cells can be obtained by a method of the third aspect of the invention. The population of primed granulopoietic cells can be as defined elsewhere in this disclosure (for example, with reference to the biological activity of the primed cells or their expression of particular markers).

[0125] GM-CSF can be used under cell culture conditions for a priming step at a concentration of 1-1000 ng / mL, 2-500 ng / mL, 3-250 ng / mL, 4-200 ng / mL. GM-CSF can be used at a concentration of 5-150 ng / mL, for example, at a concentration of 10-130 ng / mL.

[0126] TNF can be used under cell culture conditions for a priming step at a concentration of 0.001-10 ng / mL, 0.002-5 ng / mL, 0.003-2.5 ng / mL, 0.004-2 ng / mL. TNF can be used at a concentration of 0.005-1.5 ng / mL, for example, at a concentration of 0.01-1 ng / mL.

[0127] IFN-α can be used under cell culture conditions for a priming step at a concentration of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, 4-20 ng / mL. IFN-α can be used at a concentration of 5-15 ng / mL, for example, at a concentration of 10 ng / mL.

[0128] IFN-β can be used under cell culture conditions for a step Petition 870250101699, dated 06 / 11 / 2025, page 30 / 275 25 / 243 priming at a concentration of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, 4-20 ng / mL. IFN-β can be used at a concentration of 5-15 ng / mL, for example, at a concentration of 10 ng / mL.

[0129] IL-15 can be used under cell culture conditions for a priming step at a concentration of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, 4-20 ng / mL. IL-15 can be used at a concentration of 5-15 ng / mL, for example, at a concentration of 10 ng / mL.

[0130] IL-18 can be used under cell culture conditions for a priming step at a concentration of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, 4-20 ng / mL. IL-18 can be used at a concentration of 5-15 ng / mL, for example, at a concentration of 10 ng / mL.

[0131] IL-3 can be used under cell culture conditions for a priming step at a concentration of 1-1000 ng / mL, 2-500 ng / mL, 3-250 ng / mL, 4-200 ng / mL. IL-3 can be used at a concentration of 5-150 ng / mL, for example, at a concentration of 10-130 ng / mL.

[0132] In suitable embodiments, priming involves culturing a population of granulopoietic 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 0.011 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.

[0133] In a suitable embodiment, primed cells can be cultured in the presence of GM-CSF, G-CSF, SCF, TPO, and IL-15. By way of example only, cells can 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. Petition 870250101699, dated 06 / 11 / 2025, page 31 / 275 26 / 243

[0134] In a suitable embodiment, primed cells can be cultured in the presence of GM-CSF, G-CSF, SCF, TPO, and TNF. By way of example only, cells can 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.

[0135] In a suitable embodiment, primed cells can be cultured in the presence of GM-CSF and IL-3. By way of example only, cells can 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.

[0136] In a suitable embodiment, primed cells can be cultured in the presence of GM-CSF and IL-15. By way of example only, cells can 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.

[0137] In a suitable embodiment, primed cells can be cultured in the presence of GM-CSF and IL-18. By way of example only, cells can 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.

[0138] In a suitable embodiment, primed cells can be cultured in the presence of GM-CSF and IL-16. By way of example only, cells can 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.

[0139] In a suitable embodiment, primed cells can be cultured in the presence of GM-CSF and TNF. By way of example only, cells can be cultured in the presence of GM-CSF at a concentration of Petition 870250101699, dated 06 / 11 / 2025, page 32 / 275 27 / 243 approximately 130 ng / mL and TNF at a concentration of approximately 1 ng / mL.

[0140] In a suitable embodiment, primed cells can be cultured in the presence of GM-CSF, G-CSF, SCF, TPO, and IFN-α. By way of example only, cells can 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.

[0141] The priming step may last any suitable period of time. For example, the priming step may last 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 last 1-96 hours, 2-90 hours, 3-84 hours, 6-78 hours, 12-72 hours, 18-54 hours, or 24-48 hours. Suitablely, priming may comprise culture incorporating the cytokines discussed above, for example, at the concentrations set out above, for a period of one, two, or three days. In particular, priming may involve culturing the aforementioned priming cytokine combinations for two days.

[0142] A priming step may be suitably incorporated into any appropriate stage of a method of the invention. That said, priming will typically occur during the period when progenitor cells are cultured under conditions that promote the differentiation of progenitor cells into granulopoietic cells. For example, priming may begin on the first day of progenitor cell culture, on the second day of progenitor cell culture, on the third day of progenitor cell culture, on the fourth day of progenitor cell culture, or on the fifth day of progenitor cell culture under conditions that promote their differentiation into granulopoietic cells.

[0143] Alternatively, in a suitable embodiment, a priming step may occur after the granulopoietic cells have been produced and, optionally, after the granulopoietic cells have been harvested. For example, the Petition 870250101699, dated 06 / 11 / 2025, page 33 / 275 28 / 243 priming can occur before or after cryopreservation of a population of granulopoietic cells according to the invention.

[0144] By way of example only, in the case of priming steps performed over two days, priming may occur on days 3 and 4 of the culture conditions that promote the differentiation of progenitor cells into granulopoietic cells, on days 4 and 5 of such a culture, or on days 5 and 6 of such a culture. For the avoidance of doubt, any of the priming protocols described above may be appropriately performed on days 3 and 4, days 4 and 5, or days 5 and 6 of the culture conditions that promote the differentiation of progenitor cells into granulopoietic cells.

[0145] The priming steps developed by the inventors do not appear to significantly influence the immunomodulatory capacity of granulopoietic cell populations. Consequently, in embodiments where it is exclusively desired to make use of the immunomodulatory activities of granulopoietic cells, it may be preferable to exclude the priming steps from the methods by which granulopoietic cell populations are produced.

[0146] The methods of the first aspect of the invention result in the production of populations of granulopoietic cells, which are the subject of the second aspect of the invention. Similarly, the methods of the third aspect of the invention relate to the priming of granulopoietic cells and result in populations of cells according to the fourth aspect of the invention. Granulopoietic cells are also used in the pharmaceutical compositions, medical uses, and treatment methods of the invention. Except where the context may require otherwise, the following definitions are applicable to granulopoietic cells in each of these aspects of the invention, or in any other situation where granulopoietic cells, or populations of such cells, are referred to.

[0147] Any of the cells or cell populations disclosed in this document may be derived from a mammal, such as a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Properly, the cell or cell population is derived from a human. Thus, the cell may be a human cell or the cell population may be a population of cells Petition 870250101699, dated 06 / 11 / 2025, page 34 / 275 29 / 243 human. In particular, a granulopoietic cell, or population of granulopoietic cells, disclosed in this document may be derived from a mammal, such as a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Appropriately, the granulopoietic cell or population of granulopoietic cells is derived from a human. Thus, the granulopoietic cell may be a human granulopoietic cell. The population of granulopoietic cells may be a population of human granulopoietic cells.

[0148] To be considered granulopoietic within the terms of the present invention, a cell must be capable of giving rise to granulocytes (e.g., neutrophils) or to granulocyte precursor cells of the granulocytic lineage. In fact, a suitable granulopoietic cell can give rise to such cells. For the avoidance of doubt, granulocytes themselves should be considered granulopoietic for the purposes of the present invention, although in many embodiments granulopoietic cells are not granulocytes, but rather cells capable of giving rise to granulocytes. Appropriately, granulopoietic cells in the context of the present invention can be taken as excluding other cell lineages, for example, excluding monocyte lineages and / or lymphocyte lineages.

[0149] Suitable granulopoietic cell populations in the context of the present invention can be defined with reference to their expression of 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 desired, enriched, based on their expression of specific cell surface marker profiles.

[0150] The following definitions, based on appropriate marker expression profiles, can be used alone or in combination to identify suitable populations of granulopoietic cells.

[0151] Unless otherwise specified (for example, in lists reciting or or and / or), references in this disclosure to cells being positive or negative for the expression of a specified number of markers should be taken as requiring that the cells in question have Petition 870250101699, dated 06 / 11 / 2025, page 35 / 275 30 / 243 the recited expression (positive or negative) of each of the referred markers. Thus, by way of example, the reference to a cell, or population of cells, as CD15+ CD66b+ should be taken as meaning that the cell is positive for the expression of CD15 and CD66b and that the cell population comprises cells that are CD15+ as well as cells that are CD66b+.

[0152] This disclosure includes definitions of populations, or subpopulations, of cells with reference to a recited expression (positive or negative) of a number of specified markers.

[0153] In a suitable embodiment, such definitions may be taken as requiring that the population, or subpopulation, in question comprise cells that are positive or negative (as required by the definition) for the cited markers. For example, in the case of a population defined as positive for the expression of the first marker, negative for the expression of a second marker, and positive for the expression of a third marker, this requirement may be met by a population of cells comprising cells positive for the first marker, while also comprising cells negative for the second marker and further comprising cells positive for the third marker. In such an embodiment, the population, or subpopulation, of cells may be heterogeneous with respect to the cells that have the cited expression (positive or negative).Appropriately, cells in which each exhibits the required expression with respect to each of the cited markers may constitute the largest group of cells within such a population or subpopulation. Appropriately, cells in which each exhibits the required expression with respect to each of the cited markers may constitute the majority of cells within such a population or subpopulation. Appropriately, cells in which each exhibits the required expression with respect to each of the cited markers may provide 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 within such a population or subpopulation. Petition 870250101699, dated 06 / 11 / 2025, page 36 / 275 31 / 243

[0154] In one embodiment, in a given population or subpopulation, a cell in that population or subpopulation may express at least 2, 3, 4, or 5 of the markers mentioned. In one embodiment, in a given population or subpopulation, each of the cells in the population or subpopulation may express at least 2, 3, 4, or 5 of the markers mentioned.

[0155] In a suitable embodiment, such definitions may be taken as requiring that the population, or subpopulation, in question consist of cells that are positive or negative (as required by the definition) for the cited markers. In such embodiment, the population, or subpopulation, of cells is homogeneous with respect to cells that have the cited expression (positive or negative).

[0156] In a suitable embodiment, a granulopoietic cell population comprises cells that are Lin- (i.e., negative for a cocktail of common leukocyte lineage markers, defined for the present purposes as negative for the expression of each of CD3, CD16, CD19, CD20, CD14, and CD56). For example, a suitable granulopoietic cell population may comprise 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. By way of example, a suitable granulopoietic cell population may comprise at least 90% Lin- cells. A suitable granulopoietic cell population may comprise approximately 95-99% Lin- cells. Appropriately, a granulopoietic cell population comprises approximately 97% Lin- cells.

[0157] Alternatively, or additionally, a suitable population of granulopoietic cells comprises CD34+ cells. For example, such a population of granulopoietic cells may comprise less than 50%, less than 45%, less than 40%, or less than 35% of CD34+ cells. By way of example, such a population of granulopoietic cells may comprise less than 30% of CD34+ cells. In such an embodiment, the proportion of CD34+ cells may be approximately 525%. Suitablely, a population of granulopoietic cells comprises Petition 870250101699, dated 06 / 11 / 2025, page 37 / 275 32 / 243 approximately 14% CD34+ cells.

[0158] Alternatively, or additionally, a suitable population of granulopoietic cells comprises CD38+ cells. For example, such a population of granulopoietic cells may comprise at least 10%, at least 15%, or at least 20% of CD38+ cells. In such an embodiment, the proportion of CD38+ cells may be between approximately 10% and 80%, as well as between approximately 10% and 30%. Suitablely, a population of granulopoietic cells comprises approximately 12% of CD38+ cells.

[0159] Alternatively, or additionally, a suitable population of granulopoietic cells comprises cells with a hematopoietic stem cell (HSC) phenotype (defined for the present purposes as Lin-CD34+CD38CD45RA-CD90+). For example, such a population of granulopoietic cells may comprise less than 5%, less than 4%, less than 3%, or less than 2% of cells with an HSC phenotype. By way of example, such a population of granulopoietic cells may comprise less than 1% of cells with an HSC phenotype. A suitable population of granulopoietic cells may comprise approximately 0.01–0.15% of cells with an HSC phenotype. Appropriately, a population of granulopoietic cells comprises approximately 0.04% of cells with an HSC phenotype.

[0160] Alternatively, or additionally, an adequate granulopoietic cell population comprises less than 1% of cells with a long-term repopulation hematopoietic stem cell (LT-HSC) phenotype (defined for the present purposes as Lin-CD34+CD38-CD45RA-CD90+CD49f+). For example, such a granulopoietic cell population may comprise less than 5%, less than 4%, less than 3%, or less than 2% of cells with an LT-HSC phenotype. As an example, such a granulopoietic cell population may comprise less than 1% of cells with an LT-HSC phenotype. An adequate granulopoietic cell population may comprise approximately 0.010.05% of cells with an LT-HSC phenotype. Appropriately, a population of granulopoietic cells comprises approximately 0.02% of cells with a Petition 870250101699, dated 06 / 11 / 2025, page 38 / 275 33 / 243 LT-HSC phenotype.

[0161] Alternatively, or additionally, a suitable granulopoietic cell population comprises cells with a lymphoid-primed multipotent progenitor (LMPP) phenotype (defined for the present purposes as Lin-CD34+CD38-CD45RA+). For example, such a granulopoietic cell population may comprise less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, or less than 25% of cells with an LMPP phenotype. As an example, such a granulopoietic cell population may comprise less than 20% of cells with an LMPP phenotype. A suitable granulopoietic cell population may comprise approximately 2-15% of cells with an LMPP phenotype. Appropriately, a granulopoietic cell population comprises approximately 5% of cells with an LMPP phenotype.

[0162] Alternatively, or additionally, a suitable population of granulopoietic cells comprises cells with a multipotent progenitor (MPP) phenotype (defined for the present purposes as Lin-CD34+CD38-CD45RA). For example, such a population of granulopoietic cells may comprise less than 30%, less than 25%, less than 20%, or less than 15% of cells with an MPP phenotype. By way of example, such a population of granulopoietic cells may comprise less than 10% of cells with an MPP phenotype. A suitable population of granulopoietic cells may comprise approximately 1-6% of cells with an MPP phenotype. Suitablely, a population of granulopoietic cells comprises approximately 2% of cells with an MPP phenotype.

[0163] In a suitable embodiment, a granulopoietic cell population may comprise 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% cells with an HSC phenotype as defined above (e.g., Petition 870250101699, dated 06 / 11 / 2025, page 39 / 275 34 / 243 approximately 0.04% of cells with an HSC phenotype) and / or less than 1% of cells with an LT-HSC phenotype as defined above (e.g., approximately 0.02% of cells with an LT-HSC phenotype) and / or less than 20% of cells with an LMPP phenotype as defined above (e.g., approximately 5% of cells with an LMPP phenotype) and / or less than 10% of cells with an MPP phenotype as defined above (e.g., approximately 2.5% of cells with an MPP phenotype).

[0164] In a suitable embodiment, a granulopoietic cell population may comprise more than 90% Lin- cells (e.g., approximately 97% Lin- cells) and less than 30% CD34+ cells (e.g., approximately 14% CD34+ cells) and more than 10% CD38+ cells (e.g., approximately 12% CD38+ cells) and less than 1% cells with an HSC phenotype as defined above (e.g., approximately 0.04% cells with an HSC phenotype) and less than 1% cells with an LT-HSC phenotype as defined above (e.g., approximately 0.02% cells with an LT-HSC phenotype) and less than 20% cells with an LMPP phenotype as defined above (e.g., approximately 5% cells with an LMPP phenotype) and less than 10% cells with a MPP phenotype as defined above (e.g., approximately 2.5% of cells with an MPP phenotype).

[0165] Alternatively, or additionally, an adequate population of granulopoietic cells may comprise a ratio of CD15- to CD15+ cells that is approximately 1:1.

[0166] An adequate population of granulopoietic cells may comprise approximately 25-75%, or 35-60 CD15- cells. For example, an adequate population of granulopoietic cells may comprise approximately 50% CD15- cells.

[0167] An adequate population of granulopoietic cells may comprise approximately 30-70%, or 40-65%, of CD15+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 50% of Petition 870250101699, dated 06 / 11 / 2025, page 40 / 275 35 / 243 CD15+ cells.

[0168] An adequate population of granulopoietic cells may comprise approximately 5-25%, 5-20%, 7-18%, or 10-15% of CD15+CD66b+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 12% of CD15+CD66b+ cells.

[0169] An adequate population of granulopoietic cells may comprise approximately less than 30% or less than 25% of CD11b+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 10-25% or 15-25% of CD11b+ cells, for example, approximately 19% of CD11b+ cells.

[0170] An adequate population of granulopoietic cells may comprise at least 30%, at least 35%, at least 40%, or at least 45% CD71+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 60% CD71+ cells.

[0171] An adequate population of granulopoietic cells may comprise approximately 60-95%, or 65-90% of CD49d+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 75% of CD49d+ cells.

[0172] An adequate population of granulopoietic cells may comprise less than 5%, less than 4%, less than 3%, or less than 2% of CD10+ cells. An adequate population of granulopoietic cells may comprise approximately 0.03-2% of CD10+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 0.5% of CD10+ cells.

[0173] An adequate population of granulopoietic cells may comprise approximately 1-120%, or 2-15% of CD177+ cells. An adequate population of granulopoietic cells may comprise approximately 6% of CD177+ cells.

[0174] An adequate population of granulopoietic cells may comprise less than 20% or less than 15% CD62L+ cells. For example, an adequate population of granulopoietic cells may comprise between approximately 2 Petition 870250101699, dated 06 / 11 / 2025, page 41 / 275 36 / 243 15%, for example, approximately 8% of CD62L+ cells.

[0175] An adequate population of granulopoietic cells may comprise approximately 40-85%, or 50-75%, of CD54+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 63% of CD54+ cells.

[0176] An adequate population of granulopoietic cells may comprise approximately 2-15%, or approximately 5-10% of CD63+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 7% of CD63+ cells.

[0177] An adequate population of granulopoietic cells may comprise approximately 70-90%, or 75-85% CD18+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 80% CD18+ cells.

[0178] An adequate population of granulopoietic cells may comprise approximately 35-55% HLA-DR+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 47% HLA-DR+ cells.

[0179] An adequate population of granulopoietic cells may comprise approximately 6-8% CD115+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 5% CD115+ cells.

[0180] An adequate population of granulopoietic cells may comprise approximately 5-30% CD40+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 15% CD40+ cells.

[0181] An adequate population of granulopoietic cells may comprise approximately 5-30% CD64+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 15% CD64+ cells.

[0182] An adequate population of granulopoietic cells may comprise Petition 870250101699, dated 06 / 11 / 2025, page 42 / 275 37 / 243 approximately 20-55% of cells are CD32+. For example, an adequate population of granulopoietic cells may comprise approximately 40% CD32+ cells.

[0183] An adequate population of granulopoietic cells may comprise approximately 4-9% CXCR2+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 6% CXCR2+ cells.

[0184] An adequate population of granulopoietic cells may comprise approximately 0.04-1% CD16+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 0.25% CD16+ cells.

[0185] An adequate population of granulopoietic cells may comprise approximately 2-15% CD14+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 8% CD14+ cells.

[0186] An adequate population of granulopoietic cells may comprise approximately 0.5-4% CD68+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 1.5% CD68+ cells.

[0187] An adequate population of granulopoietic cells may comprise approximately 2-18% CD206+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 10% CD206+ cells.

[0188] The fifth aspect of the invention provides an isolated population of granulopoietic cells comprising: • more than 90% of Lin- cells (for example, approximately 97% of Lin- cells); • less than 30% CD34+ cells (for example, approximately 14% CD34+ cells); • more than 30% of CD38+ cells (for example, approximately 65%) Petition 870250101699, dated 06 / 11 / 2025, page 43 / 275 38 / 243 of CD38+ cells); • less than 1% of cells with an HSC phenotype (e.g., approximately 0.04% of cells with an HSC phenotype); • less than 1% of cells with an LT-HSC phenotype (e.g., approximately 0.02% of cells with an LT-HSC phenotype; • less than 20% of cells with an LMPP phenotype (e.g., approximately 5% of cells with an LMPP phenotype); and • less than 10% of cells with an MPP phenotype (e.g., approximately 2.5% of cells with an MPP phenotype).

[0189] The sixth aspect of the invention provides an isolated population of granulopoietic cells comprising: • a first subpopulation of cells that are CD15+ CD64+ CD18+ CD49d+ CD71+ • a second subpopulation of cells that are CD15- CD11b+ / CD18+ CD49d+ CD32+ HLA-DR- • a third subpopulation of cells that are CD15- CD11b- HLA-DR+ CD18+ CD49d+ and CD71+.

[0190] A population of granulopoietic cells according to the sixth aspect of the invention may further comprise a fourth subpopulation of cells that are CD15- CD11 b+ HLA-DR+.

[0191] It will be appreciated that, having been informed of the markers expressed by these cell subpopulations, one or more of these subpopulations can be readily isolated from within the cell populations of the sixth aspect of the invention. This gives rise to other aspects of the invention.

[0192] In a seventh aspect, the invention provides a population of granulopoietic cells that are CD15+ CD64+ CD18+ CD49d+ CD71+. A suitable population of such cells (which may also constitute a first subpopulation of cells in the context of the sixth aspect of the invention) may also be positive for one, more than one, or all of the selected markers from the group consisting of: CD177, CD11b, CD71, CD66b, HLA-DR, CD115, CD49d, CD40, CD62L, CD54, Petition 870250101699, dated 06 / 11 / 2025, page 44 / 275 39 / 243 CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1,4-1BBL, OX40L, PD-L1, and CD14. A cell population of the seventh aspect of the invention may be negative for the markers CD16 and / or CD62L (in addition to the required or optional expression or lack of expression of the other markers discussed above). Suitably, the cell population, or subpopulation, is heterogeneous for the cited marker profile (which may suitably include the optional constituents referred to in this document). Suitably, a cell population, or subpopulation, according to this aspect of the invention is homogeneously positive for CD15 and heterogeneous with respect to the other markers of the cited marker profile (which may suitably include the optional constituents referred to in this document). Suitably, the cell population, or subpopulation, is homogeneous for the cited marker profile (which may suitably include the optional constituents referred to in this document).

[0193] The first subpopulation of cells present in a population of granulopoietic cells according to the sixth aspect of the invention, or cells according to the seventh aspect of the invention, expresses markers that resemble those expressed by committed neutrophil precursors. However, the cells disclosed according to this aspect of the invention are CD64+ and may be CD16- and / or CD62L-. This is in contrast to neutrophil precursors found in circulation and at times of homeostasis, which are CD64- CD16+ and CD62L+. The expression of CD64 by CD15+ CD64+ CD18+ CD49d+ CD71+ cells thus provides a useful means by which the cells disclosed in this document can be distinguished from those of natural occurrence, as well as the lack of expression of CD16 and / or CD62L.A cell, or a population of cells, that are CD15+ CD64+ CD18+ CD49d+ CD71+ and also CD16- and / or CD62L- can be distinguished as one that was produced by the method according to the invention, rather than a naturally occurring granulopoietic cell, or population of such cells.

[0194] The inventors identified that the cells of a first subpopulation of cells present in a population of granulopoietic cells according to Petition 870250101699, dated 06 / 11 / 2025, page 45 / 275 40 / 243 The sixth aspect of the invention, or cells of the seventh aspect of the invention, demonstrate cytocidal activity that makes them particularly effective in terms of their medical uses. In fact, such cells appear to constitute the main source of cytocidal activity in cell populations according to the sixth aspect of the invention. Thus, such cells can be particularly useful in clinical contexts where it is necessary to kill cells (such as cancer cells, infected cells or cellular infectious agents) to achieve a therapeutic effect.

[0195] The first subpopulation of cells present in a population of granulopoietic cells according to the sixth aspect of the invention, or cells of the seventh aspect of the invention, may express 4-1BBL and / or OX40L. These markers are ligands for T cells and NK cells, and their expression by these cells may indicate that the cells will have immunomodulatory activities. Similarly, the first subpopulation of cells present in a population of granulopoietic cells according to the sixth aspect of the invention, or cells of the seventh aspect of the invention, may express CD38 and / or CD40 and / or CD54, other co-stimulatory molecules associated with functional interactions with immune cells, such as T cells. Consequently, such cells, or pharmaceutical compositions comprising such cells, may be effective in biological or therapeutic applications by modulating the activity of such types of non-granulocytic inflammatory cells.

[0196] In addition to expressing markers indicative of immunomodulatory capacity, this cell population also expresses molecules (in particular CD11b, CD18, Mac1 and CD32) that suggest they possess direct cytocidal activity. This may make them suitable for uses where it is desired to therapeutically kill cells, such as cancerous or infected cells.

[0197] In an eighth aspect, the invention provides a population of cells that are CD15- CD11b+ / - CD18+ CD49d+ CD32+ HLA-DR-. A suitable population of such cells (which may also constitute a second subpopulation of cells in the context of the sixth aspect of the invention) may also be positive for one, more than one, or all of the selected markers from the group consisting of: CD177, Petition 870250101699, dated 06 / 11 / 2025, page 46 / 275 41 / 243 CD11b, CD71, CD66b, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14. Appropriately, the population, or subpopulation, of cells is heterogeneous for the cited marker profile (which may appropriately include the optional constituents referred to in this document). Appropriately, a population, or subpopulation, of cells according to this aspect of the invention is homogeneously negative for CD15 and HLA-DR, and heterogeneous with respect to the other markers of the cited marker profile (which may appropriately include the optional constituents referred to in this document). Appropriately, a population, or subpopulation, of cells according to this modality is homogeneously negative for CD15, HLA-DR, and CD11b, and heterogeneous with respect to the other markers of the cited marker profile (which may appropriately include the optional constituents referred to in this document).Appropriately, a population, or subpopulation, of cells according to this embodiment is homogeneously positive for CD11b and homogeneously negative for CD15 and HLA-DR, and heterogeneous with respect to the other markers of the cited marker profile (which may appropriately include the optional constituents referred to in this document). Appropriately, the population, or subpopulation, of cells is homogeneous for the cited marker profile (which may appropriately include the optional constituents referred to in this document).

[0198] The second subpopulation of cells present in a population of granulopoietic cells according to the sixth aspect of the invention, or cells according to the eighth aspect of the invention, expresses markers such as Mac-1 (comprising CD11b and CD18) and CD32, which are consistent with a high capacity for cytotoxic activity. Consequently, these cells may also be beneficial in medical uses or treatment methods where direct cytocidal activity is required, such as the killing of cancerous or infected cells. These cells may also express molecules such as 4-1BBL and / or OX40L, indicating their immunomodulatory potential and suitability for use in biological or therapeutic applications requiring such activity. The cells of this Petition 870250101699, dated 06 / 11 / 2025, page 47 / 275 Group 42 / 243 may also express CXCR2, which can be elevated by their exposure to IL-3 during the methods according to the invention, a marker that may contribute to increased chemotaxis (in response to agents such as IL-8) and targeting of these cells to the TME.

