Immunomodulatory Cells and Compositions

Abstract

There is provided a granulopoietic cell, or a population of such cells, for use to modulate a therapeutic immune response. The therapeutic immune response may be a non-granulocytic immune response. The granulopoietic cell, or population of such cells, may be used to amplify the therapeutic immune response, such as a non-granulocytic immune response. The granulopoietic cell or population of cells may be CD62L-. The granulopoietic cell or population of cells may have a marker expression profile: CD10-, CD11b-, CD16-, CD62L-, CD66b-, CD177-, CD15+, CD38+, CD49d+, CD54+, CD63+. The granulocytic cell or population of cells may be used in the treatment of cancer, or in the treatment of an infection.

Description

Immunomodulatory Cells and Compositions Field of invention

[0001] The present invention relates to cells for use in modulating, such as amplifying, a therapeutic immune response and to methods of treatment using such cells. The therapeutic immune response may be a non-granulocytic immune response. The invention also relates to pharmaceutical compositions. The invention further relates to screening methods and methods useful in the culture of immune cells. Fundamentals

[0002] The immune response plays a vital role in the body's fight against cancer or infections. The native response can be enhanced by the use of immunotherapies, which are seen as increasingly important for use in such therapeutic contexts.

[0003] However, cancers and many infectious agents have adopted strategies that allow them to evade or reduce the effects of the immune system. Infectious agents may be present in the body's own cells, thus avoiding immune surveillance. Tumors may be adapted to be immunologically cold and may create an immunosuppressive tumor microenvironment (TME) that can render antitumor immune responses ineffective.

[0004] The present invention addresses one or more of the problems mentioned above. Summary of the invention

[0005] In a first aspect, the invention provides a granulopoietic cell, or optionally a population of such cells, for use in modulating a therapeutic immune response. Appropriately, the therapeutic immune response is a non-granulocytic immune response. In a suitable embodiment, the granulopoietic cell or population of such cells is for use in amplifying the therapeutic immune response, as a non-granulocytic immune response. Petition 870250102349, dated 07 / 11 / 2025, page 10 / 209 2 / 177

[0006] In a second aspect, the invention provides a treatment method comprising modulating a therapeutic immune response, the method comprising providing a granulopoietic cell, or optionally a population of such cells, to a subject in need of such treatment. The therapeutic immune response may be a non-granulocytic immune response. The treatment method may comprise amplifying the immune response.

[0007] In a third aspect, the invention provides a granulopoietic cell, or optionally a population of such cells, for use in the manufacture of a medicament for use in modulating a therapeutic immune response. The therapeutic immune response to be modulated may be a non-granulocytic immune response. The therapeutic immune response may be amplified.

[0008] In a fourth aspect, the invention provides a pharmaceutical composition comprising an enriched population of granulopoietic cells.

[0009] In a fifth aspect, the invention provides a method for promoting the therapeutic activity of non-granulocytic immune cells, the method comprising incubating a non-granulocytic immune cell with a granulopoietic cell.

[0010] In a sixth aspect, the invention provides a method for selecting an appropriate treatment regimen for a patient, the method comprising: • Identify whether the patient has an impaired non-granulocytic immune response; wherein • if the patient is identified as having an impaired non-granulocytic immune response, then treatment with a granulopoietic cell 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 granulopoietic cell is Petition 870250102349, dated 07 / 11 / 2025, page 11 / 209 3 / 177 selected.

[0011] In a seventh aspect, the invention provides a method for selecting an appropriate treatment regimen for a patient, the method comprising: • Incubate a patient's non-granulocytic immune cell with a granulopoietic cell; wherein • if the activity of the patient's non-granulocytic immune cell is increased in response to incubation, then treatment with a granulopoietic cell is selected as an appropriate treatment; and • if the activity of the patient's non-granulocytic immune cell is increased in response to incubation, then treatment with a therapy other than a granulopoietic cell is selected.

[0012] In an eighth aspect, the invention provides a method for increasing the survival of immune cells in culture, the method comprising culturing the immune cells in the presence of a feeder layer of granulopoietic cells.

[0013] In a ninth aspect, the invention provides a method for increasing the proliferation of immune cells in culture, the method comprising culturing the immune cells in the presence of a feeder layer of granulopoietic cells.

[0014] In a tenth aspect, the invention provides a method for identifying whether a granulopoietic cell is suitable for use in cancer treatment by beneficially modulating the tumor microenvironment, the method comprising: • to assess whether the granulopoietic cell, or a cell derived from the granulopoietic cell, is capable of expressing pro-inflammatory cytokines; and / or • to assess whether the granulopoietic cell, or a cell derived from the granulopoietic cell, is capable of stimulating the expression of pro-inflammatory cytokines by non-granulocytic immune cells; Petition 870250102349, dated 07 / 11 / 2025, page 12 / 209 4 / 177 and identify whether a granulopoietic cell is suitable for use in cancer treatment by beneficially modulating the tumor microenvironment based on this assessment.

[0015] In an eleventh aspect, the invention provides a method for identifying whether a granulopoietic cell is suitable for use in cancer treatment by increasing immune cell recruitment in a tumor and / or immune cell activation, the method comprising: • to assess whether the granulopoietic cell, or a cell derived from the granulopoietic cell, is capable of expressing a chemokine associated with the promotion of cell trafficking; and / or • to assess whether the granulopoietic cell, or a cell derived from the granulopoietic cell, is capable of stimulating the expression of degranulation markers by non-granulocytic immune cells; and to identify whether a granulopoietic cell is suitable for use in cancer treatment, increasing immune cell recruitment in a tumor and / or immune cell activation based on this assessment.

[0016] In a twelfth aspect, the invention provides a method for identifying whether a granulopoietic cell is suitable for use in cancer treatment by directly promoting the death of cancer cells, the method comprising: • Incubate the granulopoietic cell, or a cell derived from the granulopoietic cell, with cells from a cancerous cell line; and • evaluate whether the granulopoietic cell, or a cell derived from the granulopoietic cell, is capable of increasing the death of cancerous cell line cells to a greater extent than the death of non-cancerous cells; and to identify whether a granulopoietic cell is suitable for use in cancer treatment, directly promoting the death of cancerous cells based on this assessment. Petition 870250102349, dated 07 / 11 / 2025, page 13 / 209 5 / 177

[0017] In a thirteenth aspect, the invention provides a method for identifying whether a granulopoietic cell is suitable for use in the treatment of infection by directly promoting the death of cellular infectious agents or infected cells, the method comprising: • Incubate the granulopoietic cell, or a cell derived from the granulopoietic cell, with a sample of a cellular infectious agent or infected cells; and • evaluate whether the granulopoietic cell, or a cell derived from the granulopoietic cell, is capable of increasing the death of the cellular infectious agent or infected cells; and to identify whether a granulopoietic cell is suitable for use in treating infection, directly promoting the death of infectious cellular agents or infected cells based on this assessment.

[0018] In a fourteenth aspect, the invention provides a method for identifying whether a granulopoietic cell is suitable for use in treatment by amplifying a therapeutic immune response, the method comprising: • Incubate the granulopoietic cell, or a cell derived from the granulopoietic cell, with immune cells; and • evaluate whether the granulopoietic cell is able to increase the activation of immune cells; and to identify whether a granulopoietic cell is suitable for use in treatment, amplifying a therapeutic immune response based on this assessment.

[0019] Granulopoietic cells suitable for use according to the present invention can be prepared by a method comprising: • To cultivate a population of progenitor cells under cell culture conditions that promote progenitor cell differentiation, including the presence of: • G-CSF, Petition 870250102349, dated 07 / 11 / 2025, page 14 / 209 6 / 177 • GM-CSF, • IL-3 and • TNF; to produce a population of granulopoietic cells. Brief description of the figures

[0020] Figure 1 illustrates the effect of granulopoietic cells on the activation of blood-derived CD8+ T cells assessed with reference to the expression of degranulation markers or co-stimulatory molecules.

[0021] Figure 2 illustrates the effect of granulopoietic cells on the activation of blood-derived CD4+ T cells assessed with reference to the expression of co-stimulatory molecules.

[0022] Figure 3 illustrates the effect of granulopoietic cells on the activation of αβ T cells evaluated with reference to T cell proliferation.

[0023] Figure 4 illustrates the effect of granulopoietic cells on the activation of blood-derived NK and NKT cells assessed with reference to cell survival.

[0024] Figure 5 illustrates the effect of granulopoietic cells on the activation of blood-derived natural killer (NK) cells and natural killer T (NKT) cells assessed with reference to the expression of degranulation markers or co-stimulatory molecules.

[0025] Figure 6 illustrates the effect of granulopoietic cells on the activation of CD8+ T cells, CD4+ T cells and NK cells, assessed with reference to the expression of degranulation markers or co-stimulatory molecules.

[0026] Figure 7 illustrates the effect of granulopoietic cells on the activation of peripheral blood mononuclear cells (PBMCs), with reference to cytokine expression.

[0027] Figure 8 illustrates the effect of granulopoietic cells on the activation of tumor-infiltrating lymphocytes. Petition 870250102349, dated 07 / 11 / 2025, page 15 / 209 7 / 177 (TILs), with reference to cytokine expression.

[0028] Figure 9 illustrates the effect of granulopoietic cells on the activation of immune cells, with reference to immune cell trafficking.

[0029] Figure 10 illustrates the effect of granulopoietic cells on the activation of immune cells, with reference to cytocidal activity.

[0030] Figure 11 illustrates the expression of chemokines by granulocytes formed in the differentiation of granulopoietic cells.

[0031] Figure 12 illustrates the expression of ligands for co-stimulatory molecules by granulocytes formed in the differentiation of granulopoietic cells.

[0032] Figure 13 illustrates the relative proportions of granulopoietic cell subpopulations produced by methods of the invention without priming or with multiple priming steps.

[0033] Figure 14 further characterizes the first subpopulation of granulopoietic cells identified.

[0034] Figure 15 further characterizes the second subpopulation of granulopoietic cells identified.

[0035] Figure 16 further characterizes the third subpopulation of granulopoietic cells identified.

[0036] Figure 17 further characterizes the fourth subpopulation of granulopoietic cells identified. Detailed description of the invention

[0037] The present invention is based, at least to some extent, on the inventors' conclusion that granulopoietic cells can be used to modulate a therapeutic immune response.

[0038] In particular, the inventors have identified that granulopoietic cells of the types described in this document can increase the activation or recruitment of immune cells and, particularly, of non-granulocytic immune cells, of a Petition 870250102349, dated 07 / 11 / 2025, page 16 / 209 8 / 177 in a way that allows for the amplification of a therapeutic immune response. This achievement allows such granulopoietic cells, which the inventors have designated Immunomodulatory Alpha-Neutrophil Precursors (IMANPs), to be used to strengthen immunotherapeutic treatments in a range of conditions, including (among others) cancer therapies. By amplifying the immune response, the medical uses and treatment methods of the invention are able to make otherwise immunologically cold tumors warm and therefore responsive to treatment.

[0039] The amplification that occurs in relation to a therapeutic immune response is not simply due to the generation of high numbers of granulocytes as a result of the administration of granulopoietic cells. Instead, granulopoietic cells appear to be able to significantly increase the activation of non-granulocytic immune cells, and particularly T cells and NK cells, thereby increasing the immune response obtained. 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 by activated 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 show an increased degree of recruitment to the TME, as well as increased cytocidal activity (particularly increased tumor cell killing activity).

[0040] These properties suggest that granulopoietic cell populations can be used therapeutically in cancer treatment and that such treatment can also be used to strengthen other cell-based immunotherapies. Petition 870250102349, dated 07 / 11 / 2025, page 17 / 209 9 / 177

[0041] Furthermore, the granulopoietic cells to be used according to the invention may themselves be capable of differentiating into granulocytes with the ability to kill cancer cells. In this way, treatments according to the invention are capable of achieving a dual mode of action, both amplifying a non-granulocytic immune response and giving rise to granulocytes that are capable of directly killing cancer cells.

[0042] The granulopoietic cells used in the medical uses and treatment methods of the invention express many markers that suggest they have a differentiation stage approximately corresponding to that of a myeloblast or promyelocyte. However, unlike naturally occurring cells, the granulopoietic cells considered useful in medical uses and treatment methods can be characterized with respect to their absence of CD62L expression.

[0043] The inventors have demonstrated that granulopoietic cells suitable for use in the medical applications of the invention, or in the methods of the invention, are capable of amplifying immune responses through a number of different mechanisms. In particular, granulopoietic cells can increase the activation of immune cells and enhance the activities (such as cell trafficking and cytocidal activity) necessary to achieve a successful therapeutic immune response.

[0044] Surprisingly, the inventors found that these effects can be achieved using granulopoietic cells that are allogeneic with reference to the subject who will receive the granulopoietic cell therapeutically.

[0045] Modulation may be the modulation of a therapeutic immune response of a subject to whom granulopoietic cells are administered, for example, in a pharmaceutical composition of the invention. Alternatively, or additionally, a pharmaceutical composition of the invention may comprise an immune cell. Petition 870250102349, dated 07 / 11 / 2025, page 18 / 209 10 / 177 (preferably a non-granulocytic immune cell) in addition to the recited granulopoietic cells, and the modulation of an immune response may be in relation to such an additional immune cell composition. Preferably, the modulation is the amplification of a therapeutic immune response.

[0046] The invention will now be further described with reference to the following paragraphs. Granulopoietic cells

[0047] In several of its aspects, the present invention relates to medical uses, methods and products employing granulopoietic cells. These granulopoietic cells are capable of amplifying a non-granulocytic therapeutic immune response, as considered in more detail elsewhere in the descriptive report.

[0048] 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. Appropriately, a granulopoietic cell in this context is one that gives rise to a granulocyte or to a granulocyte precursor cell of the granulocytic lineage. 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 (and preferably cells that give rise to granulocytes). Preferably, a granulopoietic cell is not a neutrophil.

[0049] Other useful ways in which relevant granulopoietic cells can be defined are presented below.

[0050] Granulopoietic cells suitable for medical uses and methods of the invention can be defined with reference to their marker profiles. For example, in one embodiment Petition 870250102349, dated 07 / 11 / 2025, page 19 / 209 11 / 177 adequate, the granulopoietic cell does not express one or more selected markers from the group consisting of: CD10; CD11b; CD16; CD62L; CD66b; and CD177. An adequate granulopoietic cell may not express 1, 2, 3, 4, 5, or all 6 of these markers (i.e., an adequate granulopoietic cell may be CD10-e / or CD11b-e / or CD16-e / or CD62L-e / or CD66b-e / or CD177).

[0051] In a suitable embodiment, the granulopoietic cell is CD62L-. The lack of CD62L expression may be useful in identifying granulopoietic cells well suited for use according to the various aspects of the invention.

[0052] Additionally, or alternatively, a granulopoietic cell may be CD66b-. The lack of CD66b expression suggests a differentiation stage corresponding to that of a myeloblast and may be useful in identifying granulopoietic cells well suited for use according to the various aspects of the invention.

[0053] In a suitable embodiment, the granulopoietic cell expresses one or more markers from the group consisting of: CD15; CD38; CD49d; CD54; and CD63. A suitable granulopoietic cell may express 1, 2, 3, 4, or all 5 of these markers (i.e., a suitable granulopoietic cell may be CD15+ and / or CD38+ and / or CD49d+ and / or CD54+ and / or CD63+).

[0054] The granulopoietic cells suitable for use in the various aspects of the invention may be CD10-, CD11b-, CD16-, CD62L, CD66b-, CD177-, CD15+, CD38+, CD49d+, CD54+, CD63+. A further aspect of the invention provides a granulopoietic cell that is CD10-, CD11b-, CD16-, CD62L-, CD66b-, CD177-, CD15+, CD38+, CD49d+, CD54+, CD63+.

[0055] The inventors have identified two additional populations of granulopoietic cells that can be used in various aspects of the invention and that can be distinguished based on their marker expression profiles. The cells of Petition 870250102349, dated 07 / 11 / 2025, page 20 / 209 The cells in the first population are CD11bhi, CD15+, CD66b+, CD177+, CD18hi, CD16, CD34-, CD38-, and CD49d-. The cells in the second population are CD34+ / -, CD38+ / -, CD15+ / -, CD49d+, CD18+, CD66b-, CD177-, and CD16-. Of these, it is believed that the first population represents a more mature group of cells than the second population.

[0056] A further aspect of the invention provides a granulopoietic cell that is CD11bhiCD15+CD66b+CD177+CD18hiCD16-CD34CD38-CD49d-. A further aspect of the invention provides a granulopoietic cell that is CD34+ / -, CD38+ / -, CD15+ / -, CD49d+, CD18+, CD66b-, CD177-, CD16-.

[0057] As described in more detail below, a suitable isolated population of granulopoietic cells that can be employed in the medical uses, methods or products of the invention may comprise: • a first subpopulation of cells that are CD15+ CD64+ CD18+ CD49d+ CD71+ • a second subpopulation of cells that are CD15CD11b+ / - CD18+ CD49d+ CD32+ HLA-DR- • a third subpopulation of cells that are CD15- CD11bHLA-DR+ CD18+ CD49d+ and CD71+.

[0058] This adequate population of granulopoietic cells may further comprise a fourth subpopulation of cells that are CD15- CD11b+ HLA-DR+.

[0059] Details of additional characteristic marker profiles that can be used to distinguish these populations (and subpopulations) of useful granulopoietic cells, as well as methods by which they can be produced, are further described in other parts of this descriptive report.

[0060] Those skilled in the art will be well aware of the appropriate methods by which cell populations can be isolated and, if desired, enriched based on their expression of specific cell surface marker profiles.

[0061] Granulopoietic cells suitable for use in Petition 870250102349, dated 07 / 11 / 2025, page 21 / 209 13 / 177 medical uses and methods of the invention can be defined with reference to its potency. In a suitable embodiment, the granulopoietic cell is a unipotent cell.

[0062] Granulopoietic cells suitable 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 has a differentiation stage corresponding to that between a myeloblast and a granulocyte. Suitablely, the granulopoietic cell has a differentiation stage corresponding to that between a myeloblast and a band cell. For example, the granulopoietic cell may have a differentiation stage corresponding to that between a myeloblast and a metamyelocyte. Suitablely, the granulopoietic cell has a differentiation stage corresponding to that between a myeloblast and a myelocyte. Suitablely, the granulopoietic cell has a differentiation stage corresponding to that between a myeloblast and a promyelocyte.

[0063] In a suitable embodiment, the granulopoietic cell has a differentiation stage corresponding to a myeloblast. In a suitable embodiment, the granulopoietic cell has a differentiation stage corresponding to a promyelocyte. In a suitable embodiment, the granulopoietic cell has a differentiation stage corresponding to a myelocyte. In a suitable embodiment, the granulopoietic cell has a differentiation stage corresponding to a metamyelocyte. In a suitable embodiment, the granulopoietic cell has a differentiation stage corresponding to a band cell. In a suitable embodiment, the granulopoietic cell has a differentiation stage corresponding to a granulocyte.

[0064] As presented elsewhere in the descriptive report, the granulopoietic cells suitable for use in the various aspects of the invention can be derived from cells Petition 870250102349, dated 07 / 11 / 2025, page 22 / 209 14 / 177 artificial stem cells, such as iPSCs. It will be appreciated that such granulopoietic cells 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 the preceding paragraphs should be interpreted accordingly.

[0065] Appropriately, the granulopoietic cell is selected from the group consisting of: a myeloblast; a promyelocyte; a myelocyte; a metamyelocyte; a band cell; and a granulocyte. Appropriately, the granulopoietic cell is selected from the group consisting of: a myeloblast; a promyelocyte; a myelocyte; a metamyelocyte; and a band cell. Appropriately, the granulopoietic cell is selected from the group consisting of: a myeloblast; a promyelocyte; a myelocyte; and a metamyelocyte. Appropriately, the granulopoietic cell is selected from the group consisting of: a myeloblast; a promyelocyte; and a myelocyte. Appropriately, the granulopoietic cell is selected from the group consisting of: a myeloblast; and a promyelocyte.

[0066] In a suitable embodiment, the granulopoietic cell is a myeloblast. In a suitable embodiment, the granulopoietic cell is a promyelocyte. In a suitable embodiment, the granulopoietic cell is a myelocyte. In a suitable embodiment, the granulopoietic cell is a metamyelocyte. In a suitable embodiment, the granulopoietic cell is a band cell. In a suitable embodiment, the granulopoietic cell is a granulocyte.

[0067] Appropriately, the granulopoietic cell is committed to the neutrophil lineage. In such a mode, a suitable granulopoietic cell can be selected from Petition 870250102349, dated 07 / 11 / 2025, page 23 / 209 15 / 177 group consisting of: a neutrophilic promyelocyte; a neutrophilic myelocyte; a neutrophilic metamyelocyte; a neutrophilic band cell; and a neutrophil.

[0068] As further set forth elsewhere in this descriptive report, the granulopoietic cells 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 cells 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 cells may be capable of giving rise to granulocytes that have desirable expression profiles of molecules such as chemokines or costimulatory receptor ligands. Enriched populations of granulopoietic cells

[0069] A granulopoietic cell 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.

[0070] By way of example only, such an enriched population may be a cell population in which granulopoietic 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%, at least 0.8%, at least 0.9%, or at least 1% of the total cell population. Such an enriched population may also 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 population of cells in which granulopoietic cells comprise at least 15%, at least 20%, at least 25%, or at least 25%. Petition 870250102349, dated 07 / 11 / 2025, page 24 / 209 16 / 177 less than 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.

[0071] The granulopoietic 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-. Pharmaceutical compositions of the invention

[0072] The fourth aspect of the invention provides a pharmaceutical composition comprising an enriched population of granulopoietic cells. The enriched population of granulopoietic cells incorporated into a pharmaceutical composition of the invention may be as described above.

[0073] Suitablely, the granulopoietic cells present in a pharmaceutical composition of the invention may be CD62L-.

[0074] 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.

[0075] Suitably, the pharmaceutical composition comprises a granulocyte-macrophage colony-stimulating factor (GM-CSF), a granulocyte colony-stimulating factor (GCSF), 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 pharmaceutical composition comprises IFN-gamma and a GM-CSF. Preferably, the pharmaceutical composition comprises TNF-alpha. In a particularly preferred manner, the Petition 870250102349, dated 07 / 11 / 2025, page 25 / 209 17 / 177 pharmaceutical composition comprises 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 pharmaceutical composition comprises 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 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.

[0076] In a suitable embodiment, a pharmaceutical composition of the invention comprises (or additionally comprises) a granulocyte in addition to granulopoietic cells. In a suitable embodiment, a pharmaceutical composition of the invention comprises a neutrophil in addition to granulopoietic cells. A therapeutic immune response

[0077] In the context of the present invention, a therapeutic immune response should be understood as an immune response that contributes to or achieves a desired therapeutic outcome. In a suitable embodiment, a therapeutic immune response may be an immune response that leads (directly or indirectly) to the death of cancerous cells, thus enabling the treatment of cancer. In a suitable embodiment, a therapeutic immune response may be an immune response that leads (directly or indirectly) to the death of infected cells or cellular infectious agents, thus enabling the treatment of an infection.

[0078] A therapeutic immune response can involve the action of any cells of the immune system. A non-therapeutic immune response Petition 870250102349, dated 07 / 11 / 2025, page 26 / 209 18 / 177 Granulocytic response may involve the action of any immune system cells other than granulocytes. By way of example only, a therapeutic immune response that may be amplified by the medical uses, treatment methods or pharmaceutical compositions of the invention may involve the action of one or more cell types selected from the group 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. Amplification of a therapeutic immune response

[0079] The amplification of an immune response can be demonstrated by an increase in one or more of the following: 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 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.

[0080] Alternatively, or additionally, the amplification of a therapeutic immune response can be evaluated with reference to the outcome to be achieved by the therapeutic immune response.

[0081] For example, in the case of an immune response Petition 870250102349, dated 07 / 11 / 2025, page 27 / 209 19 / 177 therapeutic to be used in cancer treatment, the amplification of the immune response can be demonstrated by an increase in the effectiveness of cancer treatment. Such an increase in effectiveness can 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 recurrence; a reduction in the number of recurrence incidents; a reduction in the number of metastasis incidents; and / or a prevention or delay of metastasis.

[0082] In the case of a therapeutic immune response to be 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 clear the infection. Host cells and host immune responses

[0083] 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, granulopoietic cells in accordance with any of the various aspects of the invention. Except where the context requires otherwise, all references to immune cells or immune responses in connection with the various aspects and embodiments of the invention should be taken as applicable to host immune cells or host immune responses. Increased activation of immune cells

[0084] A granulopoietic cell suitable for use according to the various aspects of the present invention may be able to increase the activation of immune cells. In particular, such a cell may be able to increase the activation of Petition 870250102349, dated 07 / 11 / 2025, page 28 / 209 20 / 177 host immune cells. Therefore, such a cell may be able to amplify a therapeutic host immune response by increasing the activation of host immune cells.

