Immunomodulatory cells and compositions

Abstract

Granulocyte-producing cells, or populations of such cells, are provided for use in modulating therapeutic immune responses. The therapeutic immune response may be a non-granulocyte immune response. Granulocyte-producing cells, or populations of such cells, can be used to amplify therapeutic immune responses, such as non-granulocyte immune responses. The granulocyte-producing cells or populations of cells may be CD62L-. The granulocyte-producing cells or populations of cells may have marker expression profiles: CD10-, CD11b-, CD16-, CD62L-, CD66b-, CD177-, CD15+, CD38+, CD49d+, CD54+, CD63+. The granulocyte-producing cells or populations of cells can be used in the treatment of cancer or infections.

Description

[Technical Field]

[0001] The present invention relates to cells for use in modulating, for example, amplifying, a therapeutic immune response, and to methods of therapy using such cells. The therapeutic immune response may be a nongranulocyte immune response. The present invention also relates to pharmaceutical compositions. The present invention further relates to screening methods and methods useful for cell culture of immune cells. [Background technology]

[0002] The immune response plays a crucial role in the body's fight against cancer or infection. This natural response can be enhanced through the use of immunotherapy, and its use in such therapeutic situations is becoming increasingly important.

[0003] However, cancer and many infectious pathogens employ strategies that allow them to evade or reduce the effects of the immune system. Infectious pathogens may reside within the body's own cells, thereby evading immune surveillance. Tumors can adapt to an immunologically "cold" state and create an immunosuppressive tumor microenvironment (TME) that can neutralize the anti-tumor immune response.

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

[0005] In a first aspect, the present invention provides granulocyte-producing cells, or optionally populations of such cells, for use in modulating a therapeutic immune response. Preferably, the therapeutic immune response is a nongranulocyte immune response. In a preferred embodiment, the granulocyte-producing cells or populations of such cells are intended for use in amplifying a therapeutic immune response, such as a nongranulocyte immune response.

[0006] In a second aspect, the present invention provides a method of treatment comprising modulating a therapeutic immune response, comprising providing granulocyte-producing cells, or optionally a population of such cells, to a subject requiring such treatment. The therapeutic immune response may be a non-granulocyte immune response. The method of treatment may also comprise amplifying the immune response.

[0007] In a third aspect, the present invention provides granulocyte-producing cells, or optionally populations of such cells, for use in the manufacture of agents for use in modulating a therapeutic immune response. The therapeutic immune response to be modulated may be a non-granulocyte immune response. The therapeutic immune response may be amplified.

[0008] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a concentrated population of granulocyte-producing cells.

[0009] In a fifth aspect, the present invention provides a method for promoting the therapeutic activity of nongranulocyte immune cells, the method comprising incubating nongranulocyte immune cells with granulocyte-producing cells.

[0010] In a sixth aspect, the present invention provides a method for selecting an appropriate treatment regimen for a patient, the method being: This includes identifying whether the patient has impaired nongranulocyte immune response, If the patient is identified as having impaired non-granulocyte immune response, treatment with granulocyte-producing cells is selected as the appropriate treatment. If the patient is identified as having no impairment in the non-granulocyte immune response, treatment with therapies other than granulocyte-producing cells will be selected.

[0011] In a seventh aspect, the present invention provides a method for selecting an appropriate treatment regimen for a patient, the method being: This includes incubating the patient's nongranulocyte immune cells together with granulocyte-producing cells. If the activity of non-granulocyte immune cells in the patient increases in response to incubation, then treatment with granulocyte-producing cells is selected as the appropriate treatment. If the activity of the patient's non-granulocyte immune cells increases in response to incubation, treatment with therapies other than granulocyte-producing cells will be selected.

[0012] In an eighth aspect, the present invention provides a method for increasing the viability of immune cells in culture, the method comprising culturing immune cells in the presence of a feeder layer of granulocyte-producing cells.

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

[0014] A tenth aspect of the present invention provides a method for determining whether granulocyte-producing cells are suitable for use in the treatment of cancer by beneficially modulating the tumor microenvironment, the method being • To evaluate whether the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, can express inflammatory cytokines, and / or • To evaluate whether the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, can stimulate the expression of inflammatory cytokines by non-granulocyte immune cells, Based on this evaluation, we will identify whether granulocyte-producing cells are suitable for use in cancer treatment by beneficially modulating the tumor microenvironment. Includes.

[0015] In an eleventh aspect, the present invention provides a method for determining whether granulocyte-producing cells are suitable for use in the treatment of cancer by increasing the recruitment of immune cells to tumors and / or immune cell activation, the method is • To evaluate whether the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, can express chemokines associated with promoting cell transport, and / or • To evaluate whether the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, can stimulate the expression of degranulation markers by non-granulocyte immune cells, Based on this evaluation, we will determine whether granulocyte-producing cells are suitable for use in the treatment of cancer by increasing the recruitment of immune cells to tumors and / or immune cell activation. Includes.

[0016] In a twelfth aspect, the present invention provides a method for determining whether granulocyte-producing cells are suitable for use in the treatment of cancer by directly promoting the death of cancer cells, the method being: • Incubating the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, together with cells of a cancer cell line. • To evaluate whether the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, can increase cell death in cancer cell lines to a greater extent than the death of non-cancer cells, Based on this evaluation, we will determine whether granulocyte-producing cells are suitable for use in cancer treatment by directly promoting the death of cancer cells. Includes.

[0017] In a thirteenth aspect, the present invention provides a method for determining whether granulocyte-producing cells are suitable for use in treating infectious diseases by directly promoting the death of cell-infecting pathogens or infected cells, the method being: • Incubating the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, together with a sample of a cell-infecting pathogen or infected cells. • To evaluate whether the granulocyte-producing cells, or cells derived from such granulocyte-producing cells, can increase the death of cell-infecting pathogens or infected cells, Based on this evaluation, we will determine whether granulocyte-producing cells are suitable for use in treating infections by directly promoting the death of cell-infecting pathogens or infected cells. Includes.

[0018] In a fourteenth aspect, the present invention provides a method for determining whether granulocyte-producing cells are suitable for use in therapy by amplifying a therapeutic immune response, the method being: • Incubating the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, together with immune cells. • To evaluate whether the granulocyte-producing cells can increase the activation of the immune cells, Based on this evaluation, we will determine whether granulocyte-producing cells are suitable for use in therapy by amplifying therapeutic immune responses. Includes.

[0019] Granulocyte-producing cells suitable for use according to the present invention are: ·below: · G-CSF, GM-CSF, · IL-3 and TNF This can be prepared by a method that includes culturing a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells, including the presence of a certain substance, to generate a population of granulocyte-producing cells. [Brief explanation of the drawing]

[0020] [Figure 1] This study demonstrates the effect of granulocyte-producing cells on the activation of blood-derived CD8+ T cells, as evaluated by the expression of degranulation markers or costimulatory molecules. [Figure 2] This study demonstrates the effect of granulocyte-producing cells on the activation of blood-derived CD4+ T cells, as evaluated by the expression of co-stimulatory molecules. [Figure 3] This shows the effect of granulocyte-producing cells on the activation of αβT cells, which was evaluated in relation to T cell proliferation. [Figure 4] This study demonstrates the effect of granulocyte-producing cells on the activation of blood-derived NK cells and NKT cells, which were evaluated in terms of cell survival. [Figure 5] This study demonstrates the effect of granulocyte-producing cells on the activation of blood-derived natural killer (NK) cells and natural killer T (NKT) cells, as evaluated by the expression of degranulation markers or co-stimulatory molecules. [Figure 6]This study demonstrates the effect of granulocyte-producing cells on the activation of blood-derived CD8+ T cells, CD4+ T cells, and NK cells, as evaluated by the expression of degranulation markers or costimulatory molecules. [Figure 7] This study demonstrates the effect of granulocyte-producing cells on the activation of peripheral blood mononuclear cells (PBMCs) in relation to cytokine expression. [Figure 8] This study demonstrates the effect of granulocyte-producing cells on the activation of tumor-infiltrating lymphocytes (TILs) in relation to cytokine expression. [Figure 9] This study demonstrates the effect of granulocyte-producing cells on the activation of immune cells in relation to immune cell transport. [Figure 10] This study demonstrates the effect of granulocyte-producing cells on the activation of immune cells in terms of cell destructive activity. [Figure 11] This shows the expression of chemokines by granulocytes formed during the differentiation of granulocyte-producing cells. [Figure 12] This shows the expression of ligands for co-stimulatory molecules by granulocytes formed during the differentiation of granulocyte-producing cells. [Figure 13] This shows the relative proportion of subpopulations of granulocyte-producing cells generated by the method of the present invention, either without priming or using various priming steps. [Figure 14] Further characterizing the first subpopulation of identified granulocyte-producing cells. [Figure 15] Further characterizing the second subgroup of identified granulocyte-producing cells. [Figure 16] Further characterizing the third subgroup of identified granulocyte-producing cells. [Figure 17] Further characterizing the fourth subgroup of identified granulocyte-producing cells. [Modes for carrying out the invention]