[0199] In a ninth aspect, the invention provides a population of cells that are CD15- CD11b- HLA-DR+ CD18+ CD49d+ and CD71+. A suitable population of such cells (which may also constitute a third subpopulation of cells in the context of the sixth aspect of the invention) may also be positive for one, more than one, 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. Suitably, the population, or subpopulation, of cells is heterogeneous for the aforementioned marker profile (which may suitably include the optional constituents referred to in this document).Appropriately, a population, or subpopulation, of cells according to this aspect of the invention is homogeneously negative for CD15 and CD11b and homogeneously positive for HLA-DR, and heterogeneous with respect to the other markers of the cited marker profile (which may appropriately include the optional constituents referred to in this document). Appropriately, the population, or subpopulation, of cells is homogeneous for the cited marker profile (which may appropriately include the optional constituents referred to in this document).

[0200] The third subpopulation of cells present in a population of granulopoietic cells according to the sixth aspect of the invention, or cells according to the ninth aspect of the invention, expresses markers indicative of a relatively low level of differentiation. Accordingly, these cells may also be CD34+. The cells in this group may also express markers such as 4-1BBL and / or OX40L and / or CD40 and / or CD54 that indicate their suitability for use in applications requiring immunomodulation of non-granulocytic immune cells. Although the cells in this group do not express markers indicative of direct cytocidal activity, they may have the ability to differentiate further and express markers such as CD11b and CD15 that Petition 870250101699, dated 06 / 11 / 2025, page 48 / 275 43 / 243 would confer such activity. Consequently, these cells could be employed in medical uses or treatment methods where in vivo signals would induce such differentiation, leading to the ability to kill deleterious cell types.

[0201] In a tenth aspect, the invention provides a population of granulopoietic cells that are CD15-CD11b+ HLA-DR+. A suitable population of such cells (which may also constitute an optional fourth subpopulation of cells in the context of the sixth aspect of the invention) may also be positive for one, more than one, 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. Suitably, the population, or subpopulation, of cells is heterogeneous for the cited marker profile (which may suitably include the optional constituents referred to in this document).Appropriately, a population, or subpopulation, of cells according to this aspect of the invention is homogeneously negative for CD15 and homogeneously positive for HLA-DR and CD11b, and heterogeneous with respect to the other markers of the cited marker profile (which may appropriately include the optional constituents referred to in this document). Appropriately, the population, or subpopulation, of cells is homogeneous for the cited marker profile (which may appropriately include the optional constituents referred to in this document).

[0202] These cells according to the tenth aspect of the invention, which may optionally also be present in granulopoietic cell populations according to the sixth aspect of the invention, express markers that are similar to those that would be expected from activated myeloid cells. The cells may also express markers such as CD14 and / or CD11b and / or CD206. They may be suitable for use in applications where direct cytocidal or immunomodulatory activity is desired.

[0203] A granulopoietic cell from a population produced by a method of the invention, or present in a composition of the invention, may be CD64+, CD16e- or CD62L-. For example, the granulopoietic cell may be CD64+. The cell Petition 870250101699, dated 06 / 11 / 2025, page 49 / 275 44 / 243 Granulopoietic cells can be CD64+ and CD16-. The granulopoietic cell can be CD64+ and CD62L-. The granulopoietic cell can be CD16- and CD62L-. Appropriately, the granulopoietic cell is CD64+, CD16-, and CD62L-. The expression of CD64 and the lack of expression of CD16 and CD62L by granulopoietic cells of the invention contrast with neutrophils found in circulation and at times of homeostasis, which are CD64-, CD16+, and CD62L+. The expression of CD64 thus provides a useful means by which the granulopoietic cell disclosed in this document can be distinguished from those of natural occurrence, as does the lack of expression of CD16 and / or CD62L. A granulopoietic cell that is CD64+, CD16-, and / or CD62L- can be distinguished as one that was produced by a method according to the invention, rather than a naturally occurring granulopoietic cell or population of such cells.

[0204] Thus, in one aspect, the invention provides a granulopoietic cell that is a CD64+ granulopoietic cell, or a population of such cells. Suitably, the CD64+ granulopoietic cell is a CD64+ and CD16- granulopoietic cell. The CD64+ granulopoietic cell may be a CD64+ and CD62L- granulopoietic cell. The CD64+ granulopoietic cell may be a CD64+, CD16- and CD62L- granulopoietic cell. Cell populations with each of these expression profiles are provided according to this aspect of the invention.

[0205] In a related aspect, the invention provides a granulopoietic cell that is a CD16 granulopoietic cell. The CD16- granulopoietic cell can be a CD16 and CD62L granulopoietic cell.

[0206] In another related aspect, the invention provides a granulopoietic cell that is a CD62L- granulopoietic cell.

[0207] In an eleventh aspect, the invention provides a pharmaceutical composition comprising a population of granulopoietic cells. Suitably, the population of granulopoietic cells may be a population according to the second, or fourth to tenth aspects of the invention.

[0208] In a twelfth aspect, the invention provides the use of a Petition 870250101699, dated 06 / 11 / 2025, page 50 / 275 45 / 243 population of granulopoietic cells in the manufacture of a medicinal product. Suitablely, the population of granulopoietic cells may be a population according to the second, or fourth to tenth aspects of the invention.

[0209] In a thirteenth aspect, the invention provides a pharmaceutical composition of the sixth aspect of the invention, or a population of granulopoietic cells according to the second, or fourth to tenth aspects of the invention, for use as a medicament.

[0210] In a fourteenth aspect, the invention provides a method of treating a disease or disorder in a subject, the method comprising administering a pharmaceutical composition according to the sixth aspect of the invention, or a population of granulopoietic cells according to the second, or fourth to tenth aspects of the invention, to the subject.

[0211] In one aspect, the invention provides a pharmaceutical composition of the invention for use in a method of treating a disease or disorder in a subject.

[0212] In one aspect, the invention provides a pharmaceutical composition of the invention for use in medicine.

[0213] In one aspect, the invention provides a method of treating a disease or disorder in a subject comprising administering a pharmaceutical composition of the invention to the subject.

[0214] In one aspect, the invention provides a pharmaceutical composition of the invention for use in a method of treating cancer in a subject.

[0215] In one aspect, the invention provides a method of treating cancer in a subject comprising administering a pharmaceutical composition of the invention to the subject.

[0216] In one aspect, the invention provides the use of a pharmaceutical composition of the invention in the manufacture of a medicament for treating cancer in a subject.

[0217] In one aspect, the invention provides a pharmaceutical composition of the invention for use in a method of treating an infection in a subject. Petition 870250101699, dated 06 / 11 / 2025, page 51 / 275 46 / 243

[0218] In one aspect, the invention provides a method of treating an infection in a subject comprising administering a pharmaceutical composition of the invention to the subject.

[0219] In one aspect, the invention provides the use of a pharmaceutical composition of the invention in the manufacture of a medicament for treating an infection in a subject.

[0220] In one aspect, the invention provides a pharmaceutical composition of the invention, for use in amplifying a non-granulocytic therapeutic immune response.

[0221] In one aspect, the invention provides a treatment method comprising amplifying a non-granulocytic therapeutic immune response, the method comprising delivering a pharmaceutical composition of the invention to a subject in need of such treatment.

[0222] In one aspect, the invention provides a pharmaceutical composition of the invention for use in the manufacture of a medicament for use in amplifying a non-granulocytic therapeutic immune response.

[0223] The present invention is based, at least to some extent, on the inventors' development of a method capable of generating populations of granulopoietic cells with properties that are highly beneficial in a wide range of therapeutic contexts. The methods of the first aspect of the invention result in the production of populations of granulopoietic cells, which are the subject of the second aspect of the invention. Similarly, the methods of the third aspect of the invention relate to the priming of granulopoietic cells and result in cell populations according to the fourth aspect of the invention. Granulopoietic cells are also used in the pharmaceutical compositions, medical uses and treatment methods of the invention.

[0224] The granulopoietic cell populations described in this document, such as those that can be produced using the methods of the invention, are capable of amplifying the therapeutic immune response of non-granulocytic immune cells. In suitable embodiments, the cell populations Petition 870250101699, dated 06 / 11 / 2025, page 52 / 275 47 / 243 Granulopoietic cells amplify an immune response from non-granulocytic immune cells. Such populations of granulopoietic cells, or pharmaceutical compositions comprising such populations, can be used to treat a range of conditions, including (but not limited to) cancer and the treatment of infections. These populations and compositions can also be used to augment immunotherapeutic treatments in a range of conditions, including (but not limited to) cancer therapies and the treatment of infections.

[0225] As established above, granulopoietic cell populations may comprise cells expressing markers such as 4-1BBL and / or OX40L and / or CD40 and / or CD54, associated with interaction with non-granulocytic immune cells. Such cells, or pharmaceutical compositions comprising such cells, may be employed in medical uses or treatment methods requiring beneficial immunomodulatory activity.

[0226] Alternatively, or additionally, suitable populations of granulopoietic cells may comprise cells expressing markers such as Mac-1 (or its constituents CD11b and CD18) or CD32, which are indicative of a capacity for direct cytocidal activity. Such cells, or pharmaceutical compositions comprising such cells, may be employed in medical uses or treatment methods that require cell death, such as cancerous or infected cells.

[0227] The methods of the invention make use of progenitor cell populations as the starting material from which granulopoietic cells are produced. As noted above, some embodiments of the methods of the invention may also incorporate an optional step of culturing a population of stem cells to produce a population of progenitor cells.

[0228] Similar to the granulopoietic cell populations discussed above, progenitor cells and progenitor cell populations, in the context of this disclosure, can be usefully defined by means of their expression of marker profiles and phenotypes. The following definitions, based on appropriate marker expression profiles, can be used alone or Petition 870250101699, dated 06 / 11 / 2025, page 53 / 275 48 / 243 in combination to identify suitable populations of progenitor cells. Except where the context requires otherwise, they should be considered applicable to progenitor cells as referred to in any embodiment of the invention.

[0229] In a suitable embodiment, a progenitor cell population comprises cells that are Lin- (as defined above). For example, a suitable progenitor cell population may comprise 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% of Lin- cells. By way of example, a suitable progenitor cell population may comprise at least 98% of Lin- cells. A suitable progenitor cell population may comprise approximately 98-99% of Lin- cells. Suitablely, a progenitor cell population comprises approximately 99% of Lin- cells.

[0230] Alternatively, or additionally, a suitable progenitor cell population comprises CD34+ cells. For example, such a progenitor cell population may comprise between approximately 5-90% or approximately 10-85% CD34+ cells. As an example, such a progenitor cell population may comprise between approximately 15-80% CD34+ cells. In such an embodiment, the proportion of CD34+ cells may be between approximately 20-70%. Suitablely, a progenitor cell population comprises approximately 43% CD34+ cells.

[0231] Alternatively, or additionally, a suitable progenitor cell population comprises CD38+ cells. For example, such a progenitor cell population may comprise approximately 10-65%, approximately 15-60%, or approximately 20-55% CD38+ cells. As an example, such a progenitor cell population may comprise approximately 25-50% CD38+ cells. In such an embodiment, the proportion of CD38+ cells may be between approximately 30% and 41%. Suitablely, a progenitor cell population comprises approximately 35% of Petition 870250101699, dated 06 / 11 / 2025, page 54 / 275 49 / 243 CD38+ cells.

[0232] Alternatively, or additionally, a suitable population of progenitor cells comprises cells with an HSC phenotype. For example, such a population of progenitor cells may comprise less than 5%, less than 4%, less than 3%, or less than 2% of cells with an HSC phenotype. By way of example, such a population of progenitor cells may comprise less than 1% of cells with an HSC phenotype. A suitable population of progenitor cells may comprise approximately 0.01–0.7% of cells with an HSC phenotype. Suitablely, a population of progenitor cells comprises approximately 0.3% of cells with an HSC phenotype.

[0233] Alternatively, or additionally, a suitable progenitor cell population comprises cells with an LT-HSC phenotype. For example, such a progenitor cell population may comprise less than 5%, less than 4%, less than 3%, or less than 2% of cells with an LT-HSC phenotype. As an example, such a progenitor cell population may comprise less than 1% of cells with an LT-HSC phenotype. A suitable progenitor cell population may comprise approximately 0.01–0.03% of cells with an LT-HSC phenotype. Appropriately, a progenitor cell population comprises approximately 0.02% of cells with an LT-HSC phenotype.

[0234] Alternatively, or additionally, a suitable progenitor cell population comprises cells with an LMPP phenotype. For example, such a progenitor cell population may comprise less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, or less than 45% of cells with an LMPP phenotype. As an example, such a progenitor cell population may comprise less than 40% of cells with an LMPP phenotype. A suitable progenitor cell population may comprise approximately 5-30% of cells with an LMPP phenotype. Appropriately, a progenitor cell population comprises approximately 13% of cells with an LMPP phenotype.

[0235] Alternatively, or additionally, an adequate population of Petition 870250101699, dated 06 / 11 / 2025, page 55 / 275 50 / 243 progenitor cells comprise cells with an MPP phenotype. For example, such a progenitor cell population may comprise less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, or less than 45% of cells with an MPP phenotype. As an example, such a progenitor cell population may comprise less than 40% of cells with an MPP phenotype. An adequate progenitor cell population may comprise approximately 1-35% of cells with an MPP phenotype. Appropriately, a progenitor cell population comprises approximately 13% of cells with an MPP phenotype.

[0236] In a suitable embodiment, a progenitor cell population may comprise 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% cells with an HSC phenotype as defined above (e.g., approximately 0.3% cells with an HSC phenotype) and / or less than 1% cells with an LT-HSC phenotype as defined above (e.g., approximately 0.02% cells with an LT-HSC phenotype) and / or less than 40% cells with an LMPP phenotype as defined above (e.g., approximately 13% cells with an LMPP phenotype) and / or less than 40% of cells with an MPP phenotype as defined above (e.g., approximately 13% of cells with an MPP phenotype).

[0237] In a suitable embodiment, a progenitor cell population may comprise 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% cells with an HSC phenotype as defined above (e.g., approximately 0.3% cells with an HSC phenotype), less than 1% cells with an LT-HSC phenotype as defined above (e.g., approximately 0.02% cells with a LT-HSC phenotype). Petition 870250101699, dated 06 / 11 / 2025, page 56 / 275 51 / 243 of LT-HSC), less than 40% of cells with an LMPP phenotype as defined above (e.g., approximately 13% of cells with an LMPP phenotype) and less than 40% of cells with an MPP phenotype as defined above (e.g., approximately 13% of cells with an MPP phenotype).

[0238] Alternatively, or additionally, a suitable population of progenitor cells may comprise a ratio of CD15- to CD15+ cells that is approximately 2:1.

[0239] An adequate population of progenitor cells may comprise approximately 60-95% CD15- cells. For example, an adequate population of progenitor cells may comprise approximately 71% CD15- cells.

[0240] An adequate population of progenitor cells may comprise approximately 10-50% CD15+ cells. For example, an adequate population of progenitor cells may comprise approximately 35% CD15+ cells.

[0241] An adequate population of progenitor cells may comprise approximately 0.02-1% of CD15+CD66b+ cells. For example, an adequate population of progenitor cells may comprise approximately 0.04-0.47% or 0.24% of CD15+CD66b+ cells.

[0242] An adequate population of progenitor cells may comprise less than 20% CD11b+ cells. For example, an adequate population of progenitor cells may comprise approximately 2-6% or approximately 3% CD11b+ cells.

[0243] An adequate population of progenitor cells may comprise approximately 25-60% CD71+ cells. For example, an adequate population of progenitor cells may comprise approximately 33% CD71+ cells.

[0244] An adequate population of progenitor cells may comprise approximately 90-100% CD49d+ cells. For example, an adequate population of progenitor cells may comprise approximately 95% CD49d+ cells.

[0245] An adequate population of progenitor cells may comprise approximately 0.01-1.5% CD10+ cells. For example, an adequate population of progenitor cells may comprise approximately 0.5% CD10+ cells. Petition 870250101699, dated 06 / 11 / 2025, page 57 / 275 52 / 243

[0246] An adequate population of progenitor cells may comprise approximately 0.25-3% of CD177+ cells. For example, an adequate population of progenitor cells may comprise approximately 1% of CD177+ cells.

[0247] An adequate population of progenitor cells may comprise approximately 20-60% or 40-60% CD62L+ cells. For example, an adequate population of progenitor cells may comprise approximately 46% CD62L+ cells.

[0248] An adequate population of progenitor cells may comprise approximately 1-17% CD54+ cells. For example, an adequate population of progenitor cells may comprise approximately 6% CD54+ cells.

[0249] An adequate population of progenitor cells may comprise approximately 2-20% CD63+ cells. For example, an adequate population of progenitor cells may comprise approximately 5% CD63+ cells.

[0250] An adequate population of progenitor cells may comprise approximately 70-90% CD18+ cells. For example, an adequate population of progenitor cells may comprise approximately 87% CD18+ cells.

[0251] The granulopoietic cell populations or pharmaceutical compositions of the invention can be used in a wide range of therapeutic applications, as disclosed in this document. In particular, the cell populations or pharmaceutical compositions can be used to enhance the activation or recruitment of host immune cells and, particularly, non-granulocytic immune cells, in a manner that allows for the amplification of a therapeutic host immune response. This embodiment enables such compositions to be used to augment immunotherapeutic treatments in a range of conditions, including (but not limited to) cancer therapies. By amplifying the host immune response, the compositions, medical uses, and treatment methods of the invention are able to render otherwise immunologically cold tumors warm and therefore responsive to treatment.

[0252] The amplification that occurs in relation to a therapeutic host immune response is not simply due to the generation of high numbers of Petition 870250101699, dated 06 / 11 / 2025, page 58 / 275 53 / 243 granulocytes and non-granulocytic immune cells, for example, as a result of administering the compositions of the invention. Instead, granulopoietic cells and compositions comprising said granulopoietic cells are capable of markedly increasing the activation of non-granulocytic immune cells and, particularly, T cells such as CD8, CD4 and γδ T cells; monocytes; macrophages; dendritic cells (DCs) and NK cells. Meanwhile, non-granulocytic immune cells are capable of significantly increasing the activation of granulopoietic cells. As discussed in more detail below, and as demonstrated in the Examples, this is capable of causing increased expression of degranulation markers, co-stimulatory molecules and cytokines through activation.

[0253] The inventors' surprising discovery that granulopoietic cell populations according to the present invention are capable of amplifying (and preferably serve to amplify) the therapeutic immune response of non-granulocytic immune cells also gives rise to other aspects and embodiments of the invention. As used in this document, an immune response encompasses any response of an immune cell to its environment. Immune cells are constantly responding to their environment, including in vitro, and therefore are constantly generating immune responses even during homeostasis. A therapeutic immune response can be an immune response that can contribute to the eradication of disease.A therapeutic immune response may include increased activation of an immune cell, increased expression of a cell degranulation marker by an immune cell, increased expression of a co-stimulatory molecule by an immune cell, or increased expression of a cytokine by an immune cell. Such therapeutic immune responses may occur in vitro or in vivo.

[0254] Thus, the inventors surprisingly showed that granulopoietic cells may be able to promote (preferably promote) the proliferation and / or survival of non-granulocytic immune cells, including NK cells and T cells, thereby allowing for increased ex vivo expansion of these types of Petition 870250101699, dated 06 / 11 / 2025, page 59 / 275 54 / 243 cells and improving their persistence in vivo. The inventors also showed that granulopoietic cells may be able to increase (preferably increase) the expression of co-stimulatory molecules, including 4-1BB and OX40, on non-granulocytic immune cells, such as NK cells and T cells, including γδ T cells, thus improving their therapeutic efficacy. The inventors also surprisingly discovered that non-granulocytic immune cells may be able to increase (preferably increase) the expression of co-stimulatory molecules, including CD54, on granulopoietic cells, thus improving the therapeutic efficacy of granulopoietic cells.

[0255] Compositions comprising granulopoietic cells and non-granulocytic immune cells may therefore be useful for therapy. Such compositions comprise cells with amplified therapeutic immune responses, which, in turn, may amplify a therapeutic immune response of the host, for example, after administration to a subject.

[0256] Advantageously, such compositions can help to successfully eradicate a tumor (e.g., cancer) by providing a combination of immune cells suitable for this purpose. This can be particularly advantageous in cases where a subject's own immune cells may be defective. Furthermore, the present invention may allow the production of such a composition without conventional manufacturing difficulties and / or without adverse immunogenic effects.

[0257] Consequently, in one aspect, the invention provides a composition comprising a population of granulopoietic cells and a non-granulocytic immune cell. Advantageously, such compositions can help to successfully eradicate a tumor (e.g., cancer) by providing a combination of immune cells suitable for this purpose. This can be particularly advantageous in cases where a subject's own immune cells may be defective. Furthermore, the present invention can enable the production of such a composition without conventional manufacturing difficulties and / or without adverse immunogenic effects.

[0258] T cells comprising an αβ T cell receptor (also referred to as αβ T cells) are generally considered the central cell type. Petition 870250101699, dated 06 / 11 / 2025, page 60 / 275 55 / 243 involved in the coordination of immune responses. However, the inventors surprisingly showed that granulopoietic cells can amplify the therapeutic immune responses of non-granulocytic immune cells in the absence of αβ T cells.

[0259] Consequently, in one aspect, the invention provides a composition comprising a population of granulopoietic cells and a non-granulocytic immune cell, wherein the composition does not comprise an αβ T cell. For example, the composition may comprise a population of granulopoietic cells and a terminally differentiated non-granulocytic immune cell, wherein the composition does not comprise an αβ T cell.

[0260] In one aspect, the invention provides a composition comprising a granulopoietic cell and a non-granulocytic immune cell, wherein the granulopoietic cell is capable of modulating (preferably modulating) the therapeutic immune response of the non-granulocytic immune cell.

[0261] In one aspect, the invention provides a composition comprising a population of granulopoietic cells and a non-granulocytic immune cell, wherein the population of granulopoietic cells is capable of amplifying (preferably amplifies) the therapeutic immune response of the non-granulocytic immune cell.

[0262] In one aspect, the invention provides a kit comprising: (a) the composition according to the invention; or (b) a population of granulopoietic cells and a non-granulocytic immune cell (for example, a terminally differentiated non-granulocytic immune cell); and (c) optionally, instructions for their use (for example, in the treatment of cancer).

[0263] In one aspect, the invention provides a method for manufacturing a composition (for example, a composition of the invention), the method comprising: cultivating PBMCs in the presence of granulopoietic cells, thereby forming the composition; and optionally depleting αβ T cells before, during or after the Petition 870250101699, dated 06 / 11 / 2025, page 61 / 275 56 / 243 culture.

[0264] In one aspect, the invention provides a method for manufacturing a composition (for example, a composition of the invention), the method comprising: culturing αβ T cell-depleted PBMCs under conditions that promote the differentiation of progenitor cells present in the αβ T cell-depleted PBMCs into granulopoietic cells, thereby forming the composition.

[0265] In one aspect, the invention provides a composition that can be obtained by a method of the invention.

[0266] In one aspect, the invention provides a composition of the invention for use in a method of treating a disease or disorder in a subject.

[0267] In one aspect, the invention provides a composition of the invention for use in medicine.

[0268] In one aspect, the invention provides a method of treating a disease or disorder in a subject comprising administering a composition of the invention to the subject.

[0269] In one aspect, the invention provides the use of a composition of the invention in the manufacture of a medicament.

[0270] In one aspect, the invention provides a composition of the invention for use in a method of treating cancer in a subject.

[0271] In one aspect, the invention provides a method of treating cancer in a subject comprising administering a composition of the invention to the subject.

[0272] In one aspect, the invention provides the use of a composition of the invention in the manufacture of a medicament for treating cancer in a subject.

[0273] In one aspect, the invention provides a composition of the invention for use in a method of treating an infection in a subject.

[0274] In one aspect, the invention provides a method of treating an infection in a subject comprising administering a composition of the invention to the subject.

[0275] In one aspect, the invention provides the use of a composition of the invention in the manufacture of a medicament for treating an infection in a subject. Petition 870250101699, dated 06 / 11 / 2025, page 62 / 275 57 / 243

[0276] In a therapeutic application, the composition can modulate (preferably amplify) a therapeutic immune response of the subject, such as a non-granulocytic therapeutic immune response of the subject.

[0277] In one aspect, the invention provides a composition of the invention, for use in modulating a non-granulocytic therapeutic immune response.

[0278] In one aspect, the invention provides a treatment method comprising modulating a non-granulocytic therapeutic immune response, the method comprising delivering a composition of the invention to a subject in need of such treatment.

[0279] In one aspect, the invention provides a composition of the invention, for use in amplifying a non-granulocytic therapeutic immune response.

[0280] In one aspect, the invention provides a treatment method comprising amplifying a non-granulocytic therapeutic immune response, the method comprising delivering a pharmaceutical composition of the invention to a subject in need of such treatment.

[0281] In one aspect, the invention provides a composition of the invention for use in the manufacture of a medicament for use in modulating a non-granulocytic therapeutic immune response.

[0282] In one aspect, the invention provides a composition of the invention for use in the manufacture of a medicament for use in amplifying a non-granulocytic therapeutic immune response.

[0283] The present invention is based, at least to some extent, on the inventors' discovery that the granulopoietic cells described herein may be able to amplify (preferably amplify) the therapeutic immune response of non-granulocytic immune cells. Advantageously, this may allow such granulopoietic cells to be combined with non-granulocytic immune cells to provide a composition that can be used to treat a range of conditions, including (but not limited to) cancer. Said compositions may also be used to augment immunotherapeutic treatments in a range of conditions, including (but not limited to) Petition 870250101699, dated 06 / 11 / 2025, page 63 / 275 58 / 243 cancer therapies.

[0284] The amplification of an immune response (e.g., a therapeutic immune response) can 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 co-stimulatory 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 trafficking by immune cells; increased cytocidal activity by immune cells; and / or increased tumor cell killing activity by immune cells.

[0285] The aforementioned compositions can also be used to enhance the activation or recruitment of host immune cells and, in particular, non-granulocytic immune cells, in a manner that allows for the amplification of a therapeutic host immune response. This embodiment may enable such compositions to be used to enhance immunotherapeutic treatments in a range of conditions, including (but not limited to) cancer. By amplifying the host immune response, the compositions, medical uses, and treatment methods of the invention may be able to render otherwise immunologically cold tumors warm and therefore responsive to treatment.

[0286] In one embodiment, the amplification that occurs in relation to a therapeutic host immune response is not simply due to the generation of elevated numbers of granulocytes and non-granulocytic immune cells, for example, as a result of administration of the compositions of the invention. Instead, granulopoietic cells and compositions comprising said granulopoietic cells may be able to markedly increase the activation of non-granulocytic immune cells and, particularly, T cells, such as γδ T cells; monocytes; macrophages; and NK cells. Meanwhile, non-granulocytic immune cells may be able to significantly increase the activation of granulopoietic cells. As discussed in more detail below, and as demonstrated in the Examples, this may be able to elicit an increase in Petition 870250101699, dated 06 / 11 / 2025, page 64 / 275 59 / 243 expression of degranulation markers, co-stimulatory molecules, and cytokines by activated granulopoietic and non-granulocytic cells. It can also increase the proliferation and survival of activated non-granulocytic cells, leading to increased accumulation of such cells. The inventors also demonstrated that activated non-granulocytic immune cells can show an increased degree of recruitment to the TME, as well as increased cytocidal activity (particularly increased tumor cell killing activity).