[0085] It will be appreciated that activated immune cells are primarily responsible for providing the desired activity in a therapeutic immune response. Consequently, the ability of medical uses and treatment methods to increase immune cell 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 immune cell activation can, without limitation, be advantageous in the treatment of cancer or infections.

[0086] Suitably, a granulopoietic cell suitable for use in the present invention can increase the activation of immune cells, so that the expression by immune cells of one or more degranulation markers is increased. Suitably, a granulopoietic cell suitable for use in the present invention can increase the activation of immune cells, so that the expression by immune cells of one or more co-stimulatory molecules is increased. Suitably, a granulopoietic cell suitable for use in the present invention can increase the activation of immune cells, so that the proliferation of immune cells is increased. Suitably, a granulopoietic cell suitable for use in the present invention can increase the activation of immune cells, so that the abundance of immune cells is increased.Suitablely, a granulopoietic cell suitable for use in the present invention can increase the activation of immune cells, so that the survival of immune cells is increased. Suitablely, a granulopoietic cell suitable for use in the present invention can increase the activation of immune cells, so that... Petition 870250102349, dated 07 / 11 / 2025, page 29 / 209 21 / 177 the expression by immune cells of one or more cytokines is increased. Suitablely, a granulopoietic cell suitable for use in the present invention can increase the activation of immune cells, so that the trafficking of immune cells is increased. Suitablely, a granulopoietic cell suitable for use in the present invention can increase the activation of immune cells, so that the cytocidal activity of immune cells is increased.

[0087] Suitablely, a granulopoietic cell suitable for use according to the present invention may increase the activation of immune cells by signal 2 (co-stimulation). Alternatively, or additionally, a granulopoietic cell suitable for use according to the present invention may increase the activation of immune cells by signal 3 (cytokine stimulation). A granulopoietic cell suitable for use according to the present invention may have the ability to increase the activation of immune cells by both signal 2 and signal 3.

[0088] 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. Therefore, the inventors' data (presented in the Examples) illustrating that granulopoietic cells suitable for use according to the invention are capable of providing these signals provide a clear indication of their suitability for use in amplifying therapeutic immune responses that will be relevant in cancer treatment.

[0089] Granulopoietic cells suitable for use according to the present invention may exhibit some or all of the properties presented above.

[0090] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of a pharmaceutical composition of the invention), for example, for use according to the invention, is an amount sufficient to increase the activation of immune cells, such as host immune cells. The extent Petition 870250102349, dated 07 / 11 / 2025, page 30 / 209 22 / 177 of the increase, relevant host immune cells and adequate indicators of increased activation can be considered in the preceding paragraphs and / or those that follow. Increased T cell activation

[0091] A granulopoietic cell suitable for use according to the present invention can increase T cell activation. In particular, a granulopoietic cell suitable for use according to the present invention can increase the activation of host T cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by increasing the activation of host T cells.

[0092] It will be appreciated that increasing the activation of 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. Therefore, the use of a granulopoietic cell to increase the activation of host T cells 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 T cell activation can, without limitation, be advantageous in the treatment of cancer or in the treatment of infections.

[0093] A T cell, such as a host T cell, whose activation can be increased, can be selected from the group 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.

[0094] Without limitation, increased T cell activation Petition 870250102349, dated 07 / 11 / 2025, page 31 / 209 23 / 177 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 death) by T cells; an increase in T cell proliferation; an increase in T cell survival; and an increase in T cell abundance. Changes in these properties associated with increased T cell activation exposed to granulopoietic cells suitable for use according to the present invention are demonstrated in the Examples. Further relevant considerations regarding these various properties are presented elsewhere in this descriptive report.

[0095] 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.

[0096] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of a pharmaceutical composition of the invention), for example, for use according to the invention, is an amount sufficient to increase the activation of T cells, such as host T 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 870250102349, dated 07 / 11 / 2025, page 32 / 209 24 / 177 follow. Increased activation of CD8+ T cells

[0097] A granulopoietic cell suitable for use according to the present invention can enhance the activation of CD8+ T cells, such as host CD8+ T cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by enhancing the activation of host CD8+ T cells.

[0098] Increased CD8+ T cell activation may be 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, 41BB and / or OX40); and an increase in CD8+ T cell proliferation. Further relevant considerations regarding these various properties are presented elsewhere in this descriptive report.

[0099] CD8+ T cells, whose activation is increased, may be CD8+ T cells from peripheral blood or may be CD8+ T cells infiltrated by a tumor.

[0100] 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. Increased activation of CD4+ T cells

[0101] A granulopoietic cell suitable for use according to the present invention can increase the activation of Petition 870250102349, dated 07 / 11 / 2025, page 33 / 209 25 / 177 CD4+ T cells, like host CD4+ T cells. Therefore, such a granulopoietic cell may be able to amplify a therapeutic host immune response by increasing the activation of host CD4+ T cells.

[0102] Increased 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. Additional relevant considerations regarding these various properties are presented elsewhere in this descriptive report.

[0103] CD4+ T cells, whose activation is increased, may be CD4+ T cells from peripheral blood or may be CD4+ T cells infiltrated by a tumor.

[0104] 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 100% or more. Quantification of the increase in CD4+ T cell activation according to such a modality can make use of comparison with an appropriate control. Increased activation of NK T cells

[0105] A granulopoietic cell suitable for use according to the present invention can enhance the activation of NK T cells, such as host NK T cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by enhancing the activation of host NK T cells.

[0106] Increased activation of NK T cells may be associated with one or more of the following: an increase in expression Petition 870250102349, dated 07 / 11 / 2025, p. 34 / 209 26 / 177 by NK T cells of a degranulation marker (including, but not limited to, CD107a); an increase in the expression by NK T cells of a co-stimulatory molecule (including, but not limited to, 4-1BB and / or OX40); and an increase in NK T cell survival. Additional relevant considerations regarding these various properties are presented elsewhere in this descriptive report.

[0107] NK T cells, whose activation is increased, may be peripheral blood NK T cells or may be tumor-infiltrated NK T cells.

[0108] 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. Increased activation of NK cells

[0109] A granulopoietic cell suitable for use according to the present invention can enhance NK cell activation. In particular, a granulopoietic cell suitable for use according to the present invention can enhance the activation of host NK cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by enhancing the activation of host NK cells.

[0110] 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 cells Petition 870250102349, dated 07 / 11 / 2025, page 35 / 209 27 / 177 physiologically stressed cells, such as tumor cells and virus-infected cells. Therefore, the use of a granulopoietic cell to increase the activation of host NK cells 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 NK cell activation can, without limitation, be advantageous in the treatment of cancer or in the treatment of infections.

[0111] NK cells, whose activation is increased, may be peripheral blood NK cells or may be tumor-infiltrated NK cells.

[0112] Without limitation, increased 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; 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 T 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 NK cell activation exposed to granulopoietic cells suitable for use according to the present invention are demonstrated in the Examples.Additional relevant considerations regarding these various properties are presented elsewhere in this descriptive report.

[0113] 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%, Petition 870250102349, dated 07 / 11 / 2025, page 36 / 209 28 / 177 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 NK cell activation according to this modality may make use of comparison with an appropriate control.

[0114] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of a pharmaceutical composition of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the activation of NK cells, such as host NK cells. The extent of the increase and appropriate indicators of increased activation may be considered in the preceding and / or following paragraphs. Increased activation of PBMCs

[0115] A granulopoietic cell suitable for use according to the present invention can increase the activation of PBMCs. In particular, such a granulopoietic cell may be able to increase the activation of host PBMCs. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by increasing the activation of host PBMCs.

[0116] 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 additional immune cells. Consequently, the use of a granulopoietic cell to enhance PBMC activation will be beneficial in almost all circumstances where a therapeutically effective immune response is required. In particular, the Petition 870250102349, dated 07 / 11 / 2025, page 37 / 209 29 / 177 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.

[0117] PBMCs whose activation should be increased include, among others, those selected from the group consisting of: peripheral blood T cells (such as: peripheral blood CD8+ T cells; peripheral blood CD4+ T cells; peripheral blood NK T cells; peripheral blood αβ T cells; or peripheral blood γδ T cells); and peripheral blood NK cells.

[0118] Increased PBMC activation 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 PBMCs exposed to granulopoietic cells suitable for use according to the present invention is shown in the Examples. Further relevant considerations regarding these various properties are presented elsewhere in this descriptive report.

[0119] PBMC activation can be increased by at least 5%. For example, PBMC activation can be increased by at least 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%, by less than 90%, at least 95%, or at least 100% or more. Quantifying the increase in PBMC activation according to this modality may involve comparison with an appropriate control.

[0120] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of a pharmaceutical composition of the invention), for example, for use according to the invention, is an amount sufficient to increase the activation of PBMCs, such as host PBMCs. The extent of the increase and Petition 870250102349, dated 07 / 11 / 2025, page 38 / 209 30 / 177 suitable indicators of increased activation can be considered in the preceding paragraphs and / or those that follow. Increased activation of TILs

[0121] A granulopoietic cell suitable for use according to the present invention can enhance the activation of TILs. In particular, such a cell may be able to enhance the activation of host TILs. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by enhancing the activation of host TILs.

[0122] For the purposes of the present invention, 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 TIL activation can play a highly advantageous role in cancer treatment.

[0123] In particular, the inventors have determined that a granulopoietic cell 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.

[0124] Increased TIL activation, like increased activation of tumor-infiltrated T cells or tumor-infiltrated NK cells, can be demonstrated by any appropriate activation marker. By way of example only, the Petition 870250102349, dated 07 / 11 / 2025, page 39 / 209 31 / 177 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 cells suitable for use according to the present invention is demonstrated in the Examples. Further relevant considerations regarding these various properties are presented elsewhere in this descriptive report.

[0125] TIL activation can be increased by at least 5%. For example, TIL activation can be increased by at least 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%, by less than 90%, at least 95%, or at least 100% or more. Quantifying the increase in TIL activation according to this modality may involve comparison with an appropriate control.

[0126] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of a pharmaceutical composition of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the activation of TILs, such as host TILs. The extent of the increase and appropriate indicators of increased activation may be considered in the preceding and / or following paragraphs. Increased expression of degranulation markers

[0127] A granulopoietic cell suitable for use of Petition 870250102349, dated 07 / 11 / 2025, page 40 / 209 32 / 177 according to the present invention may be able to increase the expression of degranulation markers by immune cells. In particular, such a granulopoietic cell may be able to increase the expression of degranulation markers by host immune cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by increasing the expression of degranulation markers by host immune cells.

[0128] 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. Therefore, it will be appreciated that the increased expression of degranulation markers, such as CD107, provides an indication that the therapeutic immune activity of such cells has been increased and the therapeutic immune response amplified accordingly.

[0129] In a suitable embodiment, a degranulation marker, whose expression by host immune cells is increased, is selected from the group 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.

[0130] The increase in the expression of degranulation markers can be evaluated and, if desired, quantified, by any appropriate method.

[0131] 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%, Petition 870250102349, dated 07 / 11 / 2025, page 41 / 209 33 / 177 at least 85%, at least 90%, at least 95%, or at least 100% or more. Quantifying the increase in the expression of degranulation markers according to this modality may make use of comparison with an appropriate control.

[0132] The expression of degranulation markers can be increased in selected host immune cells from the group consisting of: T cells and NK cells. In the case of increased expression of degranulation markers in a T cell, such a T cell can be selected from the group consisting of: a CD8+ T cell; an NK T cell; an αβ T cell; and a γδ T cell.

[0133] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of 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 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. Increased expression of co-stimulatory molecules

[0134] A granulopoietic cell 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, such a granulopoietic cell may be able to increase the expression of co-stimulatory molecules by host immune cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by increasing the expression by host immune cells of a co-stimulatory molecule.

[0135] Co-stimulatory molecules act to amplify or neutralize the activation signals provided to T cells that cause T cell differentiation. Cell differentiation Petition 870250102349, dated 07 / 11 / 2025, page 42 / 209 34 / 177 T cell activation is a key process in the therapeutic immune response, giving rise to the production of cytotoxic T cells or helper T cells. Increased expression of costimulatory molecules can thus direct the functional differentiation of T cells, causing the therapeutic immune response to be amplified. The use of a granulopoietic cell to increase the expression of costimulatory molecules 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 the activation of costimulatory molecules can, without limitation, be advantageous in the treatment of cancer or infections. In a suitable modality, a costimulatory molecule, whose expression by host immune cells is increased, is selected from the group 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 co-stimulatory molecules can 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 co-stimulatory molecules can be increased. In particular, the expression by host immune cells of 4-1BB and OX40 can be increased.

[0136] The expression of a co-stimulatory molecule can be evaluated and, if desired, quantified by any appropriate method.

[0137] 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%, Petition 870250102349, dated 07 / 11 / 2025, page 43 / 209 35 / 177 at least 85%, at least 90%, at least 95%, or at least 100% or more. Quantifying the increase in the expression of a co-stimulatory molecule according to this modality may make use of comparison with an appropriate control.

[0138] The expression of the co-stimulatory molecule can be increased in selected host immune cells from the group consisting of: T cells and NK cells. In the case of increased expression of the co-stimulatory molecule in a T cell, such a T cell can be selected from the group 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.

[0139] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of 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 host immune cells, of one or more co-stimulatory molecules. The co-stimulatory molecules, extent of increase, and relevant host immune cells can be considered in the preceding paragraphs. Increased cytokine expression

[0140] A granulopoietic cell suitable for use according to the present invention may be able to increase the expression of cytokines by immune cells. In particular, such a granulopoietic cell may be able to increase the expression of cytokines by host immune cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by increasing the expression of a co-stimulatory molecule by host immune cells.

[0141] Cytokines are key chemical messengers in the immune response. Cytokines signal for cell activation (targeting immune cells), immune cell differentiation, Petition 870250102349, dated 07 / 11 / 2025, page 44 / 209 36 / 177 as during T cell differentiation and immune cell proliferation, such as NK cells. The use of a granulopoietic cell 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.

[0142] For the purposes of the present invention, cytokines should be understood as encompassing chemokines, interferons, interleukins, lymphokines and TNFs.

[0143] In a suitable embodiment, a cytokine, whose expression by host immune cells is increased, is selected from the group consisting of: IFN-γ; and TNF (e.g., TNF-α). Suitablely, the expression of more than one of these co-stimulatory molecules may be increased. In particular, the expression by host immune cells of IFN-γ may be increased.

[0144] The increase in cytokine expression can be assessed and, if desired, quantified by any appropriate method.

[0145] 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.

[0146] The expression of a cytokine can be increased in selected host immune cells from the group consisting of: PBMCs; and TILs. The ability of granulopoietic cells Petition 870250102349, dated 07 / 11 / 2025, page 45 / 209 37 / 177 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.

[0147] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of 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 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. Increased traffic of immune cells

[0148] A granulopoietic cell suitable for use according to the present invention may be able to increase the trafficking of immune cells. In particular, such a granulopoietic cell may be able to increase the trafficking of host immune cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by increasing the trafficking of host immune cells.

[0149] 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 cells suitable for use according to the invention to enhance immune cell trafficking confers clear advantages in terms of facilitating an effective therapeutic immune response.

[0150] 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 cells suitable for use according to the invention can give rise to granulocytes that Petition 870250102349, dated 07 / 11 / 2025, page 46 / 209 38 / 177 express CXCL10, which is known to act as a chemoattractant for CXCR3+ immune cells. Thus, the medical uses and treatment methods of the invention, by giving rise to a population of cells expressing CXCL10, may be particularly beneficial in increasing the trafficking of CXCR3+ T cells and CXCR3+ NK cells.

[0151] The increase in immune cell traffic can be assessed and, if desired, quantified by any appropriate method.

[0152] In a suitable modality, 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 a modality may make use of comparison with an appropriate control.

[0153] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of a pharmaceutical composition of the invention), for example, for use according to 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.

[0154] In particular, increased immune cell traffic may give rise to increased immune cell recruitment in the TME. Increased recruitment of immune cells to the TME

[0155] As noted above, the inventors observed that exposure to a granulopoietic cell suitable for use according to the present invention increases cell trafficking. Petition 870250102349, dated 07 / 11 / 2025, page 47 / 209 39 / 177 immune. In particular, the inventors noted that granulopoietic cells suitable for use according to the present invention can increase the recruitment of immune cells to the TME. As demonstrated in the Examples, a granulopoietic cell of this type may be able to increase the recruitment of host immune cells to the TME. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by increasing the recruitment of host immune cells to the TME.

[0156] 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. Therefore, the ability to increase the recruitment of immune cells, such as host immune cells, to the TME through granulopoietic cells suitable for use according to the invention offers remarkable 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.

[0157] 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 cells suitable for use according to the treatment of the invention to increase such recruitment to the TME is demonstrated in the Examples.

[0158] In the Examples, the inventors also demonstrate that granulopoietic cells 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 cells suitable for use according to the invention can be particularly beneficial in Petition 870250102349, dated 07 / 11 / 2025, page 48 / 209 40 / 177 establishment of a population of granulocyte progeny cells capable of increasing the recruitment of CXCR3+ T cells and CXCR3+ NK cells to the TME.

[0159] The increase in immune cell recruitment to the TME can be assessed and, if desired, quantified by any appropriate method.

[0160] 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.

[0161] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of a pharmaceutical composition of the invention), for example, for use according to the invention, is an amount sufficient to increase the recruitment of immune cells, such as host immune cells, to the TME. The extent of the increased recruitment of immune cells to the TME, and the relevant host immune cells, can be considered in the preceding paragraphs. Increased cytocidal activity of immune cells

[0162] A granulopoietic cell suitable for use according to the present invention may be able to increase the cytocidal activity of immune cells. In particular, such a granulopoietic cell may be able to increase the cytocidal activity of host immune cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response to Petition 870250102349, dated 07 / 11 / 2025, page 49 / 209 41 / 177 increase the cytocidal activity of host immune cells.

[0163] 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 cells 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.

[0164] Increased cytocidal activity of immune cells can be observed in relation to PBMCs and, particularly, in relation to host PBMCs.

[0165] The increase in cytocidal activity of immune cells can be assessed and, if desired, quantified by any appropriate method.

[0166] 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 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 such an embodiment may make use of comparison with an appropriate control.

[0167] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of a pharmaceutical composition of the invention), for example, for use according to 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 immune cells Petition 870250102349, dated 07 / 11 / 2025, page 50 / 209 42 / 177 and the relevant host immune cells can be considered in the preceding paragraphs.

[0168] 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. Increased activity of immune cells in killing tumor cells

[0169] A granulopoietic cell suitable for use according to the present invention may be able to increase the tumor cell killing activity of immune cells. In particular, such a granulopoietic cell may be able to increase the tumor cell killing activity of host immune cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by increasing the tumor cell killing activity of host immune cells.

[0170] The use of immune cells to target and kill cancer cells forms the basis for most anticancer immunotherapies. Consequently, it will be readily appreciated that the ability of the granulopoietic cell 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.

[0171] 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.

[0172] The increased activity of immune cells in killing tumor cells can be assessed and, if desired, quantified by any appropriate method.

[0173] In a suitable modality, the death activity of Petition 870250102349, dated 07 / 11 / 2025, page 51 / 209 43 / 177 tumor cells 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 a modality may make use of comparison with an appropriate control.

[0174] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of 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. Increased proliferation of immune cells

[0175] A granulopoietic cell suitable for use according to the present invention may be able to increase the proliferation of immune cells. In particular, such a granulopoietic cell may be able to increase the proliferation of host immune cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by increasing the proliferation of host immune cells.

[0176] 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 Petition 870250102349, dated 07 / 11 / 2025, page 52 / 209 44 / 177 treatment of cancer or infections). Therefore, it will be appreciated that the ability of granulopoietic cells 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 the proliferation of immune cells, granulopoietic cells suitable for use according to the invention may be able to amplify immune responses that would otherwise not reach a therapeutic threshold or reduce the time required for a therapeutically effective quantity of immune cells to be produced.

[0177] 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 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 presented in the Examples. By way of example only, the αβ T cells can be CD4+ T cells or they can be CD8+ T cells.

[0178] The increase in immune cell proliferation can be assessed and, if desired, quantified by any appropriate method.

[0179] 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.

[0180] Appropriately, a therapeutically Petition 870250102349, dated 07 / 11 / 2025, page 53 / 209 45 / 177 effective of such granulopoietic cells (or of a pharmaceutical composition of the invention), for example, for use according to the invention, is a quantity 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. Increased survival of immune cells

[0181] A granulopoietic cell suitable for use according to the present invention may be able to increase the survival of immune cells. In particular, such a granulopoietic cell may be able to increase the survival of host immune cells. Consequently, such a granulopoietic cell may be able to amplify a therapeutic host immune response by increasing the survival of host immune cells.

[0182] 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 granulopoietic cells suitable for use according to the treatment of the invention are capable of increasing the survival of immune cells thus indicates 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 would otherwise reduce the longevity of immune cells.

[0183] 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 Petition 870250102349, dated 07 / 11 / 2025, page 54 / 209 46 / 177 NK T cells) or NK cells can be increased. Data illustrating the ability of useful granulopoietic cells according to the invention to increase the survival of NK T cells and NK cells are presented in the Examples.

[0184] The increase in immune cell survival can be assessed and, if desired, quantified by any appropriate method.

[0185] 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.

[0186] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of 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 the relevant immune cells and host immune cells can be considered in the preceding paragraphs. Increased abundance of immune cells

[0187] A granulopoietic cell suitable for use according to the present invention may be able to increase the abundance of immune cells. In particular, such a granulopoietic cell may be able to increase the abundance of host immune cells. Consequently, such a granulopoietic cell may be able to amplify a response Petition 870250102349, dated 07 / 11 / 2025, page 55 / 209 47 / 177 host immune therapy by increasing the abundance of host immune cells.

[0188] Without wishing to be bound by any hypothesis, the increase in the abundance of immune cells observed upon exposure of such cells to granulopoietic cells suitable for use according to the invention may emerge as a result of a combination of the increased proliferation and increased survival of immune cells discussed in more detail above. However, this 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.

[0189] The increase in the abundance of immune cells can be assessed and, if desired, quantified by any appropriate method.

[0190] 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 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. A Quantifying the increase in the abundance of host immune cells according to this method can make use of comparison with an appropriate control.

[0191] In a suitable embodiment, the abundance of T cells, such as host T cells, can be increased. The T cells whose abundance can be increased can be selected from the group consisting of: one αβ T cell; one CD8+ T cell; one Petition 870250102349, dated 07 / 11 / 2025, page 56 / 209 48 / 177 CD4+ T cells; one NK T cell; and one γδ T cell. In particular, the abundance of host αβ T cells can be increased, as further illustrated in the Examples. αβ T cells can be CD4+ T cells or they can be CD8+ T cells.

[0192] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of a pharmaceutical composition of the invention), for example, for use in accordance with the invention, is an amount sufficient to increase the abundance of immune cells, such as host immune cells. The extent of the increase in the abundance of immune cells and relevant host immune cells can be considered in the preceding paragraphs. Use in combination with other cellular immunotherapies.

[0193] Many of the properties of the cells suitable for use in the medical uses and methods of the invention indicate that these cells are also well suited for use in combination with other cell therapies and, particularly, for use with other cell immunotherapies.

[0194] The ability of the cells of the invention to increase the proliferation, abundance, and survival of immune cells suggests that treatments employing the cells of the invention may be particularly advantageous when used in combination with other cell therapies. These may be therapies using the host's own cells or therapies using allogeneic cells. By providing a treatment according to the invention, the cells involved in the additional cell therapy may be induced to proliferate, survive longer, and accumulate in greater abundance. The effectiveness of such therapy may thus be improved.

[0195] As noted above, the inventors identified the ability of granulopoietic cells to provide signal 2 (co-stimulation) and signal 3 (simulation of Petition 870250102349, dated 07 / 11 / 2025, page 57 / 209 49 / 177 cytokines) to other immune cells, such as those that are part of an additional cellular immunotherapy. The delivery of these signals is important in generating effective immune responses to tumors and in overcoming the immunosuppressive effects of TME. This property of granulopoietic cells suggests that they can be used in combination with additional cellular immunotherapy and that, by doing so, the proliferation, survival, and accumulation of cells involved with said additional cellular therapy can be improved.