[0021] This invention is based on the inventors' discovery that granulocyte-producing cells can be used, at least to some extent, to modulate therapeutic immune responses.

[0022] In particular, the inventors have identified that the types of granulocyte-producing cells described herein may increase the activation or recruitment of immune cells, especially non-granulocyte immune cells, in a manner that enables amplification of therapeutic immune responses. This realization makes it possible to use such granulocyte-producing cells, which the inventors have designated as “immunomodulatory alpha-neutrophil progenitor cells” (IMANP), to enhance immunotherapeutic treatments in many conditions, including (but not limited to) cancer treatment. By amplifying the immune response, the medical uses and therapeutic methods of the present invention can make otherwise immunologically “cold” tumors “hot” and thus responsive to treatment.

[0023] The amplification that occurs with respect to therapeutic immune responses is not simply due to an increase in the number of granulocytes, for example, as a result of the administration of granulocyte-producing cells. Instead, granulocyte-producing cells appear to be able to significantly increase the activation of non-granulocyte immune cells, particularly T cells and NK cells, thereby enhancing the resulting immune response. As will be discussed in more detail below and as shown in the examples, this can be achieved by activated non-granulocyte cells, which can increase the expression of degranulation markers, costimulatory molecules, and cytokines. It can also increase the proliferation and survival of activated non-granulocyte cells, leading to an increase in the accumulation of such cells. The inventors have also demonstrated that activated non-granulocyte immune cells exhibit an increased degree of recruitment to TMEs and increased cell-destructive activity (particularly increased tumor cell-killing activity).

[0024] These properties suggest that granulocyte-producing cells may be used therapeutically in the treatment of cancer, and that such therapies may also be usable to enhance other cell-based immunotherapies.

[0025] Furthermore, the granulocyte-producing cells used according to the present invention may themselves be able to differentiate into granulocytes that have the ability to kill cancer cells. Thus, the treatment according to the present invention can achieve a dual mode of action: amplifying the non-granulocyte immune response and generating granulocytes that can directly kill cancer cells.

[0026] The granulocyte-producing cells used in the medical use and therapeutic methods of the present invention express many markers that suggest they have differentiation stages that closely correspond to the differentiation stages of myeloblasts or promyelocytes. However, unlike naturally occurring cells, granulocyte-producing cells that have been found useful in medical use and therapeutic methods can be characterized in that they do not express CD62L.

[0027] The inventors have demonstrated that granulocyte-producing cells suitable for use in the medical use of the present invention or in the methods of the present invention can amplify immune responses through several different mechanisms. In particular, granulocyte-producing cells can increase the activation of immune cells and increase the activity required for a successful therapeutic immune response (such as cell transport and cell destructive activity).

[0028] Surprisingly, the inventors discovered that these effects can be achieved by using allogeneic granulocyte-producing cells in patients receiving therapeutic treatment with granulocyte-producing cells.

[0029] The modulation may, for example, be the modulation of the therapeutic immune response of the subject to which the granulocyte-producing cells in the pharmaceutical composition of the present invention are administered. Alternatively, the pharmaceutical composition of the present invention may further contain immune cells (preferably non-granulocyte immune cells) in addition to the enumerated granulocyte-producing cells, and the modulation of the immune response may be with respect to such additional immune cells in the composition. Preferably, the modulation is the amplification of the therapeutic immune response.

[0030] The present invention will now be further described with reference to the following paragraphs.

[0031] granulopoietic cells In some aspects, the present invention relates to medical uses, methods, and products using granulocyte-producing cells. These granulocyte-producing cells can amplify non-granulocyte therapeutic immune responses, as will be considered in more detail elsewhere in this specification.

[0032] For a cell to be considered "granulocyte-generating" in the terminology of this invention, it must be able to produce granulocytes (e.g., neutrophils) or granulocyte precursor cells of the granulocyte lineage. Preferably, granulocyte-generating cells in this context are those that produce granulocytes or granulocyte precursor cells of the granulocyte lineage. To avoid misunderstanding, granulocytes themselves should be considered "granulocyte-generating" for the purposes of this invention, but in many embodiments, granulocyte-generating cells are not granulocytes themselves, but cells capable of producing granulocytes (preferably cells that produce granulocytes). Preferably, granulocyte-generating cells are not neutrophils.

[0033] Other useful methods for defining related granulocyte-producing cells are listed below.

[0034] Granulocyte-producing cells suitable for use in the medical and methodological applications of the present invention may be defined with respect to their marker profiles. For example, in a preferred embodiment, granulocyte-producing cells do not express one or more markers selected from the group consisting of CD10, CD11b, CD16, CD62L, CD66b, and CD177. Preferred granulocyte-producing cells may lack the expression of one, two, three, four, five, or all six of these markers (i.e., preferred granulocyte-producing cells may lack the expression of CD10 - and / or CD11b - and / or CD16 - and / or CD62L - and / or CD66b - and / or CD177 - (This is possible.)

[0035] In a preferred embodiment, granulocyte-producing cells are CD62L -It is. The lack of CD62L expression may be useful in identifying granulocyte-producing cells well-suited for use according to various aspects of the present invention.

[0036] Furthermore, or alternatively, the granulocyte-producing cells may be CD66b - It may be. The lack of CD66b expression suggests a differentiation stage corresponding to the myeloblast differentiation stage and may be useful in identifying granulocyte-producing cells well-suited for use according to various aspects of the present invention.

[0037] In a preferred embodiment, the granulocyte-producing cells express one or more markers from the group consisting of CD15, CD38, CD49d, CD54, and CD63. Preferred granulocyte-producing cells may express 1, 2, 3, 4, or all 5 of these markers (i.e., preferred granulocyte-producing cells may be CD15 + and / or CD38 + and / or CD49d + and / or CD54 + and / or CD63 + ).

[0038] Granulocyte-producing cells suitable for use in various aspects of the present invention are CD10 - , CD11b - , CD16 - , CD62L - , CD66b - , CD177 - , CD15 + , CD38 + , CD49d + , CD54 + , CD63 + . A further aspect of the present invention is CD10 - , CD11b - , CD16 - , CD62L - , CD66b - , CD177 - , CD15 + , CD38 + , CD49d + , CD54 + , CD63 +It provides granulocyte-producing cells.

[0039] The inventors have identified two further populations of granulocyte-producing cells that can be used in various embodiments of the present invention and can be identified based on their marker expression profiles. The cells of the first population are CD11b hi CD15 + CD66b + CD177 + CD18 hi CD16 - CD34 - CD38 - CD49d - The cells of the second population are CD34. + / - CD38 + / - CD15 + / - CD49d + CD18 + CD66b - CD177 - CD16 - Of these, the first group is considered to be a more mature group of cells than the second group.

[0040] Further aspects of the present invention include CD11b hi CD15 + CD66b + CD177 + CD18 hi CD16 - CD34 - CD38 - CD49d - The present invention provides granulocyte-producing cells. Further aspects of the present invention include CD34 + / - CD38 + / - CD15 + / - CD49d + CD18 + CD66b - CD177 - CD16 - It provides granulocyte-producing cells.