[0287] Surprisingly, the inventors discovered that these effects can be achieved using granulopoietic cells and / or non-granulocytic immune cells and compositions comprising said cells that are allogeneic with reference to the subject who will receive the granulopoietic cell population or composition therapeutically.

[0288] These properties suggest that granulopoietic cells, including compositions comprising granulopoietic cells and non-granulocytic immune cells, may be used therapeutically in the treatment of cancer and that such treatment may also be used to enhance other cell-based immunotherapies.

[0289] Furthermore, the granulopoietic cells of, or to be used in accordance with, the invention may be able to differentiate (preferably differentiate) into granulocytes with the ability to kill cancer cells. In this way, the compositions and treatments according to the invention may be able to achieve a dual mode of action, both amplifying a non-granulocytic immune response and giving rise to granulocytes that are able to directly kill cancer cells.

[0290] The inventors have demonstrated that granulopoietic cells suitable for use in the medical compositions or uses of the invention, or in the methods of the invention, may be able to amplify (preferably amplify) immune responses through several different mechanisms. In particular, granulopoietic cells may increase the activation of immune cells and enhance the activities (such as cell trafficking and cytocidal activity) required to achieve Petition 870250101699, dated 06 / 11 / 2025, page 65 / 275 60 / 243 a successful therapeutic immune response.

[0291] The granulopoietic cell populations suitable for use in these aspects of the invention can be characterized according to the definitions offered elsewhere in this descriptive report. Such granulopoietic cell populations can be produced by the cell preparation methods for therapeutic use set forth earlier in this descriptive report.

[0292] Preferably, the composition comprises a population of granulopoietic cells that is capable of amplifying (preferably amplifies) the therapeutic immune response of the non-granulocytic immune cell. Thus, in one aspect, a composition is provided comprising a population of granulopoietic cells and a non-granulocytic immune cell (e.g., a terminally differentiated non-granulocytic immune cell), wherein the population of granulopoietic cells is capable of amplifying (preferably amplifies) the therapeutic immune response of the non-granulocytic immune cell.

[0293] The ability of a population of granulopoietic cells to amplify a therapeutic immune response of a non-granulocytic immune cell can be determined by any suitable means.

[0294] For example, the ability of a population of granulopoietic cells to amplify a therapeutic immune response from a non-granulocytic immune cell can be determined by an in vitro assay. For example, the ability of a population of granulopoietic cells to amplify a therapeutic immune response from a non-granulocytic immune cell can be determined by a method comprising: (a) mixing PBMCs or PBMCs depleted of αβ T cells in the presence of granulopoietic cells to form a mixture; (b) incubate the mixture; (c) to determine a therapeutic immune response of a non-granulocytic immune cell present in the mixture after incubation; and (d) to compare the therapeutic immune response of the non-granulocytic immune cell present in the mixture after incubation with a standard of Petition 870250101699, dated 06 / 11 / 2025, page 66 / 275 61 / 243 reference.

[0295] A reference standard may be any suitable control. For example, the reference standard may be the corresponding therapeutic immune response of the non-granulocytic immune cell present in PBMCs or PBMCs depleted of αβ T cells prior to mixing. The reference standard may be the corresponding therapeutic immune response of the non-granulocytic immune cell present in the mixture prior to incubation. The reference standard may be the corresponding therapeutic immune response of the non-granulocytic immune cell cultured in the absence of granulopoietic cells, but subjected to identical conditions. The reference standard may be the corresponding therapeutic immune response of the non-granulocytic immune cell cultured in the presence of fewer granulopoietic cells, but subjected to identical conditions.

[0296] Such a reference standard may be obtained using cells from the same donor or from a different donor than those used in steps (a)-(c). Preferably, the reference standard may be obtained using cells from the same donor as those used in steps (a)-(c).

[0297] The granulopoietic cell population can be considered capable of amplifying a therapeutic immune response from a non-granulocytic immune cell when a therapeutic immune response from the non-granulocytic immune cell present in the mixture after incubation is increased compared to the reference standard. Thus, the granulopoietic cell population can be considered capable of amplifying a therapeutic immune response from a non-granulocytic immune cell when a therapeutic immune response from the non-granulocytic immune cell present in the mixture after incubation is increased compared to the corresponding therapeutic immune response from the non-granulocytic immune cell present in PBMCs or PBMCs depleted of αβ T cells before mixing.Preferably, the granulopoietic cell population is considered capable of amplifying a therapeutic immune response from a non-granulocytic immune cell when a therapeutic immune response from the non-granulocytic immune cell present in the mixture after incubation is increased compared to the immune response. Petition 870250101699, dated 06 / 11 / 2025, page 67 / 275 62 / 243 corresponding therapeutic response of the non-granulocytic immune cell cultured in the absence of granulopoietic cells, but subjected to identical conditions. The population of granulopoietic cells can be considered capable of amplifying a therapeutic immune response of a non-granulocytic immune cell when a therapeutic immune response of the non-granulocytic immune cell present in the mixture after incubation is increased compared to the corresponding therapeutic immune response of the non-granulocytic immune cell cultured in the presence of fewer granulopoietic cells, but subjected to identical conditions.

[0298] Preferably, the ability of a population of granulopoietic cells to amplify a therapeutic immune response from a non-granulocytic immune cell is determined by a method comprising: (a) mixing PBMCs or PBMCs depleted of αβ T cells in the presence of granulopoietic cells to form a mixture; (b) incubate the mixture; (c) to determine a therapeutic immune response of a non-granulocytic immune cell present in the mixture after incubation; and (d) to compare the therapeutic immune response of the non-granulocytic immune cell present in the mixture after incubation with the corresponding therapeutic immune response of the non-granulocytic immune cell cultured in the absence of granulopoietic cells, but subjected to identical conditions.

[0299] Granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells may be mixed in any suitable ratio. Granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells may be mixed in a ratio of 100:1 to 0.01:1 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells. The population of granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells may be mixed 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 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells. Preferably, the granulopoietic cells and PBMCs are Petition 870250101699, dated 06 / 11 / 2025, page 68 / 275 63 / 243 mixed in a ratio of 3:1 to 0.25:1 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells.

[0300] Granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells may be mixed in a ratio less than or equal to 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 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells. Granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells can be mixed 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 granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells. Granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells can be mixed 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 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells.Preferably, granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells are mixed in a ratio of 2:1, 1:1, or 0.5:1 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells. For example, granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells can be mixed in a ratio of 2:1 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells. Granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells can be mixed in a ratio of 1:1 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells. Granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells can be mixed in a ratio of 0.5:1 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells.

[0301] The mixture can be incubated for any suitable time. For example, the mixture can be incubated for 1-240 hours. The mixture can be incubated for 1240 hours; 2-220 hours; 4-200 hours; 8-180 hours; 12-160 hours; 16-140 hours; 20-120 hours; 24-100 hours; 24-96 hours; 48-96 hours; or 48-72 hours. Preferably, the mixture is incubated for 48-96 hours.

[0302] The mixture can be incubated for 1, 2, 4, 8, 12, 16, 20, 24, 30, 36, 42, 48, Petition 870250101699, dated 06 / 11 / 2025, page 69 / 275 64 / 243 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.

[0303] Consequently, the ability of a population of granulopoietic cells to amplify a therapeutic immune response from a non-granulocytic immune cell can be determined by a method comprising: (a) mixing PBMCs or αβ T cell-depleted PBMCs in the presence of granulopoietic cells to form a mixture, wherein the mixture comprises 2:1, 1:1 or 0.5:1 of granulopoietic cells to PBMCs or αβ T cell-depleted PBMCs; (b) incubate the mixture for 72 hours; (c) to determine a therapeutic immune response of a non-granulocytic immune cell present in the mixture after incubation; and (d) to compare the therapeutic immune response of the non-granulocytic immune cell present in the mixture after incubation with a reference standard.

[0304] Preferably, the ability of a population of granulopoietic cells to amplify a therapeutic immune response from a non-granulocytic immune cell can be determined by a method comprising: (a) mixing PBMCs or αβ T cell-depleted PBMCs in the presence of granulopoietic cells to form a mixture, wherein the mixture comprises 2:1, 1:1 or 0.5:1 of granulopoietic cells to PBMCs or αβ T cell-depleted PBMCs; (b) incubate the mixture for 72 hours; (c) to determine a therapeutic immune response of a non-granulocytic immune cell present in the mixture after incubation; and (d) to compare the therapeutic immune response of the non-granulocytic immune cell present in the mixture after incubation with the corresponding therapeutic immune response of the non-granulocytic immune cell cultured in the absence of granulopoietic cells, but subjected to identical conditions. Petition 870250101699, dated 06 / 11 / 2025, page 70 / 275 65 / 243

[0305] The mixture may additionally comprise a CD3 activating agent, such as OKT3.

[0306] A therapeutic immune response of a non-granulocytic immune cell can be determined by any suitable means. For example, a therapeutic immune response of a non-granulocytic immune cell can be determined by measuring cell surface markers present on the non-granulocytic immune cell, for example, using flow cytometry. A therapeutic immune response of a non-granulocytic immune cell can be determined by measuring the level of an activation marker; the level of a degranulation marker; and / or the level of a co-stimulatory marker present on the non-granulocytic immune cell using flow cytometry. A therapeutic immune response of a non-granulocytic immune cell can be determined by measuring the proliferation and / or survival of the non-granulocytic immune cell, for example, using flow cytometry.

[0307] Consequently, a therapeutic immune response of a non-granulocytic immune cell can be determined by a method comprising: (a) wash away non-granulocytic immune cells; (b) incubate cells with live / dead staining; (c) washing cells in flow cytometry buffer and staining cell surfaces with antibodies to measure the number of non-granulocytic immune cells present; the level of an activation marker; the level of a degranulation marker; and / or the level of a co-stimulatory marker present on non-granulocytic immune cells; (d) fix the cells; and (e) analyze the cells using a flow cytometer.

[0308] The population of granulopoietic cells can be considered capable of amplifying a therapeutic immune response from a non-granulocytic immune cell when the number of non-granulocytic immune cells present; the level of an activation marker; the level of a degranulation marker; and / or the level of a co-stimulatory marker present in the non-granulocytic immune cells is Petition 870250101699, dated 06 / 11 / 2025, page 71 / 275 66 / 243 increased compared to a reference standard.

[0309] Preferably, a therapeutic immune response from a non-granulocytic immune cell can be determined by a method comprising: (a) washing non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or PBMCs depleted of αβ T cells) in PBS; (b) Incubate the cells with live / dead stain (Fixable Viability Dye eFluor 780; 1:500 dilution) and FcyR block (Human TruStain FcX; 1:50 dilution) for 20 minutes; (c) washing the cells in flow cytometry buffer and staining the cell surface with antibodies specific for CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (HCD56), CD107a (H4A3), 41BB (4B4-1) and / or (preferably e) OX40 (Ber-ACT35), wherein the antibodies are used at a dilution of 1:50, with staining performed on 50 μl / sample; (d) fix the cells using 100 μl 1X BD CellFix; and (e) analyze the cells using a flow cytometer (e.g., a MACSQuant 16 (Miltenyi)).

[0310] The population of granulopoietic cells can be considered capable of amplifying a therapeutic immune response from a non-granulocytic immune cell when the expression level of CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (HCD56), CD107a (H4A3), 4-1BB (4B4-1) and / or (preferably and) OX40 present on non-granulocytic immune cells is increased compared to a reference standard.

[0311] The data can be analyzed using any suitable software, preferably FlowLogic software. Stained cell populations are preferably analyzed by gating on single live cells.

[0312] A therapeutic immune response of a non-granulocytic immune cell can be determined by measuring cytokine production by the non-granulocytic immune cell. For example, a therapeutic immune response of an immune cell Petition 870250101699, dated 06 / 11 / 2025, page 72 / 275 67 / 243 non-granulocytic can be determined by measuring cytokine production by non-granulocytic immune cells using ELISA.

[0313] Consequently, a therapeutic immune response of a non-granulocytic immune cell can be determined by a method comprising: (a) measure the concentration of a cytokine present in the cell culture supernatant of non-granulocytic immune cells using an ELISA; and / or (b) measure the concentration of a cytokine present in the cell culture supernatant of non-granulocytic immune cells using LEGENDplex.

[0314] The population of granulopoietic cells can be considered capable of amplifying a therapeutic immune response from a non-granulocytic immune cell when the concentration of a cytokine present in the cell culture supernatant of non-granulocytic immune cells is increased compared to a reference standard.

[0315] Preferably, a therapeutic immune response from a non-granulocytic immune cell can be determined by a method comprising: (a) measure the concentration of secreted IFN-γ present in the cell culture supernatant of non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or depleted αβ T cell PBMCs) using a quantitative sandwich ELISA (e.g., Abcam; ab174443) according to the manufacturer's instructions; and / or (b) measure the concentration of CXCL10 present in the cell culture supernatant of non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or depleted αβ T cell PBMCs) using LEGENDplex (e.g., BioLegend; 740985) according to the manufacturer's instructions.

[0316] The population of granulopoietic cells can be considered capable of Petition 870250101699, dated 06 / 11 / 2025, page 73 / 275 68 / 243 to amplify a therapeutic immune response of a non-granulocytic immune cell when the concentration of an IFN-γ and / or CXCL10 (preferably e) present in the cell culture supernatant of non-granulocytic immune cells is increased compared to a reference standard.

[0317] A therapeutic immune response from a non-granulocytic immune cell can be determined by measuring cell surface markers present on the non-granulocytic immune cell and / or by measuring cytokine production by the non-granulocytic immune cell. Preferably, a therapeutic immune response from non-granulocytic immune cells is determined by a method comprising: (a) (i) washing non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or PBMCs depleted of αβ T cells) in PBS; (ii) Incubate the cells with live / dead stain (Fixable Viability Dye eFluor 780; 1:500 dilution) and FcyR block (Human TruStain FcX; 1:50 dilution) for 20 minutes; (iii) washing the cells in flow cytometry buffer and staining the cell surface with antibodies specific for CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (HCD56), CD107a (H4A3), 41BB (4B4-1) and / or (preferably e) OX40 (Ber-ACT35), wherein the antibodies are used at a dilution of 1:50, with staining performed on 50 μl / sample; (iv) fix the cells using 100 μl 1X BD CellFix; and (v) analyze the cells using a flow cytometer (e.g., a MACSQuant 16 (Miltenyi)); and / or (b) (i) measure the concentration of secreted IFN-γ present in the cell culture supernatant of non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or depleted αβ T cell PBMCs) using a quantitative sandwich ELISA (e.g., Abcam; ab174443) according to the manufacturer's instructions; and / or Petition 870250101699, dated 06 / 11 / 2025, page 74 / 275 69 / 243 (ii) measure the concentration of CXCL10 present in the cell culture supernatant of non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or depleted αβ T cell PBMCs) using LEGENDplex (e.g., BioLegend; 740985) according to the manufacturer's instructions.

[0318] A therapeutic immune response of a non-granulocytic immune cell can be determined by measuring non-granulocytic immune cell tumor death, for example, as determined by a method described in this document. The population of granulopoietic cells can be considered capable of amplifying a therapeutic immune response of a non-granulocytic immune cell when the level of non-granulocytic immune cell tumor death is increased compared to the reference standard.

[0319] The ability of a population of granulopoietic cells to amplify a therapeutic immune response from a non-granulocytic immune cell can be determined by a method comprising: (a) mixing PBMCs or αβ T cell-depleted PBMCs in the presence of granulopoietic cells to form a mixture, wherein the mixture comprises 2:1, 1:1 or 0.5:1 of granulopoietic cells to PBMCs or αβ T cell-depleted PBMCs; (b) incubate the mixture for 72 hours; (c) to determine a therapeutic immune response of a non-granulocytic immune cell present in the mixture after incubation by a method comprising: (i) washing non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or PBMCs depleted of αβ T cells) in PBS; (ii) Incubate the cells with live / dead stain (Fixable Viability Dye eFluor 780; 1:500 dilution) and FcyR block (Human TruStain FcX; 1:50 dilution) for 20 minutes; Petition 870250101699, dated 06 / 11 / 2025, page 75 / 275 70 / 243 (iii) wash the cells in flow cytometry buffer and stain the cell surface with antibodies specific for CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (HCD56), CD107a (H4A3), 41BB (4B4-1) and / or (preferably e) OX40 (Ber-ACT35), where the antibodies are used at a dilution of 1:50, with staining performed on 50 μL / sample; (iv) fix the cells using 100 μL 1X BD CellFix; and (v) analyze the cells using a flow cytometer (e.g., a MACSQuant 16 (Miltenyi)); and / or determine a therapeutic immune response of a non-granulocytic immune cell present in the mixture after incubation by a method comprising: (i) measure the concentration of secreted IFN-γ present in the cell culture supernatant of non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or αβ T cell-depleted PBMCs) using a quantitative sandwich ELISA (e.g., Abcam; ab174443) according to the manufacturer's instructions; and / or (ii) measure the concentration of CXCL10 present in the cell culture supernatant of non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or αβ T cell-depleted PBMCs) using LEGENDplex (e.g., BioLegend; 740985) according to the manufacturer's instructions; and (d) compare the therapeutic immune response of the non-granulocytic immune cell present in the mixture after incubation with a reference standard.

[0320] Preferably, the ability of a population of granulopoietic cells to amplify a therapeutic immune response from a non-granulocytic immune cell can be determined by a method comprising: a) mixing PBMCs or depleted PBMCs of αβ T cells in the presence of Petition 870250101699, dated 06 / 11 / 2025, page 76 / 275 71 / 243 granulopoietic cells to form a mixture, wherein the mixture comprises 2:1, 1:1 or 0.5:1 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells; b) Incubate the mixture for 72 hours; c) to determine a therapeutic immune response of a non-granulocytic immune cell present in the mixture after incubation by a method comprising: (i) washing non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or PBMCs depleted of αβ T cells) in PBS; (ii) Incubate the cells with live / dead stain (Fixable Viability Dye eFluor 780; 1:500 dilution) and FcyR block (Human TruStain FcX; 1:50 dilution) for 20 minutes; (iii) washing the cells in flow cytometry buffer and staining the cell surface with antibodies specific for CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (HCD56), CD107a (H4A3), 41BB (4B4-1) and / or (preferably e) OX40 (Ber-ACT35), wherein the antibodies are used at a dilution of 1:50, with staining performed on 50 μl / sample; (iv) fix the cells using 100 μl 1X BD CellFix; and (v) analyze the cells using a flow cytometer (e.g., a MACSQuant 16 (Miltenyi)); and / or determine a therapeutic immune response of a non-granulocytic immune cell present in the mixture after incubation by a method comprising: (i) measure the concentration of secreted IFN-γ present in the cell culture supernatant of non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or depleted αβ T cell PBMCs) using a quantitative sandwich ELISA (e.g., Abcam; ab174443) according to the manufacturer's instructions; and / or Petition 870250101699, dated 06 / 11 / 2025, page 77 / 275 72 / 243 (ii) measure the concentration of CXCL10 present in the cell culture supernatant of non-granulocytic immune cells (e.g., present in the mixture after incubation, or present in PBMCs or depleted αβ T cell PBMCs) using LEGENDplex (e.g., BioLegend; 740985) according to the manufacturer's instructions; and (d) compare the therapeutic immune response of the non-granulocytic immune cell present in the mixture after incubation with the corresponding therapeutic immune response of the non-granulocytic immune cell cultured in the absence of granulopoietic cells, but subjected to identical conditions.

[0321] The ability of a granulopoietic cell population to enhance a plurality of therapeutic immune responses in a non-granulocytic immune cell may indicate that the granulopoietic cell population is particularly suitable for inclusion in a composition of the invention. Consequently, the composition may comprise a granulopoietic cell population that is capable of amplifying (preferably amplifying) the level of CD3, CD4, CD8, CD56, CD107a, 4-1BB, and OX40 in the non-granulocytic immune cell compared to the reference standard. The composition may comprise a granulopoietic cell population that is capable of amplifying (preferably amplifying) the level of CD107a, 4-1BB, and OX40 in the non-granulocytic immune cell compared to the reference standard.The composition may comprise a population of granulopoietic cells that is capable of amplifying (preferably amplifying) the level of IFN-γ and CXCL10 in the non-granulocytic immune cell, for example, compared to the reference standard.

[0322] The inventors surprisingly discovered that a population of granulopoietic cells that may be able to amplify (preferably amplifies) a therapeutic immune response of a type of non-granulocytic immune cell may also be able to amplify (preferably amplifies) a therapeutic immune response of a different type of non-granulocytic immune cell. Petition 870250101699, dated 06 / 11 / 2025, page 78 / 275 73 / 243 granulocytic. Consequently, a population of granulopoietic cells can be considered capable of amplifying the therapeutic immune response of a non-granulocytic immune cell if the population of granulopoietic cells is capable of amplifying the therapeutic immune response of an NK cell and / or a T cell, for example, as determined using a method described in this document. Preferably, a population of granulopoietic cells is considered capable of amplifying the therapeutic immune response of a non-granulocytic immune cell if the population of granulopoietic cells is capable of amplifying the therapeutic immune response of an NK cell, for example, as determined using a method described in this document.

[0323] The inventors also showed that granulopoietic cells that are capable of amplifying (preferably amplifies) a particular therapeutic immune response may also be capable of amplifying (preferably amplifies) a different type of therapeutic immune response. For example, a population of granulopoietic cells that is capable of increasing (preferably increasing) cell activation may also be capable of increasing (preferably increasing) the expression of degranulation markers.Thus, a population of granulopoietic cells can be considered capable of amplifying the therapeutic immune response of a non-granulocytic immune cell if the population of granulopoietic cells is able to increase (preferably increase) NK cell activation; increase the expression of NK cell degranulation markers; increase the expression of NK cell co-stimulatory molecules; increase NK cell proliferation; increase NK cell survival; increase cytokine expression by NK cells; increase the cytocidal activity of NK cells; and / or increase the tumor cell killing activity of NK cells.Preferably, a population of granulopoietic cells is considered capable of amplifying the therapeutic immune response of a non-granulocytic immune cell if the population of granulopoietic cells is able to increase (preferably increase) the level of CD107a, 4-1BB and / or (preferably and) OX40 in an NK cell, for example, as determined. Petition 870250101699, dated 06 / 11 / 2025, page 79 / 275 74 / 243 using a method described in this document.

[0324] A population of granulopoietic cells suitable for use according to the various aspects of the present invention may be able to increase the activation of immune cells, for example, non-granulocytic immune cells. In particular, a population of granulopoietic cells may be able to increase (preferably increase) the activation of the non-granulocytic immune cell present in a composition of the invention. Consequently, a population of granulopoietic cells may be able to amplify a therapeutic immune response of a non-granulocytic immune cell by increasing the activation of the non-granulocytic immune cell.

[0325] Suitably, a population of granulopoietic cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocytic immune cells), so that the expression by immune cells of one or more degranulation markers is increased. Suitably, a population of granulopoietic cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocytic immune cells), so that the expression by immune cells of one or more co-stimulatory molecules is increased. Suitably, a population of granulopoietic cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocytic immune cells), so that the proliferation of immune cells is increased.Suitablely, a population of granulopoietic cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocytic immune cells), so that the abundance of immune cells is increased. Suitablely, a population of granulopoietic cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocytic immune cells), so that the survival of immune cells is increased. Suitablely, a population of granulopoietic cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocytic immune cells). Petition 870250101699, dated 06 / 11 / 2025, page 80 / 275 75 / 243 so that the expression of one or more cytokines by immune cells is increased. Suitablely, a population of granulopoietic cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocytic immune cells), so that the trafficking of immune cells is increased. Suitablely, a population of granulopoietic cells suitable for use in the present invention can increase the activation of immune cells (e.g., non-granulocytic immune cells), so that the cytocidal activity of immune cells is increased.

[0326] The term one or more, as used in this document, may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In an embodiment where one or more precedes a list, one or more may mean all members of the list. Similarly, the term at least one, as used in this document, may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In an embodiment where at least one precedes a list, at least one may mean all members of the list.

[0327] Suitablely, a population of granulopoietic cells suitable for use according to the present invention can enhance the activation of immune cells (e.g., non-granulocytic immune cells) by signal 2 (co-stimulation). Alternatively, or additionally, a population of granulopoietic cells suitable for use according to the present invention can enhance the activation of immune cells (e.g., non-granulocytic immune cells) by signal 3 (cytokine stimulation). A population of granulopoietic cells suitable for use according to the present invention may have the ability to enhance the activation of immune cells (e.g., non-granulocytic immune cells) by signal 2 and signal 3.

[0328] Signal 2 and signal 3 are known to be both important in generating effective immune responses to tumors and in overcoming the immunosuppressive effects of TME. Consequently, the inventors' data (presented in the Examples) illustrate that granulopoietic cells suitable for use according to the invention may be able to provide Petition 870250101699, dated 06 / 11 / 2025, page 81 / 275 76 / 243 these signals can provide a clear indication of their suitability for use in amplifying therapeutic immune responses that may be relevant in cancer treatment.

[0329] Other useful ways in which relevant populations of granulopoietic cells can be defined are presented below.

[0330] The granulopoietic cells suitable for use in the compositions, medical uses and methods of the invention can be defined with reference to their potency. The granulopoietic cell population may comprise multipotent cells. In a suitable embodiment, the granulopoietic cell population may comprise unipotent cells.

[0331] Suitable populations of granulopoietic cells for use in the various aspects of the invention can be defined with reference to their differentiation state within the granulopoiesis pathway. In a suitable embodiment, the granulopoietic cell population has a differentiation stage corresponding to that between a myeloblast and a granulocyte. Suitablely, the granulopoietic cell population has a differentiation stage corresponding to that between a myeloblast and a band cell. For example, the granulopoietic cell population may have a differentiation stage corresponding to that between a myeloblast and a metamyelocyte. Suitablely, the granulopoietic cell population has a differentiation stage corresponding to that between a myeloblast and a myelocyte. Suitablely, the granulopoietic cell population has a differentiation stage corresponding to that between a myeloblast and a promyelocyte.

[0332] In a suitable embodiment, the granulopoietic cell population has a differentiation stage corresponding to a myeloblast. In a suitable embodiment, the granulopoietic cell population has a differentiation stage corresponding to a promyelocyte. In a suitable embodiment, the granulopoietic cell population has a differentiation stage corresponding to a myelocyte. In a suitable embodiment, the granulopoietic cell population has a differentiation stage corresponding to a Petition 870250101699, dated 06 / 11 / 2025, page 82 / 275 77 / 243 metamyelocyte. In a suitable modality, the granulopoietic cell population has a differentiation stage corresponding to a band cell.