[0196] The inventors' discovery that granulopoietic cells are capable of generating granulocytes that secrete chemokines, such as CXCL10, also suggests usefulness in combination with additional cellular immunotherapy. Chemokines play a vital role in the migration, positioning, and release of immune cells during a therapeutic immune response. The ability of granulopoietic cells to give rise to chemokine-secreting granulocyte progeny cells suggests that the use of granulopoietic cells in combination with additional cellular immunotherapy may give rise to the production of granulocytes capable of beneficially enhancing the activity of the additional therapy cells.

[0197] The inventors also identified that granulocytes produced in the differentiation of granulopoietic cells suitable for use in the various aspects of the invention express ligands for co-stimulatory molecules, such as 4-1BBL and OX40L. The interaction of these ligands with their receptors plays a vital role in regulating T cell activation and generating effector T cell responses. Therefore, the expression of such receptors by granulopoietic cell progeny suggests that the use of granulopoietic cells in combination with additional cell immunotherapies will allow granulopoietic cells to produce granulocytes that positively influence the T cell responses of this Petition 870250102349, dated 07 / 11 / 2025, page 58 / 209 50 / 177 way.

[0198] Appropriately, a therapeutically effective amount of such granulopoietic cells (or of a pharmaceutical composition of the invention), for example, for use according to the invention, when combined with an additional cell therapy, is an amount sufficient to increase the survival, proliferation, and / or abundance of immune cells associated with said additional cell immunotherapy. The extent of the increase, relevant immune cells, and appropriate indicators of increased activation may be considered elsewhere in the descriptive report.

[0199] Those skilled in the art will be aware of many examples of cellular immunotherapies that can be used beneficially in combination with treatment using granulopoietic cells according to the invention. These include, among others: NK cell therapies; chimeric antigen receptor (CAR) based therapies (including CAR-T cell therapies, such as CAR-γδ T cell therapies and CAR-NK cell therapies); TIL therapies; and manipulated T cell receptor (TCR) therapies. Medical uses and treatment methods of the invention

[0200] Medical uses, treatment methods, and pharmaceutical compositions may all involve granulopoietic cells for use in treating a subject by amplifying a non-granulocytic therapeutic immune response.

[0201] The term treat or treating, as used in this document, encompasses prophylactic treatment (e.g., to prevent the onset of a disease) as well as corrective treatment (treatment of a subject already suffering from a disease). Preferably, treat or treating, as used in this document, means corrective treatment.

[0202] The term treat or treating, as used in this document, may refer to the disorder and / or a symptom of Petition 870250102349, dated 07 / 11 / 2025, page 59 / 209 51 / 177 same.

[0203] A granulopoietic cell, for example, as part of a pharmaceutical composition of the invention, can be administered to a subject in a therapeutically effective amount or a prophylactically effective amount.

[0204] Some considerations regarding specific therapeutically effective amounts, selected with respect to specific outcomes to be achieved, have been presented above. However, in general terms, a therapeutically effective amount should be taken to be any amount of the granulopoietic cells or pharmaceutical compositions of the invention which, when administered alone or in combination to a subject to treat cancer or an infection (or a symptom thereof), is sufficient to effect such treatment of the disorder or symptom thereof.

[0205] In the case where the therapeutically effective amount of granulopoietic cells or of a pharmaceutical composition of the invention is administered alone, this can amplify a native immune response, thereby helping to treat cancer or infection.

[0206] A prophylactically effective amount is any quantity of the granulopoietic cells or pharmaceutical compositions of the invention that, when administered alone or in combination to a subject, inhibits or delays the onset or recurrence of cancer or an infection (or a symptom thereof). In some embodiments, the prophylactically effective amount prevents the onset or recurrence of a cancer or an infection entirely. Inhibiting onset means decreasing the likelihood of cancer onset or infection onset (or a symptom thereof), or preventing onset entirely.

[0207] An appropriate dosage range is one that produces the desired therapeutic effect (for example, where the granulopoietic cells or pharmaceutical compositions of the invention are Petition 870250102349, dated 07 / 11 / 2025, pages 60 / 209 52 / 177 dosed in a therapeutically or prophylactically effective amount).

[0208] A typical treatment regimen may include administering 10⁶, 10⁷, 10⁸, or 10⁹ cells (e.g., granulopoietic cells) to a subject, or even 10¹², 10¹³, or 10¹⁴ cells to a subject. In an appropriate embodiment, a treatment regimen includes administering a dose of at least 1 x 10⁹ cells to a subject. Appropriately, a treatment regimen may include administering a dose of at least 2 x 10⁹ cells or at least 5 x 10⁹ cells to a subject. In an appropriate embodiment, a treatment regimen may include administering a dose of at least 1 x 10¹⁰ cells or at least 5 x 10¹⁰ cells to a subject. At least 1 x 10¹¹ or at least 2 x 10¹¹ cells may be administered to a subject. In some modalities, between 1 x 10⁹ and 3 x 10¹¹ or 1 x 10¹⁰ and 3 x 10¹¹ cells are administered to a subject. Appropriately, between 5 x 10¹⁰ and 2.5 x 10¹¹ cells are administered to a subject.

[0209] A subject for treatment may be dosed once, twice, three times, four times, five times, or six times per week. Alternatively, a subject may be dosed daily (e.g., once or twice a day). In other modalities, a subject may be dosed once a week or twice a week. Preferably, the dose is weekly. Those skilled in the art will appreciate that the dose may be adapted based on the subject's needs and the effectiveness of the medication. For example, when the medication is highly effective, the dose may be reduced.

[0210] In a suitable modality, a treatment subject is dosed weekly (e.g., once a week) with at least 2 x 109 cells or at least 2 x 1010 cells. Appropriately, a treatment subject may be dosed weekly with at least 1 x 1011 cells or at least 2 x 1011 cells.

[0211] The term of address may vary based on Petition 870250102349, dated 07 / 11 / 2025, page 61 / 209 53 / 177 The subject's response to treatment and / or the type and / or severity of the cancer or infection. For example, the subject for treatment may be dosed for at least 1 or 2 weeks. Appropriately, the subject for treatment may be dosed for at least 3 or 4 weeks. In an appropriate modality, the subject for treatment is dosed for at least 5 or 6 weeks, appropriately at least 7 or 8 weeks.

[0212] In a suitable modality, a treatment subject is dosed for 4-8 weeks with at least 2 x 109 cells, wherein said cells are administered once a week. Suitablely, a treatment subject is dosed for 8 weeks with at least 2 x 109 cells (preferably at least 2 x 1010 or 2 x 1011 cells), wherein said cells are administered once a week.

[0213] Administration may be by any suitable technique or route, including, but not limited to, intravenous injection, intra-arterial injection, intraperitoneal injection, injection into a tumor resection cavity, intrathecal injection, or combinations thereof. Suitablely, the drug may be administered intravenously.

[0214] A white blood cell growth factor may be administered with a medicament of the invention. Administration may be sequential or simultaneous (suitably simultaneous). Suitable white blood cell growth factors may include 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, fetal bovine serum (FBS), retinoic acid, lipopolysaccharide (LPS), IFN-gamma, IFN-beta or combinations thereof. Suitably, the white blood cell growth factors comprise IFN-gamma and GM-CSF. Preferably, the Petition 870250102349, dated 07 / 11 / 2025, page 62 / 209 54 / 177 white blood cell growth factors comprise TNF-alpha. Appropriately, white blood cell growth factors may 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). Appropriately, white blood cell growth factors may 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.Specific examples of the above include, among others, sargramostim from the brand name LEUKINE®, filgrastim from the brand name NEUPOGEN®, and 5 PEG-filgrastim from the brand name NEULAST A®.

[0215] In a suitable embodiment, a granulopoietic cell may be administered (e.g., sequentially or simultaneously, preferably simultaneously) with a granulocyte colony-stimulating factor; and a growth hormone; and serotonin; and an interleukin. In a suitable embodiment, a granulopoietic cell is administered (e.g., sequentially or simultaneously, preferably simultaneously) with a granulocyte colony-stimulating factor; and a growth hormone; and serotonin; and an interleukin.

[0216] In some embodiments, the granulopoietic cells or pharmaceutical compositions of the invention may be used in combination with another therapeutic product, for example, in combination with an existing cancer or infection therapy, such as Petition 870250102349, dated 07 / 11 / 2025, page 63 / 209 55 / 177 radiotherapy, chemotherapy and / or immunotherapy.

[0217] By way of example, the granulopoietic cells or pharmaceutical compositions of the invention may be used in combination with a cell-snatching therapy, such as a T-cell snatching therapy. Examples of such therapies that may be used in combination with the granulopoietic cells or pharmaceutical compositions of the invention include those selected from the group consisting of: bispecific T-cell snatchers (BiTEs); checkpoint inhibitor T-cell snatchers (CiTEs); simultaneous multi-interaction T-cell snatchers (SMiTEs); trispecific killer snatchers (TRiKEs); and CAR-T cells expressing BiTE (CART.BiTE cells).In particular, the discovery that the granulopoietic cells or pharmaceutical compositions of the invention are capable of increasing the expression by immune cells of co-stimulatory molecules, such as 4-1BB and OX40, suggests that they may be advantageously used in combination with T-cell engagement therapies, such as mono / bispecific 4-1BB agonists, or bispecific TAA / 4-1BB T-cell engagers.

[0218] Prior to administration, there may be a matching step between a medicinal product of the invention (e.g., granulopoietic cells or pharmaceutical compositions of the invention) and the subject to be treated. The matching may be based on data derived from the donor from whom the granulopoietic cell is derived and similar data obtained from the subject to be treated. The matching may be achieved based on blood group type, similarity of human leukocyte antigen (HLA) type, or combinations thereof. Treatment methods

[0219] The second aspect of the invention provides a treatment method comprising amplifying a non-granulocytic therapeutic immune response, the method comprising providing a granulopoietic cell to a subject in need of such. Petition 870250102349, dated 07 / 11 / 2025, page 64 / 209 56 / 177 treatment.

[0220] The granulopoietic cells provided may be cells according to any of the embodiments described in this descriptive report. Suitablely, the granulopoietic cells may be provided by means of a pharmaceutical composition of the invention.

[0221] Appropriately, such a subject may be a cancer patient. An appropriate patient may have any form of cancer, including those described further in this disclosure. For example, a patient may have pancreatic cancer.

[0222] Appropriately, such a patient may have an infection. An appropriate patient may have any form of infection, including those described further in this disclosure. By way of example only, a patient may have a viral infection. Pharmaceutical manufacturing

[0223] The third aspect of the invention provides a granulopoietic cell for use in the manufacture of a medicament for use in amplifying a non-granulocytic therapeutic immune response.

[0224] The granulopoietic cells used in such manufacture may be cells according to any of the embodiments described in this document. The drug manufactured according to this aspect of the invention may be a pharmaceutical composition of the invention. Cancers to be treated

[0225] The medical uses, treatment methods, or pharmaceutical compositions of the invention can all be employed in the treatment of cancer. Cancer can be treated by killing or otherwise therapeutically reducing the activity of cancer cells. This can occur as a result of the activity of non-granulocytic cells that provide effective therapeutic immunity and can also occur as a result of cell death activity. Petition 870250102349, dated 07 / 11 / 2025, page 65 / 209 57 / 177 of cancer by granulocytes produced in the differentiation of granulopoietic cells employed in the medical uses, treatment methods or pharmaceutical compositions of the invention.

[0226] In a proper modality, a cancer is a solid tumor cancer. The term solid tumor cancer refers to an abnormal malignant mass of tissue that does not contain cysts or fluid inclusions. Examples of solid tumor cancers include carcinomas, sarcomas, and lymphomas.

[0227] A solid tumor cancer may be a carcinoma. A carcinoma may be selected from one or more of an adenocarcinoma, a basal cell carcinoma, a squamous cell carcinoma, an adenosquamous carcinoma, a renal cell carcinoma, a ductal carcinoma in situ (DCIS), an invasive ductal carcinoma, an anaplastic carcinoma, a large cell carcinoma, a small cell carcinoma, or combinations thereof.A carcinoma may also be selected from among epithelial neoplasms, squamous cell neoplasms, basal cell carcinoma, transitional cell carcinomas, adenocarcinomas (such as adenocarcinoma not otherwise specified (NOS), linitis plastica, vipoma, cholangiocarcinoma, NOS hepatocellular carcinoma, adenoid cystic carcinoma, renal cell carcinoma, Grawitz tumor), adnexal and appendiceal neoplasms of the skin, mucoepidermoid neoplasms, mucinous and serous cystic neoplasms, lobular and medullary ductal neoplasms, acinar cell neoplasms, or complex epithelial neoplasms.

[0228] Alternatively, a solid tumor cancer may be a sarcoma. A sarcoma may be selected from among Askin's tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, or soft tissue sarcomas (including alveolar soft tissue sarcoma, angiosarcoma, cystosarcoma phyllodes, Petition 870250102349, dated 07 / 11 / 2025, page 66 / 209 58 / 177 dermatofibrosarcoma protuberans (DFSP), desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, malignant fibrous histiocytoma, undifferentiated pleomorphic sarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma).

[0229] Alternatively, a solid tumor may be a lymphoma, such as a B-cell lymphoma, a T-cell lymphoma, an NK-cell lymphoma, or a Hodgkin lymphoma.

[0230] In a suitable embodiment, a medical use, treatment method or pharmaceutical composition of the invention 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.

[0231] In the case where the medical use, treatment method or pharmaceutical composition of the invention is for use in the treatment of pancreatic cancer, pancreatic cancer may be a solid pancreatic tumor cancer, such as a pancreatic adenocarcinoma (for example, a pancreatic ductal adenocarcinoma). Infections to be treated

[0232] The medical uses, treatment methods, or pharmaceutical compositions of the invention can all be employed in the treatment of infections. Such infections can be treated by killing or otherwise therapeutically reducing the activity of infectious agents (such as cellular infectious agents), or by killing or otherwise therapeutically reducing the activity Petition 870250102349, dated 07 / 11 / 2025, page 67 / 209 59 / 177 of cells infected by infectious agents.

[0233] As used in this document, a cell infected by an infectious agent refers to a cell that is infected by an intracellular infectious agent. The said intracellular infectious agent may be a pathogen and the cell is therefore a cell infected by a pathogen. In a suitable embodiment, a cell may be infected by an intracellular bacterium or a virus, preferably a virus.

[0234] In a suitable modality, an infection to be treated is caused by a Gram-negative bacterium or a Gram-positive bacterium. Preferably, an infectious agent is a Gram-positive bacterium, such as a bacterium of the genus Staphylococcus.

[0235] Appropriately, an infection to be treated is caused by a bacterium selected from one or more of Staphylococcus spp., multidrug-resistant Gram-negative bacteria (MRDGN bacteria), vancomycin-resistant Enterococcus (VRE), Mycobacterium spp., bacillus-resistant Enterobacteriaceae (CRE) intestinal bacteria, Acinetobacter spp., Actinomyces spp., Propionibacterium spp., Anaplasma spp., Bacillus spp., Arcanobacterium spp., Bacteroides spp., Bartonella spp., Brucella spp., Yersinia spp., Burkholderia spp., Campylobacter spp., Streptococcus spp., Haemophilus spp., Clostridium spp. , Corynebacterium spp., Echinococcus spp., Ehrlichia spp. , Enterococcus spp., Rickettsia spp., Fusobacterium spp. , Neisseria spp., Klebsiella spp., Helicobacter spp., Escherichia spp. , Kingella spp., Legionella spp., Listeria spp., Borrelia spp., Mycoplasma spp., Chlamydia spp., Nocardia spp., Pasteurella spp., Bordetella spp., Prevotella spp. , Chlamydophila spp., Coxiella spp., Salmonella spp., Group A Streptococcus spp., Shigella spp., Staphylococcus spp. , Treponema spp., Vibrio spp., Francisella spp., Pseudomonas spp. and Ureaplasma spp. Petition 870250102349, dated 07 / 11 / 2025, pages 68 / 209 60 / 177

[0236] In a suitable embodiment, the bacterium is selected from one or more of methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Mycobacterium tuberculosis (MDR-TB), Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriae, Propionibacterium propionicus, Bacillus anthracis, Arcanobacterium haemolyticum, Bacillus cereus, Yersinia pestis, Mycobacterium ulcerans, Campylobacter jejuni, Bartonella bacilliformis, Bartonella henselae, Haemophilus ducreyi, Clostridium difficile, Corynebacterium diphtheria, Burkholderia mallei, Neisseria gonorrhoeae, Klebsiella granulomatis, Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae, Helicobacter pylori, Escherichia coli (e.g., O157:H7, O111, and O104:H4), Kingella kingae, Legionella pneumophila, Listeria monocytogenes, Burkholderia pseudomallei, Neisseria meningitis, Mycoplasma pneumoniae,Mycoplasma genitalium, Chlamydia trachomatis, Bordetella pertussis, Streptococcus pneumoniae, Chlamydophila psittaci, Coxiella burnetii, Treponema pallidum, Clostridium tetani, Chlamydophila pneumoniae, Vibrio cholera, Mycobacterium tuberculosis, Salmonella enterica subsp. enterica, serovartyphi, Ureaplasma urealyticum and Francisella tularensis. Preferably Mycobacterium tuberculosis.

[0237] Preferably, in a suitable embodiment, the bacterium is selected from one or more of methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Gram-negative bacteria (MRDGN bacteria), vancomycin-resistant Enterococcus (VRE), multidrug-resistant Mycobacterium tuberculosis (MDR-TB), and carbapenem-resistant Enterobacteriaceae (CRE) intestinal bacteria.

[0238] Appropriately, an infection to be treated is caused by a virus selected from one or more selected families. Petição 870250102349, de 07 / 11 / 2025, pág. 69 / 209 61 / 177 dentre Adenoviridae, Picornaviridae, Herpesviridae, Coronaviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, Togaviridae e Bunyaviridae.

[0239] In a suitable embodiment, the virus may be selected from one or more of HIV-1 (human immunodeficiency virus), HIV-2, Junin virus, BK virus, Machupo virus, Sabiá virus, Varicella zoster virus (VZV), Alphavirus, Colorado tick fever virus (CTFV), rhinovirus, Crimean-Congo hemorrhagic fever virus, cytomegalovirus, dengue virus, Ebola virus (EBOV), Parvovirus B19, human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV-7), enteroviruses (e.g., EV71), Coxsackie A virus, Sin Nombre virus, Heartland virus, Hanta virus, Hendra virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpes simplex virus 1 and 2 (HSV-1 and HSV-2), human bocavirus (HBoV), Human metapneumovirus (hMPV), human papillomavirus, human parainfluenza virus (HPIV), Epstein-Barr virus (EBV), Lassa virus, lymphocytic choriomeningitis virus (LCMV), Marburg virus, measles virus,Middle East Respiratory Syndrome coronavirus, Molluscum Contagiosum virus (MCV), Monkeypox virus, Mumps virus, Nipah virus, Norovirus, Poliovirus, JC virus, Respiratory Syncytial Virus (RSV), Rhinovirus, Rift Valley Fever virus, Rotavirus, Rubella virus, SARS coronavirus, SARS-CoV-2, Variola major, Variola minor, Venezuelan Equine Encephalitis virus, Guanarito virus, West Nile virus, Yellow Fever virus, and Zika virus.

[0240] Appropriately, an infection to be treated is caused by a fungus selected from one or more of Aspergillus spp., Piedraia spp., Blastomyces spp., Candida spp., Fonsecaea spp., Coccidioides spp., Cryptococcus spp., Cryptosporidium spp., Geotrichum spp., Histoplasma spp., Microsporidia phylum, Paracoccidioides spp., Pneumocystis spp., Sporothrix spp., Petition 870250102349, dated 07 / 11 / 2025, pp. 70 / 209 62 / 177 Trichophyton spp., Epidermophyton spp., Hortaea spp., Malassezia spp., Trichosporon spp. from the order Mucorales.

[0241] In a suitable embodiment, the pathogen is a fungus selected from one or more of Aspergillus fumigatus, Aspergillus flavus, Piedraia hortae, Blastomyces dermatitidis, Candida albicans, Fonsecaea pedrosoi, Coccidioides immitis, Coccidioides posadasii, Cryptococcus neoformans, Geotrichum candidum, Histoplasma capsulatum, Paracoccidioides brasiliensis, Pneumocystis jirovecii, Sporothrix schenckii, Trichophyton tonsurans, Epidermophyton floccosum, Hortaea werneckii, and Trichosporon beigelii.

[0242] Um macroparasita pode ser um ou mais selecionados dentre Angiostrongylus spp., Entamoeba Anisakis spp., Ascaris spp., Babesia spp., Balantidium spp., Baylisascaris spp., Blastocystis spp., Capillaria spp., Trypanosoma spp., Clonorchis spp., Ancylostoma spp., Cyclospora spp., Taenia spp., Desmodesmus spp., Dientamoeba spp., Dracunculus spp,. Enterobius spp., Fasciola spp., superfamília Filarioidea, Giardia spp., Gnathostoma spp., Necator spp., Hymenolepis spp., Isospora spp., Leptospira spp., Wuchereria spp., Rhinosporidium spp., Brugia spp., Plasmodium spp., Onchocerca spp., Opisthorchis spp., Paragonimus spp., Naegleria spp., Schistosoma spp., Strongyloides spp., Toxocara spp., Toxoplasma spp., Trichinella spp., Trichomonas spp. e Trichuris spp.

[0243] In a suitable embodiment, the macroparasite is selected from one or more of Entamoeba histolytica, Ascaris lumbricoides, Balantidium coli, Trypanosoma brucei, Trypanosoma cruzi, Clonorchis sinensis, Cyclospora cayetanensis, Taenia solium, Desmodesmus armatus, Dientamoeba fragilis, Dracunculus medinensis, Enterobius vermicularis, Fasciolopsis buski, Giardia lamblia, Necator americanus, Hymenolepis nana, Hymenolepis diminuta, Isospora belli, Wuchereria bancrofti, Rhinosporidium seeberi, Brugia malayi, Plasmodium vivax, Plasmodium falciparum, Petition 870250102349, dated 07 / 11 / 2025, p. 71 / 209 63 / 177 Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi Onchocerca volvulus, Opisthorchis viverrini, Opisthorchis felineus, Naegleria fowleri, Strongyloides stercoralis, Toxoplasma gondii, Trichinella spiralis, Trichuris trichiura, and Trichomonas vaginalis.

[0244] In a suitable embodiment, the infectious agent is an antibiotic-resistant bacterium (e.g., MRSA), preferably a multi-antibiotic-resistant bacterium. An antibiotic-resistant bacterium may be resistant to beta-lactams, such as methicillin.

[0245] Antibiotic resistance can be assessed using any technique known in the art, such as the Kirby-Baure method, Stokes method, Etest and / or agar and broth dilution methods for determining the minimum inhibitory concentration (MIC).

[0246] In a suitable embodiment, a bacterium is resistant to one or more of the following: a penicillin, a penicillinase-resistant penicillin, a cephalosporin, a beta-lactamase inhibitor, a tetracycline, and combinations thereof, or pharmaceutically acceptable salts thereof.

[0247] In a suitable embodiment, a bacterium is resistant to one or more of the following: vancomycin, nafcillin, oxacillin, teicoplanin, penicillin, methicillin, flucloxacillin, dicloxacillin, cefazolin, cephalothin, cephalexin, cefuroxime, clindamycin, cefazolin, amoxicillin / clavulanate, ampicillin / sulbactam, lincomycin, erythromycin, trimethoprim, sulfamethoxazole, daptomycin, linezolid, rifampicin, ciprofloxacin, gentamicin, tetracycline, doxycycline, minocycline, tigecycline, and combinations thereof or pharmaceutically acceptable salts thereof. In a suitable embodiment, a bacterium may be resistant to vancomycin and / or teicoplanin, or pharmaceutically acceptable salts thereof. Petition 870250102349, dated 07 / 11 / 2025, page 72 / 209 64 / 177

[0248] A multidrug-resistant bacterium is resistant to at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 antibiotics (e.g., chemical antibiotics).

[0249] In a suitable embodiment, a granulocyte produced in the differentiation of a suitable granulopoietic cell kills an infectious agent by phagocytosis from a cell infected by the infectious agent. For example, in a suitable embodiment, a granulocyte produced in the differentiation of a suitable granulopoietic cell kills a virus by phagocytosis from a cell infected by the virus. In a suitable embodiment, a granulocyte produced in the differentiation of a suitable granulopoietic cell kills a bacterium by phagocytosis from a cell infected by the bacterium. In a suitable embodiment, a granulocyte produced in the differentiation of a suitable granulopoietic cell kills an infectious agent by releasing one or more factors that kill the infectious agent. For example, in a suitable embodiment, a granulocyte produced in the differentiation of a suitable granulopoietic cell kills a virus by releasing one or more factors that kill the virus.In one suitable embodiment, a granulocyte produced upon differentiation from a suitable granulopoietic cell kills a bacterium by releasing one or more factors that kill the bacterium. In some embodiments, a granulocyte produced upon differentiation from a suitable granulopoietic cell kills an infectious agent by a combination of the above. Granulopoietic cells capable of giving rise to granulocytes with desirable cytocidal activity.