[0041] As will be described in more detail below, preferred isolated populations of granulocyte-producing cells that can be used in the medical use, method, or product of the present invention are: • The first subgroup of cells that are CD15+CD64+CD18+CD49d+CD71+ • A second subgroup of cells that are CD15-CD11b+ / -CD18+CD49d+CD32+HLA-DR- • A third subgroup of cells that are CD15-CD11b-HLA-DR+CD18+CD49d+ and CD71+ It may include.

[0042] Such a suitable population of granulocyte-producing cells may further include a fourth subpopulation of cells that are CD15-CD11b+HLA-DR+.

[0043] Further details of characteristic marker profiles that may be used to identify these useful granulocyte-producing cell populations (and subpopulations), as well as methods by which they may be generated, are described further elsewhere in this specification.

[0044] Those skilled in the art will be well aware of suitable methods for isolating and, if necessary, enriching a population of cells based on the expression of a specific profile of cell surface markers.

[0045] Granulocyte-producing cells suitable for use in the medical and methodological applications of the present invention may be defined in terms of their efficacy. In preferred embodiments, granulocyte-producing cells are unipotent cells.

[0046] Preferred granulocyte-producing cells for use in various embodiments of the present invention may be defined with respect to their differentiation state within the granulocyte-producing pathway. In preferred embodiments, granulocyte-producing cells have a differentiation stage corresponding to the differentiation stage between myeloblasts and granulocytes. Preferably, granulocyte-producing cells have a differentiation stage corresponding to the differentiation stage between myeloblasts and band neutrophils. For example, granulocyte-producing cells may have a differentiation stage corresponding to the differentiation stage between myeloblasts and metamyelocytes. Preferably, granulocyte-producing cells have a differentiation stage corresponding to the differentiation stage between myeloblasts and myelocytes. Preferably, granulocyte-producing cells have a differentiation stage corresponding to the differentiation stage between myeloblasts and promyelocytes.

[0047] In preferred embodiments, granulocyte-producing cells have a differentiation stage corresponding to myeloblasts. In preferred embodiments, granulocyte-producing cells have a differentiation stage corresponding to promyelocytes. In preferred embodiments, granulocyte-producing cells have a differentiation stage corresponding to myelocytes. In preferred embodiments, granulocyte-producing cells have a differentiation stage corresponding to metamyelocytes. In preferred embodiments, granulocyte-producing cells have a differentiation stage corresponding to band neutrophils.

[0048] In a preferred embodiment, granulocyte-producing cells have a differentiation stage corresponding to granulocytes.

[0049] As described elsewhere in this specification, granulocyte-producing cells suitable for use in various embodiments of the present invention may be derived from artificial stem cells such as iPSCs. Such granulocyte-producing cells may not be identical to naturally occurring cells in the granulocyte-producing pathway, but it will be understood that they may share structural (e.g., marker expression) or functional (e.g., potency) characteristics with such naturally occurring cells. References in the preceding paragraph to cells having a differentiation stage "corresponding" to the cell type specified therein should be interpreted accordingly.

[0050] Preferably, granulocyte-producing cells are selected from the group consisting of myeloblasts, promyelocytes, myelocytes, metamyelocytes, band neutrophils, and granulocytes. Preferably, granulocyte-producing cells are selected from the group consisting of myeloblasts, promyelocytes, myelocytes, metamyelocytes, and band neutrophils. Preferably, granulocyte-producing cells are selected from the group consisting of myeloblasts, promyelocytes, myelocytes, and metamyelocytes. Preferably, granulocyte-producing cells are selected from the group consisting of myeloblasts, promyelocytes, and myelocytes. Preferably, granulocyte-producing cells are selected from the group consisting of myeloblasts and promyelocytes.

[0051] In a preferred embodiment, the granulocyte-producing cell is a myeloblast. In a preferred embodiment, the granulocyte-producing cell is a promyelocyte. In a preferred embodiment, the granulocyte-producing cell is a myelocyte. In a preferred embodiment, the granulocyte-producing cell is a metamyelocyte. In a preferred embodiment, the granulocyte-producing cell is a band neutrophil. In a preferred embodiment, the granulocyte-producing cell is a granulocyte.

[0052] Preferably, the granulocyte-producing cells are committed to the neutrophil lineage. In such embodiments, the preferred granulocyte-producing cells may be selected from the group consisting of neutrophil promyelocytes, neutrophil myelocytes, neutrophil metamyelocytes, neutrophil band neutrophils, and neutrophils.

[0053] As further described elsewhere in this specification, granulocyte-producing cells that can be used in various embodiments of the present invention may also be defined with respect to granulocytes that they can induce differentiation of. Preferred examples of granulocyte-producing cells may be able to produce granulocytes that have the ability to kill cancer cells and / or infected cells. Alternatively, or further, preferred granulocyte-producing cells may be able to produce granulocytes that have a desirable expression profile of molecules such as chemokines or costimulatory receptor ligands.

[0054] Concentrated population of granulocyte-producing cells Granulocyte-producing cells that can be used in various embodiments of the present invention may be provided in the form of an enriched population of such granulocyte-producing cells.

[0055] As a mere example, such a fermented population may be a population of cells in which granulocyte-producing cells constitute at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, or at least 1% of the total cell population. Such a fermented population may further be a population of cells in which granulocyte-producing cells constitute at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% of the total cell population. In fact, a concentrated population may be a population of cells in which granulocyte-producing cells constitute at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or substantially 100% of the total cell population present.

[0056] The granulocyte-producing cells of such a concentrated population may be as defined in any preferred embodiment described elsewhere in this specification. For example, the granulocyte-producing cells of the concentrated population may be CD62L - It is possible.

[0057] Pharmaceutical composition of the present invention A fourth aspect of the present invention provides a pharmaceutical composition comprising a concentrated population of granulocyte-producing cells. The concentrated population of granulocyte-producing cells incorporated into the pharmaceutical composition of the present invention may be as considered above.

[0058] Preferably, the granulocyte-producing cells present in the pharmaceutical composition of the present invention are CD62L - It is possible.

[0059] 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.

[0060] Preferably, the pharmaceutical composition contains granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone, serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, serum (e.g., fetal bovine serum [FBS]), retinoic acid, lipopolysaccharide (LPS), IFN-gamma, IFN-beta, or a combination thereof. Preferably, the pharmaceutical composition contains IFN-gamma and GM-CSF. Preferably, the pharmaceutical composition contains TNF-alpha. Particularly preferred, the pharmaceutical composition comprises granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), growth hormone, serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, and fetal bovine serum (FBS). Preferably, the pharmaceutical composition includes granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone, serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, fetal bovine serum (FBS), retinoic acid, lipopolysaccharide (LPS), IFN-gamma, and IFN-beta.

[0061] In preferred embodiments, the pharmaceutical composition of the present invention comprises (or further comprises) granulocytes in addition to granulocyte-producing cells. In preferred embodiments, the pharmaceutical composition of the present invention comprises neutrophils in addition to granulocyte-producing cells.

[0062] Therapeutic immune response In the context of the present invention, a therapeutic immune response should be interpreted as an immune response that contributes to or achieves a desired therapeutic outcome. In a preferred embodiment, a therapeutic immune response may be an immune response that leads (directly or indirectly) to the death of cancer cells, thereby enabling the treatment of cancer. In a preferred embodiment, a therapeutic immune response may be an immune response that leads (directly or indirectly) to the death of infected cells or cell-infecting pathogens, thereby enabling the treatment of an infectious disease.

[0063] A therapeutic immune response may involve the action of any cell in the immune system. A "non-granulocyte immune response" may involve the action of any cell in the immune system other than granulocytes. As merely an example, a therapeutic immune response that can be amplified by the medical use, method of treatment, or pharmaceutical composition of the present invention may involve T cells (CD8 + T cells, CD4 + This may include the action of one or more cell types selected from the group consisting of T cells, NK T cells, αβ T cells, γδ T cells, peripheral blood T cells, and tumor-infiltrating T cells (but not limited to these), NK cells, monocytes, macrophages, dendritic cells (DCs), and B cells.