[0333] In a suitable modality, the population of granulopoietic cells has a differentiation stage corresponding to a granulocyte.

[0334] As set out elsewhere in the descriptive report, granulopoietic cell populations suitable for use in the various aspects of the invention may be derived from artificial stem cells, such as iPSCs. It will be appreciated that such granulopoietic cell populations may not be identical to naturally occurring cells of the granulopoietic pathway, but may share structural (e.g., marker expression) or functional (e.g., potency) characteristics with such naturally occurring cells. Reference to cells with differentiation stages corresponding to named cell types in this disclosure should be interpreted accordingly.

[0335] Suitably, the granulopoietic cell population is selected from the group comprising (or consisting of): a myeloblast; a promyelocyte; a myelocyte; a metamyelocyte; a band cell; and a granulocyte. Suitably, the granulopoietic cell population is selected from the group comprising (or consisting of): a myeloblast; a promyelocyte; a myelocyte; a metamyelocyte; and a band cell. Suitably, the granulopoietic cell population is selected from the group comprising (or consisting of): a myeloblast; a promyelocyte; a myelocyte; and a metamyelocyte. Suitably, the granulopoietic cell population is selected from the group comprising (or consisting of): a myeloblast; a promyelocyte; and a myelocyte. Appropriately, the population of granulopoietic cells is selected from the group comprising (or consisting of): a myeloblast; and a promyelocyte.

[0336] In a suitable embodiment, the granulopoietic cell population is a myeloblast. In a suitable embodiment, the granulopoietic cell population is a promyelocyte. In a suitable embodiment, the granulopoietic cell population is a myelocyte. In a suitable embodiment, the Petition 870250101699, dated 06 / 11 / 2025, page 83 / 275 78 / 243 The population of granulopoietic cells is a metamyelocyte. In a suitable representation, the population of granulopoietic cells is a band cell. In a suitable representation, the population of granulopoietic cells is a granulocyte.

[0337] Appropriately, the granulopoietic cell population may be committed to the neutrophil lineage. In such an embodiment, an appropriate granulopoietic cell population may comprise or consist of cells selected from the group comprising (or consisting of): a neutrophilic promyelocyte; a neutrophilic myelocyte; a neutrophilic metamyelocyte; a neutrophilic band cell; and a neutrophil.

[0338] As further set out elsewhere in this descriptive report, the granulopoietic cell populations that may be employed in the various aspects of the invention may also be defined with reference to the granulocytes that they are capable of giving rise to upon differentiation. Suitable examples of granulopoietic cell populations may be capable of giving rise to granulocytes that have the ability to kill cancerous cells and / or the ability to kill infectious agents or cells infected by infectious agents. Alternatively, or additionally, suitable granulopoietic cell populations may be capable of giving rise to granulocytes that have desirable expression profiles of molecules such as chemokines or costimulatory receptor ligands.

[0339] The inventors have surprisingly shown that granulopoietic cell populations cultured in the presence of non-granulocytic immune cells can have an amplified therapeutic immune response. Consequently, granulopoietic cell populations suitable for use in the compositions, medical uses and methods of the invention can be characterized by having an amplified therapeutic immune response. For example, granulopoietic cell populations suitable for use in the compositions, medical uses and methods of the invention can be characterized by one or more of the following: increased activation; increased expression of degranulation markers; increased expression of co-stimulatory molecules; increased proliferation; Petition 870250101699, dated 06 / 11 / 2025, page 84 / 275 79 / 243 increased survival; increased cytokine expression; increased cytocidal activity; or increased tumor cell killing activity, for example, compared with the corresponding therapeutic immune response of non-granulocytic immune cells cultured in the absence of granulopoietic cells, as described in this document; or compared with a reference standard, as determined by a method described in this document.Preferably, the composition comprises a population of granulopoietic cells characterized by one or more of the following: increased activation; increased expression of degranulation markers; increased expression of co-stimulatory molecules; increased proliferation; increased survival; increased cytokine expression; increased cytocidal activity; or increased tumor cell killing activity, for example, compared to the corresponding therapeutic immune response of the granulopoietic cell population cultured in the absence of a non-granulocytic immune cell, as described in this document; or compared to a reference standard, as determined by a method described in this document.

[0340] A population of granulopoietic cells with an amplified therapeutic immune response may be a population of granulopoietic cells with increased activation. Consequently, the composition may comprise a population of granulopoietic cells with increased activation. The increased activation of granulopoietic cell populations may be associated with increased expression of one or more selected markers from the group comprising (or consisting of): CD54, CD40, CD11b and Mac1. The compositions of the invention may therefore comprise a population of granulopoietic cells with increased expression of CD54, CD40, CD11b and / or Mac1, for example, compared to a population of uncultured granulopoietic cells in the presence of a non-granulocytic immune cell, but subjected to identical conditions.The compositions of the invention may comprise a population of granulopoietic cells with increased CD40 expression, for example, compared to a population of uncultured granulopoietic cells. Petition 870250101699, dated 06 / 11 / 2025, page 85 / 275 80 / 243 presence of a non-granulocytic immune cell, but subjected to identical conditions. The compositions of the invention may comprise a population of granulopoietic cells with increased expression of CD11b, for example, compared to a population of uncultured granulopoietic cells in the presence of a non-granulocytic immune cell, but subjected to identical conditions. The compositions of the invention may comprise a population of granulopoietic cells with increased expression of Mac1, for example, compared to a population of uncultured granulopoietic cells in the presence of a non-granulocytic immune cell, but subjected to identical conditions. Preferably, the compositions of the invention comprise a population of granulopoietic cells with increased expression of CD54, for example, compared to a population of uncultured granulopoietic cells in the presence of a non-granulocytic immune cell, but subjected to identical conditions.

[0341] The activation (e.g., as determined by CD54 expression) of such granulopoietic cell populations can be increased by at least 5%. For example, the activation of granulopoietic cell populations can be increased 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. Quantifying the increase in activation of granulopoietic cell populations according to this modality can make use of comparison with an appropriate control, for example, a population of non-cultured granulopoietic cells in the presence of a non-granulocytic immune cell, but subjected to identical conditions.

[0342] The compositions of the invention may comprise a population of granulopoietic cells with increased expression of CD54; a population of granulopoietic cells with increased expression of CD40; a population of granulopoietic cells with increased expression of CD11b; and / or a population Petition 870250101699, dated 06 / 11 / 2025, page 86 / 275 81 / 243 of granulopoietic cells with increased Mac1 expression, for example, compared to a population of uncultured granulopoietic cells in the presence of a non-granulocytic immune cell, but subjected to identical conditions. In some embodiments, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% of the granulopoietic cell populations in the composition express CD54, for example, as determined by flow cytometry. Preferably, at least about 45% of the granulopoietic cell populations in the composition express CD54, for example, as determined by flow cytometry.

[0343] The granulopoietic cell population can be obtained from any suitable source. For example, the granulopoietic cell population can be obtained from a sample of PBMCs or a sample of umbilical cord blood. The PBMC sample or umbilical cord blood sample can be obtained (e.g., obtained) from a donor. Preferably, the granulopoietic cell population can be obtained (e.g., obtained) from a sample of PBMCs depleted of αβ T cells. The granulopoietic cell population can be obtained from (e.g., differentiated in vitro from) a stem cell, such as a hematopoietic stem cell or iPSC.

[0344] The term obtainable, as used in this document, encompasses the term obtained. In one embodiment, obtainable means obtained.

[0345] The term donor, as used in this document, refers to a subject (properly a human subject) from whom a sample can be obtained (e.g., harvested). Any suitable sample from which a population of granulopoietic cells and / or non-granulocytic immune cells can be obtained from the donor. The donor may be selected based on one or more of the following characteristics: sex, age, medical history, and / or blood type. A donor may be selected if the donor is a healthy donor. A donor may be selected if the donor does not have cancer and does not have an infection. For example, a donor may be selected if the donor does not have cancer. A donor may be selected if the donor does not Petition 870250101699, dated 06 / 11 / 2025, page 87 / 275 82 / 243 has an infection. A donor may be selected if the donor is a man. A donor may be selected if the donor is 18-55 years old and preferably 18-35 (more preferably 18-24). Appropriately, a donor may be selected if the donor is a man aged 18-55 and preferably 18-35 (more preferably 18-24). Alternatively, a donor may be selected if the donor is a woman. A donor may be selected if the donor is over 40 years old. Appropriately, a donor may be selected if the donor is a woman over 40 years old.

[0346] A population of granulopoietic cells can be obtained from a hematopoietic cell. The term hematopoietic cell, as used in this document, refers to a cell that is capable of differentiating (preferably differentiating) into a granulopoietic cell or a population of granulopoietic cells. The term hematopoietic cell thus encompasses a hematopoietic stem cell as well as a precursor cell (e.g., differentiated from a hematopoietic stem cell), wherein said precursor cell is capable of differentiating (preferably differentiating) into a granulopoietic cell or a population of granulopoietic cells. The precursor cell may be referred to in this document as a granulopoietic precursor cell. Suitable examples of such precursor cells have been defined elsewhere in the descriptive report in the context of the production of granulopoietic cell populations.A hematopoietic cell according to the present invention may refer to a hematopoietic stem cell, a granulopoietic precursor cell, or combinations thereof. In one embodiment, a hematopoietic cell is a cell of the hematopoiesis pathway or a cell equivalent thereto. In another embodiment, the hematopoietic cell is an induced pluripotent stem cell (iPSC) or a cell equivalent thereto. A stem cell may be obtained from umbilical cord blood.

[0347] In one embodiment, an iPSC can be obtained from a somatic cell from a donor. The generation of iPSCs is a well-known technique in Petition 870250101699, dated 06 / 11 / 2025, page 88 / 275 83 / 243 technique, see Yu et al (2007), Science, 318:1917-1920, whose teaching is incorporated into this document by reference. Consequently, the granulopoietic cell population can be obtained from an induced pluripotent stem cell (iPSC) or hematopoietic stem cell (HSC). Preferably, the granulopoietic cell population can be obtained from an HSC. The granulopoietic cell population can be obtained (e.g., acquired) by a method of obtaining a granulopoietic cell population described in this document.

[0348] Granulopoietic cells obtainable (e.g., obtained) by the above methods are capable of amplifying (preferably amplifying) the therapeutic immune response of non-granulocytic immune cells. Granulopoietic cells obtainable (e.g., obtained) by the above methods amplify the therapeutic immune response of non-granulocytic immune cells.

[0349] The population of granulopoietic cells present in a composition of the invention may be a heterogeneous population of granulopoietic cells, that is, comprising a plurality of different types or subtypes of granulopoietic cells, or it may be a homogeneous population of granulopoietic cells, that is, comprising a single type of granulopoietic cell. Preferably, the population of granulopoietic cells is a heterogeneous population of granulopoietic cells.

[0350] As used in this document, the term non-granulocytic immune cell refers to any cell of the immune system that is not a granulocytic cell (i.e., not a granulocyte). Consequently, a non-granulocytic immune cell can be any immune cell that is not a neutrophil, an eosinophil, or a basophil.

[0351] The non-granulocytic immune cell may be a dendritic cell, a blood-derived myeloid cell, a monocyte, a macrophage, a natural killer (NK) cell, a B cell, or a T cell, for example, a γδ T cell. Preferably, the non-granulocytic immune cell is a dendritic cell, a blood-derived myeloid cell, a monocyte, a macrophage, an NK cell, a B cell, or a γδ T cell. Particularly preferably, the immune cell Petition 870250101699, dated 06 / 11 / 2025, page 89 / 275 84 / 243 non-granulocytic is a γδ T cell (e.g., a Vδ1+ or νδ2+ γδ T cell) or an NK cell.

[0352] Non-granulocytic immune cells suitable for use in the compositions, medical uses and methods of the invention may be characterized by having an amplified therapeutic immune response. For example, non-granulocytic immune cells suitable for use in the compositions, medical uses and methods of the invention may be characterized by one or more of the following: increased activation; increased expression of degranulation markers; increased expression of co-stimulatory molecules; increased proliferation; increased survival; increased cytokine expression; increased cytocidal activity; or increased tumor cell killing activity, for example, compared with the corresponding therapeutic immune response of non-granulocytic immune cells cultured in the absence of granulopoietic cells; or compared with a reference standard.Preferably, the composition comprises a non-granulocytic immune cell characterized by one or more of the following: increased activation; increased expression of degranulation markers; increased expression of co-stimulatory molecules; increased proliferation; increased survival; increased cytokine expression; increased cytocidal activity; or increased tumor cell killing activity, for example, compared with the corresponding therapeutic immune response of the non-granulocytic immune cell cultured in the absence of a granulopoietic cell population; or compared with a reference standard.

[0353] For example, the non-granulocytic immune cell may have increased expression of one or more selected markers from: CD3, CD4, CD8, CD56, CD107a, 4-1BB, and OX40. Preferably, the non-granulocytic immune cell has increased expression of one or more selected markers from: CD107a, 41BB, and OX40. The non-granulocytic immune cell may have increased expression of CD107a. The non-granulocytic immune cell may have increased expression of 4-1BB. The non-granulocytic immune cell may have increased expression of OX40. The non-granulocytic immune cell may have increased expression of CXCL10. The immune cell Petition 870250101699, dated 06 / 11 / 2025, page 90 / 275 85 / 243 non-granulocytic immune cells may secrete increased concentrations of CXLC10. Non-granulocytic immune cells may have increased expression of IFN-γ. Non-granulocytic immune cells may secrete increased concentrations of IFN-γ. Non-granulocytic immune cells may have increased proliferation. Non-granulocytic immune cells may have increased tumor killing capacity. The increase may be an increase compared to a non-granulocytic immune cell cultured in the absence of a granulopoietic cell population, but subjected to identical conditions.

[0354] The inventors have surprisingly discovered that granulopoietic cell populations, as described in this document, are capable of amplifying (preferentially amplifying) the therapeutic immune response of NK cells. For example, the inventors have shown that granulopoietic cell populations, as described in this document, increase NK cell proliferation, thus overcoming the problem of limited ex vivo expansion of NK cells. The inventors have shown that granulopoietic cells, as described in this document, increase NK cell survival, thus overcoming the problem of limited in vivo survival of NK cells. Furthermore, the inventors have shown that granulopoietic cell populations, as described in this document, potentially increase the expression of 4-1BB and OX40 in NK cells, thus increasing NK cell cytotoxicity.

[0355] Consequently, the composition may comprise a population of granulopoietic cells and an NK cell. An NK cell may be any suitable NK cell. The NK cell may be an NK cell that is CD3- and CD56+. For example, the NK cell may be an NK cell that is CD3-, CD56dim and / or CD16+, for example, CD3-, CD56dim and CD16+. The NK cell may be an NK cell that is CD3-, CD56bright and / or CD16-, for example, CD3-, CD56bright and CD16-. An NK cell can be an NK cell that is CD3-, CD56+, CD7+, CD127-, NKp46+, T-bet+, and / or Eomes+, for example, CD3-, CD56+, CD7+, CD127-, NKp46+, T-bet+, and Eomes+. Without being limited by theory, it is believed that CD56dim and CD16+ NK cells are predominantly found in the blood, while CD56bright and Petition 870250101699, dated 06 / 11 / 2025, page 91 / 275 86 / 243 CD16- NK cells are predominantly found in lymph. The NK cell can be obtained by the method described in Oyer et al. Biol Blood Marrow Transplant 21 (2015) 632-639, which is incorporated herein by reference in its entirety.

[0356] A population of granulopoietic cells suitable for use according to the present invention can enhance NK cell activation. Consequently, the composition may comprise an NK cell with enhanced activation. Without limitation, the enhanced NK cell activation may be associated with one or more of the following: an increase in NK cell expression of a degranulation marker (including, but not limited to, CD107a); an increase in NK cell expression of a co-stimulatory molecule (including, but not limited to, 4-1BB and / or OX40); an increase in NK cell expression of a cytokine (including, but not limited to, IFN-γ and / or TNF); an increase in NK cell trafficking; an increase in NK cell recruitment to the TME; an increase in cytocidal activity (including, but not limited to, tumor cell killing) by NK cells; an increase in NK cell proliferation; an increase in NK cell survival; and an increase in NK cell abundance.The changes in these properties associated with increased activation of NK cells exposed to granulopoietic cell populations suitable for use according to the present invention are demonstrated in the Examples. Other relevant considerations regarding these various properties are set forth elsewhere in this descriptive report.

[0357] NK cell activation can be increased by at least 5%. For example, NK cell activation can be increased 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 according to such a modality can make use of comparison with an appropriate control. Petition 870250101699, dated 06 / 11 / 2025, page 92 / 275 87 / 243

[0358] As used in this document, the term an appropriate control may refer to a non-granulocytic immune cell that has not been cultured in the presence of a population of granulopoietic cells, but has been subjected to identical conditions.

[0359] In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the NK cells in the composition express 4-1BB, for example, as determined by flow cytometry. Preferably, at least about 10% of the NK cells in the composition express 4-1BB, for example, as determined by flow cytometry. In some embodiments, at least about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, or 35% of the NK cells in the composition express OX40, for example, as determined by flow cytometry. Preferably, at least about 5% of the NK cells in the composition express OX40, for example, as determined by flow cytometry. Specifically, at least about 10% of the NK cells in the composition should express 4-1BB and at least about 5% of the NK cells in the composition should express OX40.

[0360] The inventors have surprisingly discovered that granulopoietic cell populations, as described in this document, are capable of amplifying (preferably amplifying) the therapeutic immune response of T cells. For example, the inventors have shown that granulopoietic cell populations, as described in this document, increase the expression of 41BB and OX40 on CD4+ and CD8+ T cells and increase the expression of 4-1BB and CD25 on γδ T cells, thus improving the effector function of these cells. Consequently, the composition may comprise a granulopoietic cell population and a T cell. A T cell may be any suitable T cell. The T cell may be an αβ T cell or a γδ T cell. An αβ T cell is a T cell comprising an αβ T cell receptor (TCR) on its cell surface. Meanwhile, a γδ T cell is a T cell that comprises a γδ TCR on its cell surface. Preferably, the T cell is a γδ T cell.More preferentially, the γδ T cell is a Vδ1 or Vδ2 γδ T cell. Petition 870250101699, dated 06 / 11 / 2025, page 93 / 275 88 / 243 Preferably, the T cell is not an αβ T cell.

[0361] A population of granulopoietic cells suitable for use according to the present invention can increase T cell activation. Consequently, the composition may comprise a T cell with increased activation. Without limitation, the increased T cell activation may be associated with one or more of the following: an increase in T cell expression of a degranulation marker (including, but not limited to, CD107a); an increase in T cell expression of a co-stimulatory molecule (including, but not limited to, 4-1BB and / or OX40); an increase in T cell expression of a cytokine; an increase in T cell trafficking; an increase in T cell recruitment to the TME; an increase in cytocidal activity (including, but not limited to, tumor cell killing) by T cells; an increase in T cell proliferation; an increase in T cell survival; and an increase in T cell abundance.The changes in these properties associated with increased activation of T cells exposed to granulopoietic cell populations suitable for use according to the present invention are demonstrated in the Examples. Other relevant considerations regarding these various properties are set forth elsewhere in this descriptive report.

[0362] T cell activation can be increased by at least 5%. For example, T cell activation can be increased 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 T cell activation according to such a modality can make use of comparison with an appropriate control.

[0363] A population of granulopoietic cells suitable for use according to the present invention can increase the activation of CD8+ T cells. Consequently, the composition can comprise a CD8+ T cell with increased activation. The increased activation of CD8+ T cells can be Petition 870250101699, dated 06 / 11 / 2025, page 94 / 275 89 / 243 associated with one or more of the following: an increase in CD8+ T cell expression of a degranulation marker (including, but not limited to, CD107a); an increase in CD8+ T cell expression of a co-stimulatory molecule (including, but not limited to, 4-1BB and / or OX40); and an increase in CD8+ T cell proliferation. Other relevant considerations regarding these various properties are set forth elsewhere in this descriptive report.

[0364] The activation of such CD8+ T cells can be increased by at least 5%. For example, the activation of CD8+ T cells can be increased 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 CD8+ T cell activation according to such a modality can make use of comparison with an appropriate control.

[0365] A population of granulopoietic cells suitable for use according to the present invention can enhance the activation of CD4+ T cells. Consequently, the composition may comprise a CD4+ T cell with enhanced activation. The enhanced activation of CD4+ T cells may be associated with one or more of the following: an increase in the expression by CD4+ T cells of a co-stimulatory molecule (including, but not limited to, 4-1BB and / or OX40); and an increase in the proliferation of CD4+ T cells. Other relevant considerations regarding these various properties are set forth elsewhere in this descriptive report.

[0366] The activation of such CD4+ T cells can be increased by at least 5%. For example, the activation of CD4+ T cells can be increased 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 Petition 870250101699, dated 06 / 11 / 2025, page 95 / 275 90 / 243 100% or more. Quantifying the increase in CD4+ T cell activation according to this modality may require comparison with an appropriate control.

[0367] A population of granulopoietic cells suitable for use according to the present invention can increase the activation of γδ T cells (e.g., Vδ1+γδ T cells or Vδ2+γδ T cells). Consequently, the composition may comprise a γδ T cell (e.g., a Vδ1+γδ T cell or a Vδ2+γδ T cell) with increased activation. Increased activation of Vδ1+γδ T cells may be associated with increased expression of 4-1BB and / or increased expression of CD25 on the cell surface. Increased activation of Vδ2+γδ T cells may be associated with increased expression of 4-1BB on the cell surface. Increased activation of Vδ1+ and Vδ2+ γδ T cells may be associated with increased proliferation and / or survival of Vδ1 and Vδ2+ γδ T cells, respectively.

[0368] The activation of such γδ T cells can be increased by at least 5%. For example, the activation of γδ T cells can be increased 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 γδ T cell activation according to such a modality can make use of comparison with an appropriate control.

[0369] In some embodiments, at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4% or 5% of the Vδ1+ γδ T cells in the composition express 4-1BB, for example, as determined by flow cytometry. Preferably, at least about 0.5% of the Vδ1+ γδ T cells in the composition express 4-1BB, for example, as determined by flow cytometry. In some modalities, at least approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% of the Vδ1+ γδ T cells in the composition express CD25, for example, as determined by flow cytometry. Petition 870250101699, dated 06 / 11 / 2025, page 96 / 275 91 / 243 Preferably, at least about 1% of the νδ1+ γδ T cells in the composition express CD25, for example, as determined by flow cytometry. Preferably, at least about 0.5% of the Vδ1+ γδ T cells in the composition express 4-1BB, for example, as determined by flow cytometry, and at least about 1% of the Vδ1+ γδ T cells in the composition express CD25, for example, as determined by flow cytometry. Particularly preferably, at least about 5% of the Vδ1+ T cells in the composition express 4-1BB, for example, as determined by flow cytometry, and at least about 30% of the Vδ1+ γδ T cells in the composition express CD25, for example, as determined by flow cytometry.

[0370] In some modalities, at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5% or 10% of the Vδ2+ γδ T cells in the composition express 4-1BB, for example, as determined by flow cytometry. Preferably, at least about 0.5% of the Vδ2+ γδ T cells in the composition express 4-1BB, for example, as determined by flow cytometry. Specifically, at least about 10% of the Vδ2+ γδ T cells in the composition express 4-1BB, for example, as determined by flow cytometry.

[0371] A dendritic cell can be any suitable dendritic cell. For example, a dendritic cell can be a classic or conventional dendritic cell (cDC), a plasmacytoid dendritic cell (pDC), or a monocyte-derived cell with dendritic cell-like properties (moDC). A cDC can be a type 1 cDC (cDC1) or a type 2 cDC (cDC2). Without being limited by theory, cDC1 cells are believed to present exogenous antigens to MHC class I to induce naive CD8+ T cells to acquire the effector function of cytotoxic T cells (CTLs), while cDC2 cells perform priming of naive CD4+ T cells through antigen presentation to MHC class II. Meanwhile, pDCs are believed to have a dedicated function of secreting type I interferon (IFN).

[0372] Consequently, a dendritic cell can be a dendritic cell Petition 870250101699, dated 06 / 11 / 2025, page 97 / 275 92 / 243 which is CD11c+, HLA-DR+ and / or CD141+, for example, CD11c+, HLA-DR+ and CD141+. This expression profile may be characteristic of a cDC1 cell. A dendritic cell may be a dendritic cell (e.g., a cDC1 cell) that is CD11c+, HLA-DR+, CD141+, CLEC9A+ and / or CADM1+, for example, CD11c+, HLA-DR+, CD141+, CLEC9A+ and CADM1+. A dendritic cell may be a dendritic cell that is CD11c+, HLA-DR+, CD1c+ and / or CD11b+, for example, CD11c+, HLA-DR+, CD1c+ and CD11b+. This expression profile may be characteristic of a cDC2 cell. A dendritic cell can be a dendritic cell (e.g., a cDC2 cell) that is 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+. A dendritic cell can also be a dendritic cell that is HLA-DR+, CD303+, and / or CD123+, for example, HLA-DR+, CD303+, and CD123+. This expression profile may be characteristic of a pDC.A dendritic cell can be a dendritic cell (e.g., a pDC) that is HLA-DR+, CD303+, CD123+, CD11c+ (e.g., CD11cint), MHCII+ (e.g., MHClo), Bst2+, and / or B220+, for example, HLA-DR+, CD303+, CD123+, CD11c+ (e.g., CD11cint), MHCII+ (e.g., MHClo), Bst2+, and B220+, such as HLA-DR+, CD303+, CD123+, CD11cint, MHClo, Bst2+, and B220+. A dendritic cell can be a dendritic cell that is CD11c+, CD11b+, CD1a+, and / or CD1c+, for example, CD11c+, CD11b+, CD1a+, and CD1c+. This expression profile may be characteristic of a moDC. A dendritic cell may be a dendritic cell (e.g., a moDC) that is CD11c+, CD11b+, CD1a+, CD1c+, CD206+, CD209+ and / or CD172A+, CD11c+, CD11b+. CD1a+, CD1c+, CD206+, CD209+ and CD172A+.

[0373] A population of granulopoietic cells suitable for use according to the present invention can increase the activation of dendritic cells. Consequently, the composition can comprise a dendritic cell with increased activation. The increased activation of dendritic cells can be associated with increased expression of CD83, CD86 and / or CD80.

[0374] The activation of such dendritic cells can be increased by at least Petition 870250101699, dated 06 / 11 / 2025, page 98 / 275 93 / 243 5%. For example, dendritic cell activation can be increased 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. Quantifying the increase in dendritic cell activation according to such a modality can make use of comparison with an appropriate control.