[0250] Appropriately, the granulopoietic cells for use in the various aspects of the invention may be able to differentiate to give rise to granulocytes that have cytocidal activity which may further contribute to a therapeutic immune response. In particular, such cells may produce granulocytes that are capable of killing cancerous cells, Petition 870250102349, dated 07 / 11 / 2025, page 73 / 209 65 / 177 infected cells or cellular infectious agents.

[0251] The inventors have developed a number of ways in which granulocytes with such cytocidal activity can be identified.

[0252] For example, a granulopoietic cell, for use in accordance with the invention, may be one that has the ability to differentiate to produce granulocytes with the capacity to kill at least 5% of cancerous cells in a cancer killing assay, the cancer killing assay comprising: a. mixing granulocytes with cancerous cells to form a mixture; b. incubate the aforementioned mixture; and c. measure the percentage of dead cancer cells in the aforementioned mixture.

[0253] Appropriately, in such an embodiment, the % of cancer cells killed in the said mixture is the maximum % of cancer cells killed 48 hours after the mixture is formed. The granulocytes thus produced may have the capacity to kill at least 10%, 20%, 30%, 40%, 50%, 51.5%, 60%, 70% or 80% of cancer cells in the cancer killing assay.

[0254] In a suitable embodiment, the assay mixture comprises 1:1, 5:1 or 10:1 granulocytes to cancer cells.

[0255] Appropriately, the cancer cells used in such an assay are HeLa or PANC-1 cancer cells.

[0256] Those skilled in the art will be aware of many suitable cancer death assays that can be used in evaluating the ability to kill cancer cells. By way of example only, in a suitable embodiment, the cancer death assay is performed using an ACEA Biosciences xCELLigence RTCA DP Analyzer system® in accordance with the manufacturer's instructions and as follows: a. 6000 cancerous cells are placed at the bottom of a Petition 870250102349, dated 07 / 11 / 2025, page 74 / 209 66 / 177 plate of 16 wells; b. the cells are cultured until confluence, as determined by the stabilization of the Cell Index (CI) values ​​(i.e., the 'normalization point'); c. 60,000 granulocytes are added (that is, giving a ratio of 10 granulocytes to 1 cancer cell) and incubated at 37 °C; and d. The percentage of cancerous cells killed is the maximum percentage of cancerous cells killed 48 hours after the addition of granulocytes, as determined using the following formula: ((Non-Effector Cell Index — Effector Cell Index) / Non-Effector Cell Index) X 100.

[0257] In one embodiment, the cancer death assay is performed using a luciferase cytotoxicity assay as follows: a. the cancer cells are placed at the bottom (e.g., of a 96-well plate); b. Effector cells, such as granulopoietic cells or granulocytes differentiated from granulopoietic cells, are added to the cancer cells (e.g., 17-24 hours later at a ratio of 10:1 or 20:1 of effector cells to cancer cells) to form a mixture; c. the mixture is incubated (for example, for 48 hours at 37 °C in a 5% CO2 atmosphere); d. After incubation, a luciferase substrate (such as luciferin, preferably 5-fluoroluciferin) is added to the mixture (e.g., and incubated at room temperature until the luminescence signal is stabilized (e.g., 7-10 minutes)); and The luminescence signal is measured and the percentage of dead cancer cells is determined.

[0258] The luciferase substrate can be added in any suitable concentration range, such as 1-1000 μM, per Petition 870250102349, dated 07 / 11 / 2025, page 75 / 209 67 / 177 example, 10-500 μΜ or 100-400 μΜ.

[0259] In a suitable embodiment, the cancer death assay is performed using a luciferase cytotoxicity assay as follows: a. 1.5 x 10⁴ cancer cells are placed at the bottom of a 96-well plate; b. Effector cells, such as granulopoietic cells or granulocytes differentiated from granulopoietic cells, are added to the cancer cells 17-24 hours later in a ratio of 10:1 or 20:1 of effector cells to cancer cells to form a mixture; c. the mixture is incubated for 48 hours at 37 °C in a 5% CO2 atmosphere; d. After incubation, 100 μL of ONEglo™ reagent are added to the mixture and incubated at room temperature until the luminescence signal is stabilized (e.g., 710 minutes); and The luminescence signal is measured and the percentage of dead cancer cells is determined.

[0260] The following formula can be used to calculate the % of dead cancer cells: 100 - ((Corrected Sample Background Luminescence) / (Corrected Target Background Luminescence) * 100).

[0261] In this case, the sample may be the mixture referred to above comprising effector cells and cancer cells, and the target only may refer to a sample comprising cancer cells and no effector cells. Those skilled in the art will appreciate that the sample and the target only may have been exposed to the same steps, for example, incubations, to allow comparability. Background correction may be achieved by usual normalization techniques, for example, by subtracting any observed luminescence signal with a Petition 870250102349, dated 07 / 11 / 2025, pp. 76 / 209 68 / 177 sample of the middle only. Preferably, background correction can be achieved by subtracting the luminescence of the middle only from the luminescence values ​​of the sample or target only.

[0262] A granulopoietic cell suitable for use in the various aspects of the invention may be one that has the ability to differentiate to produce granulocytes characterized by: a. increased expression of one or more GM2A, CTSG, CAP37, ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, and PSMB2 when compared to a reference standard, where the reference standard is a neutrophil unsuitable for cancer treatment; and / or b. decreased expression of ANXA1 and / or PPP3CB when compared to a reference standard, where the reference standard is a neutrophil unsuitable for cancer treatment.

[0263] In a suitable embodiment, a granulopoietic cell suitable for use in the various aspects of the invention can be characterized by the granulocytes produced in the differentiation of the granulopoietic cell having a positively charged cell surface.

[0264] The granulopoietic cells suitable for use in the various aspects of the invention can also be identified in relation to the expression profiles of the granulocytes that they are able to produce.

[0265] In a suitable configuration, a granulopoietic cell may be able to differentiate to produce a granulocyte that is characterized by: a. Increased expression of one or more of GM2A, CTSG, CAP37, ITGB1, CYBB, SYK, DOCKS, COMP, ATG7, SLC2A1, GZMK, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, and PSMB2 when compared to a reference standard, where the reference standard is a granulocyte that does not have the ability to kill cells. Petition 870250102349, dated 07 / 11 / 2025, page 77 / 209 69 / 177 carcinogens, or an infectious agent, or cells infected by an infectious agent; and / or b. decreased expression of ANXA1 and / or PPP3CB when compared to a reference standard, where the reference standard is a granulocyte that does not have the ability to kill cancerous cells, or an infectious agent, or cells infected by an infectious agent.

[0266] Representative sequences for genes for use in such embodiments of the invention are described in the Sequence Listings and appropriate Set Accession Numbers set out in International Patent Application Numbers: PCT / GB2020 / 053197 (published as WO 2021 / 116711) and PCT / GB2020 / 053199 (published as WO 2021 / 116713), relevant disclosures of which, particularly relating to sequence listings and sequence identity suitable for use in this embodiment of the invention, are incorporated herein by reference.

[0267] Determining whether a granulocyte has increased or decreased the expression of one or more GM2A, CTSG, CAP37, ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, and PSMB2; and / or decreased the expression of ANXA1 and / or PPP3CB can be performed by measuring the expression of said markers. The expression measurement can be performed by any means known to those skilled in the art. The term measurement, as used in reference to the expression of one or more genes of the invention, encompasses measuring both negative expression (e.g., no expression) and positive expression (e.g., expression). In a suitable embodiment, the expression is positive expression.

[0268] In some modalities, expression can be measured using high-throughput techniques. For example, expression measurement can be at the transcription level (e.g., transcriptomic techniques) or translation level (e.g., techniques). Petition 870250102349, dated 07 / 11 / 2025, pp. 78 / 20970 / 177 proteomics). Alternatively, or additionally, the invention may employ the use of genomics, for example, to detect the presence or absence of single nucleotide polymorphisms (SNPs), promoter sequences, gene copy number (e.g., duplications) and / or enhancer or other relevant genetic characteristics, preferably those that determine the expression level of one or more genes of the invention. High-throughput techniques can be used to rapidly analyze entire genomes, proteomes, and transcriptomes, providing data, including expression levels, of all genes, polypeptides, and transcripts in a cell. Proteomics is a technique for analyzing the proteome of a cell (e.g., at a given point in time). The proteome differs in different cell types.Typically, proteomics is performed by mass spectrometry, including tandem mass spectrometry and gel-based techniques, including differential gel electrophoresis. Proteomics can be used to detect polypeptides expressed in a particular cell type and generate a proteomic profile to allow the identification of specific cell types. [02 69] In a suitable modality, the mRNA of a target gene can be detected and quantified by, for example, Northern blotting or by quantitative reverse transcription PCR (RTPCR). Single-cell gene expression analysis can also be performed using commercially available systems (e.g., Fluidigm Dynamic Array). Alternatively, or additionally, gene expression levels can be determined by analyzing polypeptide levels, for example, using Western blotting techniques, such as ELISA-based assays.

[0270] Thus, in a suitable embodiment, gene expression levels are determined by measuring the mRNA / cDNA levels of the genes of the present invention, such as RNA sequencing. Petition 870250102349, dated 07 / 11 / 2025, pp. 79 / 209 71 / 177 (RNA-Seq).

[0271] In a preferred embodiment, gene expression levels are determined by measuring the polypeptide levels produced by the genes of the present invention, such as by means of mass spectrometry, for example, liquid chromatography-mass spectrometry (LC-MS / MS).

[0272] In a suitable modality, a granulocyte (or stem cell) for cancer treatment can be detected using an enzyme-linked immunosorbent assay (ELISA) or a Luminex assay (commercially available from R&D Systems, USA).

[0273] Thus, in a suitable embodiment, expression measurement comprises measuring and / or comparing the expression level of one or more polypeptides by a granulocyte, wherein one or more polypeptides are selected from among: CTSG, CAP37, ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PPP3CB, ANXA1, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37 and PSMB2.

[0274] In a suitable embodiment, expression measurement comprises measuring and / or comparing the quantity of one or more polypeptides produced by a granulocyte, wherein one or more polypeptides are selected from among: CTSG, CAP37, ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PPP3CB, ANXA1, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37 and PSMB2.

[0275] In a suitable embodiment, expression measurement comprises measuring and / or comparing the expression level of one or more polypeptides by a stem cell, wherein one or more polypeptides are selected from among: CTSG, CAP37, ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PPP3CB, ANXA1, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37 and PSMB2.

[0276] In a suitable embodiment, the measurement of the expression comprises measuring and / or comparing a quantity of one or more Petition 870250102349, dated 07 / 11 / 2025, pages 80 / 209 72 / 177 polypeptides produced by a stem cell, wherein one or more polypeptides are selected from: CTSG, CAP37, ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PPP3CB, ANXA1, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37 and PSMB2.

[0277] In a suitable embodiment, the measurement expression employs a genome-wide association study, which is compared to a reference standard (for example, a reference standard from a reference population, such as a reference standard of: a suitable or unsuitable donor, or a suitable or unsuitable granulocyte, or a subject who is suitable or unsuitable for treatment with a granulopoietic cell according to the invention, or a subject who is at risk or not at risk of cancer, or combinations thereof).

[0278] The appropriate methods for establishing a baseline or reference value for comparing expression levels are conventional techniques known to those skilled in the art.

[0279] The term enhanced, as used in this document in reference to the expression of one or more genes of the invention, may refer to an expression level that is increased in a statistically significant manner when compared to a reference standard. Such a gene may be considered to be upregulated.

[0280] In a suitable embodiment, augmented means of expression greater than 1 time, 1.25 times to about 10 times or more of expression relative to a reference standard. In some embodiments, augmented means of expression greater than at least about 1.1 times, 1.2 times, 1.25 times, 1.5 times, 1.75 times, 2 times, 4 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 50 times, 75 times, 100 times, 150 times, 200 times or at least about 300 times of expression when compared to a reference standard. Petition 870250102349, dated 07 / 11 / 2025, p. 81 / 209 73 / 177

[0281] The term diminished, as used in this document in reference to the expression of one or more genes of the invention, may refer to an expression level that is statistically significantly diminished when compared to a reference standard. Such a gene may be considered negatively regulated.

[0282] In a suitable embodiment, diminished means of expression less than -1 time, -1.25 time to about -10 times or more of expression relative to a reference standard. In some embodiments, diminished means of expression less than at least about -1.1 time, -1.2 time, -1.25 time, -1.5 time, 1.75 time, -2 time, -4 time, -5 time, -10 time, -15 time, -20 time, -25 time, -30 time, -35 time, -40 time, -50 time, -75 time, -100 time, -150 time, -200 time or at least about -300 times of expression when compared to a reference standard.

[0283] The fold change difference can be in absolute terms (e.g., CPM: counts per million) or Log2CPM (a standard measure in the field) of the expression level in a sample. Preferably, the fold change is the Log2 fold change. In a suitable embodiment, a Log2 change is an increase of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, or 2.7. In a proper form, a change in Log2 is a decrease of 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1.0 or more, 1.1 or more, 1.2 or more, or 1.3 or more. A decrease may be indicated by the presence of a - symbol before the value.

[0284] In a suitable embodiment, the said fold change is measured and / or determined by RNA sequencing (RNA-Seq), for example, in toto.

[0285] The unchanged or same term, as used Petition 870250102349, dated 07 / 11 / 2025, p. 82 / 209 74 / 177 in this document, when referring to the expression of one or more genes of the invention, may refer to an expression level that is not statistically significantly different from a reference standard. Preferably, an expression level that is the same as a reference standard.

[0286] The expression level can be a mean, such as an average expression level. In a suitable embodiment, statistical significance is determined using two-way ANOVA, for example, where n is at least 3 and the data are presented as mean + / - standard error of the mean.

[0287] In a suitable embodiment, the methods of the invention comprise measuring the expression of combinations of the genes described in this document.

[0288] The term one or more, when used in the context of a gene described in this document, may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 of the genes. Preferably, the term one or more means all the genes. Similarly, the term one or more, when used in the context of a polypeptide described in this document, may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 of the polypeptides. Preferably, the term "one or more" means all polypeptides.

[0289] The expression of one or more of ITGB1, CYBB, SYK, DOCKS, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PPP3CB, ANXA1, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2 may correlate with a granulocyte's ability to kill cancer cells. These genes may therefore be referred to in this document as genes associated with the ability to kill cancer cells. Thus, the term one or more genes associated with the ability to kill cancer cells (and similar terms) may be synonymous with (and thus replaced by) the term one or more of ITGB1, CYBB, SYK, DOCKS, COMP, ATG7, SLC2A1, Petition 870250102349, dated 07 / 11 / 2025, p. 83 / 209 75 / 177 GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PPP3CB, ANXA1, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2. Thus, the term one or more polypeptides associated with the ability to kill cancer cells (and similar cells) can be synonymous with (and thus replaced by) the term one or more of ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PPP3CB, ANXA1, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2.

[0290] In a suitable embodiment, the expression of one or more of ITGB1, CYBB, SYK, DOCKS, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2 may be increased in a granulocyte that has the ability to kill cancer cells when compared to a granulocyte that does not have the ability to kill cancer cells. Alternatively, or additionally, in a suitable embodiment, the expression of ANXA1 and / or PPP3CB is decreased in a granulocyte that has the ability to kill cancer cells when compared to a granulocyte that does not have the ability to kill cancer cells.

[0291] In a suitable embodiment, the expression of S100A9 and / or S100A8 can be increased in a granulocyte of the invention when compared to a reference standard, where the reference standard is a granulocyte that does not have the ability to kill cancer cells.

[0292] The expression of one or more of ITGB1, CYBB, SYK, DOCKS, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PPP3CB, ANXA1, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2 correlates with the ability of a granulocyte to kill an infectious agent or cells infected by an infectious agent. These genes are therefore referred to in this document as genes associated with the ability to kill an infectious agent or cells infected by an infectious agent. Thus, the term one or more genes associated with the ability to kill an infectious agent or cells infected by an infectious agent Petition 870250102349, dated 07 / 11 / 2025, p. 84 / 209 76 / 177 infectious (and similar) may be synonymous with (and thus replaced by) one or more of the following terms: ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PPP3CB, ANXA1, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37 and PSMB2.

[0293] In a suitable embodiment, the expression of one or more of ITGB1, CYBB, SYK, DOCKS, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2 is increased in a granulocyte that has the ability to kill an infectious agent or cells infected by an infectious agent when compared to a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent. Alternatively or additionally, in a suitable embodiment, the expression of ANXA1 and / or PPP3CB is decreased in a granulocyte that has the ability to kill an infectious agent or cells infected by an infectious agent when compared to a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent.

[0294] In a suitable embodiment, a method of the invention may further comprise measuring the expression of one or more genes selected from: S100A9 and S100A8. In a suitable embodiment, the expression of S100A9 and / or S100A8 may be increased in a granulocyte of the invention when compared to a reference standard, where the reference standard is a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent.

[0295] The expression level of one or more genes of the invention can be compared to a reference standard. The comparison can be performed by any suitable technique known to those skilled in the art, for example, a bioinformatics technique. The expression level of the genes described in this document is suitably known in the aforementioned reference standard.

[0296] The reference standard may be a proteomic profile Petition 870250102349, dated 07 / 11 / 2025, page 85 / 209 77 / 177 (indicating an amount of polypeptide expressed by a granulocyte), a transcriptomic profile (indicating an amount of gene expression by a granulocyte, for example, measured by RNA produced by said granulocyte) or a genomic profile. A genomic profile can be used to detect the presence or absence of SNPs, promoter sequences, gene copy number (e.g., duplications) and / or enhancer or other relevant genetic characteristics, preferably those that determine the expression level of one or more genes of the invention. Those skilled in the art will appreciate that proteomic and transcriptomic profiles are measures of gene expression and will employ the appropriate reference standard depending on the technique used to measure gene expression according to the invention.For example, where proteomics is used in the practice of the present invention, the skilled in the art will employ a reference standard that is a proteomic profile; where transcriptomics is used in the practice of the present invention, the skilled in the art will employ a reference standard that is a transcriptomic profile; and where genomics is used in the practice of the present invention, the skilled in the art will employ a reference standard that is a genomic profile. A reference standard may refer to a database (e.g., a genomic database), for example, which may include data from one or more sources, such as one or more subjects and / or cells.

[0297] A reference standard is preferably a reference standard for a granulocyte that does not have the ability to kill cancer cells (e.g., a transcriptomic or proteomic profile of a granulocyte that is unsuitable for cancer treatment). Such a reference standard may be from a subject who does not have cancer (a healthy subject) or from a subject who has cancer. Preferably, such a reference standard is from a subject who does not have cancer.

[0298] In a suitable form, the expression of one or Petition 870250102349, dated 07 / 11 / 2025, pages 86 / 209 78 / 177 more than ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2 is increased when compared to a reference standard when the reference standard is a granulocyte that does not have the ability to kill cancer cells. In a suitable modality, the expression of ANXA1 and / or PPP3CB is decreased when compared to a reference standard when the reference standard is a granulocyte that does not have the ability to kill cancer cells.In a suitable embodiment, the expression of one or more of ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2 is increased when compared to a reference standard when the reference standard is a granulocyte that does not have the ability to kill cancer cells, and the expression of ANXA1 and / or PPP3CB is decreased when compared to a reference standard when the reference standard is a granulocyte that does not have the ability to kill cancer cells.

[0299] A reference standard may be a reference standard for a granulocyte that is suitable for cancer treatment (e.g., a transcriptomic or proteomic profile of a granulocyte that is suitable for cancer treatment). In a suitable embodiment, the expression of one or more of ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2 is increased or the same when compared to a reference standard when the reference standard is from a granulocyte that has the ability to kill cancer cells. In a suitable embodiment, the expression of ANXA1 and / or PPP3CB is decreased or the same when compared to a reference standard when the reference standard is from a granulocyte that has the ability to kill cancer cells.

[0300] In some embodiments, the present invention may comprise the use of a reference standard for a granulocyte. Petition 870250102349, dated 07 / 11 / 2025, page 87 / 209 79 / 177 which does not have the ability to kill cancer cells and a reference standard for a granulocyte that does have the ability to kill cancer cells.

[0301] A reference standard is preferably a reference standard for a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent that does not have the ability to kill an infectious agent or cells infected by an infectious agent (e.g., a transcriptomic or proteomic profile of a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent that does not have the ability to kill an infectious agent or cells infected by an infectious agent).

[0302] In a suitable embodiment, the expression of one or more of ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2 is increased when compared to a reference standard when the reference standard is a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent. In a suitable embodiment, the expression of ANXA1 and / or PPP3CB is decreased when compared to a reference standard when the reference standard is a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent.In a suitable embodiment, the expression of one or more of ITGB1, CYBB, SYK, DOCKS, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2 is increased when compared to a reference standard when the reference standard is a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent, and the expression of ANXA1 and / or PPP3CB is decreased when compared to a reference standard when the reference standard is a granulocyte that does not have the ability to kill an agent. Petition 870250102349, dated 07 / 11 / 2025, pages 88 / 209 80 / 177 infectious or cells infected by an infectious agent.

[0303] A reference standard may be a reference standard for a granulocyte that has the ability to kill an infectious agent or cells infected by an infectious agent (e.g., a transcriptomic or proteomic profile of a granulocyte that is suitable for infection treatment). In a suitable embodiment, the expression of one or more of ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2 is increased or the same when compared to a reference standard when the reference standard is of a granulocyte that has the ability to kill an infectious agent or cells infected by an infectious agent. In a suitable modality, the expression of ANXA1 and / or PPP3CB is decreased or the same when compared to a reference standard, when the reference standard is a granulocyte that has the ability to kill an infectious agent or cells infected by an infectious agent.

[0304] In some embodiments, the present invention may comprise the use of a reference standard for a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent and a reference standard for a granulocyte that has the ability to kill an infectious agent or cells infected by an infectious agent.

[0305] In a suitable embodiment, a granulopoietic cell suitable for use in the various aspects of the invention is capable of giving rise to granulocytes that have a positively charged cell surface.

[0306] The inventors believe that the cell surface charge of granulocytes may correlate with suitability for treating cancer and / or suitability for treating an infection, with granulocytes (e.g., neutrophils) that are more positively charged (or less negatively charged) being suitable for treating cancer and / or more effective in treating infection. Petition 870250102349, dated 07 / 11 / 2025, pages 89 / 209 81 / 177 cancer and / or being suitable for treating an infection and / or more effective in treating an infection. The cell surface charge level can be determined when compared to a reference standard, preferably where the reference standard is a granulocyte that does not have the ability to kill cancerous cells and / or does not have the ability to kill an infectious agent or cells infected by an infectious agent.