[0064] Amplification of therapeutic immune response Amplification of the immune response may 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 costimulatory molecules by immune cells involved in the immune response; increased proliferation by immune cells involved in the immune response; increased survival by immune cells involved in the immune response; increased abundance of immune cells involved in the immune response; increased expression of cytokines by immune cells involved in the immune response; increased transport by immune cells involved in the immune response; increased recruitment of immune cells involved in the immune response to TMEs; increased cytotoxicity by immune cells involved in the immune response; or increased tumor cell death activity by immune cells involved in the immune response.

[0065] Alternatively, or furthermore, amplification of the therapeutic immune response may be evaluated in relation to the outcomes achieved by the therapeutic immune response.

[0066] For example, in the case of therapeutic immune responses used in the treatment of cancer, amplification of the immune response can be demonstrated by an increase in the efficacy of the cancer treatment. Such an increase in efficacy can be demonstrated by symptom reduction, increased patient survival rate and / or survival time, reduced tumor burden, prevention or delay of recurrence, reduced severity of recurrence, reduced number of recurrences, reduced number of metastases, and / or prevention or delay of metastases.

[0067] In the case of therapeutic immune responses used to treat infectious diseases, amplification of the immune response can be demonstrated by an increase in the efficacy of the treatment for the infectious disease. Such an increase can be demonstrated by a reduction in symptoms, an increase in patient survival rate and / or survival time, a reduction in the infectious burden, and / or a reduction in the time to elimination of the infection.

[0068] Host cells and host immune response For the purposes of this disclosure, references to “host” cells (e.g., host immune cells) or “host” immune responses may be interpreted as referring to cells or immune responses of interest that are being treated, or are perceived to be being treated, by granulocyte-producing cells according to any of the various aspects of the Invention. Unless otherwise specified in the context, all references to immune cells or immune responses relating to the various aspects and embodiments of the Invention should be interpreted as applicable to host immune cells or host immune responses.

[0069] Increased activation of immune cells Granulocyte-producing cells suitable for use in various embodiments of the present invention may be able to increase the activation of immune cells. In particular, this type of cell may be able to increase the activation of host immune cells. Therefore, such cells may be able to amplify the host therapeutic immune response by increasing the activation of host immune cells.

[0070] It will be understood that these are activated immune cells primarily involved in providing the desired activity in a therapeutic immune response. Therefore, the ability of medical uses and therapeutic methods to increase the activation of immune cells would be beneficial in almost all situations where a therapeutic immune response is needed. In particular, the amplification of a therapeutic immune response by increasing the activation of immune cells may be advantageous in the treatment of cancer or infectious diseases, though not limited to these cases.

[0071] Preferably, granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells, such as by increasing the expression of one or more degranulation markers by immune cells. Preferably, granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells, such as by increasing the expression of one or more co-stimulatory molecules by immune cells. Preferably, granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells, such as by increasing the proliferation of immune cells. Preferably, granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells, such as by increasing the abundance of immune cells. Preferably, granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells, such as by increasing the survival of immune cells. Preferably, granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells, such as by increasing the expression of one or more cytokines by immune cells. Preferably, granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells, such as by increasing the transport of immune cells. Preferably, granulocyte-producing cells suitable for use in the present invention can increase the activation of immune cells, such that the cell-destructive activity of immune cells is increased.

[0072] Preferably, granulocyte-producing cells suitable for use according to the present invention may increase the activation of immune cells via "Signal 2" (co-stimulation). Alternatively, or further, granulocyte-producing cells suitable for use according to the present invention may increase the activation of immune cells via "Signal 3" (cytokine stimulation). Granulocyte-producing cells suitable for use according to the present invention may have the ability to increase the activation of immune cells via both Signal 2 and Signal 3.

[0073] Signals 2 and 3 are both known to be important in generating an effective immune response against tumors and in overcoming the immunosuppressive effects of TME. Therefore, our data (shown in the examples) demonstrating that granulocyte-producing cells suitable for use according to the present invention can provide these signals clearly demonstrate their suitability for use in amplifying therapeutic immune responses related to cancer treatment.

[0074] Granulocyte-producing cells suitable for use according to the present invention may exhibit some or all of the above characteristics.

[0075] Preferably, for example, for use according to the present invention, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) is sufficient to increase the activation of immune cells, such as host immune cells. The degree of increase, the host immune cells involved, and appropriate indicators of the increase in activation may be as considered in the preceding paragraph and / or in the following paragraph.

[0076] Increased T cell activation Granulocyte-producing cells suitable for use according to the present invention may increase T cell activation. In particular, granulocyte-producing cells suitable for use according to the present invention may increase host T cell activation. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing host T cell activation.

[0077] CD8 + and CD4 +It will be understood that increased activation of host T cells, such as CD8 cells, significantly contributes to the desired activity in the therapeutic immune response. + While cytotoxic T cells such as T cells are known to have direct cell-destroying activity, CD4 + Helper T cells, such as T cells, are known to assist in regulating the immune response by further stimulating other immune cells. Therefore, the use of granulocyte-producing cells to increase T cell activation may be beneficial in a wide range of situations where a therapeutically effective immune response is required. In particular, the amplification of a therapeutic immune response by increasing T cell activation may be advantageous in the treatment of cancer or infectious diseases, though not limited to these applications.

[0078] T cells whose activation can be increased, for example, host T cells, CD8 + T cells, CD4 + The group may be selected from T cells, NK T cells, αβ T cells, γδ T cells, peripheral blood T cells, and tumor-infiltrating T cells.

[0079] Increased T cell activation may be associated with one or more of the following: increased expression of T cell degranulation markers (including, but not limited to, CD107a); increased expression of T cell costimulatory molecules (including, but not limited to, 4-1BB and / or OX40); increased T cell cytokine expression; increased T cell transport; increased T cell recruitment to TMEs; increased T cell cytotoxicity (including, but not limited to, tumor cell death); increased T cell proliferation; increased T cell survival; and increased T cell abundance. Changes in these properties associated with increased T cell activation upon exposure to granulocyte-producing cells suitable for use according to the present invention are shown in the examples. Further relevant considerations regarding these various properties are described elsewhere in this specification.

[0080] T cell activation may increase by at least 5%. For example, T cell activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The quantification of the increase in T cell activation in such embodiments can be achieved by comparison with a suitable control.

[0081] Preferably, for use according to the present invention, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) is sufficient to increase the activation of T cells, such as host T cells. The degree of increase, and appropriate indicators of the increase in activation, may be as considered in the preceding paragraph and / or in the following paragraph.

[0082] CD8 + Increased T cell activation Granulocyte-producing cells suitable for use according to the present invention are CD8 + T cells, for example, host CD8 + It may increase T cell activation. Therefore, such granulocyte-producing cells may increase host CD8 + In some cases, increasing T cell activation can amplify the host's therapeutic immune response.

[0083] CD8 + Increased T cell activation is CD8 + Increased expression of degranulation markers by T cells (including, but not limited to, CD107a), CD8 + Increased expression of costimulatory molecules by T cells (including, but not limited to, 4-1BB and / or OX40), and CD8 + This may be associated with one or more of the increased proliferation of T cells. Further relevant considerations regarding these various characteristics are described elsewhere in this specification.

[0084] CD8 with increased activation +T The cells can be peripheral blood CD8 + T cells, or can be tumor infiltrating CD8 + T cells.

[0085] Activation of such CD8 + T cells may increase by at least 5%. For example, activation of CD8 + T cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the increase in activation of CD8 + T cells according to such embodiments can utilize comparison with a suitable control.

[0086] CD4 + Increase in activation of T cells Granulocyte - generating cells suitable for use according to the present invention can increase the activation of CD4 + T cells, e.g., host CD4 + T cells. Thus, such granulocyte - generating cells may be able to amplify the host's therapeutic immune response by increasing the activation of host CD4 + T cells.