[0375] A monocyte can be any suitable monocyte. For example, a monocyte can be a classical monocyte, an intermediate monocyte, or a non-classical monocyte. Without being limited by theory, classical monocytes are believed to be the primary monocyte population responsible for phagocytic activity and have low production of pro-inflammatory cytokines; intermediate monocytes produce pro-inflammatory cytokines such as TNFα, IL-1β, and / or IL-6; and non-classical monocytes produce anti-inflammatory cytokines and constitutively produce IL-1RA.

[0376] Consequently, a monocyte can be a monocyte that is CD14+, CD16+, or CD64+. For example, a monocyte can be a monocyte that is CD14+ (e.g., CD14Hi), CD64+, CD62L+, TNFR1+, TNFR2+ (e.g., TNFR2Lo), CD192+ (e.g., CD192Hi), and / or CXCR1+ (e.g., CXCR1Lo), such as CD14+ (e.g., CD14Hi), CD64+, CD62L+, TNFR1+, TNFR2+ (e.g., TNFR2Lo), CD192+ (e.g., CD192Hi), and CXCR1+ (e.g., CXCR1Lo), for example, CD14Hi, CD64+, CD62L+, TNFR1+, TNFR2Lo, CD192Hi, and CXCR1Lo. This expression profile can be characteristic of a classic monocyte. A monocyte can be a monocyte that is CD16+, CD14+ (e.g., CD14Hi), CD64+, HLA-DR+ (e.g., HLA-DRHi), TNFR1+ (e.g., TNFR1Hi), TNFR2+, CD192+ (e.g., CD192Lo), CX3CR1+ (e.g., CX3CR1Hi), and / or CD195+, such as CD16+, CD14+ (e.g., CD14Hi), CD64+, HLA-DR+ (e.g., HLA-DRHi), TNFR1+ (e.g., TNFR1Hi), TNFR2+, CD192+ (e.g., CD192Lo), CX3CR1+ (e.g., Petition 870250101699, dated 06 / 11 / 2025, p. 99 / 275 94 / 243 CX3CR1Hi) and CD195+, for example, CD16+, CD14Hi, CD64+, HLA-DRHi, TNFR1Hi, TNFR2+, CD192Lo, CX3CR1Hi and CD195+. This expression profile may be characteristic of an intermediate monocyte. A monocyte may be a monocyte that is CD14+ (e.g., CD14Lo), CD16+ (e.g., CD16Hi), TNFR1+ (e.g., TNFR1Lo) and / or TNFR2+ (e.g., TNFR2Hi), such as CD14+ (e.g., CD14Lo), CD16+ (e.g., CD16Hi), TNFR1+ (e.g., TNFR1Lo) and TNFR2+ (e.g., TNFR2Hi), for example, CD14Lo, CD16Hi, TNFR1Lo and TNFR2Hi. This expression profile may be characteristic of a non-classical monocyte.

[0377] A macrophage can be any suitable macrophage. For example, the macrophage can be a classically activated M1 macrophage or an alternatively activated M2 macrophage. Without being limited by theory, M1 macrophages are believed to exhibit high antigen-presenting activity and high production of pro-inflammatory cytokines, such as IL-1, IL-6, TNFα, nitric oxide, and reactive oxygen species (ROS). Meanwhile, M2 macrophages are believed to exhibit low production of inflammatory cytokines, such as IL-1, IL-6, and TNFα. It is understood that M1 and M2 macrophages can be further divided into additional subclassifications.

[0378] Consequently, a macrophage can be a macrophage that is CD11b+, CD14+, CD15+, CD16+ and / or CD68+, CD11b+, CD14+, CD15+, CD16+ and CD68+. A macrophage can be a macrophage that is 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 expression profile may be characteristic of an M1 macrophage. An M1 macrophage can 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. A macrophage can be CD115+, CD163+, CD204+, CD206+, CD209+, FceR1+, and / or VSIG4+, for example, CD115+, CD163+, CD204+, CD206+, CD209+, FceR1+, and VSIG4+. This expression profile can be characteristic of an M2 macrophage. An M2 macrophage can be characterized by... Petition 870250101699, dated 06 / 11 / 2025, page 100 / 275 95 / 243 secretion of IDO, IL-10 and / or TGFe, for example, IDO, IL-10 and TGFe.

[0379] A population of granulopoietic cells suitable for use according to the present invention can increase macrophage activation. Consequently, the composition may comprise a macrophage with increased activation. Increased macrophage activation may be associated with increased expression of CD86, CD40 and / or increased TNFα secretion.

[0380] The activation of such macrophages can be increased by at least 5%. For example, macrophage activation can be increased 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 macrophage activation according to such a modality can make use of comparison with an appropriate control.

[0381] A B cell can be any suitable B cell. For example, the B cell can be any B cell comprising a B cell receptor (BCR). The B cell can be a pro-B cell, a pre-B cell, an immature B cell, a transitional B cell, a naive B cell, a B1 cell, a memory B cell, or a plasma cell. Preferably, the B cell is a transitional B cell, a naive B cell, a memory B cell, or a plasma cell.

[0382] Consequently, the B cell may be a B cell that is CD19+, CD20+, CD34+, CD38+ and / or CD45R+, for example, CD19+, CD20+, CD34+, CD38+ and CD45R+. This expression profile may be characteristic of a pro-B cell. The B cell may be a B cell that is CD19+, CD20+, CD38+, CD40+ and / or CD45R+, for example, CD19+, CD20+, CD38+, CD40+ and CD45R+. This expression profile may be characteristic of a pre-B cell. The B cell may be a B cell that is CD19+, CD20+, CD40+, CD45R+ and / or IgM+, CD19+, CD20+, CD40+, CD45R+ and IgM+. This expression profile may be characteristic of an immature B cell. The B cell may be a B cell that is CD10+, CD19+, CD20+, CD24hi, and / or CD28hi, for example, CD10+, CD19+, CD20+, CD24hi, and CD28hi. The B cell may be a Petition 870250101699, dated 06 / 11 / 2025, page 101 / 275 96 / 243 B cell that is CD10+, CD19+, CD20+, CD24hi, CD28hi, BCL-2lo and / or CD27-, for example, CD10+, CD19+, CD20+, CD24hi, CD28hi, BCL-2lo and CD27-. This expression profile may be characteristic of a transitional B cell. The B cell may be a B cell that is CD19+, CD20+, CD23+, CD40+ and / or CD150+, for example, CD19+, CD20+, CD23+, CD40+ and CD150+. A B cell can be a B cell that is CD19+, CD20+, CD23+, CD40+, CD150+, IgM+, and / or IgD+, for example, CD19+, CD20+, CD23+, CD40+, CD150+, IgM+, and IgD+. A B cell can be a B cell that is CD19+, CD20+, CD23+, CD40+, CD150+, IgM+, IgD+, and / or CD38lo, for example, CD19+, CD20+, CD23+, CD40+, CD150+, IgM+, IgD+, and CD38lo. This expression profile may be characteristic of a naive B cell. A B cell can be a B cell that is CD19+, CD20+, CD27+, and / or IgM+, for example, CD19+, CD20+, CD27+, and IgM+. A B cell can be a B cell that is CD19+, CD20+, CD27+, IgM+, and / or IgDlo, for example, CD19+, CD20+, CD27+, IgM+, and IgDlo.This expression profile may be characteristic of a B1 cell. The B cell may be a B cell that is CD19+, CD20+, CD27+, CD40+, and / or CD150-, for example, CD19+, CD20+, CD27+, CD40+, and CD150-. The B cell may be a B cell that is CD19+, CD20+, CD27+, CD40+, CD150-, IgA+, and / or IgG+, for example, CD19+, CD20+, CD27+, CD40+, CD150-, IgA+, and IgG+. A B cell can be a B cell that is CD19+, CD20+, CD27+, CD40+, CD150-, IgA+, IgG+, CD23lo, and / or CD38-, for example, CD19+, CD20+, CD27+, CD40+, CD150-, IgA+, IgG+, CD23lo, and CD38-. This expression profile may be characteristic of a memory B cell. A B cell can also be a B cell that is CD9hi, CD27hi, CD38hi, CD40+, and / or CD95+, for example, CD9hi, CD27hi, CD38hi, CD40+, and CD95+. A B cell could be a B cell that is CD9hi, CD27hi, CD38hi, CD40+, CD95+, CXCR4+, and / or CD138+, for example, CD9hi, CD27hi, CD38hi, CD40+, CD95+, CXCR4+, and CD138+.A B cell can be a B cell that is CD9hi, CD27hi, CD38hi, CD40+, CD95+, CXCR4+, CD138+, CD19lo and / or CD20-, for example, CD9hi, CD27hi, CD38hi, CD40+, CD95+, CXCR4+, CD138+, CD19lo and CD20-. This expression profile may be characteristic of a plasma cell.

[0383] The inventors surprisingly discovered that the populations of Petition 870250101699, dated 06 / 11 / 2025, page 102 / 275 97 / 243 granulopoietic cells, as described in this document, are capable of amplifying (preferably amplifying) the therapeutic immune response of blood-derived myeloid cells. For example, the inventors have shown that granulopoietic cells, as described in this document, increase the survival and / or proliferation of blood-derived myeloid cells, thus overcoming the problem of ex vivo expansion of blood-derived myeloid cells. Consequently, the composition may comprise a population of granulopoietic cells and a blood-derived myeloid cell. A blood-derived myeloid cell may be any suitable blood-derived myeloid cell. A blood-derived myeloid cell may be a blood-derived myeloid cell that is CD11b+, CD15+, and / or CD14+, for example, CD11b+, CD15+, and CD14+. Preferably, a blood-derived myeloid cell is a CD11+ blood-derived myeloid cell.A population of granulopoietic cells suitable for use according to the present invention can increase the activation of blood-derived myeloid cells. Consequently, the composition may comprise a blood-derived myeloid cell with increased activation. The increased activation of blood-derived myeloid cells may be associated with increased CD11b expression.

[0384] The activation of such blood-derived myeloid cells can be increased by at least 5%. For example, the activation of blood-derived myeloid cells can be increased 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 macrophage activation according to such a modality can make use of comparison with an appropriate control.

[0385] A non-granulocytic immune cell can be a stem cell, precursor or progenitor, for example, a stem cell, precursor or progenitor of a dendritic cell, a monocyte, a macrophage, a natural killer (NK) cell, a Petition 870250101699, dated 06 / 11 / 2025, page 103 / 275 98 / 243 B cell or a T cell, for example, a γδ T cell. The non-granulocytic immune cell may be a stem cell, precursor, or progenitor of any non-granulocytic immune cell described in this document. Preferably, the non-granulocytic immune cell is a terminally differentiated immune cell.

[0386] Non-granulocytic immune cells can be obtained from any suitable source. For example, a non-granulocytic immune cell can be obtained from a sample of umbilical cord blood, which can be obtained (e.g., obtained) from a donor. For example, a non-granulocytic immune cell can be obtained from a sample of PBMCs, which can be obtained (e.g., obtained) from a donor. Preferably, a non-granulocytic immune cell can be obtained from a sample of PBMCs depleted of αβ T cells. A non-granulocytic immune cell can be obtained from a sample of PBMCs depleted of αβ T cells obtained from blood mobilized with G-CSF. A non-granulocytic immune cell can be obtained from (e.g., differentiated in vitro from) a stem cell, such as a hematopoietic stem cell or iPSC.

[0387] Without being limited by theory, it is believed that compositions comprising a plurality of different types of non-granulocytic immune cells may have a synergistically amplified therapeutic immune response when combined with a population of granulopoietic cells of the invention. Consequently, the composition may comprise a population of granulopoietic cells and a plurality of different types of non-granulocytic immune cells. For example, the composition may comprise at least 2, at least 3, at least 4, at least 5, or at least 6 different types of non-granulocytic immune cells. The composition may comprise 2, 3, 4, 5, or 6 different types of non-granulocytic immune cells.

[0388] The composition may comprise a plurality of different types of non-granulocytic immune cells selected from: a dendritic cell, a monocyte, a macrophage, a natural killer (NK) cell, a B cell, and a T cell (e.g., a γδ T cell). The composition may comprise a plurality Petition 870250101699, dated 06 / 11 / 2025, page 104 / 275 99 / 243 of different types of non-granulocytic immune cells selected from: a dendritic cell, a monocyte, a macrophage, a natural killer (NK) cell, a B cell, and a γδ T cell. The composition may comprise a plurality of different types of non-granulocytic immune cells selected from: a monocyte, a macrophage, an NK cell, and a γδ T cell. Thus, the composition may comprise a dendritic cell, a monocyte, a macrophage, an NK cell, a B cell, and a T cell (e.g., a γδ T cell). The composition may comprise a dendritic cell, a monocyte, a macrophage, an NK cell, a B cell, and a γδ T cell. Preferably, the composition comprises a monocyte, a macrophage, an NK cell, and a γδ T cell. More preferably, the composition comprises one NK cell and one γδ T cell.Particularly preferably, the composition comprises an NK cell and a Vδ1+ γδ T cell and / or a Vδ2+ γδ T cell, for example, an NK cell, a Vδ1+ γδ T cell and a Vδ2+ γδ T cell.

[0389] A plurality of different types of non-granulocytic immune cells can be obtained from any suitable source. For example, a plurality of different types of non-granulocytic immune cells can be obtained from the same donor or from different donors. Preferably, the plurality of different types of non-granulocytic immune cells can be obtained from the same donor. A plurality of different types of non-granulocytic immune cells can be obtained from a single source or from different sources. Preferably, the plurality of different types of non-granulocytic immune cells can be obtained from a single source. For example, a plurality of different types of non-granulocytic immune cells can be obtained from a sample of PBMCs. Preferably, the plurality of different types of non-granulocytic immune cells can be obtained from a sample of PBMCs depleted of αβ T cells.A plurality of different types of non-granulocytic immune cells can be obtained from an iPSC or a population of iPSCs.

[0390] The population of granulopoietic cells and non-granulocytic immune cells can be obtained from the same donor or from different donors. Petition 870250101699, dated 06 / 11 / 2025, page 105 / 275 100 / 243 Preferably, the population of granulopoietic cells and non-granulocytic immune cells can be obtained from the same donor. For example, the population of granulopoietic cells and non-granulocytic immune cells can be obtained from a healthy donor. Preferably, the population of granulopoietic cells and non-granulocytic immune cells can be obtained from a donor who does not have cancer. Obtaining cells from a single donor can be particularly advantageous because it allows the extraction of the entire innate immune component from that donor. Donors with 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 recruiting other immune cells to diseased tissue) can therefore be selected, and their innate immune cells included in the compositions of the invention.It is expected that the cells in these compositions will have synergistically enhanced properties (e.g., synergistically enhanced cytotoxicity and / or synergistically enhanced recruitment) compared to compositions comprising only a single cell type, which flows at least in part from the synergism between the different cell types present in the composition. In particular, as shown in this document, the granulopoietic cells of the invention have shown a particularly surprising propensity to synergistically enhance the activation, cytotoxicity, and / or recruitment of non-granulocytic immune cells.

[0391] The population of granulopoietic cells and non-granulocytic immune cells can be obtained from the same source or from different sources. For example, the population of granulopoietic cells can be obtained from a sample of isolated hematopoietic stem cells, and the non-granulocytic immune cells can be obtained from a sample of isolated PBMCs. Preferably, the population of granulopoietic cells and non-granulocytic immune cells can be obtained from the same source. For example, the population of granulopoietic cells and non-granulocytic immune cells can be obtained from an iPSC or a population of iPSCs. Preferably, the population of cells Petition 870250101699, dated 06 / 11 / 2025, page 106 / 275 101 / 243 Granulopoietic and non-granulocytic immune cells can be obtained from a sample of isolated PBMCs, for example, PBMCs from mobilized blood. Particularly preferably, the population of granulopoietic cells and non-granulocytic immune cells can be obtained from a sample of PBMCs depleted of αβ T cells, for example, PBMCs depleted of αβ T cells from mobilized blood. Consequently, in preferred embodiments, the population of granulopoietic cells and non-granulocytic immune cells can be obtained from a sample of PBMCs depleted of αβ T cells from mobilized blood obtained from a single donor. Advantageously, this allows the composition to be prepared using cells from a single source, thus providing a significantly simplified and efficient method of preparing a composition of the invention.

[0392] As used in this document, the term mobilized blood refers to blood circulating through the body that has been treated with mobilizing agents, such as Plerixafor and / or G-CSF. The term mobilizing agent refers to an agent that assists in recruiting CD34+ hematopoietic stem cells and / or progenitor cells from the bone marrow into the bloodstream. Consequently, mobilized blood has a higher concentration of CD34+ hematopoietic stem cells and / or precursor cells compared to non-mobilized blood. Mobilized blood can be collected via leukapheresis and allows for the collection of PBMCs comprising non-granulocytic immune cells and hematopoietic stem cells and / or precursor cells.

[0393] The inventors discovered that granulopoietic cells are capable of amplifying (preferentially amplifying) the therapeutic immune response of non-granulocytic immune cells when present in different ratios. Consequently, the population of granulopoietic cells and non-granulocytic immune cells can be present in the composition in any suitable ratio. The population of granulopoietic cells and non-granulocytic immune cells can be present in a ratio of 100:1 to 0.01:1 of granulopoietic cells to non-granulocytic immune cells. The population of granulopoietic cells and non-granulocytic immune cells can be present in a ratio of 100:1 to 0.01:1; Petition 870250101699, dated 06 / 11 / 2025, page 107 / 275 102 / 243 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 of granulopoietic cells to non-granulocytic immune cells. Preferably, the population of granulopoietic cells and non-granulocytic cells is present in a ratio of 3:1 to 0.25:1 of granulopoietic cells to non-granulocytic immune cells.

[0394] The population of granulopoietic cells and non-granulocytic immune cells may be present in a ratio less than or equal to 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 of granulopoietic cells to non-granulocytic immune cells. The population of granulopoietic cells and non-granulocytic 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 granulopoietic cells to non-granulocytic immune cells. The population of granulopoietic cells and non-granulocytic 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 of granulopoietic cells to non-granulocytic immune cells.Preferably, the population of granulopoietic cells and non-granulocytic immune cells is present in a ratio of 2:1, 1:1, or 0.5:1 of granulopoietic cells to non-granulocytic immune cells. For example, the population of granulopoietic cells and non-granulocytic immune cells may be present in a ratio of 2:1 of granulopoietic cells to non-granulocytic immune cells. The population of granulopoietic cells and non-granulocytic immune cells may be present in a ratio of 1:1 of granulopoietic cells to non-granulocytic immune cells. The population of granulopoietic cells and non-granulocytic immune cells may be present in a ratio of 0.5:1 of granulopoietic cells to non-granulocytic immune cells.

[0395] The compositions of the invention may be suitable for allogeneic administration. Consequently, the compositions may be substantially or entirely free of any component that causes graft-versus-host disease, for example, an αβ T cell. Then, in one embodiment, the Petition 870250101699, dated 06 / 11 / 2025, p. 108 / 275 103 / 243 composition does not comprise an αβ T cell. The term does not comprise an αβ T cell means that the composition does not comprise any, or substantially any, αβ T cells. The term substantially not, as used in this context, may mean that less than 10% of the cells in the composition may be αβ T cells; less than 5% of the cells in the composition may be αβ T cells; less than 4% of the cells in the composition may be αβ T cells; less than 3% of the cells in the composition may be αβ T cells; less than 2% of the cells in the composition may be αβ T cells; less than 1% of the cells in the composition may be αβ T cells; less than 0.1% of the cells in the composition may be αβ T cells; less than 0.01% of the cells in the composition may be αβ T cells; Less than 0.001% of the cells in the composition may be αβ T cells; or less than 0.0001% of the cells in the composition may be αβ T cells.The term "substantially not," as used in this context, may mean that the composition comprises up to about 1 x 10⁹ T αβ cells / kg of the subject to be treated; up to about 1 x 10⁸ T αβ cells / kg of the subject to be treated; up to about 1 x 10⁷ T αβ cells / kg of the subject to be treated; up to about 1 x 10⁶ T αβ cells / kg of the subject to be treated; preferably up to about 1 x 10⁵ T αβ cells / kg of the subject to be treated. The term "substantially not," as used in this context, may mean that the composition comprises about 1 x 10¹ - 1 x 10⁹ T αβ cells / kg of the subject to be treated; about 1 x 10² - 1 x 10⁸ T αβ cells / kg of the subject to be treated; approximately 1 x 10³–1 x 10⁷ αβ T cells / kg of the subject to be treated; approximately 1 x 10⁴–1 x 10⁶ αβ T cells / kg of the subject to be treated; preferably approximately 1 x 10⁴–1 x 10⁵ αβ T cells / kg of the subject to be treated.The term "substantially not," as used in this context, may mean that the composition comprises up to about 7 x 10¹⁰ αβ T cells; up to about 7 x 10⁹ αβ T cells; up to about 7 x 10⁸ αβ T cells; up to about 7 x 10⁷ αβ T cells; preferably up to about 7 x 10⁶ αβ T cells. The term "substantially not," as used in this context, may mean that the composition comprises about 7 x 10² - 7 x 10¹⁰ αβ T cells / kg of the subject to be treated; about 7 x 10³ - 7 x 10⁹ αβ T cells / kg of the subject to be treated; about... Petition 870250101699, dated 06 / 11 / 2025, page 109 / 275 104 / 243 x 104 - 7 x 108 αβ T cells / kg of the subject to be treated; approximately 7 x 105 - 7 x 107 αβ T cells / kg of the subject to be treated; preferably approximately 7 x 105 - 7 x 106 αβ T cells. Particularly preferably, the composition does not include αβ T cells.

[0396] A subject or patient, as used in this document, may be a mammal, such as a human or another mammal. Preferably, subject means a human subject. Preferably, patient means a human patient.

[0397] In one aspect, a composition is provided comprising one or more granulocytes differentiated from a population of granulopoietic cells capable of amplifying (preferably amplifying) a therapeutic immune response of a non-granulocytic cell and a non-granulocytic cell.

[0398] In several respects, the composition may be a pharmaceutical composition, wherein the pharmaceutical composition further comprises a carrier, excipient, adjuvant and / or pharmaceutically acceptable salt.

[0399] In several respects, compositions (e.g., pharmaceutical compositions) can be obtained (e.g., obtained) by a method of preparing a composition as disclosed in this document.

[0400] In one aspect, a method for preparing a composition of the invention is provided, the method comprising culturing a non-granulocytic immune cell in the presence of a population of granulopoietic cells of the invention. Preferably, the population of granulopoietic cells is capable of amplifying (preferably amplifies) the therapeutic immune response of the non-granulocytic immune cell.

[0401] As set out above, the granulopoietic cells of the invention are surprisingly capable of amplifying (preferably amplifying) the therapeutic immune response of different types of non-granulocytic immune cells. Consequently, the method may comprise culturing an NK cell in the presence of a population of granulopoietic cells, thus forming the composition. The method may comprise culturing a T cell (e.g., cell Petition 870250101699, dated 06 / 11 / 2025, page 110 / 275 105 / 243 The method preferably involves culturing an NK cell and a T cell (e.g., a T γδ cell) in the presence of a population of granulopoietic cells, thus forming the composition. Particularly preferably, the method involves culturing an NK cell and a T γδ cell (e.g., a Vδ1+ γδ T cell or a Vδ2+ γδ T cell) in the presence of a population of granulopoietic cells, thus forming the composition. The non-granulocytic immune cell and the population of granulopoietic cells can be cultured in the absence of an αβ T cell.

[0402] The inventors have surprisingly shown that particular cytokines can synergistically amplify the therapeutic immune response of a non-granulocytic immune cell. In particular, the inventors hypothesize that cytokines that signal through the common gamma chain, or gamma subunit of the interleukin-2 receptor (IL-2RG), may be particularly useful in amplifying the therapeutic immune response of non-granulocytic immune cells cultured in the presence of a population of granulopoietic cells. Such cytokines may include IL-15, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Consequently, the method may comprise culturing the population of granulopoietic cells and non-granulocytic immune cells in the presence of a cytokine that signals through IL-2RG. The method may involve culturing a population of granulopoietic cells and non-granulocytic immune cells in the presence of one or more cytokines selected from among: IL-15, IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.Preferably, the method involves culturing the population of granulopoietic cells and non-granulocytic immune cells in the presence of IL-15.

[0403] Granulopoietic and non-granulocytic cell populations can be cultured together in any suitable ratio. Granulopoietic and non-granulocytic immune cell populations can be cultured together in a ratio of 100:1 to 0.01:1 of granulopoietic cells to non-granulocytic immune cells. Granulopoietic and non-granulocytic immune cell populations can be cultured together in a ratio of 100:1 to 0.01:1; 75:1 to Petition 870250101699, dated 06 / 11 / 2025, page 111 / 275 106 / 243 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 of granulopoietic cells to non-granulocytic immune cells. Preferably, the population of granulopoietic cells and non-granulocytic cells are cultured together in a ratio of 3:1 to 0.25:1 of granulopoietic cells to non-granulocytic immune cells.

[0404] Granulopoietic cell populations and non-granulocytic immune cells can be cultured together at a ratio less than or equal to 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 of granulopoietic cells to non-granulocytic immune cells. Granulopoietic cells and non-granulocytic immune cells can 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 granulopoietic cells to non-granulocytic immune cells. The population of granulopoietic cells and non-granulocytic immune cells can 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 of granulopoietic cells to non-granulocytic immune cells.Preferably, the populations of granulopoietic cells and non-granulocytic immune cells are cultured together in a ratio of 2:1, 1:1, or 0.5:1 of granulopoietic cells to non-granulocytic immune cells. For example, the populations of granulopoietic cells and non-granulocytic immune cells can be cultured together in a ratio of 2:1 of granulopoietic cells to non-granulocytic immune cells. The populations of granulopoietic cells and non-granulocytic immune cells can be cultured together in a ratio of 1:1 of granulopoietic cells to non-granulocytic immune cells. The populations of granulopoietic cells and non-granulocytic immune cells can be cultured together in a ratio of 0.5:1 of granulopoietic cells to non-granulocytic immune cells.

[0405] A suitable source for non-granulocytic immune cells may be PBMCs. Consequently, the method may comprise culturing PBMCs in the presence of granulopoietic cells. The method may comprise: Petition 870250101699, dated 06 / 11 / 2025, page 112 / 275 107 / 243 (a) isolate PBMCs from a sample obtained from a donor; and (b) culture the PBMCs in the presence of granulopoietic cells, thus forming the composite.

[0406] To improve the suitability of the composition for allogeneic administration, components that may cause graft-versus-host disease may be removed from the composition. The method may comprise removing components that cause graft-versus-host disease from the composition. For example, the method may comprise a step of depleting αβ T cells from the composition. Consequently, the method may comprise culturing PBMCs in the presence of granulopoietic cells and depleting αβ T cells from the PBMCs. Preferably, the method comprises culturing αβ T cell-depleted PBMCs in the presence of granulopoietic cells.

[0407] The method may include: (a) isolate PBMCs from a sample obtained from a donor; (b) deplete αβ T cells from isolated PBMCs; and (c) culture the αβ T cell-depleted PBMCs in the presence of granulopoietic cells, thus forming the composite.