[0307] In a suitable embodiment, a granulocytic cell may be considered suitable for use according to the various aspects of the invention if it is capable of differentiating into a granulocyte with a positively charged (or less negatively charged) cell surface. A cell surface charge may be determined using any suitable technique known in the art. In one suitable embodiment, the cell surface charge is determined using electrophoresis. An electrophoretic mobility assay may be one described in Cell Electrophoresis edited by Johann Bauer (ISBN 0-8493-8918-6 published by CRC Press, Inc.), the teaching of which is incorporated herein in its entirety. In another embodiment, the cell surface charge may be determined using negatively and / or positively charged media.In a suitable embodiment, a granulocyte has a positive cell surface charge when it can be bound by a negatively charged medium and not a positively charged medium. In a suitable embodiment, a granulocyte has a negative cell surface charge when it can be bound by a positively charged medium and not a negatively charged medium. Such negatively and / or positively charged media can also be used to measure the concentration of a granulocyte cell in a sample. A positively charged medium can be a positively charged particle, nanoprobe or nanoparticle, or a cation exchange medium. Suitable nanoparticles can be prepared by conjugation of Iron(II,III) oxide nanoparticles (NPs). Petition 870250102349, dated 07 / 11 / 2025, pages 90 / 209 82 / 177 (FesCU) superparamagnetic with (3-Aminopropyl)triethoxysilane (APTES) to form a thin layer of silicon dioxide (SiC2) sheath on the surface of NPs by reaction with tetraethyl orthosilicate (TECS) and ammonium hydroxide (NH4CH). Fluorescein isothiocyanates (FITCs) can be incorporated into the SiC2 sheath, thus exposing the Si-bonded hydroxyl groups (SiC2-CH) and creating the negative surface charge. Branched poly(ethyleneimine) (PEI) molecules can be used not only to coat the SiC2-CH groups in a non-covalent manner, but also to expose the additional amine groups that carry the positive charges.Thus, in one suitable embodiment, a negatively charged nanoparticle is prepared by conjugating Fe3C4 nanoparticles with APTES to form a thin SiC2 shell layer on the nanoparticle surface after reaction with tetraethyl orthosilicate (TECS) and ammonium hydroxide (NH4CH) and incorporating a FITC into the SiC2 shell, thereby exposing the SiC2-CH groups (creating the negative surface charge). In another embodiment, a positively charged nanoparticle is prepared by contacting a negatively charged nanoparticle (as described in this document) with a PEI molecule (e.g., to expose additional amine groups that carry a positive charge). In one suitable embodiment, the negatively charged media (e.g., nanoparticles) may have a negative surface charge of at least -5 mV, -10 mV, -20 mV, -30 mV, or -40 mV.Preferably, negatively charged media (e.g., nanoparticles) may have a negative surface charge of at least -35 mV. In a suitable embodiment, positively charged media (e.g., nanoparticles) may have a positive surface charge of at least +5 mV, +10 mV, +20 mV, +30 mV, or +40 mV. Preferably, positively charged media (e.g., nanoparticles) may have a positive surface charge of at least +35 mV. The charge. Petition 870250102349, dated 07 / 11 / 2025, pages 91 / 209 83 / 177 of the surface of said positively or negatively charged media (e.g., nanoparticles) may refer to the surface zeta potential of the positively or negatively charged media (e.g., nanoparticles). The surface zeta potential can be measured with a dynamic light scattering particle size analyzer (e.g., the Zetasizer Nano-ZS90, Malvern, UK). Cells with the ability to kill cancer cells.

[0308] In a suitable embodiment, a granulopoietic cell suitable for use in the various aspects of the invention is capable of differentiating to produce granulocytes with the ability to kill cancerous cells.

[0309] The ability to kill cancer cells can be determined by mixing a cell (e.g., a granulocyte, such as a neutrophil) with a cancer cell and measuring (e.g., after incubation) the viability of said cancer cell. If the cancer cell is no longer viable (i.e., has been killed), the cell exhibits an ability to kill cancer cells. In a suitable embodiment, the ability to kill cancer cells is determined using a Cancer Killing Activity (CKA) assay described in this document.

[0310] In a suitable embodiment, a CKA assay comprises: a. placing cancer cells in contact with granulocytes to form a test sample (preferably in a 10:1 ratio of granulocytes to cancer cells); b. incubate the aforementioned test sample; and c. to measure the percentage of dead cancer cells in the aforementioned test sample.

[0311] In a suitable embodiment, a CKA assay comprises: a. Mixing granulocytes with cancer cells to provide a mixture (preferably in a 10:1 ratio of Petition 870250102349, dated 07 / 11 / 2025, pp. 92 / 209 84 / 177 granulocytes to cancer cells); b. incubate the aforementioned mixture; and c. measure the percentage of dead cancer cells in the aforementioned mixture

[0312] The term mixture, as used in this document, means mixing one or more components in any order, whether sequentially or simultaneously. In a suitable embodiment, mixing means bringing a first component into contact with a second component (for example, a granulocyte and a cancer cell).

[0313] The cancer cell for use in an assay may be one or more selected from a pancreatic cancer cell line, a liver cancer cell line, an esophageal cancer cell line, a stomach cancer cell line, a cervical cancer cell line, an ovarian cancer cell line, a lung cancer cell line, a bladder cancer cell line, a kidney cancer cell line, a brain cancer cell line, a prostate cancer cell line, a myeloma cancer cell line, a non-Hodgkin lymphoma (NHL) cell line, a laryngeal cancer cell line, a uterine cancer cell line, or a breast cancer cell line.Suitable cell lines are commercially available from the American Type Culture Collection UK (UK), Guernsey, Ireland, Jersey and Liechtenstein, LGC Standards, Queens Road, Teddington, Middlesex, TW11 0LY, UK. For example, a pancreatic cell line could be one or more of Capan-2, ATCC HTB80; Panc 10.05, ATCC CRL-2547; CFPAC-1, ATCC CRL-1918; HPAF-II, ATCC CRL-1997; SW 1990, ATCC CRL-2172; BxPC-3, ATCC CRL-1687; AsPC-1, ATCC CRL-1682; ATCC® TCP-1026™; SW1990, ATCC CRL-2172; SU.86.86, ATCC CRL-1837; BXPC-3, ATCC CRL-1687; Panc 10.05, ATCC CRL-2547; MIA-PaCa-2, ATCC CRL-1420; PANC-1, ATCC CRL-1469; or. Petition 870250102349, dated 07 / 11 / 2025, pp. 93 / 209 85 / 177 ATCC® TCP-2060™. Preferably, the cancer cell line is a pancreatic cancer cell line, such as PANC-1. In a suitable embodiment, the cancer cell line is a cervical cancer cell line, such as a HeLa cell.

[0314] The incubation phase can be carried out for between 1 hour and 100 hours. Suitablely, the incubation phase can be carried out for between 5 hours and 75 hours, for example, between 10 hours and 20 hours. The incubation phase can be carried out for between 6 hours and 6 days. Suitablely, the incubation phase can be carried out for between 6 hours and 2 days, for example, between 12 hours and 36 hours, as well as between 16 and 24 hours. In one suitable embodiment, the incubation phase is carried out for 24 hours. In another embodiment, the incubation phase is carried out for 48 hours. The incubation phase can be carried out at any temperature suitable for cell growth and viability, for example, at a temperature between 35 °C and 42 °C, suitablely at 37 or 39 °C. Preferably, the incubation stage is carried out at 37 or 39 °C for 24 hours. Preferably, the incubation stage is carried out for 16-24 hours at 30-40 °C (e.g., 37 °C).

[0315] The percentage of cancer cells killed can be measured by reference to the total number of initial cancer cells. The number of cancer cells killed can be measured using any suitable means, for example, by viability staining (e.g., trypan blue staining) and microscopy, or using other automated means, for example, by electronic cell detection equipment, such as the RT-CES™ system available from ACEA Biosciences, Inc. (11585 Sorrento Valley Rd., Suite 103, San Diego, CA 92121, USA). In some embodiments, the percentage of cancer cells killed can be determined within 24 hours (e.g., from incubation of a cancer cell line and a granulocyte). The percentage of cancer cells killed is preferably the maximum number of Petition 870250102349, dated 07 / 11 / 2025, pp. 94 / 209 86 / 177 cancer cells killed when performing a method of the invention. The % of cancer cells killed in said mixture may be the maximum % of cancer cells killed 48 hours after the mixture is formed.

[0316] A ratio of at least 1:1, 5:1 or 10:1 of granulocytes to cancer cells may be used. Preferably, a ratio of 5:1 of granulocytes to cancer cells is used. More preferably, a ratio of 10:1 of granulocytes to cancer cells is used.

[0317] The number of dead cancer cells can also be measured using the ACEA Biosciences xCELLigence RTCA DP Analyzer system®. The xCELLigence System is a real-time cell analyzer, allowing dynamic and label-free monitoring of cellular phenotypic changes by continuously measuring electrical impedance. Such measurements can be performed as detailed in Example 11. The system is commercially available from ACEA Biosciences 6779 Mesa Ridge Road #100, San Diego, CA 92121 USA.

[0318] In a suitable embodiment, a CKA assay is performed using an ACEA Biosciences xCELLigence RTCA DP Analyzer system® in accordance with the manufacturer's instructions and as follows: e. 6000 cancerous cells are placed at the bottom of a 16-well plate; f. the cells are cultured until confluence, as determined by the stabilization of the Cell Index (CI) values ​​(i.e., the normalization point); g. 60,000 granulocytes are added (that is, giving a ratio of 10 granulocytes to 1 cancer cell) and incubated at 37 °C; and h. The percentage of cancerous cells killed is the maximum percentage of cancerous cells killed 48 hours after the addition of granulocytes, as determined using the following formula: Petition 870250102349, dated 07 / 11 / 2025, pages 95 / 209 87 / 177 ((Cell index without effector — Cell index with effector) / Cell index without effector) X 100.

[0319] The maximum percentage of cancer cells killed may be referred to in this document as % CKA.

[0320] Preferably, the cancer cells are PANC-1 cells, which are commercially available from the American Type Culture Collection UK (UK), Guernsey, Ireland, Jersey and Liechtenstein, LGC Standards, Queens Road, Teddington, Middlesex, TW11 0LY, UK and have the catalog number ATCC CRL-1469.

[0321] In a particularly preferred embodiment, the granulocyte with the ability to kill cancer cells kills less than 15% of non-cancerous cells in the non-cancer killing activity (NCKA) assay described in this document. Preferably, a granulocyte kills less than 10% (e.g., less than 5% or less than 1%) of non-cancerous cells in the non-cancer killing activity (NCKA) assay described in this document.

[0322] The non-cancer killing activity (NCKA) assay or NCKA assay can be performed using an ACEA Biosciences xCELLigence RTCA DP Analyzer system® according to the manufacturer's instructions and as follows: a. 6000 non-cancerous cells are placed at the bottom of a 16-well plate; b. the cells are cultured until confluence, as determined by the stabilization of the Cell Index (CI) values ​​(i.e., the normalization point); c. 60,000 granulocytes are added (that is, giving a ratio of 10 granulocytes to 1 non-cancerous cell) and incubated at 37 °C; and d. The percentage of dead non-cancerous cells is the maximum percentage of dead non-cancerous cells 48 hours after the addition of granulocytes, as determined using the following formula: Petition 870250102349, dated 07 / 11 / 2025, pages 96 / 209 88 / 177 ((Cell index without effector — Cell index with effector) / Cell index without effector) X 100.

[0323] Preferably, the non-cancerous cells are MCF-12F non-cancerous cells, which are commercially available from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110 USA and have the catalog number ATCC® CRL-10783™. In another embodiment, the non-cancerous cells are liver cells (e.g., primary non-transplantable liver tissue cells).

[0324] In a suitable embodiment, a granulocyte may be considered a granulocyte with the ability to kill cancer cells if it kills at least 5% of the cancer cells in a method described in this document. A granulocyte may be considered a granulocyte with the ability to kill cancer cells if it kills at least 10%, 20%, 30%, 40%, 50%, or 51.5% of the cancer cells present. In a suitable embodiment, a granulocyte may be considered a granulocyte with the ability to kill cancer cells if it kills at least 60% of the cancer cells present. In a suitable embodiment, a granulocyte may be considered a granulocyte with the ability to kill cancer cells if it kills at least 70% of the cancer cells present. For example, a granulocyte can be considered a granulocyte with the ability to kill cancerous cells if it kills at least 80% or 90% of the cancerous cells present.In a particularly preferred embodiment, a granulocyte may be considered a granulocyte with the ability to kill cancer cells if it kills at least 51.5% of the cancer cells present. The reference in this descriptive report to a granulopoietic cell with the ability to kill cancer cells may be taken as referring to a granulopoietic cell that is capable of differentiating into a granulocyte that has the ability to kill cancer cells accordingly. Petition 870250102349, dated 07 / 11 / 2025, pages 97 / 209 89 / 177 the definitions presented above.

[0325] In contrast, a granulocyte that lacks the ability to kill cancer cells or is incapable of killing cancer cells may be a granulocyte that is unable to kill at least 5% of the cancer cells in a method described in this document. A granulocyte that lacks the ability to kill cancer cells or is incapable of killing cancer cells may be a granulocyte that is unable to kill at least 10%, 20%, 30%, 40%, 50%, or 51.5% of the cancer cells present. In a suitable embodiment, a granulocyte that lacks the ability to kill cancer cells or is incapable of killing cancer cells may be a granulocyte that is unable to kill at least 60% of the cancer cells present. In a suitable modality, a granulocyte that does not have the ability to kill cancer cells or is unable to kill cancer cells may be a granulocyte that is unable to kill at least 70% of the cancer cells present.For example, a granulocyte that lacks the ability to kill cancer cells or is incapable of killing cancer cells may be a granulocyte that is unable to kill at least 80% or 90% of the cancer cells present. In a suitable embodiment, a granulocyte that lacks the ability to kill cancer cells or is incapable of killing cancer cells may be a granulocyte that is unable to kill at least 51.5% of the cancer cells present. Similarly, reference to a granulopoietic cell that lacks the ability to kill cancer cells or is incapable of killing cancer cells may be taken as referring to a granulopoietic cell that does not differentiate into a granulocyte that has the ability to kill cancer cells and / or that differentiates into a granulocyte that lacks the ability to kill cancer cells or is incapable of killing cancer cells. Petition 870250102349, dated 07 / 11 / 2025, pages 98 / 209 90 / 177 Cells with the ability to kill an infectious agent or a cell infected by an infectious agent.

[0326] In a suitable embodiment, a granulopoietic cell suitable for use in the various aspects of the invention is capable of differentiating to produce granulocytes with the ability to kill an infectious agent or a cell infected by an infectious agent.

[0327] The ability to kill an infectious agent or a cell infected by an infectious agent can be determined by mixing a cell (e.g., a granulocyte, such as a neutrophil) with an infectious agent or a cell infected by an infectious agent and measuring (e.g., after incubation) the viability of said infectious agent or cell infected by the infectious agent. If the infectious agent or cell infected by the infectious agent is no longer viable (i.e., has been killed), the cell exhibits an ability to kill an infectious agent or a cell infected by an infectious agent. In a suitable embodiment, the ability to kill an infectious agent or a cell infected by an infectious agent is determined using an Infection Killing Activity (IKA) assay described in this document.

[0328] In a suitable embodiment, an IKA test comprises: a. placing an infectious agent or a cell infected by an infectious agent in contact with granulocytes to form a test sample; b. incubate the aforementioned test sample; and c. measure the percentage of infectious agent or cells infected by the dead infectious agent in the test sample.

[0329] In a suitable embodiment, an IKA test comprises: a. to mix granulocytes with an infectious agent or a cell infected by an infectious agent to provide a Petition 870250102349, dated 07 / 11 / 2025, pages 99 / 209 91 / 177 mixture; b. incubate the aforementioned mixture; and c. measure the percentage of infectious agent or cells infected by the dead infectious agent in the said mixture.

[0330] The incubation or contact phase between a granulocyte and a cell infected with an infectious agent / infectious agent can take between 1 hour and 100 hours. Preferably, the incubation or contact phase between a granulocyte and a cell infected with an infectious agent / infectious agent can take between 5 hours and 75 hours, for example, between 10 hours and 20 hours. The incubation or contact phase between a granulocyte and an infectious agent / cell infected with an infectious agent can take between 6 hours and 6 days. Appropriately, the incubation or contact phase between a granulocyte and an infectious agent / cell infected with an infectious agent can take between 6 hours and 2 days, for example, between 12 hours and 36 hours, such as between 16 and 24 hours. In a suitable method, the incubation stage is carried out for 24 hours.In another embodiment, the incubation or contact step between a granulocyte and an infectious agent / cell infected with an infectious agent is carried out for 48 hours. The incubation or contact step between a granulocyte and an infectious agent / cell infected with an infectious agent can be carried out at any temperature suitable for cell growth and viability, for example, at a temperature between 35 °C and 42 °C, suitably at 37 or 39 °C. Preferably, the incubation or contact step between a granulocyte and an infectious agent / cell infected with an infectious agent is carried out at 37 or 39 °C for 24 hours. Preferably, the incubation or contact step between a granulocyte and an infectious agent / cell infected with an infectious agent is carried out for 16-24 hours at 30-40 °C (e.g., 37 °C).

[0331] The conditions mentioned above may be Petition 870250102349, dated 07 / 11 / 2025, pages 100 / 209 92 / 177 particularly suitable for incubating / contacting a granulocyte with a cell infected by an infectious agent.

[0332] The incubation or contact phase between a granulocyte and a cell infected with an infectious agent / infectious agent can be carried out for between 30 minutes and 24 hours (e.g., before assessing the % death rate). Preferably, the incubation or contact phase between a granulocyte and a cell infected with an infectious agent / infectious agent can be carried out for between 1-3 hours, e.g., for 2 hours. In other words, the assessment of the % death rate can be determined after contact / incubation for 2 hours. The incubation or contact phase between a granulocyte and an infectious agent / cell infected by an infectious agent can be carried out at any temperature suitable for cell growth and viability, e.g., at a temperature between 35 °C and 42 °C, ideally at 37 °C.

[0333] The above-mentioned conditions may be particularly suitable when incubating / contacting a granulocyte with an infectious agent, such as a bacterium.

[0334] In a suitable embodiment, a contact or incubation step is performed in solution. In other words, the infectious agent or cells infected with an infectious agent may be growing in solution (i.e., not adhered / growing on a surface, such as the surface of a plate).

[0335] Preferably, where the infectious agent is a bacterium, a contact or incubation step is performed in solution. In contrast, when the method employs cells infected with an infectious agent, it is preferable that said cells be growing on or attached to a surface, such as the surface of a plate.

[0336] In a suitable embodiment, the said contact or incubation step is carried out under agitation, for example, at 100-250 rpm, such as 120 rpm.

[0337] In a suitable modality, when the method employs Petition 870250102349, dated 07 / 11 / 2025, pages 101 / 209 93 / 177 cells infected with an infectious agent, the methods of the invention may comprise the use of at least a 1:1, 5:1 or 10:1 ratio of granulocytes to cells. Preferably, the methods comprise the use of a 5:1 ratio of granulocytes to cells. More preferably, the methods comprise the use of a 10:1 ratio of granulocytes to cells.

[0338] The percentage of dead cells can be measured by reference to the total number of initial cells. The number of dead cells can be measured using any suitable means, for example, by viability staining (e.g., trypan blue staining) and microscopy, or using other automated means, for example, by electronic cell detection equipment, such as the RT-CES™ system available from ACEA Biosciences, Inc. (11585 Sorrento Valley Rd., Suite 103, San Diego, CA 92121, USA). In some embodiments, the percentage of dead cells can be determined within 24 hours (e.g., from incubation of a cell and a granulocyte). The percentage of dead cells is preferably the maximum number of dead cells when performing a method of the invention.

[0339] The number of dead cells can also be measured using the ACEA Biosciences xCELLigence RTCA DP Analyzer system®. The xCELLigence System is a real-time cell analyzer, allowing dynamic and label-free monitoring of cellular phenotypic changes by continuously measuring electrical impedance. Such measurements can be performed as detailed in the Examples. The aforementioned System is commercially available from ACEA Biosciences 6779 Mesa Ridge Road #100, San Diego, CA 92121 USA.

[0340] In a suitable embodiment, where an infectious agent is a bacterium, a ratio of at least 1:10, 1:5, 1:3 or 1:2 granulocytes to colony-forming units may be used. Preferably, a ratio of 1:2 granulocytes to colony-forming units is used. More Petition 870250102349, dated 07 / 11 / 2025, pages 102 / 209 94 / 177 preferably, a 1:1 ratio of granulocytes to colony-forming units is used.

[0341] In a suitable embodiment, the ability to kill an infectious agent or a cell infected by an infectious agent is determined using an MRSA assay described in this document.

[0342] In a suitable embodiment, the MRSA test comprises: a. mix granulocytes with MRSA cells to form a mixture; b. incubate the aforementioned mixture; and c. measure the percentage of dead MRSA cells in the mixture.

[0343] The MRSA test can be performed as follows: a. Mix 100 µl of a solution containing 1 x 10⁷ CFU / ml of MRSA USA300 strain in RPMI 1640 with 100 µl of a solution containing 1 x 10⁷ granulocytes / ml; b. Incubate the mixture at 37 °C while stirring at 120 rpm; c. collect a sample within 2 hours (diluting in sterile RPMI as needed) and plate on Tryptic Soy Agar; d. Incubate the plated sample at 37 °C for 24 hours; e. counting the bacterial colonies; and f. quantify the total CFU content; and g. Calculate the % of dead MRSA cells based on the CFU content in steps a and b using the formula ((effector CFU content — effector CFU content) / effector CFU content) X 100.

[0344] In a particularly preferred embodiment, the term "capable of killing an infectious agent or a cell infected by an infectious agent," as used in this document, further means that a granulocyte kills less than 15% of healthy (uninfected) cells in the healthy (uninfected) cell assay described in this document. Preferably, a granulocyte kills less than 10% (per Petition 870250102349, dated 07 / 11 / 2025, pages 103 / 209 95 / 177 example, less than 5% or less than 1% of healthy (uninfected) cells in the healthy (uninfected) cell assay described in this document.

[0345] The healthy (uninfected) cell assay can be performed using an ACEA Biosciences xCELLigence RTCA DP Analyzer system® in accordance with the manufacturer's instructions and as follows: a. 6000 healthy (uninfected) cells are placed at the bottom of a 16-well plate; b. the cells are cultured until confluence, as determined by the stabilization of the Cell Index (CI) values ​​(i.e., the normalization point); c. 60,000 granulocytes are added (that is, giving a ratio of 10 granulocytes to 1 pathogen-uninfected cell) and incubated at 37 °C; and d. The percentage of healthy (uninfected) dead cells is the maximum percentage of pathogen-uninfected cells that have died 48 hours after the addition of granulocytes, as determined using the following formula: ((Non-Effector Cell Index — Effector Cell Index) / Non-Effector Cell Index) X 100.

[0346] Preferably, healthy (uninfected) cells are MCF-12F, which are commercially available from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110 USA and have the catalog number ATCC® CRL10783™. Alternatively, healthy (uninfected) cells are liver cells (e.g., non-transferable primary liver tissue cells).

[0347] In a suitable embodiment, a granulocyte may be considered a granulocyte with the ability to kill an infectious agent or cells infected by an infectious agent if it kills at least 5% of the infectious agent or cells infected by an infectious agent in a method described in this document. A granulocyte may be considered a granulocyte Petition 870250102349, dated 07 / 11 / 2025, pp. 104 / 209 96 / 177 with the ability to kill an infectious agent or cells infected by an infectious agent if it kills at least 10%, 20%, 30%, 40%, or 50% of the infectious agent or cells infected by an infectious agent present. In a suitable embodiment, a granulocyte may be considered a granulocyte with the ability to kill an infectious agent or cells infected by an infectious agent if it kills at least 60% of the infectious agent or cells infected by an infectious agent present. In a suitable embodiment, a granulocyte may be considered a granulocyte with the ability to kill an infectious agent or cells infected by an infectious agent if it kills at least 70% of the infectious agent or cells infected by an infectious agent present.Preferably, a granulocyte can be considered a granulocyte with the ability to kill an infectious agent or cells infected by an infectious agent if it kills at least 80% or 90% of the infectious agent or cells infected by an infectious agent present. In a particularly preferred embodiment, a granulocyte can be considered a granulocyte with the ability to kill an infectious agent or cells infected by an infectious agent if it kills more than 41.23% of the infectious agent or cells infected by an infectious agent present.

[0348] In contrast, a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent, or is unable to kill an infectious agent or cells infected by an infectious agent, may be a granulocyte that is unable to kill at least 5% of an infectious agent or cells infected by an infectious agent in a method described in this document. A granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent, or is unable to kill an infectious agent or cells infected by an infectious agent Petition 870250102349, dated 07 / 11 / 2025, pages 105 / 209 97 / 177 infectious can be a granulocyte that is unable to kill at least 10%, 20%, 30%, 40%, or 50% of the infectious agent or cells infected by an infectious agent present. In a suitable embodiment, a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent, or is unable to kill an infectious agent or cells infected by an infectious agent, is a granulocyte that is unable to kill at least 60% of the infectious agent or cells infected by an infectious agent present. In a suitable embodiment, a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent, or is unable to kill an infectious agent or cells infected by an infectious agent, is a granulocyte that is unable to kill at least 70% of the infectious agent or cells infected by an infectious agent present.Preferably, a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent, or is incapable of killing an infectious agent or cells infected by an infectious agent, is a granulocyte that is not capable of killing at least 80% or 90% of the infectious agent or cells infected by an infectious agent present. In a particularly preferred embodiment, a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent, or is incapable of killing an infectious agent or cells infected by an infectious agent, is a granulocyte that is not capable of killing more than 41.23% of the infectious agent or cells infected by an infectious agent present.Similarly, a reference to a granulopoietic cell that does not have the ability to kill an infectious agent or cells infected by an infectious agent, or is incapable of killing an infectious agent or cells infected by an infectious agent, is a granulopoietic cell that does not differentiate into a granulocyte that does. Petition 870250102349, dated 07 / 11 / 2025, pp. 106 / 209 98 / 177 the ability to kill an infectious agent or cells infected by an infectious agent and / or that differentiates into a granulocyte that does not have the ability to kill an infectious agent or cells infected by an infectious agent or is incapable of killing an infectious agent or cells infected by an infectious agent.