[0087] CD4 + Increase in activation of T cells is associated with one or more of an increase in the expression of costimulatory molecules (including but not limited to 4 - 1BB and / or OX40) by CD4 + T cells, and an increase in the proliferation of CD4 + T cells. Further considerations regarding these various properties are described elsewhere in this specification.

[0088] CD4 activation increases +T The cells contain peripheral blood CD4 + Possibly T cells, or tumor-infiltrating CD4 + It could be a T cell.

[0089] Such CD4 + T cell activation may increase by at least 5%. For example, CD4 + T cell activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% or more. CD4 in such embodiments + The increase in T cell activation can be quantified by using comparison with an appropriate control.

[0090] Increased activation of NK T cells Granulocyte-producing cells suitable for use according to the present invention may increase the activation of NK T cells, for example, host NK T cells. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing the activation of host NK T cells.

[0091] Increased NK T cell activation may be associated with one or more of the following: increased expression of degranulation markers by NK T cells (including, but not limited to, CD107a), increased expression of costimulatory molecules by NK T cells (including, but not limited to, 4-1BB and / or OX40), and increased NK T cell survival. Further relevant considerations regarding these various properties are described elsewhere in this specification.

[0092] The NK T cells whose activation is increased may be peripheral blood NK T cells or tumor-infiltrating NK T cells.

[0093] Such NK T cell activation may increase by at least 5%. For example, NK T cell activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in NK T cell activation in such embodiments can be quantified by comparison with a suitable control.

[0094] Increased NK cell activation Granulocyte-producing cells suitable for use according to the present invention may increase NK cell activation. In particular, granulocyte-producing cells suitable for use according to the present invention may increase host NK cell activation. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing host NK cell activation.

[0095] Those skilled in the art will understand that NK cells play a crucial role in providing the activity necessary to achieve a therapeutic immune response. NK cells exhibit strong cytolytic activity against physiologically stressed cells, such as tumor cells and virus-infected cells. Therefore, the use of granulocyte-producing cells to increase NK cell activation would be beneficial in a wide range of situations where a therapeutic immune response is required. In particular, the amplification of a therapeutic immune response by increasing NK cell activation may be advantageous in the treatment of cancer or infectious diseases, though not limited to these cases.

[0096] The NK cells whose activation is increased may be peripheral blood NK cells or tumor-infiltrating NK cells.

[0097] Increased NK cell activation may be associated with one or more of the following: increased expression of degranulation markers by NK cells (including, but not limited to, CD107a); increased expression of costimulatory molecules by NK cells (including, but not limited to, 4-1BB and / or OX40); increased cytokine expression by NK cells; increased NK cell transport; increased recruitment of NK cells to TMEs; increased cell destructive activity by T cells (including, but not limited to, tumor cell death); increased NK cell proliferation; increased NK cell survival; and increased NK cell abundance. Changes in these properties associated with increased NK cell activation upon exposure to granulocyte-producing cells suitable for use according to the present invention are shown in the examples. Further relevant considerations regarding these various properties are described elsewhere in this specification.

[0098] NK cell activation may increase by at least 5%. For example, NK cell activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the increase in NK cell activation in such embodiments can be achieved by comparison with a suitable control.

[0099] Preferably, for example, for use according to the present invention, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) is an amount sufficient to increase the activation of NK cells, such as host NK cells. The degree of increase, and appropriate indicators of the increase in activation, may be as considered in the preceding paragraph and / or in the following paragraph.

[0100] Increased PBMC activation Granulocyte-producing cells suitable for use according to the present invention may increase the activation of host PBMCs. In particular, this type of granulocyte-producing cell may be able to increase the activation of host PBMCs. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing the activation of host PBMCs.

[0101] It will be understood that PBMCs play a crucial role in providing cells that contribute to any effective therapeutic immune response. PBMCs can be interpreted as any peripheral blood cell with a single round nucleus, such as T cells and NK cells. These cells possess a variety of functions that are important in bringing about an immune response, including the cytotoxic activity or activation of further immune cells. Therefore, the use of granulocyte-producing cells to increase PBMC activation would be beneficial in almost all situations where an effective immune response is required. In particular, the amplification of a therapeutic immune response by increasing PBMC activation may be advantageous in the treatment of cancer or infectious diseases, though not limited to these cases.

[0102] Examples of PBMCs whose activation is increased include peripheral blood T cells (e.g., peripheral blood CD8 + T cells, peripheral blood CD4 + Examples include, but are not limited to, those selected from the group consisting of T cells, peripheral blood NK T cells, peripheral blood αβ T cells, or peripheral blood γδ T cells, and peripheral blood NK cells.

[0103] Increased PBMC activation can be demonstrated by any suitable marker of activation. For example, increased PBMC activation can be demonstrated by increased cytokine expression (e.g., IFN-γ, and / or TNF). The ability of PBMCs exposed to granulocyte-producing cells suitable for use according to the present invention to increase cytokine expression is shown in the examples. Further relevant considerations regarding these various properties are described elsewhere in this specification.

[0104] PBMC activation may increase by at least 5%. For example, PBMC activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in PBMC activation in such embodiments can be quantified by comparison with a suitable control.

[0105] Preferably, for example, for use according to the present invention, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) is sufficient to increase the activation of PBMCs, such as host PBMCs. The degree of increase, and appropriate indicators of the increase in activation, may be as considered in the preceding paragraph and / or in the following paragraph.

[0106] Increased TIL activation Granulocyte-producing cells suitable for use according to the present invention may increase TIL activation. In particular, these types of cells may be able to increase host TIL activation. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing host TIL activation.

[0107] For the purposes of this invention, TILs can be interpreted as encompassing all lymphocyte cell populations that infiltrate tumor tissue. Considering this, it will be recognized that TILs play a crucial role in exerting a therapeutic immune response against tumor cells. TILs can exert specific cytotoxic antitumor activity (e.g., against CD8 cells that have entered the tumor). + (Cells), activation of other immune cells (e.g., CD4 cells in tumors) +Antitumor responses can be promoted by (cellular) TIL activation. Therefore, amplifying the therapeutic immune response by increasing TIL activation can play a very advantageous role in cancer treatment.

[0108] In particular, the inventors have determined that granulocyte-producing cells suitable for use according to the present invention may increase the activation of tumor-infiltrating T cells and / or NK cells. Such granulocyte-producing cells are tumor-infiltrating CD8 cells, as shown in the examples. + T cells and / or CD4 + This may increase the activation of T cells.

[0109] Increased TIL activation, such as increased activation of tumor-infiltrating T cells or tumor-infiltrating NK cells, can be demonstrated by any suitable marker of activation. For example, increased TIL activation can be demonstrated by increased expression of degranulation markers (e.g., CD107a, perforin, or granzyme). Alternatively, increased TIL activation can be demonstrated by increased expression of costimulatory molecules (e.g., 4-1BB, OX40, CD27, CD28, ICOS, HVEM, LIGHT, CD40L, DR3, GITR, CD30, TIM1, CD2, or CD226). The ability of TILs exposed to granulocyte-producing cells suitable for use according to the present invention to increase the expression of degranulation markers or costimulatory molecules is demonstrated in the examples. Further relevant considerations regarding these various properties are described elsewhere in this specification.

[0110] TIL activation may increase by at least 5%. For example, TIL activation may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in TIL activation in such embodiments can be quantified by comparison with a suitable control.

[0111] Preferably, for example, for use according to the present invention, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) is an amount sufficient to increase the activation of TILs, such as host TILs. The degree of increase, and appropriate indicators of the increase in activation, may be as considered in the preceding paragraph and / or as considered in the following paragraph.

[0112] Increased expression of degranulation markers Granulocyte-producing cells suitable for use according to the present invention may be able to increase the expression of degranulation markers by immune cells. In particular, this type of granulocyte-producing cell may be able to increase the expression of degranulation markers by host immune cells. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing the expression of degranulation markers by host immune cells.