[0408] αβ T cells can be depleted from PBMCs using any suitable means. For example, the PBMC αβ T cell depletion step may comprise: (a) incubate PBMCs in the presence of a biotin-conjugated anti-TCR αβ antibody and anti-biotin microspheres so that the αβ T cells present in the PBMCs bind to the biotin-conjugated anti-TCR αβ antibody; and (b) separate the antibody-bound αβ T cells from the PBMCs, for example, using magnetic activated cell sorting (MACS).

[0409] The depletion step of isolated PBMCs αβ T cells may include: (a) Incubate PBMCs in the presence of an anti-TCR αβ antibody. Petition 870250101699, dated 06 / 11 / 2025, page 113 / 275 108 / 243 conjugated with biotin (Clone BW242 / 412; 1:50 dilution) for 15 minutes at room temperature so that the αβ T cells present in PBMCs bind to the biotin-conjugated anti-TCR αβ antibody; (b) washing antibody-bound cells in MACS buffer; (c) Centrifuge the antibody-bound cells at 300 xg for 5 minutes; (d) resuspend the centrifuged cells in MACS buffer (80 μl / 1x107 cells) containing anti-biotin microspheres (20 μl / 1x107 cells); (e) Incubate the resuspended cells at 4 °C for 15 minutes so that the antibody-bound cells bind to the anti-biotin microspheres; (f) Wash the microsphere-bound cells in MACS buffer and centrifuge the microsphere-bound cells at 300 xg for 5 minutes; and (g) Resuspend up to 1.25 x 10⁸ microsphere-bound cells in 500 μl of MACS buffer and apply the microsphere-bound cells to an LD column placed in the magnetic field of the MACS MultiStand (Miltenyi Biotec).

[0410] in which unlabeled cells (αβTCR-) pass through the column and are collected.

[0411] As used in this document, the term αβ T cell depleted PBMCs and the like refers to a population of PBMCs that are substantially or entirely free of αβ T cells. Thus, in one embodiment, the term αβ T cell depleted PBMCs means a sample of PBMCs that does not comprise an αβ T cell. The term a sample of PBMCs that does not comprise an αβ T cell in this context means that the PBMCs do not comprise any, or substantially any, αβ T cells. The term "substantially not," as used in this context, 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% of the cells, less than 0.1% of the cells, less than 0.01% of the cells, less than 0.001% of the cells, less than 0.0001% of the PBMCs are αβ T cells. The term "substantially not," as used in this context, may mean that the Petition 870250101699, dated 06 / 11 / 2025, page 114 / 275 109 / 243 PBMCs comprise up to approximately 1 x 10⁹ αβ T cells / kg of the subject to be treated; up to approximately 1 x 10⁸ αβ T cells / kg of the subject to be treated; up to approximately 1 x 10⁷ αβ T cells / kg of the subject to be treated; up to approximately 1 x 10⁶ αβ T cells / kg of the subject to be treated; preferably up to approximately 1 x 10⁵ αβ T cells / kg of the subject to be treated. The term substantially not, as used in this context, may mean that PBMCs comprise approximately 1 x 10¹–1 x 10⁹ αβ T cells / kg of the subject to be treated; approximately 1 x 10²–1 x 10⁸ αβ T cells / kg of the subject to be treated; approximately 1 x 10³–1 x 10⁷ αβ T cells / kg of the subject to be treated; approximately 1 x 10⁴–1 x 10⁶ αβ T cells / kg of the subject to be treated; preferably approximately 1 x 10⁴–1 x 10⁵ αβ T cells / kg of the subject to be treated.The term "substantially not," as used in this context, may mean that PBMCs comprise up to about 7 x 10¹⁰ αβ T cells; up to about 7 x 10⁹ αβ T cells; up to about 7 x 10⁸ αβ T cells; up to about 7 x 10⁷ αβ T cells; preferably up to about 7 x 10⁶ αβ T cells. The term "substantially not," as used in this context, may mean that PBMCs comprise about 7 x 10² - 7 x 10¹⁰ αβ T cells / kg of the subject to be treated; about 7 x 10³ - 7 x 10⁹ αβ T cells / kg of the subject to be treated; about 7 x 10⁴ - 7 x 10⁸ αβ T cells / kg of the subject to be treated; approximately 7 x 10⁵ - 7 x 10⁷ αβ T cells / kg of the subject to be treated; preferably approximately 7 x 10⁵ - 7 x 10⁶ αβ T cells. In particular, PBMCs preferentially do not comprise αβ T cells.

[0412] αβ T cells can be depleted at any appropriate time, for example, before administration to a subject. For example, PBMCs can be cultured in the presence of granulopoietic cells before or after the αβ T cell depletion step of isolated PBMCs.

[0413] Granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) can be cultured together in any suitable ratio. Granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) can be cultured together in a ratio of 100:1 to 0.01:1 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells. The cell population Petition 870250101699, dated 06 / 11 / 2025, page 115 / 275 110 / 243 Granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) can be cultured 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 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells. Preferably, granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) are cultured together in a ratio of 3:1 to 0.25:1 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells.

[0414] Granulopoietic cells and PBMCs or αβ T cell depleted PBMCs (e.g., αβ T cell depleted PBMCs) can be cultured together in a ratio less than or equal to 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 of granulopoietic cells to PBMCs or αβ T cell depleted PBMCs (e.g., αβ T cell depleted PBMCs). Granulopoietic cells and PBMCs or αβ T cell-depleted PBMCs can be cultured at 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 granulopoietic cells to PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T cell-depleted PBMCs).Granulopoietic cells and PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T cell-depleted PBMCs) can 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 of granulopoietic cells to PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T cell-depleted PBMCs). Preferably, granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) are cultured together in a ratio of 2:1, 1:1, or 0.5:1 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells). For example, granulopoietic cells and PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) can be cultured together in a ratio of 2:1 of granulopoietic cells to PBMCs or PBMCs depleted of αβ T cells.Granulopoietic cells and PBMCs or cell-depleted PBMCs. Petition 870250101699, dated 06 / 11 / 2025, page 116 / 275 111 / 243 Tαβ cells (e.g., PBMCs depleted of Tαβ cells) can be cultured together in a 1:1 ratio of granulopoietic cells to PBMCs or PBMCs depleted of Tαβ cells. Granulopoietic cells and PBMCs or PBMCs depleted of Tαβ cells (e.g., PBMCs depleted of Tαβ cells) can be cultured together in a 0.5:1 ratio of granulopoietic cells to PBMCs or PBMCs depleted of Tαβ cells.

[0415] PBMCs and αβ T cell-depleted PBMCs may comprise granulopoietic cells. Without being limited by theory, it is believed that there is a higher concentration of hematopoietic cells, which are capable of differentiating (preferably differentiating) into granulopoietic cells, in PBMCs obtainable (e.g., obtained) from mobilized blood, for example, mobilized blood with G-CSF. Preferably, PBMCs or αβ T cell-depleted PBMCs may be obtained from mobilized blood, for example, mobilized blood with G-CSF. The method may comprise obtaining PBMCs or αβ T cell-depleted PBMCs from mobilized blood, for example, mobilized blood with G-CSF.

[0416] The method may comprise increasing the number of granulopoietic cells present in PBMCs or PBMCs depleted of αβ T cells. The method may comprise increasing the concentration of granulopoietic cells present in PBMCs or PBMCs depleted of αβ T cells. The number or concentration of granulopoietic cells present in PBMCs or PBMCs depleted of αβ T cells may be increased by any suitable means. Consequently, the step of culturing PBMCs or PBMCs depleted of αβ T cells in the presence of granulopoietic cells may comprise culturing PBMCs or PBMCs depleted of αβ T cells under conditions suitable for expansion and / or differentiation of hematopoietic cells present in PBMCs or PBMCs depleted of αβ T cells.

[0417] Particularly preferably, the method of preparing a composition of the invention comprises cultivating PBMCs depleted of αβ T cells (for example, obtainable from a mobilized blood sample, such as Petition 870250101699, dated 06 / 11 / 2025, page 117 / 275 112 / 243 mobilized blood with G-CSF) under conditions that promote the differentiation of progenitor cells present in PBMCs depleted of αβ T cells into granulopoietic cells, thus forming the composition. Advantageously, this allows the composition to be prepared using cells from a single source, thus providing a significantly simplified and efficient method of preparing a composition of the invention.

[0418] The method may involve culturing PBMCs depleted of αβ T cells (e.g., obtainable from a mobilized blood sample, such as mobilized blood with G-CSF) under conditions to produce a progenitor cell from stem cells present in PBMCs depleted of αβ T cells.

[0419] The method may optionally comprise depleting αβ T cells from PBMCs (e.g., obtainable from a mobilized blood sample, such as mobilized blood with G-CSF).

[0420] Consequently, the method may include: (a) deplete αβ T cells from PBMCs (e.g., obtainable from a mobilized blood sample, such as G-CSF mobilized blood); and (b) culture the αβ T cell-depleted PBMCs under conditions that promote the differentiation of progenitor cells present in the αβ T cell-depleted PBMCs into granulopoietic cells, thus forming the composite.

[0421] The method may include: (a) deplete αβ T cells from PBMCs (e.g., obtainable from a mobilized blood sample, e.g., mobilized blood with G-CSF); (b) to cultivate αβ T cell-depleted PBMCs under conditions to produce progenitor cells from stem cells present in αβ T cell-depleted PBMCs; and (c) to cultivate the progenitor cell present in αβ T cell-depleted PBMCs under conditions that promote cell differentiation. Petition 870250101699, dated 06 / 11 / 2025, page 118 / 275 113 / 243 progenitor cells present in PBMCs depleted of αβ T cells in granulopoietic cells, thus forming the composition.

[0422] The conditions that promote the differentiation of progenitor cells present in PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) into granulopoietic cells can be any suitable conditions. For example, the conditions that promote the differentiation of progenitor cells present in PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) into granulopoietic cells can be the conditions described in this document that are suitable for obtaining a population of granulopoietic cells.

[0423] Similarly, the conditions for producing progenitor cells from stem cells present in PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) can be any suitable conditions. For example, the conditions for producing progenitor cells from stem cells present in PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) can be the conditions described in this document that are used in a method for obtaining a population of granulopoietic cells comprising a stem cell culture step under culture conditions to produce the progenitor cell.

[0424] The method may further comprise cultivating PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) under conditions suitable for maintaining NK cells. The method may further comprise cultivating PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) under conditions suitable for maintaining γδ T cells. Preferably, the method comprises cultivating PBMCs or PBMCs depleted of αβ T cells (e.g., PBMCs depleted of αβ T cells) under conditions suitable for maintaining both NK cells and γδ T cells.

[0425] Consequently, the method may include: Petition 870250101699, dated 06 / 11 / 2025, page 119 / 275 114 / 243 (a) cultivate αβ T cell-depleted PBMCs under conditions that promote the differentiation of progenitor cells present in αβ T cell-depleted PBMCs into granulopoietic cells; and (b) cultivate αβ T cell-depleted PBMCs under conditions suitable for maintaining NK cells and γδ T cells present in αβ T cell-depleted PBMCs, thus forming the composite.

[0426] The method may include: (a) cultivate αβ T cell-depleted PBMCs under conditions to produce progenitor cells from stem cells present in αβ T cell-depleted PBMCs and under conditions suitable for maintaining NK cells and γδ T cells present in αβ T cell-depleted PBMCs; and (b) cultivate the progenitor cells present in αβ T cell-depleted PBMCs under conditions that promote the differentiation of the progenitor cells present in αβ T cell-depleted PBMCs into granulopoietic cells, thus forming the composite.

[0427] Suitable conditions for maintaining NK cells and γδ T cells may include culturing PBMCs or depleted αβ T cell PBMCs (e.g., depleted αβ T cell PBMCs) in the presence of one or more cytokines selected from: IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21. Suitable conditions for maintaining NK cells and γδ T cells may include culturing PBMCs or depleted αβ T cell PBMCs (e.g., depleted αβ T cell PBMCs) in the presence of one or more selected cytokines from: IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21, where one or more cytokines are present at a concentration of 1-20 ng / mL, 5-15 ng / mL, or 7.5-12.5 ng / mL. Petition 870250101699, dated 06 / 11 / 2025, page 120 / 275 115 / 243 among: IL-15, IL-2, IL-7, IL-9, IL-4 and IL-21, wherein one or more cytokines are 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, during the maintenance phase, one or more cytokines are present at a concentration of 10 ng / mL. Consequently, the method may comprise culturing PBMCs or depleted αβ T cell PBMCs (e.g., depleted αβ T cell PBMCs) in the presence of one or more cytokines selected from: IL-15, IL-2, IL-7, IL-9, IL-4, and IL-21, wherein one or more cytokines are present at a concentration of 10 ng / mL. The method may comprise culturing PBMCs or depleted αβ T cell PBMCs (e.g., depleted αβ T cell PBMCs) 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 an appropriate time.For example, the method may comprise culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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 (e.g., at a concentration of 10 ng / mL) for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. The method may involve culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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 (e.g., at a concentration of 10 ng / mL) for up to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.The method may involve culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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 (e.g., at a concentration of 10 ng / mL) for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. The method may involve culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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 (e.g., at a concentration of 10 ng / mL) for 1-10 days, 2-10 days, 3-10 days, 4-10 days, 5-9 days, 6-9 days, or 7-9 days. Preferably, the... Petition 870250101699, dated 06 / 11 / 2025, pp. 121 / 275 116 / 243 method comprises culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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 (e.g., at a concentration of 10 ng / mL) for 7-9 days.

[0428] The method may further comprise cultivating PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T cell-depleted PBMCs) under conditions suitable for NK cell activation. The method may further comprise cultivating PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T cell-depleted PBMCs) under conditions suitable for γδ T cell activation. Preferably, the method comprises cultivating PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T cell-depleted PBMCs) under conditions suitable for NK cell and γδ T cell activation.

[0429] Consequently, the method may include: (a) cultivate αβ T cell-depleted PBMCs under conditions that promote the differentiation of progenitor cells present in αβ T cell-depleted PBMCs into granulopoietic cells; and (b) cultivate αβ T cell-depleted PBMCs under conditions suitable for the activation of NK cells and γδ T cells present in αβ T cell-depleted PBMCs, thus forming the composite.

[0430] The conditions suitable for the differentiation of hematopoietic cells present in PBMCs depleted of αβ T cells may be suitable for the activation of NK cells and γδ T cells present in PBMCs depleted of αβ T cells.

[0431] The method may include: (a) cultivate αβ T cell-depleted PBMCs under conditions to produce progenitor cells from stem cells present in αβ T cell-depleted PBMCs and cultivate αβ T cell-depleted PBMCs under conditions suitable for maintaining NK cells and γδ T cells present in αβ T cell-depleted PBMCs; and Petition 870250101699, dated 06 / 11 / 2025, page 122 / 275 117 / 243 (b) to cultivate the progenitor cells present in the αβ T cell-depleted PBMCs under conditions that promote the differentiation of the progenitor cells present in the αβ T cell-depleted PBMCs into granulopoietic cells and to cultivate the αβ T cell-depleted PBMCs under conditions suitable for the activation of NK cells and γδ T cells present in the αβ T cell-depleted PBMCs, thus forming the composite.

[0432] The appropriate conditions for activation of NK cells and γδ T cells may include culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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. Suitable conditions for NK cell and γδ T cell activation may include culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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, wherein one or more cytokines are present at a concentration of 20-200 ng / mL, 50-150 ng / mL, or 75-125 ng / mL.Suitable conditions for NK cell and γδ T cell activation may include culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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, wherein one or more cytokines are present at a concentration of 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL. Preferably, during the activation phase, one or more cytokines are present at a concentration of 100 ng / mL. Consequently, the method may involve culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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, wherein one or more cytokines are present at a concentration of 100 ng / mL.The method may involve culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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 (e.g., a). Petition 870250101699, dated 06 / 11 / 2025, page 123 / 275 118 / 243 concentration of 100 ng / mL) for any appropriate time. For example, the method may comprise culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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 (e.g., at a concentration of 100 ng / mL) for at least 1 day, 2 days, 3 days, 4 days, 5 days or 6 days. The method may involve culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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 (e.g., at a concentration of 100 ng / mL) for up to 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days.The method may involve culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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 (e.g., at a concentration of 100 ng / mL) for 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days. The method may comprise culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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 (e.g., at a concentration of 100 ng / mL) for 1-6 days, 2-6 days, 3-6 days, or 4-6 days. Preferably, the method comprises culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T 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 (e.g., at a concentration of 100 ng / mL) for 4-6 days.The appropriate conditions for NK cell and γδ T cell activation may further comprise culturing PBMCs or depleted αβ T cell PBMCs (e.g., depleted αβ T cell PBMCs) in the presence of a T cell receptor activator, such as an OKT3 activator, for example, a T cell receptor antibody, such as an anti-CD3 antibody. Without being limited by theory, it is believed that culturing PBMCs or depleted αβ T cell PBMCs in the presence of a T cell receptor may synergistically enhance the expansion and activation of γδ T cells. Consequently, the method may comprise culturing PBMCs or depleted αβ T cell PBMCs (e.g., depleted αβ T cell PBMCs). Petition 870250101699, dated 06 / 11 / 2025, pp. 124 / 275 119 / 243 of αβ T cells) in the presence of a T cell receptor activator, such as an OKT3 activator, for example, a T cell receptor antibody, such as an anti-CD3 antibody.

[0433] Preferably, the method comprises culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T cell-depleted PBMCs) in the presence of IL-15. The method may further comprise culturing PBMCs or αβ T cell-depleted PBMCs (e.g., αβ T cell-depleted PBMCs) in the presence of a T cell receptor activator, such as an OKT3 activator, for example, a T cell receptor antibody, such as an anti-CD3 antibody.

[0434] The method may comprise culturing a non-granulocytic immune cell differentiated from an iPSC (e.g., an iPSC-derived γδ T cell and / or an iPSC-derived NK cell) in the presence of a population of granulopoietic cells, thus forming the composite. The method may comprise culturing a non-granulocytic immune cell in the presence of a population of granulopoietic cells differentiated from an iPSC, thus forming the composite. The method may comprise culturing a non-granulocytic immune cell differentiated from an iPSC (e.g., an iPSC-derived γδ T cell and / or an iPSC-derived NK cell) in the presence of a population of granulopoietic cells differentiated from an iPSC, thus forming the composite. The method may involve cultivating an iPSC-derived αβ T cell, an iPSC-derived γδ T cell, an iPSC-derived NK cell, or combinations thereof in the presence of an iPSC-derived granulopoietic cell, thus forming the composite.For example, the method may involve culturing an iPSC-derived NK cell in the presence of an iPSC-derived granulopoietic cell, thus forming the composite. The method may involve culturing an iPSC-derived γδ T cell in the presence of an iPSC-derived granulopoietic cell, thus forming the composite. The method may involve culturing an iPSC-derived γδ T cell and an iPSC-derived NK cell in the presence of an iPSC-derived granulopoietic cell, thus forming the composite.

[0435] The method may involve differentiating an iPSC in a population of Petition 870250101699, dated 06 / 11 / 2025, pp. 125 / 275 120 / 243 granulopoietic cells, for example, a population of granulopoietic cells as defined in this document, and culturing a non-granulocytic immune cell in the presence of the iPSC-derived granulopoietic cell. The method may comprise differentiating an iPSC into a non-granulocytic immune cell, for example, a γδ T cell and / or an NK cell, and culturing the iPSC-derived non-granulocytic immune cell in the presence of a population of granulopoietic cells. The method may comprise differentiating an iPSC into a population of granulopoietic cells and differentiating an iPSC into a non-granulocytic immune cell, for example, a γδ T cell and / or an NK cell, and culturing the iPSC-derived non-granulocytic immune cell in the presence of the iPSC-derived granulopoietic cell.

[0436] Consequently, the method may include: (a) to differentiate an iPSC into a granulopoietic cell derived from an iPSC; (b) to differentiate an iPSC into an iPSC-derived γδ T cell; (c) Differentiate an iPSC into an iPSC-derived NK cell, and coculture the iPSC-derived granulopoietic cell, iPSC-derived γδ T cell, and iPSC-derived NK cell, thus forming the composite.

[0437] iPSCs can be obtained from any suitable donor. For example, iPSCs can be obtained from a donor that produces granulocytes with the ability to kill cancer cells, as defined using an assay described in this document.

[0438] iPSCs can be obtained from any suitable source. For example, iPSCs can be obtained from a somatic cell, such as an αβ T cell or a γδ T cell. iPSCs can be obtained from a stem cell. In embodiments where the method comprises differentiating an iPSC into a γδ T cell, the iPSC can be obtained from a γδ T cell. In embodiments where the method comprises differentiating an iPSC into an αβ T cell, the iPSC can be obtained from an αβ T cell. Petition 870250101699, dated 06 / 11 / 2025, pp. 126 / 275 121 / 243

[0439] Consequently, the method may include: (a) to differentiate an iPSC into a granulopoietic cell derived from an iPSC; (b) to differentiate an iPSC obtainable from a γδ T cell into an iPSC-derived γδ T cell; and (c) to differentiate an iPSC into an iPSC-derived NK cell, and to co-culture the iPSC-derived granulopoietic cell, iPSC-derived αβ T cell, and iPSC-derived NK cell, thus forming the composite.

[0440] The method may include: (a) to differentiate an iPSC obtainable from a γδ T cell into a granulopoietic cell derived from an iPSC; (b) to differentiate an iPSC obtainable from a γδ T cell into an iPSC-derived γδ T cell; and (c) to differentiate an iPSC obtainable from a γδ T cell into an iPSC-derived NK cell, and to co-culture the iPSC-derived granulopoietic cell, iPSC-derived αβ T cell, and iPSC-derived NK cell, thus forming the composite.

[0441] Cell culture additives can enhance the amplification of therapeutic immune responses. Consequently, populations of granulopoietic cells and non-granulocytic immune cells can be cultured in the presence of any suitable cell culture additive, such as a growth factor, a cytokine, or a chemokine. For example, populations of granulopoietic cells and non-granulocytic immune cells can be cultured in the presence of a granulocyte-macrophage colony-stimulating factor (GMCSF), a granulocyte colony-stimulating factor (G-CSF), a growth hormone; serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, an interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, serum (e.g., fetal bovine serum [FBS]), retinoic acid, lipopolysaccharide (LPS), IFN Petition 870250101699, dated 06 / 11 / 2025, pp. 127 / 275 122 / 243 gamma, IFN-beta, or combinations thereof. The population of granulopoietic cells and non-granulocytic immune cells can be cultured in the presence of IFN-gamma and a GM-CSF. The population of granulopoietic cells and non-granulocytic immune cells can be cultured in the presence of TNF-alpha. The population of granulopoietic cells and non-granulocytic immune cells can be cultured in the presence of a granulocyte colony-stimulating factor (GCSF), and a growth hormone, and serotonin, and vitamin C, and vitamin D, and glutamine (Gln), and arachidonic acid, and AGE-albumin, and an interleukin, and TNF-alpha, and Flt-3 ligand, and thrombopoietin and fetal bovine serum (FBS).The population of granulopoietic cells and non-granulocytic immune cells can be cultured in the presence of a granulocyte-macrophage colony-stimulating factor (GMCSF), and a granulocyte colony-stimulating factor (G-CSF), and a growth hormone, and serotonin, and vitamin C, and vitamin D, and glutamine (Gln), and arachidonic acid, and AGE-albumin, and an interleukin ligand, and TNF-alpha, and Flt3 ligand, and thrombopoietin, and fetal bovine serum (FBS), and retinoic acid, and lipopolysaccharide (LPS), and IFN-gamma and IFN-beta. The population of granulopoietic cells and non-granulocytic immune cells can be cultured in the presence of an anti-CD3 agonist, such as an anti-OKT3 antibody. The population of granulopoietic cells and non-granulocytic immune cells can be cultured in the presence of an anti-OKT3 antibody.

[0442] A population of granulopoietic cells and non-granulocytic immune cells that can be used in the various aspects of the invention can be provided in the form of an enriched population of such granulopoietic cells and non-granulocytic immune cells. In one aspect, the invention provides a pharmaceutical composition comprising an enriched population of granulopoietic cells and non-granulocytic immune cells.

[0443] By way of example only, such an enriched population may be a population of cells in which granulopoietic cells and non-granulocytic immune cells comprise 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%, by Petition 870250101699, dated 06 / 11 / 2025, pp. 128 / 275 123 / 243 minus 0.8%, at least 0.9%, or at least 1% of the total cell population. Such an enriched population may further be a cell population in which granulopoietic cells comprise 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, an enriched population can be a cell population in which granulopoietic cells comprise 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.

[0444] The granulopoietic cells and non-granulocytic immune cells of such an enriched population may be as defined in any appropriate embodiment set forth elsewhere in the descriptive report. For example, the granulopoietic cells of an enriched population may be CD62L-.

[0445] In one aspect of the invention, a pharmaceutical composition comprising an enriched population of granulopoietic cells and non-granulocytic immune cells is provided. The enriched population of granulopoietic cells and non-granulocytic immune cells incorporated into a pharmaceutical composition of the invention may be as described above.

[0446] Suitably, the granulopoietic cells present in a pharmaceutical composition of the invention may be CD62L-. Suitably, the granulopoietic cells present in a pharmaceutical composition of the invention may be CD16-. Suitably, the granulopoietic cells present in a pharmaceutical composition of the invention may be CD10-. Suitably, the granulopoietic cells present in a pharmaceutical composition of the invention may be CD16-, CD10- and CD62L-.

[0447] 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. Petition 870250101699, dated 06 / 11 / 2025, page 129 / 275 124 / 243

[0448] Suitably, the compositions (e.g., pharmaceutical compositions) of the invention may comprise a granulocyte-macrophage colony-stimulating factor (GM-CSF), a granulocyte colony-stimulating factor (G-CSF), a growth hormone; serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, an interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, serum (e.g., fetal bovine serum [FBS]), retinoic acid, lipopolysaccharide (LPS), IFN-gamma, IFN-beta, or combinations thereof. Suitably, the compositions (e.g., pharmaceutical compositions) comprise IFN-gamma and a GM-CSF. Preferably, the compositions (e.g., pharmaceutical compositions) comprise TNF-alpha.Particularly preferably, the compositions (e.g., pharmaceutical compositions) comprise a granulocyte-macrophage colony-stimulating factor (GM-CSF), and a granulocyte colony-stimulating factor (G-CSF), and a growth hormone, and serotonin, and vitamin C, and vitamin D, and glutamine (Gln), and arachidonic acid, and AGE-albumin, and an interleukin, and TNF-alpha, and Flt-3 ligand, and thrombopoietin, and fetal bovine serum (FBS). Preferably, the compositions (e.g., pharmaceutical compositions) comprise a granulocyte-macrophage colony-stimulating factor (GM-CSF), and a granulocyte colony-stimulating factor (G-CSF), and a growth hormone, and serotonin, and vitamin C, and vitamin D, and glutamine (Gln), and arachidonic acid, and AGE-albumin, and an interleukin, and TNF-alpha, and Flt-3 ligand, and thrombopoietin, and fetal bovine serum (FBS), and retinoic acid, and lipopolysaccharide (LPS), and IFN-gamma, and IFN-beta.