[0349] An infectious agent may refer to a bacterium, a fungus, a virus, a macroparasite (e.g., a helminth), or a combination thereof. Preferably, an infectious agent is a bacterium or a virus. For example, in a suitable embodiment, an infectious agent is a bacterium. In an alternative embodiment, an infectious agent is a virus. Properly, an infectious agent is a pathogen. Cells capable of expressing chemokines

[0350] Granulopoietic cells that can be employed in the aspects of the invention described in this document include those capable of giving rise to granulocytes capable of expressing desirable chemokines. By way of example only, the inventors have shown that granulopoietic cells suitable for use in the various aspects of the present invention are capable of differentiating and giving rise to granulocytes that secrete CXCL10. Cells capable of expressing costimulatory receptor ligands

[0351] The granulopoietic cells that can be employed in the aspects of the invention described in this document include those capable of giving rise to granulocytes capable of expressing advantageous ligands for costimulatory receptors. By way of example only, the inventors have shown that granulopoietic cells suitable for use in the various aspects of the present invention are capable of differentiating and giving rise to granulocytes that express ligands for costimulatory receptors, such as 4-1BBL and OX40L. Methods of producing granulopoietic cells (or populations) Petition 870250102349, dated 07 / 11 / 2025, pages 107 / 209 99 / 177 of such cells), which can be used therapeutically and therapeutically useful granulopoietic cells.

[0352] In one aspect, the invention provides a method for preparing granulopoietic 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; to produce a population of granulopoietic cells.

[0353] This method may optionally comprise an additional step of purifying the produced granulopoietic cell population and / or formulating this cell population for medical use.

[0354] In one aspect, the invention provides a population of granulopoietic cells prepared for therapeutic use by a method of the first aspect of the invention.

[0355] The granulopoietic cells 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.

[0356] The granulopoietic cells produced by the methods of the invention can optionally be cryopreserved once produced. It is known that granulocytes, such as neutrophils, do not 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 process of Petition 870250102349, dated 07 / 11 / 2025, pages 108 / 209 100 / 177 freezing and thawing. Consequently, the granulopoietic cell populations of the invention offer significant advantages, compared with mature granulocytic cells, in applications where it is desired to cryopreserve cells before their use for therapy.

[0357] The granulopoietic cells produced by the methods of the invention may optionally be formulated for medical use once produced. Suitable methods for formulating cell populations intended for therapeutic use will be well known to those skilled in the art and may be used in formulating the granulopoietic cell populations of the invention, optionally to give rise to pharmaceutical compositions of the invention.

[0358] The characteristics of the granulopoietic cells 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.

[0359] 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.

[0360] The following paragraphs present details of useful embodiments of methods for producing granulopoietic cells or populations of such cells. These include useful embodiments of 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.

[0361] 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. In a suitable embodiment, IMDM is a form of the medium comprising elevated glucose, Petition 870250102349, dated 07 / 11 / 2025, pp. 109 / 209 101 / 177 glutamine, HEPES, sodium pyruvate and may optionally contain phenol red.

[0362] The methods make use of granulocyte colony-stimulating factor cytokine (G-CSF) as a supplement.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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 presented in this document.

[0367] The methods make use of granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine as a supplement.

[0368] 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.

[0369] Appropriately, GM-CSF is supplied at a concentration of 0.05 μg / mL or less. For example, GM-CSF can Petition 870250102349, dated 07 / 11 / 2025, pp. 110 / 209 102 / 177 to be supplied at a concentration of 0.04 μg / mL or less, 0.03 μg / mL or less, or 0.02 μg / mL or less.

[0370] Suitablely, GM-CSF is supplied at a concentration of approximately 0.001 μg / mL to 0.05 μg / mL, 0.125 μg / mL to 0.04 μg / mL, 0.00167 μg / mL to 0.03 μg / mL, 0.0025 μg / mL to 0.02 μg / mL or 0.005 μg / mL to 0.015 μg / mL. In a suitable embodiment, GM-CSF is supplied at a concentration of approximately 0.01 μg / mL. In fact, in a suitable embodiment, GM-CSF is supplied at a concentration of 0.01 μg / mL.

[0371] Examples of suitable forms of GM-CSF that can be used in this way include products produced by Peprotech and BioTechne and the GMP product produced by BioTechne, details of which are presented in this document.

[0372] The methods make use of the cytokine interleukin-3 (IL-3) as a supplement.

[0373] Appropriately, IL-3 is supplied at a concentration of 0.013 μg / mL or more. For example, IL-3 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.

[0374] Appropriately, IL-3 is supplied at a concentration of 0.65 μg / mL or less. For example, IL-3 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.

[0375] Appropriately, IL-3 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, IL-3 is delivered at a concentration of approximately 0.13 μg / mL. In fact, in a suitable embodiment, IL-3 is delivered at a concentration of 0.13 μg / mL.

[0376] 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 by PeproTech or BioTechne, whose Petition 870250102349, dated 07 / 11 / 2025, pages 111 / 209 Details 103 / 177 are presented in this document.

[0377] 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 for 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.

[0378] The methods make use of the cytokine tumor necrosis factor (TNF) as a supplement. The terms TNF and TNF-alpha are used interchangeably in this document.

[0379] 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.

[0380] 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.

[0381] 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.

[0382] 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 Petition 870250102349, dated 07 / 11 / 2025, pp. 112 / 209 104 / 177 presented in this document.

[0383] In a suitable embodiment, TNF is delivered to cells for a period between 12 and 36 hours, ideally a 24-hour period under cell culture conditions. For example, TNF may be delivered to cells for the final 24 hours of the period during which they are in culture. TNF may be delivered to cells on the fourth to fifth day of cell culture conditions that promote progenitor cell differentiation. TNF may be delivered to cells on the fifth day of cell culture conditions that promote progenitor cell differentiation. TNF may be delivered to cells on the fourth day of cell culture conditions that promote progenitor cell differentiation.

[0384] The methods may optionally make use of cytokine stem cell factor (SCF) as a supplement.

[0385] 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.

[0386] 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.

[0387] 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.

[0388] 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, whose Petition 870250102349, dated 07 / 11 / 2025, pp. 113 / 209 Details 105 / 177 are presented in this document.

[0389] The methods may optionally make use of the cytokine thrombopoietin (TPO) as a supplement.

[0390] 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.

[0391] 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.

[0392] 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.

[0393] 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, details of which are presented in this document.

[0394] 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.

[0395] The methods can make appropriate use of insulin at a concentration between about 0.1 g / L and about 5 g / L, by Petition 870250102349, dated 07 / 11 / 2025, pp. 114 / 209 106 / 177 For example, at a concentration of approximately 1.0 g / L, as a supplement. These cell culture methods and 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 cell culture methods and media can make 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.

[0396] The methods may optionally make use of HSA as a supplement.

[0397] Appropriately, HSA can be supplied in a concentration between 0.1% and 5%. For example, HSA supplied as a supplement can be supplied in a concentration of approximately 1%.

[0398] 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.

[0399] 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. Petition 870250102349, dated 07 / 11 / 2025, pages 115 / 209 107 / 177

[0400] 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. Methods according to the first aspect of the invention may comprise culturing the population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells for a period of 1 to 7 days. For example, such methods may comprise culturing the cells under the 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 5 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 setting, progenitor cells are cultured for 6 days under conditions to produce a population of granulopoietic cells.

[0401] In a suitable embodiment of a method of the invention, Petition 870250102349, dated 07 / 11 / 2025, pages 116 / 209 108 / 177 progenitor cells can be cultured at an initial seeding density of approximately 1x105 to 10x106 cells per cm2.

[0402] 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, at least 13 times, at least 14 times or at least 15 times. The methods presented in the Examples achieve a granulopoietic cell population that is approximately 3.5 times greater than the initial population of progenitor cells.

[0403] In a suitable embodiment, a method is practiced with respect 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.

[0404] In a suitable embodiment, a method 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, Petition 870250102349, dated 07 / 11 / 2025, pages 117 / 209 109 / 177 • IL-3, • IL-6, and • TPO.

[0405] 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 detail a protocol that the inventors used to achieve an approximately 75-fold increase in the number of progenitor cells compared to the initial stem cell population.

[0406] 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.

[0407] Consequently, a method of preparing cells for therapeutic use according to such embodiments of the invention may comprise: a) To cultivate a population of stem cells under cell culture conditions to produce progenitor cells, including the presence of: • SCF, • Flt-3 ligand, • IL-3, • IL-6, and • TPO; Petition 870250102349, dated 07 / 11 / 2025, pages 118 / 209 110 / 177 to produce a population of progenitor cells; and b) cultivate the progenitor cell population under cell culture conditions that promote progenitor cell differentiation, including the presence of: • G-CSF, • GM-CSF, • IL-3 and • TNF; to produce a population of granulopoietic cells; and optionally c) collect the granulopoietic cells.

[0408] 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 stem cell population to the granulopoietic cell population 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, 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 detail 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.

[0409] A method according to such embodiments of the invention may involve a total time period in culture 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.

[0410] The SCF can optionally be supplied as a Petition 870250102349, dated 07 / 11 / 2025, pages 119 / 209 111 / 177 supplement in embodiments of the methods of the invention comprising a step of producing a population of progenitor cells.

[0411] 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.

[0412] 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.

[0413] Appropriately, 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.

[0414] The forms of SCF discussed above are also suitable for use in such modalities.

[0415] 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.

[0416] 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.

[0417] 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.

[0418] Appropriately, 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, Petition 870250102349, dated 07 / 11 / 2025, pages 120 / 209 112 / 177 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.

[0419] 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 presented in this document.

[0420] 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.

[0421] 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.

[0422] 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.

[0423] 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.

[0424] The IL-3 forms discussed above are suitable for use in such modalities.

[0425] 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. Petition 870250102349, dated 07 / 11 / 2025, pages 121 / 209 113 / 177

[0426] 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.

[0427] 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.

[0428] 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.

[0429] 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 presented in this document.

[0430] TPO may optionally be provided as a supplement in embodiments of the invention's methods comprising a step of producing a population of progenitor cells.

[0431] 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 μg / mL or more, or 0.01 μg / mL or more.

[0432] Appropriately, TPO is supplied at a concentration of 0.1 μg / mL or less. For example, TPO may be supplied at a concentration of 0.08 μg / mL or less, 0.06 μg / mL or less, or 0.04 μg / mL or less.

[0433] Appropriately, TPO is provided at a concentration of approximately 0.002 μg / mL to 0.1 μg / mL, 0.0025 μg / mL to 0.08 μg / mL, 0.003 μg / mL to 0.06 μg / mL, 0.005 μg / mL to 0.04 μg / mL, or Petition 870250102349, dated 07 / 11 / 2025, pages 122 / 209 114 / 177 0.01 μg / mL to 0.03 μg / mL. In a suitable embodiment, TPO is delivered at a concentration of approximately 0.02 μg / mL. In fact, in a suitable embodiment, TPO is delivered at a concentration of 0.02 μg / mL.

[0434] The TPO forms discussed above are also suitable for use in these modalities.

[0435] Suitablely, the cell culture conditions that promote the production of progenitor cells used in a method 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.

[0436] Thus, in a suitable embodiment, the cell culture conditions that promote the production of progenitor cells used in a method of the invention may comprise: SCF at a concentration of approximately 0.2 qg / mL; and Flt-3 Ligand at a concentration of approximately 0.2 qg / mL; and IL-3 at a concentration of approximately 0.015 qg / mL; and IL-6 at a concentration of approximately 0.015 qg / mL; and TPO at a concentration of approximately 0.02 qg / mL; and 1x ITS; and HSA at approximately 1%. The cell culture medium may comprise IMDM, optionally with Glutamax supplementation.

[0437] 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, among others, 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.

[0438] In a suitable embodiment, the cell culture conditions used in stem cell culture to produce progenitor cells further comprise the presence of at least one supplement selected from the group consisting of: ITS and HSA. Petition 870250102349, dated 07 / 11 / 2025, pages 123 / 209 115 / 177 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.

[0439] ITS may optionally be provided as a supplement in embodiments of the invention's methods comprising a step of producing a population of progenitor cells.

[0440] Such embodiments of the methods 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 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 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.

[0441] HSA may optionally be provided as a supplement in embodiments of the invention's methods comprising a step of producing a population of progenitor cells.

[0442] 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%.

[0443] 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.

[0444] 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. Petition 870250102349, dated 07 / 11 / 2025, pages 124 / 209 116 / 177 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. Stem cells can 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.

[0445] 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.

[0446] 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 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. [04 47] A method of the invention for preparing cells for therapeutic use may comprise: Petition 870250102349, dated 07 / 11 / 2025, pages 125 / 209 117 / 177 (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. [04 48] 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. [04 49] For example, 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, SCF, TPO, GCSF, ITS and HSA for 1-6 days, or preferably 5 days, to obtain a population of granulopoietic cells. Petition 870250102349, dated 07 / 11 / 2025, pages 126 / 209 118 / 177

[0450] 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.

[0451] 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 (as HSCs) are seeded at a density of 5x10⁵ cells / mL and 5x10⁵ cells / cm².

[0452] Cells can be seeded into any suitable culture receptacle. For example, cells can be seeded into a G-Rex 6M or G-Rex 10M culture receptacle. Cells can be transferred to a new culture receptacle at any suitable time. Cells can be transferred sequentially to culture receptacles. Petition 870250102349, dated 07 / 11 / 2025, pages 127 / 209 119 / 177 cell surface area increasing. 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 culture to produce progenitor cells. For example, stem cells (such as HSCs) can be transferred from a smaller GRex to a G-Rex 100M 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 receptacle such as a G-Rex 500M, on day 4 of expansion. In a suitable modality, progenitor cells can be transferred to a new culture receptacle 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.

[0453] According to such modalities, a suitable method of preparing cells for therapeutic use may comprise: (a) seeding stem cells (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 days to obtain a granulopoietic cell population, wherein the cell culture medium comprising IMDM, SCF, TPO, GCSF, ITS, and HSA is replenished on day 3 of Petition 870250102349, dated 07 / 11 / 2025, pages 128 / 209 120 / 177 differentiation.

[0454] A suitable method for preparing cells for therapeutic use may include: (a) seeding stem cells (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.

[0455] 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.

[0456] A suitable method for preparing granulopoietic cells for therapeutic use comprises 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.

[0457] A method may also comprise a priming step of granulopoietic cells for therapeutic use, by Petition 870250102349, dated 07 / 11 / 2025, pages 129 / 209 121 / 177 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, IFNα, IFN-β, IL-15 and IL-18.

[0458] A method 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. The primed granulopoietic cell population may be as defined elsewhere in this disclosure (e.g., with reference to the biological activity of the primed cells or their expression of particular markers).

[0459] 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.

[0460] 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.

[0461] 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.

[0462] IFN-β can be used under cell culture conditions for a priming step at concentrations 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 Petition 870250102349, dated 07 / 11 / 2025, pages 130 / 209 122 / 177 of 10 ng / mL.

[0463] 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.

[0464] 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.

[0465] IL-3 can be used under cell culture conditions for a priming step at a concentration of 1-1000 ng / mL, 2500 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.

[0466] 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 0.01-1.0 ng / mL, IFN-α at a concentration of approximately 10 ng / mL, IFN-β at a concentration of approximately 10 ng / mL, IL-15 at a concentration of approximately 10 ng / mL, IL-18 at a concentration of approximately 10 ng / mL and IL-3 at a concentration of approximately 130 ng / mL.

[0467] In a suitable embodiment, primed cells can be cultured in the presence of GM-CSF, G-CSF, SCF, TPO and IL-15. Just as an example, 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 Petition 870250102349, dated 07 / 11 / 2025, pages 131 / 209 123 / 177 approximately 130 ng / mL and IL-15 at a concentration of approximately 10 ng / mL.

[0468] 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.

[0469] 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.

[0470] 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.

[0471] 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.

[0472] 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.

[0473] In a suitable arrangement, primed cells can be cultured in the presence of GM-CSF and TNF. Only Petition 870250102349, dated 07 / 11 / 2025, pages 132 / 209 124 / 177 as an example, cells can be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL and TNF at a concentration of approximately 1 ng / mL.

[0474] 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.

[0475] 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 presented above, for a period of one, two, or three days. Specifically, priming may involve culturing with the aforementioned priming cytokine combinations for two days.

[0476] A priming step can be appropriately incorporated into any suitable stage of a method. 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. Petition 870250102349, dated 07 / 11 / 2025, pages 133 / 209 125 / 177 progenitor cells or on the fifth day of progenitor cell culture under conditions that promote their differentiation into granulopoietic cells.

[0477] 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, priming may occur before or after cryopreservation of a population of granulopoietic cells according to the invention.

[0478] 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.

[0479] 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.

[0480] Except where the context may require otherwise, the following definitions are applicable to granulopoietic cells in any of these aspects of the invention, or in any other situation where granulopoietic cells, or populations of such cells, are referred to.

[0481] To be considered granulopoietic in terms of Petition 870250102349, dated 07 / 11 / 2025, pp. 134 / 209 126 / 177 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.

[0482] 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.

[0483] The following definitions, based on appropriate marker expression profiles, can be used alone or in combination to identify suitable populations of granulopoietic cells.

[0484] 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 number of specified markers should be taken as requiring that the cells in question have the recited expression (positive or negative) of each of the markers referred to. 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 population of cells Petition 870250102349, dated 07 / 11 / 2025, pages 135 / 209 127 / 177 comprises cells that are CD15+, as well as cells that are CD66b+.

[0485] This disclosure includes definitions of populations, or subpopulations, of cells with reference to a quoted expression (positive or negative) of a number of specified markers.

[0486] 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 870250102349, dated 07 / 11 / 2025, pages 136 / 209 128 / 177

[0487] 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.

[0488] 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).

[0489] 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. Adequately, a granulopoietic cell population comprises approximately 97% Lin- cells.

[0490] 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% CD34+ cells. By way of example, such a population of Petition 870250102349, dated 07 / 11 / 2025, pages 137 / 209 129 / 177 granulopoietic cells may comprise less than 30% CD34+ cells. In such a configuration, the proportion of CD34+ cells may be between approximately 5-25%. Appropriately, a granulopoietic cell population comprises approximately 14% CD34+ cells.

[0491] 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.

[0492] 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+CD38-CD45RA-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.

[0493] 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 Petition 870250102349, dated 07 / 11 / 2025, pages 138 / 209 130 / 177 5%, less than 4%, less than 3%, or less than 2% of cells with an LT-HSC phenotype. For 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 0.01-0.05% of cells with an LT-HSC phenotype. Appropriately, a granulopoietic cell population comprises approximately 0.02% of cells with an LT-HSC phenotype.

[0494] Alternatively, or additionally, a suitable granulopoietic cell population comprises cells with a lymphoid-primed multipotent progenitor (LMPP) phenotype (defined for the present purposes as LinCD34+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. By way of 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 215% of cells with an LMPP phenotype. Appropriately, a granulopoietic cell population comprises approximately 5% of cells with an LMPP phenotype.

[0495] 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. Appropriately, a population of Petition 870250102349, dated 07 / 11 / 2025, pages 139 / 209 131 / 177 granulopoietic cells comprise approximately 2% of cells with an MPP phenotype.

[0496] 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., approximately 0.04% 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 20% cells with an LMPP phenotype as defined above (e.g., approximately 5% 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).

[0497] 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). Petition 870250102349, dated 07 / 11 / 2025, pages 140 / 209 132 / 177

[0498] Alternatively, or additionally, an adequate population of granulopoietic cells may comprise a ratio of CD15- to CD15+ cells that is approximately 1:1.

[0499] 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.

[0500] 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 CD15+ cells.

[0501] 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.

[0502] 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.

[0503] 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.

[0504] An adequate population of granulopoietic cells may comprise approximately 60-95%, or 65-90% CD49d+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 75% CD49d+ cells.

[0505] An adequate population of granulopoietic cells may comprise less than 5%, less than 4%, less than 3%, or less Petition 870250102349, dated 07 / 11 / 2025, pages 141 / 209 133 / 177 of 2% CD10+ cells. An adequate population of granulopoietic cells may comprise approximately 0.03-2% CD10+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 0.5% CD10+ cells.

[0506] 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.

[0507] 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 2-15%, for example, approximately 8% CD62L+ cells.

[0508] 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.

[0509] 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.

[0510] 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.

[0511] 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.

[0512] An adequate population of granulopoietic cells may comprise approximately 6-8% CD115+ cells. For example, Petition 870250102349, dated 07 / 11 / 2025, pages 142 / 209 134 / 177 an adequate population of granulopoietic cells may comprise approximately 5% of CD115+ cells.

[0513] 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.

[0514] 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.

[0515] An adequate population of granulopoietic cells may comprise approximately 20-55% CD32+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 40% CD32+ cells.

[0516] 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.

[0517] An adequate population of granulopoietic cells may comprise approximately 0.04-1% of CD16+ cells. For example, an adequate population of granulopoietic cells may comprise approximately 0.25% of CD16+ cells.

[0518] 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.

[0519] 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.

[0520] An adequate population of granulopoietic cells may comprise approximately 2-18% CD206+ cells. For example, an adequate population of granulopoietic cells may Petition 870250102349, dated 07 / 11 / 2025, pages 143 / 209 135 / 177 comprise approximately 10% of CD206+ cells.

[0521] A suitable isolated population of granulopoietic cells comprises: • 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% CD38+ cells (for example, approximately 65% ​​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).

[0522] A suitable isolated population of granulopoietic cells comprises: • a first subpopulation of cells that are CD15+ CD64+ CD18+ CD49d+ CD71+ • a second subpopulation of cells that are CD15CD11b+ / - CD18+ CD49d+ CD32+ HLA-DR- • a third subpopulation of cells that are CD15- CD11bHLA-DR+ CD18+ CD49d+ and CD71+.

[0523] An adequate population of granulopoietic cells may also comprise a fourth subpopulation of cells that are CD15- CD11b+ HLA-DR+.

[0524] 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. Petition 870250102349, dated 07 / 11 / 2025, pages 144 / 209 136 / 177

[0525] A suitable subpopulation of granulopoietic cells that are CD15+ CD64+ CD18+ CD49d+ CD71+ 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, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14. Such a suitable cell population 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). Appropriately, the cell population, or subpopulation, is heterogeneous for the cited marker profile (which may appropriately include the optional constituents referred to in this document).Suitablely, a population, or subpopulation, of cells according to this embodiment 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). Suitablely, the population, or subpopulation, of cells is homogeneous for the cited marker profile (which may suitably include the optional constituents referred to in this document).

[0526] The first subpopulation of cells expresses markers that resemble those expressed by committed neutrophil precursors. However, the cells disclosed according to this embodiment of the invention are CD64+ and may be CD16- and 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 Petition 870250102349, dated 07 / 11 / 2025, pages 145 / 209 137 / 177, also CD16- and / or CD62L-, can be distinguished as one that was produced by a method as described in this document, rather than a naturally occurring granulopoietic cell or population of such cells.

[0527] The inventors have identified that cells in this subpopulation 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 the isolated populations of granulopoietic cells described above. Thus, such cells may 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.

[0528] The first cell subpopulation can express 41BBL 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 cell subpopulation can 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.

[0529] In addition to expressing markers indicative of immunomodulatory capacity, this cell population also expresses molecules (particularly CD11b, CD18, Mac1 and CD32) that suggest they possess direct cytocidal activity. This may make it suitable for uses where it is desired to therapeutically kill cells, such as cancerous or infected cells.

[0530] An adequate population of granulopoietic cells Petition 870250102349, dated 07 / 11 / 2025, pages 146 / 209 138 / 177 may comprise cells that are CD15-, CD11b+ / -, CD18+, CD49d+, CD32+, and HLA-DR-. A suitable subpopulation of cells that are CD15- CD11b+ / - CD18+ CD49d+ CD32+ HLA-DR- may also be positive for one, more than one, or all of the selected markers from the group consisting of: CD177, CD11b, CD71, CD66b, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14. 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 embodiment of the invention is homogeneously negative for CD15 and HLA-DR and heterogeneous with respect to the other markers of the aforementioned marker profile (which may suitably 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).

[0531] The second subpopulation of cells 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. Cells in this group may also express CXCR2, which may 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. Petition 870250102349, dated 07 / 11 / 2025, pages 147 / 209 139 / 177 for the TME.

[0532] A suitable population of cells that are CD15CD11b- HLA-DR+ CD18+ CD49d+ and CD71+ may also be 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, 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 embodiment 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 suitably 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).