[0113] Degranulation supports therapeutic immune activity, CD8 + This is a crucial process in the cell-destructive activity of immune cells such as T cells or NK cells. Therefore, it would be understood that an increase in the expression of degranulation markers such as CD107 provides an indicator that the therapeutic immune activity of such cells has increased, and that the therapeutic immune response has been amplified accordingly.

[0114] In preferred embodiments, the degranulation marker whose expression is increased by host immune cells is selected from the group consisting of CD107a, perforin, and granzymes. Preferably, the expression of two or more of these degranulation markers may be increased. For example, the expression of at least two such degranulation markers may be increased. In particular, the expression of CD107a by host immune cells may be increased.

[0115] Increased expression of degranulation markers can be evaluated by any appropriate method and quantified as needed.

[0116] In a preferred embodiment, the expression of the degranulation marker increases by at least 5%. For example, the expression of the degranulation marker may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in the expression of the degranulation marker in such embodiments can be quantified by comparison with a suitable control.

[0117] Degranulation marker expression may increase in host immune cells selected from a group consisting of T cells and NK cells. If degranulation marker expression is increased in T cells, such T cells may be CD8 + The cells may be selected from a group consisting of T cells, NK T cells, αβT cells, and γδT cells.

[0118] Preferably, for example, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) for use according to the present invention is an amount sufficient to increase the expression of one or more degranulation markers by immune cells, such as host immune cells. The degranulation markers, the degree of increase, and the host immune cells involved may be as considered in the preceding paragraph.

[0119] Increased expression of co-stimulatory molecules Granulocyte-producing cells suitable for use according to the present invention may be able to increase the expression of co-stimulatory molecules by immune cells. In particular, this type of granulocyte-producing cell may be able to increase the expression of co-stimulatory molecules by host immune cells. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing the expression of co-stimulatory molecules by host immune cells.

[0120] Co-stimulatory molecules act to amplify or suppress the activation signals provided to T cells that trigger T cell differentiation. T cell differentiation is a crucial process in therapeutic immune responses, leading to the generation of cytotoxic T cells or helper T cells. Therefore, increasing the expression of co-stimulatory molecules can amplify therapeutic immune responses by inducing functional differentiation of T cells. The use of granulocyte-producing cells to increase the expression of co-stimulatory molecules would be beneficial in a wide range of situations where a therapeutically effective immune response is required. In particular, the amplification of therapeutic immune responses by increasing the activation of co-stimulatory molecules may be advantageous in the treatment of cancer or infectious diseases, though not limited to these cases.

[0121] In a preferred embodiment, the co-stimulatory molecules whose expression is increased by host immune cells are selected from the group consisting of 4-1BB, OX40, CD27, CD28, ICOS, HVEM, LIGHT, CD40L, DR3, GITR, CD30, TIM1, CD2, and CD226. Preferably, the expression of two or more of these co-stimulatory molecules may be increased. For example, the expression of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen such co-stimulatory molecules may be increased. In particular, the expression of both 4-1BB and OX40 by host immune cells may be increased.

[0122] The expression of co-stimulatory molecules can be evaluated by any appropriate method and quantified as needed.

[0123] In a preferred embodiment, the expression of the co-stimulatory molecule increases by at least 5%. For example, the expression of the co-stimulatory molecule may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The increase in the expression of the co-stimulatory molecule in such embodiments can be quantified by comparison with a suitable control.

[0124] The expression of costimulatory molecules may increase in host immune cells selected from a group consisting of T cells and NK cells. When the expression of costimulatory molecules increases in T cells, such T cells may be CD8 + T cells, CD4 + The group may be selected from T cells, NK T cells, αβ T cells, γδ T cells, peripheral blood T cells, and tumor-infiltrating T cells.

[0125] Preferably, for example, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) for use according to the present invention is an amount sufficient to increase the expression of one or more co-stimulatory molecules by immune cells, such as host immune cells. The co-stimulatory molecules, the degree of increase, and the host immune cells involved may be as considered in the preceding paragraph.

[0126] Increased cytokine expression Granulocyte-producing cells suitable for use according to the present invention may be able to increase cytokine expression by immune cells. In particular, this type of granulocyte-producing cell may be able to increase cytokine expression by host immune cells. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing the expression of co-stimulatory molecules by host immune cells.

[0127] Cytokines are crucial chemical messengers in the immune response. They signal for cell activation (inducing immune cells), differentiation of immune cells such as T cells, and proliferation of immune cells such as NK cells. The use of granulocyte-producing cells to increase cytokine activation would be beneficial in almost any situation where a therapeutic immune response is required. In particular, the amplification of a therapeutic immune response by increasing cytokine activation could be advantageous in the treatment of cancer or infectious diseases, though not limited to these areas.

[0128] For the purposes of this invention, cytokines should be interpreted as encompassing chemokines, interferons, interleukins, lymphokines, and TNF.

[0129] In preferred embodiments, the cytokines whose expression by host immune cells is increased are selected from the group consisting of IFN-γ and TNF (e.g., TNF-α). Preferably, the expression of two or more of these costimulatory molecules may be increased. In particular, the expression of IFN-γ by host immune cells may be increased.

[0130] Increased cytokine expression can be evaluated by any appropriate method and quantified as needed.

[0131] In a preferred embodiment, cytokine expression increases by at least 5%. For example, cytokine expression may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of cytokine expression in such embodiments can be achieved by comparison with a suitable control.

[0132] Cytokine expression may be increased in host immune cells selected from the group consisting of PBMCs and TILs. The ability of granulocyte-producing cells suitable for use according to the present invention to increase cytokine (e.g., IFN-γ) expression by PBMCs and TILs is shown in the examples.

[0133] Preferably, for example, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) for use according to the present invention is an amount sufficient to increase the expression of one or more cytokines by immune cells, such as host immune cells. The cytokines, the degree of increase, and the host immune cells involved may be as considered in the preceding paragraph.

[0134] Increased transport of immune cells Granulocyte-producing cells suitable for use according to the present invention may be able to increase immune cell transport. In particular, this type of granulocyte-producing cell may be able to increase the transport of host immune cells. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing the transport of host immune cells.

[0135] The transport of immune cells plays a crucial role in their ability to access sites where they need to exert therapeutic activity, such as tumors or infection sites. Therefore, it will be understood that the ability of granulocyte-producing cells suitable for use according to the present invention to increase immune cell transport provides a clear advantage in promoting effective therapeutic immune responses.

[0136] With respect to PBMCs, and especially with respect to host PBMCs, an increase in cell transport may be observed. As mentioned elsewhere, the inventors have found that granulocyte-producing cells suitable for use according to the present invention are CXCR3 + We demonstrated that it may be possible to generate granulocytes expressing CXCL10, which is known to act as a chemotactic factor for immune cells. Therefore, the medical use and therapeutic method of the present invention involves generating a population of cells expressing CXCL10, thereby enabling CXCR3 + T cells and CXCR3 + It may be particularly beneficial in increasing NK cell transport.

[0137] Increased immune cell transport can be assessed by any appropriate method and quantified as needed.

[0138] In a preferred embodiment, the transport of immune cells increases by at least 5%. For example, the transport of immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the transport of immune cells in such embodiments can be achieved by comparison with a suitable control.

[0139] Preferably, for example, for use according to the present invention, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) is sufficient to increase the transport of immune cells, such as host immune cells. The degree of increased transport and the host immune cells involved may be as considered in the preceding paragraph.

[0140] In particular, increased transport of immune cells may lead to increased recruitment of immune cells to TMEs.

[0141] Increased mobilization of immune cells to TME As described above, the inventors noted that exposure to granulocyte-producing cells suitable for use according to the present invention increases immune cell transport. In particular, the inventors noted that granulocyte-producing cells suitable for use according to the present invention may increase the recruitment of immune cells to the TME. As demonstrated in the examples, this type of granulocyte-producing cell may increase the recruitment of host immune cells to the TME. Therefore, such granulocyte-producing cells may amplify the host therapeutic immune response by increasing the recruitment of host immune cells to the TME.