[0449] In the context of the present invention, a therapeutic host immune response should preferably be understood as an immune response that contributes to or achieves a desired therapeutic outcome. In a suitable embodiment, a therapeutic host immune response may be an immune response that leads (directly or indirectly) to the death of cancer cells, thus enabling cancer treatment. In a suitable embodiment, a therapeutic host immune response may be an immune response that leads Petition 870250101699, dated 06 / 11 / 2025, page 130 / 275 125 / 243 (directly or indirectly) to the death of infected cells or cellular infectious agents, thus allowing the treatment of an infection.

[0450] A therapeutic host immune response may involve the action of any cells of the immune system. A non-granulocytic immune response may involve the action of any cells of the immune system except granulocytes. Non-granulocytic immune responses may be beneficially amplified by any suitable population of granulopoietic cells according to the invention, or any suitable pharmaceutical composition comprising such a population of granulopoietic cells. Suitable compositions may utilize populations of granulopoietic cells without including non-granulocytic immune cells. Likewise, compositions comprising a population of granulopoietic cells and a non-granulocytic immune cell (or cells) may amplify a therapeutic host immune response, for example, after administration to a subject.By way of example only, a therapeutic host immune response that can be amplified by the compositions (e.g., pharmaceutical compositions), medical uses, or treatment methods of the invention may involve the action of one or more cell types selected from the group comprising (or consisting of): T cells (including, but not limited to, CD8+ T cells; CD4+ T cells; NK T cells; αβ T cells; γδ T cells; peripheral blood T cells; and tumor-infiltrated T cells); NK cells; monocytes; macrophages; dendritic cells (DCs); and B cells.

[0451] The amplification of an immune response (e.g., a therapeutic host immune response) can be demonstrated 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 co-stimulatory 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 Petition 870250101699, dated 06 / 11 / 2025, page 131 / 275 126 / 243 in the immune response; increased trafficking by immune cells involved in the immune response; increased recruitment in the TME of immune cells involved in the immune response; increased cytocidal activity by immune cells involved in the immune response; or increased tumor cell killing activity by immune cells involved in the immune response.

[0452] Alternatively, or additionally, the amplification of a therapeutic host immune response can be evaluated with reference to the outcome to be achieved by the therapeutic immune response.

[0453] For example, in the case of a therapeutic host immune response to be used in cancer treatment, amplification of the immune response may be demonstrated by an increase in the effectiveness of cancer treatment. Such an increase in effectiveness may be demonstrated by a reduction in symptoms; an increase in the rate and / or duration of patient survival; a reduction in tumor burden; prevention or delay of recurrence; a reduction in the severity of relapse; a reduction in the number of recurrence incidents; a reduction in the number of metastasis incidents; and / or a prevention or delay of metastasis.

[0454] In the case of a therapeutic host immune response being used in the treatment of infection, amplification of the immune response may be demonstrated by an increase in the effectiveness of the infection treatment. Such an increase may be demonstrated by a reduction in symptoms; an increase in the rate and / or duration of patient survival; a reduction in the infection burden; and / or a reduction in the time to infection clearance.

[0455] For the purposes of this disclosure, references to host cells (such as host immune cells) or a host immune response may be taken as referring to the cells or immune response of a subject receiving treatment with, or purportedly receiving treatment with, populations of granulopoietic cells or compositions in accordance with any of the various aspects of the invention. Except where the context otherwise requires, all references to immune cells or immune responses in connection with the various aspects and embodiments of the invention should be taken as Petition 870250101699, dated 06 / 11 / 2025, page 132 / 275 127 / 243 applicable to host immune cells or host immune responses.

[0456] A population of granulopoietic cells or composition suitable for use according to the various aspects of the present invention may be able to increase (preferably enhance) the activation of host immune cells. Consequently, such a cell may be able to amplify (preferably enhance) a therapeutic host immune response by increasing the activation of host immune cells. It will be appreciated that it is the activated immune cells that are primarily responsible for providing the desired activity in a therapeutic immune response. Consequently, the ability of medical uses and treatment methods to increase the activation of host immune cells will be beneficial in almost all circumstances where a therapeutically effective immune response is required.In particular, amplifying a therapeutic immune response by increasing the activation of host immune cells can, without limitation, be advantageous in the treatment of cancer or infections.

[0457] Granulopoietic cell populations suitable for use according to the present invention may exhibit some or all of the properties set forth above.

[0458] Appropriately, a therapeutically effective amount of such a population of granulopoietic cells (or of a composition [e.g., a pharmaceutical composition] of the invention comprising a population of granulopoietic cells), for example, for use in accordance with the invention, is an amount sufficient to increase the activation of immune cells, such as host immune cells. The extent of the increase, relevant host immune cells, and suitable indicators of increased activation may be considered in the preceding and / or following paragraphs.

[0459] A population of granulopoietic cells or composition suitable for use according to the present invention can enhance the activation of host T cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a Petition 870250101699, dated 06 / 11 / 2025, page 133 / 275 128 / 243 therapeutic host immune response, increasing the activation of host T cells.

[0460] It will be appreciated that increasing the activation of host T cells, such as CD8+ and CD4+ T cells, will contribute significantly to the desired activity in a therapeutic immune response. Cytotoxic T cells, such as CD8+ T cells, are known to have direct cytocidal activity, while helper T cells, such as CD4+ T cells, are known to help coordinate the immune response by further stimulating other immune cells. Consequently, the use of a granulopoietic cell population or composition to increase host T cell activation will be beneficial in a wide range of circumstances where a therapeutically effective immune response is required. In particular, amplifying a therapeutic host immune response by increasing host T cell activation may, without limitation, be advantageous in the treatment of cancer or in the treatment of infections.

[0461] A host T cell, whose activation can be increased, may be selected from the group comprising (or consisting of): a CD8+ T cell; a CD4+ T cell; an NK T cell; an αβ T cell; a γδ T cell; a peripheral blood T cell; and a tumor-infiltrated T cell.

[0462] Appropriately, a therapeutically effective amount of such a population of granulopoietic cells (or of a composition [e.g., a pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the activation of host T cells. The extent of the increase and suitable indicators of increased activation may be considered in the preceding and / or following paragraphs.

[0463] A population of granulopoietic cells or composition suitable for use according to the present invention can increase the activation of host CD8+ T cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing T cell activation. Petition 870250101699, dated 06 / 11 / 2025, page 134 / 275 129 / 243 Host CD8+.

[0464] A population of granulopoietic cells or composition suitable for use according to the present invention can enhance the activation of host CD4+ T cells, such as host CD4+ T cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the activation of host CD4+ T cells.

[0465] A population of granulopoietic cells or composition suitable for use according to the present invention can enhance the activation of host NK T cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by enhancing the activation of host NK T cells.

[0466] Increased NK T cell activation may be associated with one or more of the following: an increase in NK T cell expression of a degranulation marker (including, but not limited to, CD107a); an increase in NK T cell expression of a co-stimulatory molecule (including, but not limited to, 41BB and / or OX40); and an increase in NK T cell survival. Other relevant considerations regarding these various properties are set out elsewhere in this descriptive report.

[0467] Host NK T cells, whose activation is increased, may be peripheral blood NK T cells or may be tumor-infiltrated NK T cells.

[0468] The activation of such NK T cells can be increased by at least 5%. For example, NK T cell activation can be increased 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 T cell activation according to such a modality can make use of comparison with an appropriate control. Petition 870250101699, dated 06 / 11 / 2025, pp. 135 / 275 130 / 243

[0469] A population of granulopoietic cells or composition suitable for use according to the present invention can increase the activation of host γδ T cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the activation of host γδ T cells.

[0470] A population of granulopoietic cells or composition suitable for use according to the present invention can enhance the activation of host NK cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the activation of host NK cells.

[0471] Those skilled in the art will appreciate that NK cells play an important role in providing the activity necessary to achieve a therapeutic immune response. NK cells show strong cytolytic activity against physiologically stressed cells, such as tumor cells and virus-infected cells. Consequently, the use of a granulopoietic cell population to enhance NK cell activation will be beneficial in a wide range of circumstances where a therapeutically effective immune response is required. In particular, amplifying a therapeutic immune response by increasing NK cell activation may, without limitation, be advantageous in the treatment of cancer or in the treatment of infections.

[0472] Host NK cells, whose activation is increased, may be peripheral blood NK cells or may be tumor-infiltrated NK cells.

[0473] Appropriately, a therapeutically effective amount of such a granulopoietic cell population (or of a composition [e.g., a pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the activation of host NK cells. The extent of the increase and suitable indicators of increased activation may be considered in the preceding paragraphs and / or those that follow. Petition 870250101699, dated 06 / 11 / 2025, page 136 / 275 131 / 243 follow.

[0474] A population of granulopoietic cells or composition suitable for use according to the present invention can increase the activation of host monocytes or macrophages. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the activation of host monocytes or macrophages.

[0475] A population of granulopoietic cells or composition suitable for use according to the present invention can enhance the activation of host PBMCs. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by enhancing the activation of host PBMCs.

[0476] It will be appreciated that PBMCs play an essential role in supplying the cells that contribute to any effective therapeutic immune response. PBMCs can be taken to refer to any peripheral blood cell with a single round nucleus, such as T cells and NK cells. These cells have a variety of fundamental functions to drive the immune response, including cytocidal activity or activation of other immune cells. Consequently, the use of a granulopoietic cell population or composition to enhance the activation of host PBMCs will be beneficial in almost all circumstances where a therapeutically effective immune response is required. In particular, amplifying a therapeutic immune response by increasing the activation of host PBMCs can, without limitation, be advantageous in the treatment of cancer or in the treatment of infections.

[0477] The host PBMCs whose activation should be increased include, but are not limited to, those selected from the group comprising (or consisting of): peripheral blood T cells (such as: peripheral blood CD8+ T cells; peripheral blood CD4+ T cells; peripheral blood NK cells; peripheral blood αβ T cells; or peripheral blood γδ T cells); and peripheral blood NK cells. Petition 870250101699, dated 06 / 11 / 2025, page 137 / 275 132 / 243 peripheral.

[0478] Increased activation of host PBMCs can be demonstrated by any appropriate activation marker. By way of example only, increased PBMC activation can be demonstrated by increased cytokine expression (such as: IFN-γ; and / or TNF). The ability to increase cytokine expression by host PBMCs exposed to granulopoietic cell populations suitable for use according to the present invention is shown in the Examples. Other relevant considerations regarding these various properties are set forth elsewhere in this descriptive report.

[0479] Host PBMC activation can be increased by at least 5%. For example, PBMC activation can be increased 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 host PBMC activation according to such a modality can make use of comparison with an appropriate control.

[0480] Appropriately, a therapeutically effective amount of such a population of granulopoietic cells (or of a composition [e.g., a pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the activation of host PBMCs. The extent of the increase and appropriate indicators of increased activation may be considered in the preceding and / or following paragraphs.

[0481] A population of granulopoietic cells or composition suitable for use according to the present invention can increase the activation of host TILs. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the activation of host TILs. Petition 870250101699, dated 06 / 11 / 2025, page 138 / 275 133 / 243

[0482] For the purposes of the present invention, host TILs can be understood as encompassing all populations of lymphocytic cells that have invaded tumor tissue. With this in mind, it will be recognized that TILs play a fundamental role in the component that exerts a therapeutic immune response against tumor cells. TILs can exert specific cytotoxic antitumor activity (e.g., CD8+ cells that have entered the tumor) and can promote an antitumor response through the activation of other immune cells (such as CD4+ cells within the tumor). Consequently, amplifying a therapeutic immune response by increasing the activation of host TILs can play a highly advantageous role in cancer treatment.

[0483] In particular, the inventors have determined that a population of granulopoietic cells suitable for use according to the present invention can enhance the activation of tumor-infiltrated T cells and / or NK cells. Such granulopoietic cells can enhance the activation of tumor-infiltrated CD8+ T cells and / or CD4+ T cells, as demonstrated in the Examples.

[0484] Increased activation of host TILs, such as increased activation of tumor-infiltrating T cells or tumor-infiltrating NK cells, can be demonstrated by any appropriate activation marker. By way of example only, increased TIL activation can be demonstrated by increased expression of degranulation markers (such as: CD107a; perforin; or granzymes). Alternatively, or additionally, increased TIL activation can be demonstrated by increased expression of co-stimulatory molecules (such as: 4-1BB; OX40; CD27; CD28; ICOS; HVEM; LIGHT; CD40L; DR3; GITR; CD30; TIM1; CD2; or CD226). The ability to increase the expression of degranulation markers or co-stimulatory molecules by TILs exposed to granulopoietic cell populations suitable for use according to the present invention is demonstrated in the Examples.Other relevant considerations regarding these various properties are set forth elsewhere in this descriptive report. Petition 870250101699, dated 06 / 11 / 2025, page 139 / 275 134 / 243

[0485] TIL activation can be increased by at least 5%. For example, TIL activation can be increased 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. Quantifying the increase in TIL activation according to such a modality can make use of comparison with an appropriate control.

[0486] Appropriately, a therapeutically effective amount of a population of such granulopoietic cells (or of a composition [e.g., a pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the activation of host TILs. The extent of the increase and appropriate indicators of increased activation may be considered in the preceding and / or following paragraphs.

[0487] A population of granulopoietic cells suitable for use according to the present invention may be able to increase the expression of degranulation markers by immune cells. In particular, granulopoietic cells may be able to increase (preferably increase) the expression of degranulation markers by the non-granulocytic immune cell present in a composition of the invention. A population of granulopoietic cells or composition may be able to increase (preferably increase) the expression of degranulation markers by host immune cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the expression of degranulation markers by host immune cells.

[0488] Degranulation is a key process in the cytocidal activity of immune cells, such as CD8+ T cells or NK cells, which sustains their therapeutic immune activity. Consequently, it will be appreciated that the increased expression of Petition 870250101699, dated 06 / 11 / 2025, pp. 140 / 275 135 / 243 degranulation markers, such as CD107, provide an indication that the therapeutic immune activity of such cells has been increased and the therapeutic immune response amplified accordingly.

[0489] In a suitable embodiment, a degranulation marker, whose expression by host immune cells is increased, is selected from the group comprising (or consisting of): CD107a; perforin; and granzymes. Suitablely, the expression of more than one of these degranulation markers may be increased. For example, the expression of at least 2 of these degranulation markers may be increased. In particular, the expression by host immune cells of CD107a may be increased.

[0490] The increase in the expression of degranulation markers can be evaluated and, if desired, quantified, by any appropriate method.

[0491] In a suitable embodiment, the expression of a degranulation marker is increased by at least 5%. For example, the expression of a degranulation marker may be increased 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 degranulation marker expression according to such an embodiment may make use of comparison with an appropriate control.

[0492] The expression of degranulation markers can be increased in non-granulocytic immune cells present in a composition of the invention or host immune cells selected from the group comprising (or consisting of): T cells and NK cells. In the case of the expression of degranulation markers being increased in a T cell, such T cell can be selected from the group comprising (or consisting of): a CD8+ T cell; an NK T cell; an αβ T cell; and a γδ T cell.

[0493] Appropriately, a therapeutically effective quantity of such a population of granulopoietic cells (or of a composition [for example, a Petition 870250101699, dated 06 / 11 / 2025, page 141 / 275 136 / 243 pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is a sufficient quantity to increase the expression by immune cells, such as host immune cells, of one or more degranulation markers. The degranulation markers, extent of increase and relevant host immune cells can be considered in the preceding paragraphs.

[0494] A population of granulopoietic cells suitable for use according to the present invention may be able to increase the expression by immune cells of a co-stimulatory molecule. In particular, granulopoietic cells may be able to increase (preferably increase) the expression of co-stimulatory molecules by the non-granulocytic immune cell present in a composition of the invention. A population of granulopoietic cells or composition may be able to increase (preferably increase) the expression of co-stimulatory molecules by host immune cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the expression by host immune cells of a co-stimulatory molecule.

[0495] Co-stimulatory molecules act to amplify or neutralize activation signals delivered to T cells that cause T cell differentiation. T cell differentiation is a key process in the therapeutic immune response, giving rise to the production of cytotoxic T cells or helper T cells. Increasing the expression of co-stimulatory molecules can thus direct the functional differentiation of T cells, causing the therapeutic immune response to be amplified. Using a population of granulopoietic cells to increase the expression of co-stimulatory molecules will be beneficial in a wide range of circumstances where a therapeutically effective immune response is needed. In particular, amplifying a therapeutic immune response by increasing the activation of co-stimulatory molecules can, without limitation, be advantageous in the treatment of cancer or infections.

[0496] In a suitable embodiment, a co-stimulatory molecule, whose Petition 870250101699, dated 06 / 11 / 2025, page 142 / 275 137 / 243 expression by non-granulocytic immune cells and / or host immune cells is increased, is selected from the group comprising (or consisting of): 4-1BB; OX40; CD27; CD28; ICOS; HVEM; LIGHT; CD40L; DR3; GITR; CD30; TIM1; CD2; and CD226. Appropriately, the expression of more than one of these costimulatory 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 of these costimulatory molecules may be increased. In particular, the expression by non-granulocytic immune cells and / or host immune cells of 4-1BB and OX40 may be increased.

[0497] The expression of a co-stimulatory molecule can be evaluated and, if desired, quantified by any appropriate method.

[0498] In a suitable embodiment, the expression of a co-stimulatory molecule is increased by at least 5%. For example, the expression of a co-stimulatory molecule may be increased 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 the expression of a co-stimulatory molecule according to such an embodiment may make use of comparison with an appropriate control.

[0499] The expression of the costimulatory molecule can be increased in non-granulocytic immune cells and / or host immune cells selected from the group comprising (or consisting of): T cells and NK cells. In the case of increased expression of the costimulatory molecule in a T cell, such T cell can be selected from the group comprising (or consisting of): a CD8+ T cell; a CD4+ T cell; an NK T cell; an αβ T cell; a γδ T cell; a peripheral blood T cell; and a tumor-infiltrated T cell.

[0500] Appropriately, a therapeutically effective quantity of such granulopoietic cells (or of a composition [for example, a composition Petition 870250101699, dated 06 / 11 / 2025, page 143 / 275 138 / 243 pharmaceutical] of the invention), for example, for use in accordance with the invention, is a quantity sufficient to increase the expression by immune cells, such as non-granulocytic immune cells or host immune cells, of one or more costimulatory molecules. The costimulatory molecules, extent of increase and relevant host immune cells can be considered in the preceding paragraphs.

[0501] A population of granulopoietic cells suitable for use according to the present invention may be able to increase the expression of cytokines by immune cells. In particular, granulopoietic cells may be able to increase (preferably increase) the expression of cytokines by the non-granulocytic immune cell present in a composition of the invention. A population of granulopoietic cells or composition may be able to increase (preferably increase) the expression of cytokines by host immune cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the expression by host immune cells of a co-stimulatory molecule.

[0502] Cytokines are the main chemical messengers in the immune response. Cytokines signal for cell activation (targeting immune cells), immune cell differentiation, such as during T cell differentiation, and immune cell proliferation, such as NK cells. Using a population of granulopoietic cells to increase cytokine activation will be beneficial in almost all circumstances where a therapeutically effective immune response is needed. In particular, amplifying a therapeutic immune response by increasing cytokine activation can, without limitation, be advantageous in cancer treatment or infection treatment.

[0503] For the purposes of the present invention, cytokines should be understood as encompassing chemokines, interferons, interleukins, lymphokines and TNFs.

[0504] In a suitable embodiment, a cytokine whose expression by non-granulocytic immune cells and / or host immune cells is increased is Petition 870250101699, dated 06 / 11 / 2025, pp. 144 / 275 139 / 243 selected from the group comprising (or consisting of): IFN-γ; and TNF. Appropriately, the expression of more than one of these co-stimulatory molecules may be increased. In particular, the expression of IFN-γ by non-granulocytic immune cells and / or host immune cells may be increased.

[0505] The increase in cytokine expression can be assessed and, if desired, quantified by any appropriate method.

[0506] In a suitable embodiment, the expression of a cytokine is increased by at least 5%. For example, the expression of a cytokine may be increased 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 according to such an embodiment may make use of comparison with an appropriate control.

[0507] The expression of a cytokine can be increased in selected host immune cells from the group comprising (or consisting of): PBMCs; and TILs. The ability of granulopoietic cell populations suitable for use according to the invention to increase the expression by PBMCs and TILs of cytokines (such as IFN-γ) is demonstrated in the Examples.

[0508] Suitably, a therapeutically effective amount of such a granulopoietic cell population (or of a composition [e.g., a pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the expression by immune cells, such as non-granulocytic immune cells and / or host immune cells, of one or more cytokines. The cytokines, extent of increase, and relevant host immune cells can be considered in the preceding paragraphs.

[0509] A granulopoietic cell population suitable for use in accordance with the present invention may be capable of increasing immune cell trafficking. In particular, a granulopoietic cell population of this type may Petition 870250101699, dated 06 / 11 / 2025, pages 145 / 275 140 / 243 be able to increase (preferably increase) the trafficking of host immune cells. Consequently, such a population of granulopoietic cells may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the trafficking of host immune cells.

[0510] The trafficking of immune cells plays a vital role in their ability to access sites, such as tumor sites or infection sites, where they are needed to exert their therapeutic activity. Therefore, it will be appreciated that the ability of granulopoietic cell populations or compositions suitable for use according to the invention to enhance immune cell trafficking confers clear advantages in terms of facilitating an effective therapeutic immune response.

[0511] Increased cell trafficking can be observed in relation to PBMCs and, particularly, in relation to host PBMCs. As noted elsewhere, the inventors have demonstrated that granulopoietic cell populations suitable for use according to the invention can give rise to granulocytes expressing CXCL10, which is known to act as a chemoattractant for CXCR3+ immune cells. Thus, the medical uses and treatment methods of the invention, giving rise to a population of cells expressing CXCL10, can be particularly beneficial in increasing the trafficking of CXCR3+ T cells and CXCR3+ NK cells.

[0512] The increase in immune cell traffic can be assessed and, if desired, quantified by any appropriate method.

[0513] In a suitable embodiment, immune cell trafficking is increased by at least 5%. For example, immune cell trafficking may be increased 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 immune cell trafficking according to such an embodiment may make use of comparison with an appropriate control. Petition 870250101699, dated 06 / 11 / 2025, pp. 146 / 275 141 / 243

[0514] Appropriately, a therapeutically effective amount of such granulopoietic cell populations (or of a composition [e.g., a pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the trafficking of immune cells, such as host immune cells. The extent of the increase in trafficking and the relevant host immune cells can be considered in the preceding paragraphs.

[0515] In particular, increased immune cell traffic may give rise to increased immune cell recruitment in the TME.

[0516] As noted above, the inventors observed that exposure to a population of granulopoietic cells suitable for use according to the present invention increases immune cell trafficking. In particular, the inventors observed that populations of granulopoietic cells or compositions suitable for use according to the present invention can increase the recruitment of immune cells to the TME. As demonstrated in the Examples, a population of granulopoietic cells or composition of this type may be able to increase (preferably increase) the recruitment to the TME of host immune cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the recruitment of host immune cells to the TME.

[0517] The low propensity for immune cells to enter the TME is well known. Many immune cells demonstrate little ability to penetrate tumors, and the TME has immunosuppressive properties. Consequently, the ability to increase the recruitment of immune cells, such as host immune cells, to the TME through granulopoietic cell populations or compositions suitable for use according to the invention offers notable advantages in tumor treatment. By increasing the number of immune cells present in a tumor, the antitumor activity of the cells exerting the therapeutic immune response can be dramatically increased. Petition 870250101699, dated 06 / 11 / 2025, pp. 147 / 275 142 / 243

[0518] Increased recruitment of immune cells to the TME can be observed in relation to PBMCs and, particularly, in relation to host PBMCs. The ability of granulopoietic cell populations or compositions suitable for use according to the treatment of the invention to increase such recruitment to the TME is demonstrated in the Examples.

[0519] In the Examples, the inventors also demonstrate that granulopoietic cell populations and compositions suitable for use according to the invention can differentiate to give rise to granulocytes expressing CXCL10. CXCL10 is a chemoattractant for CXCR3+ immune cells, which may include CXCR3+ T cells and CXCR3+ NK cells. Thus, granulopoietic cell populations and compositions suitable for use according to the invention can be particularly beneficial in establishing a population of granulocyte progeny cells capable of increasing (preferably increasing) the recruitment of CXCR3+ T cells and CXCR3+ NK cells to the TME.

[0520] The increased recruitment of immune cells to the TME can be assessed and, if desired, quantified by any appropriate method.

[0521] In a suitable embodiment, immune cell recruitment to the TME is increased by at least 5%. For example, immune cell recruitment to the TME may be increased 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 immune cell recruitment to the TME according to such an embodiment may make use of comparison with an appropriate control.

[0522] Appropriately, a therapeutically effective amount of such a granulopoietic cell population (or of a composition [e.g., a pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the recruitment of immune cells, such as host immune cells, to the TME. The extent of Petition 870250101699, dated 06 / 11 / 2025, pp. 148 / 275 143 / 243 Increased recruitment of immune cells to the TME and in relevant host immune cells can be considered in the preceding paragraphs.

[0523] A population of granulopoietic cells suitable for use according to the present invention may be able to increase the cytocidal activity of immune cells. In particular, granulopoietic cells may be able to increase (preferably increase) the cytocidal activity of the non-granulocytic immune cell present in a composition of the invention. A population of granulopoietic cells or composition may be able to increase (preferably increase) the cytocidal activity of host immune cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the cytocidal activity of host immune cells.

[0524] Cell death of infected, cancerous, or other pathological cells is a key mechanism by which many immune cells exert their therapeutic activity. Therefore, it will be appreciated that the ability of granulopoietic cell populations and compositions suitable for use according to the invention to enhance the cytocidal activity of immune cells will offer advantages in terms of increasing the effectiveness of therapeutic immune responses that can be used to treat a large number of conditions, including cancer and infections.

[0525] Increased cytocidal activity of immune cells can be observed in relation to PBMCs and, particularly, in relation to host PBMCs.

[0526] The increase in cytocidal activity of immune cells can be assessed and, if desired, quantified by any appropriate method.

[0527] In a suitable embodiment, the cytocidal activity of immune cells is increased by at least 5%. For example, the cytocidal activity of immune cells may be increased 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 Petition 870250101699, dated 06 / 11 / 2025, pp. 149 / 275 144 / 243 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% or more. Quantification of cytocidal activity of immune cells according to this method may make use of comparison with an appropriate control.