[0533] The third subpopulation of cells expresses markers indicative of a relatively low level of differentiation. Accordingly, these cells may also be CD34+. Cells in this group may also express markers such as 4-1BBL and / or OX40L and / or CD40 and / or CD54 that indicate their suitability for use in applications requiring immunomodulation of non-granulocytic immune cells. Although cells in this group do not express markers indicative of direct cytocidal activity, they may have the capacity to differentiate further and express markers such as CD11b and CD15 that would confer such activity. Consequently, these cells may 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. Petition 870250102349, dated 07 / 11 / 2025, pages 148 / 209 140 / 177

[0534] A suitable population of granulopoietic cells that are CD15- CD11b+ HLA-DR+ may also be 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, 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 embodiment 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 aforementioned marker profile (which may suitably 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).

[0535] These cells express markers that are similar to those that would be expected for active 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.

[0536] A suitable granulopoietic cell, or population of granulopoietic cells, to be employed in the medical uses of the treatment methods of the invention may be CD64+, CD16- and / or CD62L-. For example, the granulopoietic cell may be CD64+. The granulopoietic cell may be CD64+ and CD16-. The granulopoietic cell may be CD64+ and CD62L-. The granulopoietic cell may be CD16- and CD62L-. Suitably, the granulopoietic cell is CD64+, CD16- and CD62L-. The expression of CD64 and the lack of expression of CD16 and CD62L by granulopoietic cells Petition 870250102349, dated 07 / 11 / 2025, pages 149 / 209 141 / 177 of the invention contrast with neutrophils found in circulation and during 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 well as 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.

[0537] Thus, in a suitable embodiment, a medical use or treatment method of the invention employs 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.

[0538] In a further embodiment, a medical use or treatment method of the invention employs a granulopoietic cell that is a CD16- granulopoietic cell. The CD16- granulopoietic cell may be a CD16- and CD62L- granulopoietic cell.

[0539] In a further embodiment, a medical use or treatment method of the invention employs a granulopoietic cell that is a CD62L- granulopoietic cell. Sources of granulopoietic cells

[0540] Granulopoietic cells suitable for use in the various aspects of the invention may be obtained from any suitable source. The granulopoietic cells may be allogeneic with reference to their intended recipient. They may be obtained from or derived from any suitable donor.

[0541] Any of the cells or cell populations disclosed in this document may be derived from a mammal, Petition 870250102349, dated 07 / 11 / 2025, pages 150 / 209 142 / 177 such as a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Appropriately, the cell or population of cells is derived from a human. Thus, the cell may be a human cell or the population of cells may be a population of human cells. Particularly, 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.

[0542] Granulopoietic cells can be obtained from any suitable source. For example, granulopoietic cells can be obtained (e.g., 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, granulopoietic cells can be obtained (e.g., obtained) from a sample of PBMCs depleted of αβ T cells. Granulopoietic cells can be obtained from (e.g., differentiated in vitro from) a stem cell, such as a hematopoietic stem cell or iPSC.

[0543] The term obtainable, as used in this document, encompasses the term obtained. In one embodiment, obtainable means obtained.

[0544] The term donor, as used in this document, may refer to a subject (properly a human subject) from whom a sample is obtainable (e.g., obtained). Any suitable sample from which a granulopoietic cell and / or non-granulocytic immune cell is obtainable may be obtained from the donor. The donor may be selected Petition 870250102349, dated 07 / 11 / 2025, pages 151 / 209 143 / 177 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 healthy. 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 is male. 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 male aged 18-55 and preferably 18-35 (more preferably 18-24). Alternatively, a donor may be selected if the donor is female. A donor may be selected if the donor is over 40 years of age.Appropriately, a donor can be selected if the donor is a woman over 40 years of age.

[0545] A granulopoietic cell suitable for use in the various embodiments of the invention can be produced by in vitro differentiation of a stem cell. The term stem cell, as used in this document, encompasses any cell capable of differentiating into a granulopoietic cell (and preferably a granulopoietic cell capable of generating neutrophils). For example, the term stem cell may encompass totipotent, pluripotent, multipotent, or unipotent cells. In a suitable embodiment, the term stem cell 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 into a granulocyte (preferably a neutrophil). Preferably, the term stem cell, as used in this document, does not encompass a human embryonic stem cell. Petition 870250102349, dated 07 / 11 / 2025, pages 152 / 209 144 / 177

[0546] A stem cell can be part of a stem cell culture.

[0547] A stem cell can be a natural stem cell or an artificial stem cell. In a suitable embodiment, a natural stem cell can be a cell of the hematopoiesis pathway or a cell equivalent to it. In a suitable embodiment, granulopoietic cells are derived from an artificial stem cell that is an induced pluripotent stem cell (iPSC) or a cell equivalent to it.

[0548] In a suitable embodiment, an iPSC can be obtained from a somatic cell, such as a somatic cell from a donor. The generation of iPSCs is a well-known technique in the art, see Yu et al. (2007), Science, 318:1917-1920, the teaching of which is incorporated herein by reference.

[0549] In another embodiment, an iPSC can be obtained from a stem cell (e.g., obtained from a donor), as from a hematopoietic stem cell. Preferably, an iPSC is obtained from a hematopoietic stem cell or a precursor cell described in this document.

[0550] In a suitable embodiment, a stem cell is a nuclear transfer embryonic stem cell (NT-ESC) or equivalent thereof. In a suitable embodiment, an NT-ESC is obtainable by injecting the nucleus of a donor cell into a somatic cell from which the original nucleus has been removed. The generation of NT-ESCs is a well-known technique in the art, see Tachibana M, Amato P, Sparman M, et al. (2013), Cell, 154(2): 465-466, whose teachings are incorporated herein by reference.

[0551] A stem cell can be immortalized. Those skilled in the art are familiar with immortalization techniques, which include, among other things, the introduction of a viral gene that Petition 870250102349, dated 07 / 11 / 2025, pages 153 / 209 145 / 177 disrupts the cell cycle (e.g., the E1 gene of adenovirus type 5) and artificially expresses telomerase. Immortization advantageously allows the preparation of a cell line that can be stably cultured in vitro. Thus, in one aspect, the invention provides an immortalized cell line obtainable (e.g., obtained) from a selected stem cell, as well as a stable stem cell culture. Suitably, an immortalized cell line or stable stem cell culture is obtainable (e.g., obtained) by a method of the present invention.

[0552] The term stable, as used in reference to a stem cell culture or cell line, means that the cell culture or cell line has been modified so that it is more receptive to in vitro cell culture than an unmodified cell (i.e., a cell obtained from a donor and subjected directly to in vitro cell culture). Such a stable cell culture or cell line is therefore capable of undergoing more rounds of replication (preferably for extended periods of time) when compared to an unmodified cell. Methods for promoting the therapeutic activity of non-granulocytic immune cells.

[0553] The fifth aspect of the invention provides methods for promoting the therapeutic activity of non-granulocytic immune cells, in which a non-granulocytic immune cell is incubated with a granulopoietic cell.

[0554] A method according to this aspect of the invention can be practiced in vitro or in vivo. Suitablely, the method is practiced in vivo. A method according to this aspect of the invention can be used to promote the therapeutic activity of non-granulocytic immune cells before their administration to a patient as a therapeutic agent.

[0555] A method according to this aspect of the invention Petition 870250102349, dated 07 / 11 / 2025, pages 154 / 209 146 / 177 can be applied to any non-granulocytic immune cells. The method can be applied to non-granulocytic host immune cells. Appropriately, the method is applied to NK cells.

[0556] The increase in therapeutic activity can be demonstrated by an increase in activation, according to any of the parameters discussed further in this document. Methods using granulopoietic cells to improve immune cell culture.

[0557] In an eighth aspect, the invention provides a method for increasing the survival of immune cells in culture, the method comprising culturing the immune cells in the presence of a feeder layer of granulopoietic cells.

[0558] In a ninth aspect, the invention provides a method for increasing the proliferation of immune cells in culture, the method comprising culturing the immune cells in the presence of a feeder layer of granulopoietic cells.

[0559] The immune cells cultured in a method of the eighth or ninth aspect of the invention may be selected from the group consisting of: a T cell; and an NK cell. In an embodiment in which the cultured immune cell comprises a T cell, the cell may be selected from the group 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.

[0560] The methods of the eighth aspect of the invention may be well suited for use in NK or NK T cell culture. The methods of the ninth aspect of the invention may be well suited for use in αβ T cell culture. Methods for selecting appropriate treatment regimens

[0561] The sixth aspect of the invention provides a method for selecting an appropriate treatment regimen for a patient. This method involves: Petition 870250102349, dated 07 / 11 / 2025, pages 155 / 209 147 / 177 • identify 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 granulopoietic cell 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 granulopoietic cell is selected.

[0562] Those skilled in the art will be aware of many suitable methods by which the impairment (or lack thereof) of a patient's non-granulocytic immune response can be assessed.

[0563] Such methods may be of particular relevance in the case of a patient suspected of having an impaired non-granulocytic immune response. A patient with, or suspected of having, an impaired non-granulocytic immune response may be a patient with a disease or receiving treatment resulting in immune suppression.

[0564] The seventh aspect of the invention provides an alternative or additional method for selecting an appropriate treatment regimen for a patient. This method involves: • Incubate a patient's non-granulocytic immune cell with a granulopoietic cell; wherein • if the activation of the patient's non-granulocytic immune cell is increased in response to incubation, then treatment with a granulopoietic cell is selected as an appropriate treatment; and • if the activation of the patient's non-granulocytic immune cell is increased in response to incubation, then treatment with a therapy other than a granulopoietic cell is selected.

[0565] The activation of non-granulocytic cells of a Petition 870250102349, dated 07 / 11 / 2025, pages 156 / 209 148 / 177 patient may be evaluated with reference to any appropriate indication for activation and by any appropriate means, including (but not limited to) the indications and means discussed further in this descriptive report.

[0566] In methods according to the sixth or seventh aspects of the invention, if treatment with a granulopoietic cell is selected as an appropriate treatment, this treatment can be implemented using granulopoietic cells as considered in any of the aspects or embodiments of the invention. Such cells can be provided by means of a pharmaceutical composition of the invention. Screening methods of the invention

[0567] The tenth, eleventh, twelfth, thirteenth, and fourteenth aspects of the invention relate to screening methods for identifying granulopoietic cells suitable for therapeutic use. Respectively, the tenth aspect provides a method for identifying whether a granulopoietic cell is suitable for use in cancer treatment by beneficially modulating the tumor microenvironment; the eleventh aspect provides a method for identifying whether a granulopoietic cell is suitable for use in cancer treatment by increasing immune cell recruitment in a tumor and / or immune cell activation; in the twelfth aspect, the invention provides a method for identifying whether a granulopoietic cell is suitable for use in cancer treatment by directly promoting cancer cell death; the thirteenth aspect provides a method for identifying whether a granulopoietic cell is suitable for use in infection treatment.directly promoting the death of infectious cellular agents or infected cells, and the fourteenth aspect provides a method for identifying whether a granulopoietic cell is suitable for use in treatment by amplifying a therapeutic immune response.

[0568] In a suitable modality, a method according to Petition 870250102349, dated 07 / 11 / 2025, pages 157 / 209 149 / 177 The tenth aspect of the invention may comprise evaluating the expression of selected pro-inflammatory cytokines from the group consisting of: IFN-γ and TNF.

[0569] Appropriately, a method according to the eleventh aspect of the invention may comprise evaluating the expression of the chemokine CXL10.

[0570] In a suitable embodiment, a method according to the eleventh aspect of the invention may comprise evaluating the expression of degranulation markers selected from the group consisting of: CD107a; perforin; and granzymes.

[0571] Suitably, a method according to the twelfth aspect of the invention may involve positively identifying the granulopoietic cell as suitable for use in cancer treatment, directly promoting the death of cancer cells in the case where the death rate of cancer cells incubated with the granulopoietic cell, or a cell derived from the granulopoietic cell, is at least three times greater than the death rate of non-cancerous cells.

[0572] A method according to the thirteenth aspect of the invention may involve positively identifying the granulopoietic cell as suitable for use in the treatment of infection when the death rate of infectious cellular agents or infected cells incubated with the granulopoietic cell, or a cell derived from the granulopoietic cell, is at least three times greater than the death rate of uninfected cells.

[0573] A method according to the fourteenth aspect of the invention may involve identifying a granulopoietic cell as suitable for use in treatment when the activation of immune cells is increased in accordance with any of the considerations presented in relation to this disclosure. The granulopoietic cell may be incubated with any form of immune cells. For example, the granulopoietic cell may be incubated with non-granulocytic cells. The immune cells may Petition 870250102349, dated 07 / 11 / 2025, pages 158 / 209 150 / 177 to be derived from an individual requiring therapy.

[0574] In the event that a screening method according to any of these aspects of the invention identifies a granulopoietic cell as suitable for use in treatment, the method may comprise a further step of identifying the donor from whom the granulopoietic cell was taken or derived as a donor capable of providing therapeutically effective granulopoietic cells. Alternatively, or additionally, the method may comprise a further step of obtaining a stem cell from the donor from whom the granulopoietic cell was taken or derived. The stem cell may be a naturally occurring cell, such as a hematopoietic stem cell, or it may be an artificial stem cell, such as an iPSC. Such a stem cell may be stored. Such a stem cell may be used to produce other therapeutically effective granulopoietic stem cells, such as for incorporation into pharmaceutical compositions of the invention.

[0575] The invention will now be further described with reference to the following Examples. Examples Materials and methods Co-culture of PBMCs with neutrophils from donors of patients with IMANp or PDAC:

[0576] PBMCs from a healthy donor were cultured with granulopoietic cells (designated IMANps by the inventors, as shown in the Figures) (n=4) or neutrophils derived from the blood of a donor with pancreatic ductal adenocarcinoma (PDAC) (n=1) in different ratios indicated (2:1, 1:1 or 0.5:1 granulopoietic cell:PBMC, or 1:1 or 0.5:1 donor neutrophil:PBMC). PBMCs were labeled with far-red cell trace dye (CTFR) before co-culture for identification and proliferation analysis. Co-cultures were performed in the presence or absence of anti-CD3 stimulation (OKT3; 1 μg / ml). After 72 h, the Petition 870250102349, dated 07 / 11 / 2025, pages 159 / 209 151 / 177 proliferation and / or activation of different T and NK cell populations were investigated by flow cytometry. Digestion of patient biopsy with PDAC and co-culture with IMANp:

[0577] The biopsy from the patient with PDAC was digested using the Miltenyi Human Tumor Dissociation Kit (130-095-929) according to the manufacturer's protocol. Briefly, the tumor biopsy was cut into small pieces (2-4 mm) before being transferred to a gentleMACS C tube containing 4.7 ml of RPME, 200 µl of Enzyme H, 20 µl of Enzyme R, and 25 µl of Enzyme A. The C tube was then tightly closed and secured upside down in the gentleMACS dissociator sleeve (Miltenyi). The program for difficult tumors (37C_h_TDK_3) was selected and run. After the program finished, the C tube was detached, and the sample was removed and passed through a 70 µm cell filter before being washed in complete medium. The digested tumor cells were labeled with far-red cell trace dye (CTFR) before co-culture for identification purposes. CTFR-labeled tumor digestion was then co-cultured with IMANp (n=2) at a ratio of 2:1 (tumor digestion:IMANp).Co-cultures were performed in the presence or absence of anti-CD3 stimulation (OKT3; 1 pg / ml). After 72 h, the activation of different T and NK cell populations was investigated by flow cytometry. Flow cytometry:

[0578] PBMCs were labeled with far-red cellular trace dye (CTFR) prior to co-culture for identification and proliferation analysis. Granulopoietic stem cells (IMANp) or patient donor neutrophils were not labeled prior to co-culture. After 72h culture, cells were washed in PBS and incubated with live / dead stain (Fixable Viability Dye eFluor 780; 1:500 dilution) and FcyR block (Human TruStain FcX; 1:50 dilution) for 20 minutes. Cells were then washed in flow cytometry buffer and surface stained with specific antibodies for CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T4). Petition 870250102349, dated 07 / 11 / 2025, pp. 160 / 209 152 / 177 T8), CD56 (HCD56), CD107a (H4A3), 4-1BB (4B4-1), and OX40 (BerACT35). In other experiments, granulocytes (here designated IMANs) produced during the differentiation of granulopoietic cells were surface-stained for expression of 4-1BBL (5F4) and OX40L (11C3.1). All antibodies were used at a 1:50 dilution, with staining performed on 50 μL / sample. After surface staining, cells were fixed using 100 μL 1X BD CellFix before being acquired on a MACSQuant 16 (Miltenyi). Data were analyzed using FlowLogic software. Analysis of stained populations was performed by blocking single live cells. Quantification of cytokines / chemokines in cell culture supernatants:

[0579] Cell culture supernatants were collected and the concentration of secreted IFN-γ was measured by quantitative sandwich ELISA (Abcam; ab174443) according to the manufacturer's instructions. Alternatively, the concentration of CXCL10 was measured by LEGENDplex (BioLegend; 740985) according to the manufacturer's instructions. Table 1 - Reagents used in the Examples Supplier Material No. Cat. No. Human anti-CD3 antibody (OKT3) BioLegend 317326 Far-red cell trace proliferation kit Thermo Fisher Scientific C34564 eFluor780 Fixable Viability Stain Thermo Fisher Scientific 65-0865-14 PBS Thermo Fisher Scientific 15374875 Human TruStain FcX BioLegend 422302 Petition 870250102349, dated 07 / 11 / 2025, pages 161 / 209 153 / 177 Material Supplier Cat. No. BD CellFIX BD 340181 Tumor dissociation kit Miltenyi 130-095-929 IFN-γ ELISA Abcam ab174443 LEGENDplex Hu proinflam. Chemokine Panel BioLegend 740985 Anti-Human CD8 (RPA-T8) BV605 BioLegend 301040 Anti-Human CD4 (RPA-T4) BV510 BioLegend 300546 Anti-Human CD3 (OKT3) PerCP-Cy5.5 BioLegend 317336 Anti-Human CD56 HCD56 BioLegend 318306 Anti-Human CD107a (H4A3) PE-Cy7 BioLegend 328618 Anti-Human 4-1BB (4B4-1) BV421 BioLegend 309820 Human Anti-OX40 (BerAct35) FITC BioLegend 350006 Anti-Human 4-1BBL (5F4) PE-Cy7 BioLegend 311512 Anti-human OX40L (11C3.1) PE BioLegend 326308 Example 1 Co-culture with IMANp granulopoietic cells increases the activation of blood-derived CD8+ T cells.

[0580] Method: PBMCs from a healthy donor were cultured Petition 870250102349, dated 07 / 11 / 2025, pages 162 / 209 154 / 177 with granulopoietic stem cells (n=4) or neutrophils derived from PDAC donor blood (n=1) in different ratios as indicated. Co-cultures were performed in the presence or absence of anti-CD3 stimulation (OKT3; 1 μg / ml). After 72 h, CD8+ T cell activation was investigated by flow cytometry. PBMCs were labeled with far-red cell trace dye (CTFR) before co-culture, and CD8+ cells were blocked as live singlets, CTFR+ CD3+ CD8+. CD8+ cell activation was investigated by measuring the expression of degranulation markers, such as CD107a, and co-stimulatory molecules, such as 4-1BB and OX40, on the cell surface.

[0581] Results: The results show the % expression of CD107a, 4-1BB and OX40 in unstimulated CD8+ T cells (Figure 1A) and anti-CD3 stimulated cells (Figure 1B).

[0582] (Figure 1A) Co-culture with granulopoietic cells and non-neutrophils from patient donors increased the activation of blood-derived CD8+ T cells, as demonstrated by the increased expression of CD107a, 4-1BB, and OX40 on CD8+ T cells in the absence of TCR stimulation. (Figure 1B) Stimulation with anti-CD3 increased the expression of CD107a, 4-1BB, and OX40 on CD8+ T cells, and the expression of these activation markers was further increased in the presence of granulopoietic cells, but not neutrophils, from patient donors. These results indicate that the presence of granulopoietic cells potentiates activation as indicated by degranulation (CD107a) and expression of additional activation markers (co-stimulatory molecules OX40 and 4-1BB) of activated CD8+ T cells. OX40 and 4-1BB are co-stimulatory markers expressed on activated T cells.The binding of these costimulatory receptors to activated CD8 T cells should enhance the effector function of these cells (e.g., increase cytotoxicity and IFN-γ production).

[0583] Furthermore, the data suggest that granulopoietic cells are providing signal 2 (co-stimulation) and / or Petition 870250102349, dated 07 / 11 / 2025, pages 163 / 209 155 / 177 signal 3 (cytokine stimulation) of T cell activation.

[0584] The data also suggest that granulopoietic cells can be used in combination therapy with T-cell triggers, for example, mono / bispecific 4-1BB agonist, or bispecific T-cell trigger TAA / 41BB. Example 2 Co-culture with granulopoietic cells increases the activation of blood-derived CD4+ T cells.

[0585] Method: PBMCs from a healthy donor were cultured with granulopoietic cells (n=4) or neutrophils derived from PDAC donor blood (n=1) at different indicated ratios. Co-cultures were performed in the presence or absence of anti-CD3 stimulation (OKT3; 1 μg / ml). After 72 h, CD4+ T cell activation was investigated by flow cytometry. PBMCs were labeled with far-red cell trace dye (CTFR) before co-culture, and CD4+ cells were blocked as live singlets, CTFR+CD3+CD4+. CD4 cell activation was investigated by measuring the expression of co-stimulatory molecules (particularly 4-1BB and OX40) on the cell surface.

[0586] Results: The results show the % expression of 4-1BB and OX40 in unstimulated CD4+ T cells (Figure 2A) and anti-CD3 stimulated cells (Figure 2B). (Figure 2A) Co-culture with granulopoietic cells, and not neutrophils from patient donors, increased the activation of blood-derived CD4+ T cells, as demonstrated by the increased expression of 4-1BB and OX40 in CD4+ T cells in the absence of TCR stimulation. (Figure 2B) Stimulation with anti-CD3 increased the expression of 4-1BB and OX40 in CD4+ T cells, and the expression of these activation markers was further potentiated in the presence of granulopoietic cells, but not neutrophils from patient donors. These results indicate that the presence of granulopoietic cells potentiates the activation (OX40 and 4-1BB) of Petition 870250102349, dated 07 / 11 / 2025, pages 164 / 209 156 / 177 CD4 T cells. OX40 and 4-1BB are costimulatory markers expressed on activated T cells. Binding of these costimulatory receptors to CD4 T cells should enhance the effector function of these cells (e.g., increase cytokine production).

[0587] The data suggest that granulopoietic cells are providing signal 2 (co-stimulation) and / or signal 3 (cytokine stimulation) for T cell activation. Example 3 Co-culture with granulopoietic cells enhances the proliferation and accumulation of αβ T cells.

[0588] Method: PBMCs from a healthy donor were cultured with granulopoietic cells (n=4) or neutrophils derived from PDAC donor blood (n=1) in a 1:1 ratio. Co-cultures were performed in the presence of anti-CD3 stimulation (OKT3; 1 μg / ml). After 72 h, CD4+ and CD8+ T cell proliferation was investigated by flow cytometry. PBMCs were labeled with far-red cell trace dye (CTFR) (Invitrogen; C34572) before co-culture, and T cells were blocked as live singlets, CTFR+CD3+CD8+, or CD3+CD4+. Proliferative cells were identified as having reduced median fluorescence intensity (MFI) of CTFR, which occurs when cells divide and the dye is diluted.

[0589] Results: The results show (Figure 3A) % proliferation of CD4+ and CD8+ T cells and (Figure 3B) absolute counts of both cell types after 72h culture. Co-culture with granulopoietic cells, and not neutrophils from patient donors, increased the proliferation of αβ T cells (as demonstrated by the increased proliferation of anti-CD3-stimulated CD4 and CD8 T cells) and the accumulation of such cells (as demonstrated by the increased absolute counts of CD4 and CD8 T cells present after 72h culture). Granulopoietic cells are able to amplify TCR-driven proliferation of αβ T cells and increase cell accumulation. Petition 870250102349, dated 07 / 11 / 2025, pages 165 / 209 157 / 177 immune.

[0590] The data suggest that granulopoietic cells are providing signal 2 (co-stimulation) and / or signal 3 (cytokine stimulation) for T cell activation. Example 4 Co-culture with granulopoietic cells promotes the survival of blood-derived NK cells and NKT cells.

[0591] Method: PBMCs from a healthy donor were cultured with granulopoietic cells (n=4) or neutrophils derived from PDAC donor blood (n=1) at different indicated ratios. After 72 h, absolute NK and NKT cell counts were quantified by flow cytometry. PBMCs were labeled with far-red cell trace dye (CTFR) before co-culture and blocked as live singlets, CTFR+. NK cells were blocked as CD3-CD56+ and NKT cells were blocked as CD3+CD56+.