[0142] It is well known that immune cells have a low tendency to enter TMEs. Many immune cells have shown little ability to enter tumors, and TMEs possess immunosuppressive properties. Therefore, the ability of granulocyte-producing cells suitable for use according to the present invention to increase the recruitment of immune cells, such as host immune cells, to TMEs offers significant advantages in tumor treatment. By increasing the number of immune cells present within the tumor, the antitumor activity of cells that exert a therapeutic immune response can be dramatically increased.

[0143] With respect to PBMCs, and particularly with respect to host PBMCs, an increase in immune cell recruitment to TMEs may be observed. The ability of the present invention to increase such recruitment of granulocyte-producing cells to TMEs, suitable for therapeutic use, is demonstrated in the examples.

[0144] In the examples, the inventors also demonstrate that granulocyte-producing cells suitable for use according to the present invention may differentiate to produce granulocytes expressing CXCL10. CXCL10 is CXCR3 + T cells and CXCR3 + CXCR3 may contain NK cells. + It is a chemotactic factor for immune cells. Therefore, granulocyte-producing cells suitable for use according to the present invention are CXCR3 to TME. + T cells and CXCR3 + This may be particularly useful in establishing a population of granulocyte progeny cells that can increase NK cell recruitment.

[0145] The increase in immune cell recruitment to TME can be assessed by any appropriate method and quantified as needed.

[0146] In preferred embodiments, the recruitment of immune cells to TMEs increases by at least 5%. For example, the recruitment of immune cells to TMEs may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the recruitment of immune cells to TMEs in such embodiments can be achieved by comparison with a suitable control.

[0147] Preferably, for example, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) for use according to the present invention is an amount sufficient to increase the recruitment of immune cells, such as host immune cells, to the TME. The degree of increase in the recruitment of immune cells to the TME, and the host immune cells involved, may be as considered in the preceding paragraph.

[0148] Increased cell-destroying activity of immune cells Granulocyte-producing cells suitable for use according to the present invention may be able to increase the cytodestructive activity of immune cells. In particular, this type of granulocyte-producing cell may be able to increase the cytodestructive activity of host immune cells. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing the cytodestructive activity of host immune cells.

[0149] The cell death of infected cells, cancer cells, or other diseased cells is a crucial mechanism by which many immune cells exert their therapeutic activity. Therefore, it will be understood that the ability of granulocyte-producing cells suitable for use according to the present invention to increase the cell-destroying activity of immune cells offers advantages in increasing the effectiveness of therapeutic immune responses that can be used to treat many conditions, including cancer and infectious diseases.

[0150] Regarding PBMCs, particularly host PBMCs, an increase in the cell-destroying activity of immune cells may be observed.

[0151] The increase in the cell-destructive activity of immune cells can be evaluated by any appropriate method and quantified as necessary.

[0152] In a preferred embodiment, the cytotoxic activity of immune cells increases by at least 5%. For example, the cytotoxic activity of immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the cytotoxic activity of immune cells in such embodiments can be achieved by comparison with a suitable control.

[0153] Preferably, for example, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) for use according to the present invention is an amount sufficient to increase the cytodestructive activity of immune cells, such as host immune cells. The degree of increase in the cytodestructive activity of immune cells and the host immune cells involved may be as considered in the preceding paragraph.

[0154] In particular, increased cellular destructiveness of immune cells may lead to increased tumor cell death activity of immune cells, especially host immune cells.

[0155] Increased tumor cell death activity of immune cells Granulocyte-producing cells suitable for use according to the present invention may be able to increase the tumor cell-killing activity of immune cells. In particular, this type of granulocyte-producing cell may be able to increase the tumor cell-killing activity of host immune cells. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing the tumor cell-killing activity of host immune cells.

[0156] The use of immune cells to target and kill cancer cells forms the basis of most anti-cancer immunotherapies. Therefore, it will be readily apparent that the ability of granulocyte-producing cells suitable for use according to the present invention to increase the tumor cell-killing activity of immune cells, such as host immune cells, provides a clear and desirable advantage in anti-cancer treatment.

[0157] With regard to PBMCs, and particularly with respect to host PBMCs, an increase in the tumor cell killing activity of immune cells may be observed. Such an increase is shown in the results provided in the examples.

[0158] The increased tumor cell death activity of immune cells can be evaluated by any appropriate method and quantified as needed.

[0159] In preferred embodiments, the tumor cell killing activity of immune cells increases by at least 5%. For example, the tumor cell killing activity of immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the tumor cell killing activity of immune cells in such embodiments can be achieved by comparison with a suitable control.

[0160] Preferably, for example, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) for use according to the present invention is an amount sufficient to increase the tumor cell-killing activity of immune cells, such as host immune cells. The degree of increase in the tumor cell-killing activity of immune cells and the host immune cells involved may be as considered in the preceding paragraph.

[0161] Increased proliferation of immune cells Granulocyte-producing cells suitable for use according to the present invention may be able to increase the proliferation of immune cells. In particular, this type of granulocyte-producing cell may be able to increase the proliferation of host immune cells. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing the proliferation of host immune cells.

[0162] Immune cell-based therapies rely on the development of appropriate immune cells in therapeutically effective quantities to deliver the necessary therapeutic immune response (e.g., in the treatment of cancer or infection). Therefore, the ability of granulocyte-producing cells suitable for use according to the present invention to increase the proliferation of immune cells, such as host immune cells, will be extremely beneficial in achieving this. For example, by increasing the proliferation of immune cells, granulocyte-producing cells suitable for use according to the present invention may amplify immune responses that would otherwise not reach the therapeutic threshold, or they may shorten the time required to generate therapeutically effective quantities of immune cells.

[0163] In preferred embodiments, the proliferation of T cells, such as host T cells, may increase. Preferred T cells include αβT cells and CD8 cells. + T cells, CD4 + The group may be selected from T cells, NK T cells, and γδ T cells. In particular, the proliferation of αβ T cells may increase, as demonstrated by the data shown in the examples. For example, αβ T cells are CD4 + It may also be a T cell, or CD8 + These may also be T cells.

[0164] Increased proliferation of immune cells can be assessed by any appropriate method and quantified as necessary.

[0165] Preferably, the proliferation of host immune cells may increase by at least 5%. For example, the proliferation of host immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The quantification of the increase in host immune cell proliferation in such embodiments can be achieved by comparison with a suitable control.

[0166] Preferably, for example, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) for use according to the present invention is an amount sufficient to increase the proliferation of immune cells, such as host immune cells. The degree of increase in immune cell proliferation and the host immune cells involved may be as considered in the preceding paragraph.

[0167] Increased survival of immune cells Granulocyte-producing cells suitable for use according to the present invention may be able to increase the survival of immune cells. In particular, this type of granulocyte-producing cell may be able to increase the survival of host immune cells. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing the survival of host immune cells.

[0168] It is well known that immune cells have a limited lifespan and undergo rapid metabolic turnover in the body. This is exacerbated in situations such as TME, where immunosuppressive conditions further shorten the lifespan of immune cells entering tumors. Our discovery that granulocyte-producing cells suitable for therapeutic use of the present invention can increase the survival of immune cells, therefore, indicates that therapies utilizing such granulocyte-producing cells can offer advantages in extending the period during which immune cells can produce an effective therapeutic immune response. This may be particularly beneficial in the treatment of conditions such as cancer, where an immunosuppressive environment shortens the lifespan of immune cells in other ways.

[0169] In preferred embodiments, the survival of T cells (e.g., NK T cells) or NK cells may be increased. For example, the survival of host T cells (e.g., NK T cells) or NK cells may be increased. Data demonstrating the ability of the useful granulocyte-producing cells according to the present invention to increase the survival of NK T cells and NK cells are shown in the examples.

[0170] The increase in immune cell survival can be assessed by any appropriate method and quantified as needed.

[0171] Preferably, the survival of host immune cells may increase by at least 5%. For example, the survival of host immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. The quantification of the increase in host immune cell survival by such embodiments can be achieved by comparison with a suitable control.