[0528] Appropriately, a therapeutically effective amount of such a population of granulopoietic cells (or of a composition [e.g., a pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the cytocidal activity of immune cells, such as host immune cells. The extent of the increase in cytocidal activity of the relevant immune cells and host immune cells can be considered in the preceding paragraphs.

[0529] In particular, the cytocidal increase of immune cells can give rise to increased cell-killing activity of tumor cells, immune cells, and especially host immune cells.

[0530] A population of granulopoietic cells suitable for use according to the present invention may be able to increase the tumor cell killing activity of immune cells. The tumor cell killing activity of which the immune cells to be increased may be in vitro cells, or in vivo cells, for example, immune cells present in a subject who is provided with a suitable population of granulopoietic cells. In particular, the granulopoietic cells may be able to increase (preferably increase) the tumor cell killing activity of a non-granulocytic immune cell present in a suitable composition of the invention. A population of granulopoietic cells or composition may be able to increase (preferably increase) the tumor cell killing activity of host immune cells.Consequently, such a population or composition of granulopoietic cells may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the tumor cell-killing activity of host immune cells.

[0531] The use of immune cells to target and kill cancer cells Petition 870250101699, dated 06 / 11 / 2025, pages 150 / 275 145 / 243 forms the basis for most anticancer immunotherapy. Consequently, it will be readily appreciated that the ability of the granulopoietic cell population and compositions suitable for use according to the invention to enhance the tumor cell-killing activity of immune cells, such as host immune cells, provides clear and desirable advantages in anticancer treatments.

[0532] Increased tumor cell killing activity of immune cells can be observed in relation to PBMCs and, particularly, in relation to host PBMCs. Such increases are demonstrated in the results provided in the Examples.

[0533] The increased activity of immune cells in killing tumor cells can be assessed and, if desired, quantified by any appropriate method.

[0534] In a suitable embodiment, the tumor cell killing activity of immune cells is increased by at least 5%. For example, the tumor cell killing activity of immune cells may be increased 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 tumor cell killing activity of immune cells according to such an embodiment may make use of comparison with an appropriate control.

[0535] Appropriately, a therapeutically effective amount of such granulopoietic cell populations (or of a composition [e.g., a pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the tumor cell killing activity of immune cells, such as host immune cells. The extent of the increase in tumor cell killing activity of the relevant immune cells and host immune cells can be considered in the preceding paragraphs.

[0536] A population of granulopoietic cells suitable for use of Petition 870250101699, dated 06 / 11 / 2025, pp. 151 / 275 146 / 243 according to the present invention may be able to increase the proliferation of immune cells. In particular, granulopoietic cells may be able to increase (preferably increase) the proliferation of non-granulocytic immune cells present in a composition of the invention. A population of granulopoietic cells or composition may be able to increase (preferably increase) the proliferation of host immune cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the proliferation of host immune cells.

[0537] Immune cell-based therapies depend on the development of therapeutically effective quantities of adequate immune cells to be able to provide the necessary therapeutic immune response (e.g., in the treatment of cancer or infections). Therefore, it will be appreciated that the ability of granulopoietic cell populations and compositions suitable for use according to the invention to increase the proliferation of immune cells, such as host immune cells, is highly beneficial in achieving this. For example, by increasing immune cell proliferation, granulopoietic cells and compositions suitable for use according to the invention may be able to amplify (preferably amplify) immune responses that would otherwise not reach a therapeutic threshold or reduce the time required for the therapeutically effective quantity of immune cells to be produced.

[0538] In a suitable embodiment, the proliferation of T cells, such as host T cells, can be increased. Suitable T cells can be selected from the group comprising (or consisting of): an αβ T cell; a CD8+ T cell; a CD4+ T cell; an NK T cell; and a γδ T cell. In particular, the proliferation of αβ T cells can be increased, as demonstrated by the data set forth in the Examples. By way of example only, the αβ T cells can be CD4+ T cells or they can be CD8+ T cells.

[0539] The increase in immune cell proliferation can be assessed and, if desired, quantified by any appropriate method. Petition 870250101699, dated 06 / 11 / 2025, pp. 152 / 275 147 / 243

[0540] Appropriately, the proliferation of host immune cells can be increased by at least 5%. For example, the proliferation of host immune cells can be increased 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 the proliferation of host immune cells according to such a modality can make use of comparison with an appropriate control.

[0541] Appropriately, a therapeutically effective amount of such granulopoietic cell populations (or of a composition [e.g., a pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the proliferation of immune cells, such as host immune cells. The extent of the increase in the proliferation of relevant immune cells and host immune cells can be considered in the preceding paragraphs.

[0542] A population of granulopoietic cells suitable for use according to the present invention may be able to increase the survival of immune cells. In particular, granulopoietic cells may be able to increase (preferably increase) the survival of the non-granulocytic immune cell present in a composition of the invention. A population of granulopoietic cells or composition may be able to increase (preferably increase) the survival of host immune cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response, increasing the survival of host immune cells.

[0543] It is well known that immune cells have a limited lifespan, being rapidly transformed within the body. This is increased in contexts such as TME, where immunosuppressive conditions further reduce the lifespan of immune cells that enter the tumor. The inventors' discovery that the cells Petition 870250101699, dated 06 / 11 / 2025, pp. 153 / 275 148 / 243 Granulopoietic cells and compositions suitable for use in accordance with the treatment of the invention are capable of increasing the survival of immune cells, thus indicating that treatments using such granulopoietic cells may offer advantages in terms of prolonging the period during which immune cells are able to generate an effective therapeutic immune response. This may be of particular value in the treatment of conditions such as cancer, where an immunosuppressive environment reduces the longevity of immune cells.

[0544] In a suitable embodiment, the survival of T cells (such as NK T cells) or NK cells can be increased. For example, the survival of host T cells (such as NK T cells) or NK cells can be increased. Data illustrating the ability of granulopoietic cells and useful compositions according to the invention to increase the survival of NK T cells and NK cells are presented in the Examples.

[0545] The increase in immune cell survival can be assessed and, if desired, quantified by any appropriate method.

[0546] Appropriately, the survival of host immune cells can be increased by at least 5%. For example, the survival of host immune cells can be increased 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 host immune cell survival according to such a modality can make use of comparison with an appropriate control.

[0547] Suitably, a therapeutically effective amount of such granulopoietic cell populations (or of a composition [e.g., a pharmaceutical composition] of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the survival of immune cells, such as host immune cells. The extent of the increase in the survival of relevant immune cells and host immune cells may Petition 870250101699, dated 06 / 11 / 2025, pp. 154 / 275 149 / 243 to be considered in the preceding paragraphs.

[0548] A population of granulopoietic cells suitable for use according to the present invention may be able to increase the abundance of immune cells. In particular, a population of granulopoietic cells or composition of this type may be able to increase (preferably increase) the abundance of host immune cells. Consequently, such a population of granulopoietic cells or composition may be able to amplify (preferably amplify) a therapeutic host immune response by increasing the abundance of host immune cells.

[0549] Without wishing to be bound by any hypothesis, the increase in the abundance of immune cells observed upon exposure of such cells to granulopoietic cell populations and compositions suitable for use according to the invention may arise as a result of a combination of the increased proliferation and increased survival of immune cells discussed in more detail above. However, it offers real benefits in terms of the medical uses and methods of the invention. By increasing the abundance of immune cells capable of participating in a therapeutic immune response, the medical uses and treatment methods of the invention have the ability to amplify such a therapeutic immune response both in terms of extent and duration. This will clearly provide benefits in many therapeutic contexts.

[0550] The increase in the abundance of immune cells can be assessed and, if desired, quantified by any appropriate method.

[0551] Appropriately, the abundance of host immune cells can be increased by at least 5%. For example, the abundance of host immune cells can be increased 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 le...

Claims

1. Method of preparing cells for therapeutic use, the method characterized in that it comprises: • cultivating a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells comprising the presence of: • G-CSF, • GM-CSF, • IL-3 and • TNF; to produce a population of granulopoietic cells.

2. Method according to claim 1, characterized in that the cell culture conditions that promote the differentiation of progenitor cells further comprise at least one supplement from the group consisting of: SCF; TPO; ITS; and HSA.

3. Method according to claim 2, characterized in that the cell culture condition that promotes the differentiation of progenitor cells comprises: • GM-CSF at a concentration of approximately 0.01 pg / mL; and • G-CSF at a concentration of approximately 0.13 pg / mL; and • SCF at a concentration of approximately 0.13 pg / mL; and • TPO at a concentration of approximately 0.13 pg / mL; and • IL-3 at a concentration of approximately 0.13 pg / mL; and • TNF at a concentration of approximately 0.001 pg / mL; and • ITS; and • HSA at approximately 1%.

4. A method, according to any one of claims 1 to 3, characterized in that the progenitor cells are cultured for 4 to 6 days under conditions to produce a population of granulopoietic cells.

5. Method, according to claim 4, characterized by the fact that Petition 870250080961, dated 09 / 09 / 2025, page 18 / 32 2 / 14, the progenitor cells are cultured for 4 or 5 days under conditions to produce a population of granulopoietic cells.

6. A method according to any of the preceding claims, characterized in that GM-CSF and IL-3 are supplied to the cells during the final 48 hours of the period during which they are in culture.

7. A method, according to any of the preceding claims, characterized in that TNF is supplied to the cells during the final 24 hours of the period during which they are in culture.

8. A method, according to any preceding claim, characterized in that it further comprises a step of cultivating a population of stem cells under cell culture conditions to produce the progenitor cell population: • wherein the cell culture conditions for the production of progenitor cells comprise the presence of • SCF, • Flt-3 ligand, • IL-3, • IL-6, and • TPO.

9. Method according to claim 8, characterized in that the cell culture conditions for the production of progenitor cells comprise: • SCF at a concentration of approximately 0.2 pg / mL; and • Flt-3 ligand at a concentration of approximately 0.2 pg / mL; and • IL-3 at a concentration of approximately 0.015 pg / mL; and • IL-6 at a concentration of approximately 0.015 pg / mL; and • TPO at a concentration of approximately 0.02 pg / mL; and • ITS; and • HSA at approximately 1%.

10. Method, according to claim 5 or 6, characterized by the fact that stem cells are cultured for 8-9 days under conditions to produce a population of progenitor cells.

11. Method according to claim 10, characterized in that stem cells are cultured for 8 days under conditions to produce a population of progenitor cells.

12. Method, according to any one of claims 8 to 11, characterized in that the stem cells are HSCs.

13. A method, according to any preceding claim, characterized in that it further comprises a purification step of the granulopoietic cell population produced.

14. A method, according to any preceding claim, characterized in that it further comprises a step of formulating the population of granulopoietic cells produced for medical use.

15. A method, according to any preceding claim, characterized in that it further comprises preparing granulopoietic cells for therapeutic use by a method comprising culturing the granulopoietic cell population 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. Method according to claim 15, characterized in that it further comprises a purification step of the population of initiated granulopoietic cells produced and / or formulation of this cell population for medical use.

17. Cell culture medium for use in a method, defined in any one of claims 1 to 16, characterized in that the medium comprises: G-CSF at a concentration of approximately 0.13 pg / mL; and SCF at a concentration of approximately 0.13 pg / mL; and TPO at a concentration of approximately 0.13 pg / mL; and ITS; and HSA at approximately 1%.

18. Cell culture medium, according to claim 17, characterized in that it further comprises: GM-CSF at a concentration of approximately 0.01 pg / mL; and IL-3 at a concentration of approximately 0.13 pg / mL.

19. Cell culture medium, according to claim 17 or 18, characterized in that it further comprises: TNF at a concentration of approximately 0.001 pg / mL.

20. Cell culture medium, for use in a method according to any one of claims 8 to 19, characterized in that the medium comprises: SCF at a concentration of approximately 0.2 pg / mL; and Flt-3 ligand at a concentration of approximately 0.2 pg / mL; and IL-3 at a concentration of approximately 0.015 pg / mL; and IL-6 at a concentration of approximately 0.015 pg / mL; and TPO at a concentration of approximately 0.02 pg / mL; and ITS; and HSA at approximately 1%.

21. A population of granulopoietic cells characterized in that it is prepared for therapeutic use by a method as defined in any one of claims 1 to 14.

22. A population of granulopoietic cells characterized in that it is prepared for therapeutic use by a method defined in any one of claims 1 to 20.

23. A population of granulopoietic cells, according to claim 21 or 22, characterized in that it comprises: • a first subpopulation of cells that are CD15+ CD64+ CD18+ CD49d+ CD71+; • a second subpopulation of cells that are CD15- CD11b+ / - CD18+ CD49d+ CD32+ HLA-DR-; and • a third subpopulation of cells that are CD15- CD11b- HLA-DR+ CD18+ CD49d+ and CD71+.

24. Isolated population of granulopoietic cells, characterized in that it comprises: • a first subpopulation of cells that are CD15+ CD64+ CD18+ CD49d+ CD71+; Petition 870250080961, dated 09 / 09 / 2025, page 21 / 32 5 / 14 • a second subpopulation of cells that are CD15- CD11b+ / - CD18+ CD49d+ CD32+ HLA-DR-; and • a third subpopulation of cells that are CD15- CD11b- HLA-DR+ CD18+CD49d+ and CD71+.

25. A population of granulopoietic cells, according to claim 23 or 24, characterized in that it further comprises: • a fourth subpopulation of cells that are CD15- CD11b+ HLA-DR+.

26. Population of granulopoietic cells characterized by the fact that they are CD15+ CD64+ CD18+ CD49d+ CD71+.

27. A population of granulopoietic cells, according to any one of claims 23 to 26, characterized in that they are CD64+ and / or CD16+ and / or CD62L-.

28. Granulopoietic cell population, according to any one of claims 23 to 27, characterized in that the first and / or second cell subpopulation are also positive for one, more than one, or all of the selected markers from the group consisting of: CD177, CD11b, CD71, CD66b, HLA-DR, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mad, 4-1BBL, OX40L, PD-L1, and CD14.

29. A population of granulopoietic cells, according to any one of claims 23 to 28, characterized in that the third cell subpopulation is also positive for one, more than one, or all of the selected markers from the group consisting of: CD177, CD71, CD66b, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mad, 41BBL, OX40L, PD-L1, and CD14.

30. Granulopoietic cell characterized by the fact that it is a CD64+ granulopoietic cell, or a population of such cells.

31. A granulopoietic cell, or a population of such cells, according to claim 30, characterized in that it is a CD64+ and CD16- granulopoietic cell.

32. Granulopoietic cell, or a population of such cells, according to Petition 870250080961, dated 09 / 09 / 2025, page 22 / 32 6 / 14, with claim 30 or 31, characterized in that it is a CD64+ and CD62L- granulopoietic cell.

33. Granulopoietic cell, or a population of such cells, according to any one of claims 30 to 32, characterized in that it is a CD64+, CD16- and CD62L- granulopoietic cell.

34. Granulopoietic cell characterized by the fact that it is a CD16- granulopoietic cell, or a population of such cells.

35. Granulopoietic cell, according to claim 34, or a population of such cells, characterized in that it is a CD16- and CD62L- granulopoietic cell.

36. Granulopoietic cell characterized by the fact that it is CD62L-, or a population of such cells.

37. Isolated population of granulopoietic cells, characterized in that it comprises: • more than 90% Lin cells (e.g., approximately 97% Lin cells); • less than 30% CD34+ cells (e.g., approximately 14% CD34+ cells); • more than 30% CD38+ cells (e.g., approximately 65% ​​CD38+ cells); • less than 1% cells with an HSC phenotype (e.g., approximately 0.04% cells with an HSC phenotype); • less than 1% of cells with an LT-HSC phenotype (e.g., approximately 0.02% of cells with an LT-HSC phenotype); • less than 20% of cells with an LMPP phenotype (e.g., approximately 5% of cells with an LMPP phenotype); and • less than 10% of cells with an MPP phenotype (e.g., approximately 2.5% of cells with an MPP phenotype).

38. Pharmaceutical composition characterized by comprising a population of granulopoietic cells, defined in any of the claims 23 to 37.

39. Pharmaceutical composition, according to claim 38, or population of granulopoietic cells, according to any one of claims 23 to 37, characterized in that it is to be used as a medicament.

40. Pharmaceutical composition or population of granulopoietic cells for use according to claim 39, characterized in that it is in the treatment of cancer.

41. Pharmaceutical composition or population of granulopoietic cells for use according to claim 40, characterized in that it is for use in the treatment of one or more of the following: pancreatic cancer, liver cancer, esophageal cancer, stomach cancer, cervical cancer, ovarian cancer, lung cancer, bladder cancer, kidney cancer, brain cancer, prostate cancer, myeloma cancer, non-Hodgkin lymphoma (NHL), laryngeal cancer, uterine cancer or breast cancer.

42. Pharmaceutical composition or population of granulopoietic cells for use according to claim 41, characterized in that it is for use in the treatment of pancreatic cancer.

43. Pharmaceutical composition or population of granulopoietic cells for use according to claim 39, characterized in that it is for use in the treatment of an infection.

44. Pharmaceutical composition or population of granulopoietic cells for use according to claim 39, characterized in that it promotes a non-granulocytic immune response.

45. Use of a population of granulopoietic cells, defined in any one of claims 23 to 37, characterized in that it is applied in the manufacture of a medicament.

46. ​​A method for treating a disease or disorder in a subject, the method being characterized in that it comprises administering a pharmaceutical composition according to the sixth aspect of the invention, or a population of granulopoietic cells according to the second, or fourth to tenth aspects of the invention, to the subject.

47. A method for treating cancer in a subject, characterized by the fact that it comprises administering a pharmaceutical composition of the invention to the subject.

48. Use of a pharmaceutical composition of the invention in the manufacture of a medicament characterized by being for the treatment of cancer in a subject.

49. A method for treating an infection in a subject, characterized by comprising administering a pharmaceutical composition of the invention to the subject.

50. Use of a pharmaceutical composition of the invention in the manufacture of a medicament characterized by being for treating an infection in a subject.

51. Pharmaceutical composition of the invention, characterized by being for use in amplifying a non-granulocytic therapeutic immune response.

52. Treatment method characterized in that it comprises amplifying a non-granulocytic therapeutic immune response, the method comprising delivering a pharmaceutical composition of the invention to a subject in need of such treatment.

53. Pharmaceutical composition of the invention characterized by being for use in the manufacture of a medicament for use in amplifying a non-granulocytic therapeutic immune response.

54. Composition characterized by the fact that it comprises a population of granulopoietic cells, as defined in this document, and a non-granulocytic immune cell.

55. Composition characterized by comprising a population of granulopoietic cells, as defined in this document, and a non-granulocytic immune cell, wherein the composition does not include an αβ T cell.

56. Composition characterized in that it comprises a granulopoietic cell, as defined in this document, and a non-granulocytic immune cell, wherein the granulopoietic cell is capable of modulating (preferably modulating) the therapeutic immune response of the non-granulocytic immune cell.

57. A composition characterized in that it comprises a population of granulopoietic cells, as defined in this document, and a non-granulocytic immune cell, wherein the population of granulopoietic cells is capable of amplifying (preferably amplifies) the therapeutic immune response of the non-granulocytic immune cell.

58. Kit characterized in that it comprises: (a) the composition according to the invention; or (b) a population of granulopoietic cells, as defined in this document, and a non-granulocytic immune cell (for example, a terminally differentiated non-granulocytic immune cell); and (c) optionally, instructions for use thereof (for example, in cancer treatment).

59. Method for manufacturing a composition (for example, a composition of the invention), the method characterized in that it comprises: cultivating PBMCs in the presence of granulopoietic cells as defined in this document, thereby forming the composition; and optionally depleting αβ T cells before, during or after cultivation.

60. Method for manufacturing a composition (for example, a composition of the invention), characterized in that it comprises: cultivating αβ T cell-depleted PBMCs under conditions that promote the differentiation of progenitor cells present in the αβ T cell-depleted PBMCs into granulopoietic cells by a method as defined herein, thereby forming the composition.

61. A method for treating a disease or disorder in a subject, characterized by the fact that it comprises administering a composition of the invention to the subject.

62. Composition of the invention, characterized by being for use in modulating a non-granulocytic therapeutic immune response.

63. Composition of the invention, characterized by being for use in Petition 870250080961, dated 09 / 09 / 2025, page 26 / 32 10 / 14 to amplify a non-granulocytic therapeutic immune response.

64. Treatment method characterized in that it comprises modulating a non-granulocytic therapeutic immune response, the method comprising delivering a composition of the invention to a subject in need of such treatment.

65. A treatment method characterized in that it comprises amplifying a non-granulocytic therapeutic immune response, the method comprising delivering a composition of the invention to a subject in need of such treatment.

66. Composition of the invention characterized by being for use in the manufacture of a medicament for use in modulating a non-granulocytic therapeutic immune response.

67. Composition of the invention characterized by being for use in the manufacture of a medicament for use in amplifying a non-granulocytic therapeutic immune response.

68. Method for preparing a composition of the invention, the method characterized in that it comprises culturing a non-granulocytic immune cell in the presence of a population of granulopoietic cells of the invention.

69. A composition characterized in that it comprises a population of granulopoietic cells, as defined herein, and a non-granulocytic immune cell (e.g., a terminally differentiated non-granulocytic immune cell), wherein the population of granulopoietic cells is capable of amplifying (preferably amplifies) the therapeutic immune response of the non-granulocytic immune cell.

70. Composition characterized by the fact that it comprises one or more differentiated granulocytes from a population of granulopoietic cells, as defined in this document, capable of amplifying (preferably amplifying) a therapeutic immune response of a non-granulocytic cell and a non-granulocytic cell. Petition 870250080961, dated 09 / 09 / 2025, page 27 / 32 11 / 14 71. Treatment method, characterized in that it comprises modulating a non-granulocytic therapeutic immune response, the method comprising delivering a composition of the invention to a subject in need of such treatment.

72. Treatment method, characterized in that it comprises amplifying a non-granulocytic therapeutic immune response, the method comprising delivering a composition of the invention to a subject in need of such treatment.

73. Method for promoting the therapeutic activity of non-granulocytic immune cells, the method characterized by the fact that it comprises incubating a non-granulocytic immune cell with a population of granulopoietic cells, as defined in this document.

74. Method for increasing the survival of immune cells in culture, the method characterized by the fact that it comprises culturing immune cells in the presence of a feeder layer of granulopoietic cells, as defined in this document.

75. Method for increasing the proliferation of immune cells in culture, the method characterized by the fact that it comprises cultivating immune cells in the presence of a feeder layer of granulopoietic cells, as defined in this document.

76. Method for selecting an appropriate treatment regimen for a patient, the method characterized in that it comprises: • identifying whether the patient has an impaired non-granulocytic immune response; and • if the patient is identified as having an impaired non-granulocytic immune response, then treatment with a population of granulopoietic cells as defined herein is selected as an appropriate treatment; and if the patient is identified as not having an impaired non-granulocytic immune response, then treatment with a therapy other than a population of granulopoietic cells, as defined in this document, is selected.

77. Method for selecting an appropriate treatment regimen for a patient, the method characterized in that it comprises: • incubating a non-granulocytic immune cell from the patient with a population of granulopoietic cells as defined herein; wherein • if the activation of the non-granulocytic immune cell from the patient is increased in response to incubation, then treatment with a population of granulopoietic cells, as defined herein, is selected as an appropriate treatment; and • if the activation of the non-granulocytic immune cell from the patient is increased in response to incubation, then treatment with a therapy other than a population of granulopoietic cells, as defined herein, is selected.

78. A method for identifying whether a population of granulopoietic cells, as defined in this document, is suitable for use in cancer treatment by beneficially modulating the tumor microenvironment or not, the method characterized in that it comprises: • evaluating whether the population of granulopoietic cells, as defined in this document, or a cell derived from the population of granulopoietic cells, is capable of expressing pro-inflammatory cytokines; and / or • evaluating whether the population of granulopoietic cells, as defined in this document, or a cell derived from the population of granulopoietic cells, is capable of stimulating the expression of pro-inflammatory cytokines by non-granulocytic immune cells; and identifying whether a population of granulopoietic cells, as defined in this document, is suitable for use in cancer treatment by beneficially modulating the tumor microenvironment based on this evaluation.

79. Method for identifying whether a population of granulopoietic cells, as defined in this document, is suitable for use in Petition 870250080961, dated 09 / 09 / 2025, page.29 / 32 13 / 14 cancer treatment, increasing immune cell recruitment in a tumor and / or immune cell activation, or not, the method characterized by the fact that it comprises: • evaluating whether the granulopoietic cell population, as defined in this document, or a cell derived from the granulopoietic cell population, is capable of expressing a chemokine associated with promoting cell trafficking; and / or • evaluating whether the granulopoietic cell population, as defined in this document, or a cell derived from the granulopoietic cell population, is capable of stimulating the expression of degranulation markers by non-granulocytic immune cells; and identifying whether a granulopoietic cell population, as defined in this document, is suitable for use in cancer treatment, increasing immune cell recruitment in a tumor and / or immune cell activation based on this evaluation.

80. Method for identifying whether a population of granulopoietic cells, as defined herein, is suitable for use in cancer treatment by directly promoting the death of cancerous cells, or not, the method characterized in that it comprises: • incubating the population of granulopoietic cells as defined herein, or a cell derived from the population of granulopoietic cells, with cells of a cancerous cell line; and • evaluating whether the population of granulopoietic cells, as defined herein, or a cell derived from the population of granulopoietic cells, is capable of increasing the death of cancerous cell line cells to a greater extent than the death of non-cancerous cells; and identifying whether a population of granulopoietic cells, as defined herein, is suitable for use in cancer treatment by directly promoting the death of cancerous cells based on this evaluation.

81. Method for identifying whether a population of cells Petition 870250080961, dated 09 / 09 / 2025, page 81.30 / 32 14 / 14 granulopoietic cells, as defined herein, are suitable for use in the treatment of infection by directly promoting the death of cellular infectious agents or infected cells, or not, the method characterized by the fact that it comprises: • incubating the population of granulopoietic cells as defined herein, or a cell derived from the population of granulopoietic cells, with a sample of a cellular infectious agent or infected cells; and • evaluating whether the population of granulopoietic cells as defined herein, or a cell derived from the population of granulopoietic cells, is capable of increasing the death of the cellular infectious agent or infected cells; and identifying whether a population of granulopoietic cells, as defined herein, is suitable for use in the treatment of infection by directly promoting the death of cellular infectious agents or infected cells based on this evaluation.

82. Method for identifying whether a population of granulopoietic cells, as defined herein, is suitable for use in treatment by amplifying a therapeutic immune response, or not, the method characterized in that it comprises: • incubating the population of granulopoietic cells as defined herein, or a cell derived from the population of granulopoietic cells, with immune cells; and • evaluating whether the population of granulopoietic cells as defined herein, or the cell derived from the population of granulopoietic cells, is capable of increasing the activation of immune cells; and identifying whether a population of granulopoietic cells, as defined herein, is suitable for use in treatment by amplifying a therapeutic immune response based on this evaluation.

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