[0592] Results: The results show absolute counts of NK cells (Figure 4A) and NKT cells (Figure 4B) in both PBMC donors. In both donors, co-culture with granulopoietic cells, rather than with blood neutrophils from PDAC patients, promoted the survival of NK and NKT cells, as demonstrated by the absolute counts.

[0593] The results demonstrate that granulopoietic cells have a favorable effect on the survival of immune cells, as exemplified by their effect on the survival of NK and NKT cells. This further suggests that medical uses or treatment methods employing granulopoietic cells can be used in conjunction with NK cell therapy, for example, as a feeder cell for the production of NK cell therapy or in combination with NK cell therapy to support the function of NK cell therapy in vivo. Example 5 Petition 870250102349, dated 07 / 11 / 2025, pages 166 / 209 158 / 177 Co-culture with granulopoietic cells promotes the activation of blood-derived NK cells and NKT cells.

[0594] Method: PBMCs from a healthy donor were cultured with granulopoietic cells (n=4) or neutrophils derived from PDAC donor blood (n=1) at different indicated ratios. After 72 h, NK and NKT cell activation was investigated by flow cytometry. PBMCs were labeled with far-red cellular trace dye (CTFR) before co-culture and blocked as live singlets, CTFR+. NK cells were blocked as CD3-CD56+ and NKT cells were blocked as CD3+CD56+. NK and NKT cell activation was investigated by measuring the expression of degranulation markers, such as CD107a, and co-stimulatory molecules, such as 4-1BB and OX40, on the cell surface.

[0595] Results: The results show the % expression of CD107a, 4-1BB, and OX40 in NK cells (Figure 5A) and NKT cells (Figure 5B). Co-culture with granulopoietic cells, and not neutrophils from patient donors, increased the activation of blood-derived NK cells and NKT cells, as demonstrated by the increased expression of CD107a, 4-1BB, and OX40 in NK cells (Figure 5A) and NKT cells (Figure 5B). These results indicate that the presence of granulopoietic cells potentiates the degranulation (CD107a) and activation (OX40 and 4-1BB) of NK and NKT cells.

[0596] The results demonstrate that granulopoietic cells have a favorable effect on the activation of NK and NKT cells. This suggests that treatments using granulopoietic cells can be used in conjunction with NK cell therapy, for example, as a feeder cell for the production of NK cell therapy or in combination with NK cell therapy to support the function of NK cell therapy in vivo. Example 6 Co-culture with granulopoietic cells enhances activation. Petition 870250102349, dated 07 / 11 / 2025, pages 167 / 209 159 / 177 of leukocytes infiltrated by tumor (CD8, CD4 and NK cells)

[0597] Method: Tumor digestion from a patient with PDAC (n=1) was cultured ± granulopoietic cells (n=2). After 72 h, the activation of tumor-infiltrated αβ T cells and NK cells was investigated by flow cytometry. Digested tumor cells were labeled with far-red cellular trace dye (CTFR) before co-culture and were blocked as live singlets, CTFR+. Effector populations were then blocked as CD3+CD8+, CD3+CD4+, or CD3-CD56+. TIL activation was investigated by measuring the expression of degranulation markers, such as CD107a, and co-stimulatory molecules, such as 41BB and / or OX40, on the cell surface, as indicated.

[0598] Results: The results show a fold-in change in the expression of indicated activation markers in tumor-infiltrated CD8 T cells (Figure 6A), NK cells (Figure 6B), and CD4 T cells (Figure 6C). The data are shown as a fold-in increase in expression versus tumor digestion-only conditions. (Figures 6A and 6B) The data show that co-culture with granulopoietic cells increased the activation of tumor-infiltrated leukocytes (exemplified by CD8 and NK cells), as demonstrated by the increased expression of CD107a and 41BB in CD8 T cells and NK cells, indicating the ability of granulopoietic cells to promote the degranulation of cytotoxic effector cells in the TME, as well as potentiate their activation through increased 4-1BB expression.(Figure 6C) The data show that co-culture with granulopoietic cells increased the activation of tumor-infiltrated CD4 cells, as demonstrated by the increased expression of costimulatory receptors 4-1BB and OX40 on tumor-infiltrated CD4 T cells. The binding of these costimulatory receptors results in increased effector function of T cells, for example, increased cytokine production. Example 7. Co-culture with granulopoietic cells increases the production of Petition 870250102349, dated 07 / 11 / 2025, pages 168 / 209 160 / 177 cytokines per PBMC

[0599] Method: PBMCs from healthy donors (n=2) were cultured with granulopoietic cells (n=4) or neutrophils derived from the blood of a PDAC donor (n=1) at different indicated ratios. Co-cultures were performed in the presence of anti-CD3 stimulation (OKT3; 1 μg / ml). After 72 h, the supernatants were collected and the concentration of secreted cytokines, such as IFN-γ, was measured by quantitative sandwich ELISA (ab174443) according to the manufacturer's instructions.

[0600] Results: The results show (Figure 7) the concentration of IFN-γ detected in supernatants. Co-culture with granulopoietic cells, and not neutrophils from patient donors, increased IFN-γ production by PBMCs. These data show granulopoietic cells that enhance the effector functions of T cells for potent antitumor immunity, as demonstrated by the increased IFN-γ production by PBMCs.

[0601] The data suggest that granulopoietic cells are providing signal 2 (co-stimulation) and / or signal 3 (cytokine stimulation) for T cell activation. Furthermore, they indicate that granulopoietic cells will not drive uncontrolled T cell activation, which is important in terms of the safety of medical uses or treatment methods.

[0602] The activation observed in the absence of anti-CD3 may reflect the activation of a small population of memory T cells that do not require TCR stimulation for their activation. Example 8 Co-culture with granulopoietic cells increases cytokine production by tumor-infiltrating lymphocytes (TILs).

[0603] Method: Tumor digestion from a patient with PDAC (n=1) was cultured ± granulopoietic cells (n=2). Co-cultures were performed in the presence of anti-CD3 stimulation (OKT3; 1 μg / ml). After 72 h, the supernatants were collected and the concentration of secreted cytokines, such as IFN-γ, was measured. Petition 870250102349, dated 07 / 11 / 2025, pages 169 / 209 161 / 177 by quantitative sandwich ELISA (ab174443) according to the manufacturer's instructions.

[0604] Results: (Figure 8) The results show the concentration of IFN-γ detected in cell culture supernatants. Co-culture with granulopoietic cells increased IFN-γ production by TILs. These data show granulopoietic cells reinforcing the effector functions of T cells for potent antitumor immunity, as demonstrated by the increased IFN-γ production by TILs.

[0605] The data suggest that granulopoietic cells are providing signal 2 (co-stimulation) and / or signal 3 (cytokine stimulation) for T cell activation. Example 9 Granulopoietic cells promote the recruitment of immune cells to the tumor microenvironment.

[0606] Method: Fresh patient tumor biopsy (RCC) ± granulopoietic cells were encapsulated in a tumor-on-a-chip model and co-cultured with PBMCs from matching donors. (Figure 9) PBMC recruitment to the microtumor was measured daily for 3 days by live cell imaging.

[0607] Results: The results show (Figure 9) a multiplier of immune cell infiltration into the microtumor at indicated time points versus day 0 tumor only at indicated time points. These data suggest that granulopoietic cells are immunomodulatory through their ability to recruit immune cells to the tumor microenvironment. Example 10 Granulopoietic cells promote enhanced tumor death.

[0608] Method: Fresh patient tumor biopsy (RCC) ± granulopoietic cells were encapsulated in a tumor-on-a-chip model and co-cultured with PBMCs from matching donors. (Figure 10) The cytotoxicity of the tumor cells was measured Petition 870250102349, dated 07 / 11 / 2025, pages 170 / 209 162 / 177 daily for 3 days by live cell imaging.

[0609] Results: The results show (Figure 10) % of tumor death at indicated time points. These data suggest that granulopoietic cells increased tumor cell death.

[0610] It will be recognized that the death of tumor cells is a key objective of anticancer treatments. Therefore, the increased tumor-killing activity observed in treatment with granulopoietic cells clearly indicates that the medical uses, treatment methods, and pharmaceutical compositions of the invention will be able to exert therapeutic anticancer activity. As demonstrated in the previous Examples, this is achieved by amplifying the immune response and, particularly, the effects of non-granulocytic cells on the immune response. Example 11 Granulocytes produced during the differentiation of granulopoietic cells promote the recruitment of immune cells to the tumor microenvironment through the secretion of chemokines.

[0611] Method: Granulopoietic cells were differentiated and the resulting granulocytes (IMANs) (n=4) were stimulated ± IFN-α, IFN-β or TNF (all 10 ng / ml) for 24h. Data show the concentration of chemokines, such as CXCL10, in cell culture supernatants quantified by LEGENDplex according to the manufacturer's instructions.

[0612] Results: The results show (Figure 11) that granulocytes produced in the differentiation of granulopoietic cells release CXCL10 when activated by various cytokines. These data suggest that treatment using granulopoietic cells may play an additional role in promoting the recruitment of immune cells to the tumor microenvironment through their production of granulocytes capable of releasing chemokines, such as CXCL10. CXCL10 is known to be a powerful chemoattractant for CXCR3+ T cells and NK cells. Petition 870250102349, dated 07 / 11 / 2025, pages 171 / 209 163 / 177

[0613] The data indicate that granulocytes produced in the differentiation of granulopoietic cells can be activated through many different pathways. The data suggest that it may be possible to combine granulopoietic cell therapy, and particularly the granulocytes produced as a result of such therapy, with mono / bispecific antibodies that activate innate immune cells. For example, combination with anti-CD40 mAb or bispecific anti-CD40 / TAA for combined granulopoietic cell activation and tumor targeting. Example 12 Granulocytes derived from granulopoietic cells express ligands for T-cell and NK-cell costimulatory receptors.

[0614] Method: Granulocytes produced in the differentiation of granulopoietic cells suitable for use in the medical uses or treatment methods of the invention (n=3) were analyzed for the expression of T and NK cell costimulatory receptors, 4-1BBL and OX40L, by flow cytometry.

[0615] Results: The results show (Figure 12) % expression of 4-1BBL and OX40L in granulocytes derived from granulopoietic cells from 3 individual donors. These data suggest that granulopoietic cells potentiate the effector functions of T and NK cells through co-stimulation, as demonstrated by the expression of 4-1BBL and OX40L. Materials and methods Preparation of granulopoietic cell populations (IMANp)

[0616] The preparation of granulopoietic cell populations (designated IMANps by the inventors, as shown in the Figures) from hematopoietic stem cells (HSCs) consists of three main stages after collecting leukapheresis from the donor: Isolation and cryopreservation of CD34+ cells from donor leukapheresis. - Expansion of isolated CD34+ cells (E0 to E8) over 9 days Petition 870250102349, dated 07 / 11 / 2025, pages 172 / 209 164 / 177 to generate intermediate progenitor cells. On day 9 (E8D0), the expansion medium is replaced by differentiation medium. - Differentiation of intermediate / primitive progenitor cells (D0-D5) over 5 days into a heterogeneous mixture of cells that are primarily granulopoietic progenitors, termed IMANp. - Optional cryopreservation of IMANp.

[0617] The materials used to prepare IMANp are as follows: Table 2 - Reagents used to prepare IMANp Material Supplier Cat. No. Materials for IMDM manufacturing process with high glucose, glutamine, HEPES, phenol red and sodium pyruvate content (referred to as IMDM in the remainder of the document) Life Technologies 12440053 (or equivalent) Penicillin / streptomycin Life Technologies 15140122 (or equivalent) G-CSF PeproTech EC 300-23 SCF PeproTech EC 300-07 FLT-3 PeproTech EC 300-19 TPO PeproTech EC 300-18 IL-3 PeproTech EC 200-03 IL-6 PeproTech EC 200-06 ITS Life Technologies 41400045 HSA Stratech Scientific Limited 30-2026-FIT-100ml Tissue culture grade water Merck Life Science UK Limited W3500 (or equivalent) GRex6 Well Plate Wilson Wolf 80240M Plate GRex6M Wilson Wolf 80660M Gas-Permeable Cell Culture Device GRex10M Wilson Wolf 80110, 80110-CS and 80110S Gas-Permeable Cell Culture Device G- Wilson Wolf 81100, 81100-CS and RU81100 Petition 870250102349, dated 07 / 11 / 2025, pages 173 / 209 165 / 177 Rex100M CryoStor CS10 Merck Life Science UK Limited C2874-100ML Analytical Testing Materials 96-well U-bottom plate VWR 734-2080 (or equivalent) BD Cellfix 10X BD 349202 (or equivalent) 50 ml tubes Appleton Woods AB034 (or equivalent) Efluor780 Live / Dead Staining Life Technologies 65-0865-14 (or equivalent) Solution 18, AO-DAPI Staining Reagent Chemometec 910-3018 Table 3 - GMP reagents used to prepare IMANp Cytokine Supplier of RUO Minimum Activity (IU / mg) Supplier of GMP Activity of the batch available for experiments (IU / mg) FLT3-L PeproTech > 1E+06 PeproTech1 Not available in CoA SCF PeproTech > 5E+05 PeproTech1 Not available in CoA TPO PeproTech > 1E+06 BioTechne >1E+07 2.54E+08 IL-3 PeproTech > 1E+07 PeproTech1 Not available in CoA IL-6 PeproTech > 1E+07 PeproTech1 Not available in CoA G-CSF PeproTech > 1E+07 BioLegend Not available in CoA GM-CSF PeproTech > 1E+07 BioTechne >1E+07 2.00E+07 BioTechne NA TNFa ThermoFisher Scientific > 2E+07 BioTechne >4.3E+07 4.90E+07 Alternative supplier - BioTechne Preparation of the medium

[0618] Cytokines are reconstituted in cell culture grade water with 5% HSA and aliquots are stored at -80 °C before addition to the medium. Means of expansion

[0619] CD34+ HSCs are expanded in expansion media containing Iscove-Modified Dulbecco Medium (IMDM) with cytokines including SCF, FLT-3, TPO, IL3 and IL6, as well as ITS and Petition 870250102349, dated 07 / 11 / 2025, pages 174 / 209 166 / 177 HAS, at the following concentrations: Table 4 - Components of the expansion medium Final Expansion Medium IMDM Unit with Glutamax SCF 0.2 mg / mL FLT-3 0.2 mg / mL TPO 0.02 mg / mL IL3 0.015 mg / mL IL6 0.015 mg / mL ITS 1 x HSA 1% Means of differentiation

[0620] Cells are differentiated in differentiation media containing IMDM, SCF, TPO, GCSF, ITS, and HSA, at the following concentrations: Table 5 - Constituents of the differentiation medium Final Differentiation Medium Unit IMDM with Glutamax SCF 0.13 mg / mL TPO 0.13 mg / mL G-CSF 0.13 mg / mL GM-CSF 0.01 mg / mL IL-3 0.13 mg / mL TNF 0.001 mg / mL ITS 1 x HSA 1% HSC CD34+ Expansion

[0621] Donor CD34 HSCs were thawed on Day 0 (E0) at 37 °C in a water bath and transferred to thawing medium consisting of IMDM and 1% HSA. Cell counts and viability measurements for all donor samples were performed immediately after thawing. Cells were subsequently seeded at 5e5 / mL and 5e5 / cm2 in expansion medium in G-Rex 6M or G-Rex 10M with a surface area of ​​10 cm2 in a volume of 10 mL per well.

[0622] On day 1 (E1), samples were collected for cell counting, viability, and flow cytometry analysis. Petition 870250102349, dated 07 / 11 / 2025, pages 175 / 209 167 / 177 for phenotypic characterization using progenitor and neutrophil panels. The wells were filled with 40 mL of expansion medium to increase the volume to 4 mL / cm2. In E2 and E3, the cells were left intact in G-Rex for continuous expansion.

[0623] In E4, cells were transferred to G-Rex with a larger surface area, for example, 1 G-Rex 100M seeded from 1 G-Rex 6M or G-Rex 10M. The G-Rex was carefully removed from the incubator and the expansion medium was removed to 15 mL per well. Cells were resuspended in residual volume by shaking, after which a sample was collected for cell counting, viability, and flow cytometry analysis for phenotypic characterization using progenitor, neutrophil, and mature neutrophil panel phenotypes. Cells were subsequently transferred to a G-Rex 100M and 85 mL of fresh expansion medium were added to the G-Rex. The cells were left intact at E5, and at E6, an optional sample could be collected for cell counting, viability, and flow cytometry analysis for phenotypic characterization using progenitors, neutrophils, mature neutrophils, and off-target myeloid and lymphoid panels.Furthermore, each well was fed with 100 mL of expansion medium and left for 48 hours. The cells were left intact at E7. At E8, the G-Rex was carefully removed from the incubator and the expansion medium was removed, so that the volume was 100 mL per well. The cells were resuspended by shaking and samples collected for cell counting, viability, and flow cytometry analysis for phenotypic characterization using progenitors, neutrophils, mature neutrophils, myeloid and lymphoid off-target panels. Media exchange was subsequently performed to initiate the differentiation process. Differentiation of intermediate progenitor ce...

Claims

CLAIMS 1. Granulopoietic cell, or a population of such cells, characterized in that it is for use in amplifying a therapeutic immune response in the treatment of cancer.

2. Granulopoietic cell, or a population of such cells, characterized by being for use in amplifying a non-granulocytic therapeutic immune response in the treatment of an infection.

3. Granulopoietic cell, or population of such cells, for use according to claim 1 or 2, characterized in that the cell is CD62L-.

4. Granulopoietic cell, or a population of such cells, for use in accordance with any of claims 1 to 3, characterized in that the granulopoietic cell or population of such cells has a marker expression profile: CD10-, CD11b-, CD16-, CD62L-, CD66b-, CD177-, CD15+, CD38+, CD49d+, CD54+, CD63+.

5. Granulopoietic cell, or a population of such cells, for use in accordance with any one of claims 1 to 3, characterized in that the granulopoietic cell has an expression profile of the markers CD11bhi CD15+ CD66b+ CD177+ CD18hi CD16- CD34- CD38- CD49d-.

6. Granulopoietic cell, or a population of such cells, for use in accordance with any one of claims 1 to 3, characterized in that the granulopoietic cell has an expression profile of the markers CD34+ / -, CD38+ / -, CD15+ / -, CD49d+, CD18+, CD66b-, CD177-, CD16-.

7. Granulopoietic cell, or a population of such cells, for use in accordance with any of claims 1 to 3, characterized in that the granulopoietic cell or population of such cells has a marker expression profile: CD10-, CD11b-, CD16-, CD62L-, CD66b-, CD177-, CD15+, Petition 870250102349, dated 07 / 11 / 2025, page 204 / 209 2 / 6 CD38+, CD49d+, CD54+, CD63+.

8. Granulopoietic cell, or a population of such cells, for use in accordance with any of claims 1 to 3, characterized in that the granulopoietic cell or population of such cells has a marker expression profile: CD11bhi CD15+ CD66b+ CD177+ CD18hi CD16- CD34- CD38CD4 9d-.

9. Granulopoietic cell, or a population of such cells, for use in accordance with any of claims 1 to 3, characterized in that the granulopoietic cell or population of such cells has a marker expression profile: CD34+ / -, CD38+ / -, CD15+ / -, CD49d+, CD18+, CD66b-, CD177-, CD16-.

10. Granulopoietic cell, or a population of such cells, for use in accordance with any one of claims 1 to 9, characterized in that the granulopoietic cell is capable of differentiating into granulocytes with the ability to kill cancerous cells.

11. Granulopoietic cell, or a population of such cells, for use in accordance with any one of claims 1 to 10, characterized in that the therapeutic immune response is a therapeutic immune response of the host.

12. Granulopoietic cell, or a population of such cells, for use in accordance with any one of claims 1 to 11, characterized in that the granulopoietic cell is allogeneic.

13. Granulopoietic cell, or a population of such cells, for use in accordance with any one of claims 1 to 12, characterized in that the granulopoietic cell is capable of differentiating to produce a cell that secretes CXCL10 and / or expresses a ligand for a co-stimulatory molecule selected from the group consisting of: 4-1BBL; and OX40L.

14. Granulopoietic cell, or a population of such cells, for use in accordance with claim 1 or any of claims 3 to 13 when dependent on claim 1, characterized in that the cancer is selected from the group consisting of: 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 and breast cancer.

15. Granulopoietic cell, or a population of such cells, for use according to claim 2 or any of claims 3 to 13 when dependent on claim 2, characterized in that the infection is selected from the group consisting of: a viral infection; a bacterial infection; and a fungal infection.

16. Granulopoietic cell, or a population of such cells, for use in accordance with any of claims 1 to 15, characterized in that it is for use in amplifying a non-granulocytic therapeutic immune response by increasing the activation of non-granulocytic immune cells.

17. Granulopoietic cell, or a population of such cells, for use according to claim 16, characterized in that activation increases the expression by immune cells of a degranulation marker selected from the group consisting of: CD107a; perforin and granzymes.

18. Granulopoietic cell, or a population of such cells, for use according to claim 16 or 17, characterized in that activation increases the expression by immune cells of a co-stimulatory molecule selected from the group consisting of: 4-1BB; OX40; CD27; CD28; ICOS; HVEM; LIGHT; CD40L; DR3; GITR; CD30; TIM1; CD2 and CD226.

19. Granulopoietic cell, or a population of such cells, for use in accordance with any of the claims Petition 870250102349, dated 07 / 11 / 2025, pp. 206 / 209 4 / 6 1 to 18, characterized in that it is for use in amplifying a therapeutic immune response by increasing T cell activation.

20. Granulopoietic cell, or a population of such cells, for use according to claim 19, characterized in that the T cells are selected from the group 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-infiltrating T cell.

21. Granulopoietic cell, or a population of such cells, for use in accordance with any of claims 1 to 20, characterized in that it is for use in amplifying a therapeutic immune response by increasing the activation of NK cells.

22. Granulopoietic cell, or a population of such cells, for use in accordance with any one of claims 1 to 21, characterized in that it is for use in amplifying a therapeutic immune response by increasing the activation of PBMCs.

23. Granulopoietic cell, or a population of such cells, for use in accordance with any of claims 1 to 22, characterized in that it is for use in amplifying a therapeutic immune response by increasing the activation of TILs.

24. Granulopoietic cell, or a population of such cells, for use in accordance with any of claims 1 to 23, characterized in that it is for use in amplifying a therapeutic immune response by increasing the tumor cell-destroying activity of immune cells.

25. Granulopoietic cell, or a population of such cells, for use in accordance with any of claims 1 to 24, characterized in that it is for use in Petition 870250102349, dated 07 / 11 / 2025, pp. 207 / 209 5 / 6 amplification of a therapeutic immune response by increasing the proliferation of immune cells.

26. Granulopoietic cell, or a population of such cells, for use in accordance with any one of claims 1 to 25, characterized in that it is for use in combination with additional cellular immunotherapy.

27. A method for promoting the therapeutic activity of non-granulocytic immune cells, the method being characterized in that it comprises incubating a non-granulocytic immune cell with a granulopoietic cell, or a population of such cells, as defined in any one of claims 1 to 26.

28. Pharmaceutical composition, characterized in that it comprises an enriched population of granulopoietic cells, as defined in any one of claims 1 to 26.

29. Method for selecting an appropriate treatment regimen for a patient, the method being characterized in that it comprises: • identifying whether the patient has an impaired non-granulocytic immune response; wherein • if the patient is identified as having an impaired non-granulocytic immune response, then treatment with a granulopoietic cell 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 granulopoietic cell is selected.

30. Method for selecting an appropriate treatment regimen for a patient, the method being characterized in that it comprises: • incubating a non-granulocytic immune cell from the patient Petition 870250102349, dated 07 / 11 / 2025, pp. 208 / 209 6 / 6 with a granulopoietic cell; wherein • if the activity of the patient's non-granulocytic immune cell is increased in response to incubation, then treatment with a granulopoietic cell is selected as an appropriate treatment; and • if the activity of the patient's non-granulocytic immune cell is increased in response to incubation, then treatment with a therapy other than a granulopoietic cell is selected.

31. A method for identifying whether a granulopoietic cell is suitable or not for use in treatment by amplifying a non-granulocytic therapeutic immune response, the method being characterized in that it comprises: • incubating the granulopoietic cell, or a cell derived from the granulopoietic cell, with immune cells; and • evaluating whether the granulopoietic cell is capable of increasing the activation of immune cells; and identifying whether a granulopoietic cell is suitable or not for use in treatment by amplifying a non-granulocytic therapeutic immune response based on this evaluation.

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