[0172] Preferably, for example, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) for use according to the present invention is an amount sufficient to increase the survival of immune cells, such as host immune cells. The degree of increase in immune cell survival and the host immune cells involved may be as considered in the preceding paragraph.

[0173] Increase in the abundance of immune cells Granulocyte-producing cells suitable for use according to the present invention may be able to increase the abundance of immune cells. In particular, this type of granulocyte-producing cell may be able to increase the abundance of host immune cells. Therefore, such granulocyte-producing cells may be able to amplify the host therapeutic immune response by increasing the abundance of host immune cells.

[0174] While not wishing to be bound by any hypothesis, the increase in the abundance of immune cells observed upon exposing such cells to granulocyte-producing cells suitable for use according to the present invention may result from a combination of increased proliferation and increased survival of immune cells, which are discussed in more detail above. However it occurs, it provides a practical benefit with respect to the medical uses and methods of the present invention. By increasing the abundance of immune cells capable of participating in a therapeutic immune response, the medical uses and methods of treatment of the present invention have the ability to amplify such a therapeutic immune response with respect to both the degree and the duration. This clearly provides a benefit in many therapeutic situations.

[0175] The increase in the abundance of immune cells can be evaluated by any suitable method and, if desired, quantified.

[0176] Preferably, the abundance of host immune cells may increase by at least 5%. For example, the abundance of host immune cells may increase by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more. Quantification of the increase in the abundance of host immune cells according to such embodiments can utilize a comparison with a suitable control.

[0177] In a preferred embodiment, the abundance of T cells, such as host T cells, may increase. T cells whose abundance can be increased may be selected from the group consisting of αβ T cells, CD8 + T cells, CD4 + T cells, NK T cells, and γδ T cells. In particular, as further shown in the examples, the abundance of host αβ T cells may increase. αβ T cells may be CD4 + T cells or may be CD8 + T cells.

[0178] Preferably, for use according to the present invention, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) is sufficient to increase the abundance of immune cells, such as host immune cells. The degree of increase in the abundance of immune cells, and the host immune cells involved, may be as considered in the preceding paragraph.

[0179] Use in combination with other cell immunotherapies Many of the characteristics of cells suitable for use in the medical use and methods of the present invention indicate that these cells are well-suited for use in combination with other cell therapies, particularly with further cellular immunotherapy.

[0180] The ability of the cells of the present invention to increase the proliferation, abundance, and survival of immune cells suggests that therapies using the cells of the present invention may be particularly advantageous when used in combination with other cell therapies. These may be therapies using the host's own cells or therapies using cells of the same species. By providing therapies according to the present invention, cells involved in further cell therapies may be induced to proliferate, survive longer, and increase in abundance and accumulation. This may improve the effectiveness of such therapies.

[0181] As described above, the inventors have identified the ability of granulocyte-producing cells to provide “Signal 2” (co-stimulation) and “Signal 3” (cytokine simulation) to other immune cells, such as those constituting part of further cellular immunotherapy. The provision of these signals is important in generating an effective immune response against tumors and overcoming the immunosuppressive effects of TME. This property of granulocyte-producing cells suggests that they may be used in combination with further cellular immunotherapy, thereby potentially improving the proliferation, survival, and accumulation of cells involved in such further cellular therapy.

[0182] Our discovery that granulocyte-producing cells can generate granulocytes that secrete chemokines such as CXCL10 also suggests potential utility in combination with further cellular immunotherapy. Chemokines play a crucial role in the migration, positioning, and release of immune cells during therapeutic immune responses. The ability of granulocyte-producing cells to produce chemokine-secreting granulocyte progeny suggests that the use of granulocyte-producing cells in combination with further cellular immunotherapy may be expected to generate granulocytes that can favorably enhance the activity of the cells in the further therapy.

[0183] The inventors also confirmed that granulocytes generated during the differentiation of granulocyte-producing cells suitable for use in various embodiments of the present invention express ligands for costimulatory molecules such as 4-1BBL and OX40L. The interaction of these ligands with their receptors plays a crucial role in regulating T cell activation and the generation of effector T cell responses. Therefore, the expression of such receptors by the offspring of granulocyte-producing cells suggests that the use of granulocyte-producing cells in combination with further cellular immunotherapy may enable the granulocyte-producing cells to generate granulocytes that thus positively influence T cell responses.

[0184] Preferably, for example, for use according to the present invention, the therapeutically effective amount of such granulocyte-producing cells (or the pharmaceutical composition of the present invention) is sufficient to increase the proliferation, survival, and / or abundance of immune cells associated with the further cell immunotherapy when combined with further cell therapy. The degree of increase, the immune cells involved, and appropriate indicators of the increased activation may be as considered elsewhere herein.

[0185] Those skilled in the art will recognize many examples of cellular immunotherapies that can be beneficially used in combination with therapies using granulocyte-producing cells according to the present invention. These include, but are not limited to, NK cell therapy, chimeric antigen receptor (CAR)-based therapies (including CAR-T cell therapy, e.g., CAR-γδ T cell therapy and CAR-NK cell therapy), TIL therapy, and engineered T cell receptor (TCR) therapy.

[0186] Medical use and treatment method of the present invention All medical uses, methods of treatment, and pharmaceutical compositions include granulocyte-producing cells for use in the treatment of a target by amplifying a non-granulocyte therapeutic immune response.

[0187] As used herein, the terms “to treat,” “to treat,” or “to treat” include preventive measures (e.g., to prevent the onset of a disease) and corrective measures (treatment of a subject already suffering from a disease). Preferably, as used herein, “to treat,” or “to treat” means corrective measures.

[0188] As used herein, the terms “to treat, to treat” or “to treat, to treat” may refer to both the disorder and / or its symptoms.

[0189] For example, granulocyte-producing cells as part of the pharmaceutical composition of the present invention may be administered to a subject in a therapeutically effective or prophylactically effective dose.

[0190] Several considerations regarding specific therapeutic doses selected in relation to the specific outcomes to be achieved are shown above. However, as a general rule, “therapeutic dose” should be interpreted as any amount of the granulocyte-producing cells or pharmaceutical composition of the present invention that is sufficient to perform such treatment of the disorder or its symptoms when administered to a subject alone or in combination for the treatment of cancer or infection (or its symptoms).

[0191] When a therapeutically effective amount of the granulocyte-producing cells or pharmaceutical composition of the present invention is administered alone, it may amplify the innate immune response, thereby helping to treat cancer or infection.

[0192] The “preventive effective dose” is any amount of the granulocyte-producing cells or pharmaceutical composition of the present invention that, when administered alone or in combination to a subject, suppresses or delays the onset or recurrence of cancer or an infection (or its symptoms). In some embodiments, the preventive effective dose completely prevents the onset or recurrence of cancer or an infection. “Suppressing” the onset means either reducing the likelihood of developing cancer or an infection (or its symptoms), or completely preventing the onset.

[0193] The appropriate dosage range is one that produces the desired therapeutic effect (for example, the granulocyte-producing cells or pharmaceutical composition of the present invention are administered in a therapeutically effective or prophylactically effective dose).

[0194] A typical treatment regimen is 10 6 From, 10 7 , 10 8 , or 10 9 Targeting cells (e.g., cells from a population of granulocyte-producing cells), or up to 10 12 , 10 13 Or 10 14 The treatment regimen may include administering to cells of at least 1 × 10 9 This includes administering a target dose of cells. Preferably, the treatment regimen includes at least 2 × 10 9 Cells or at least 5 × 10 9 The treatment regimen may include administering a target dose of cells. In a preferred embodiment, the treatment regimen is at least 1 × 10 10 Cells or at least 5 × 10 10 This may include administering a dose of cells to the target population. At least 1 × 10 11 or at least 2 × 10 11 The cells may be administered to the target. In some embodiments, 1 × 109 from 3×10 11 or 1×10 10 to 3×10 11 of cells are administered to the subject. Preferably, 5×10 10 to 2.5×10 11 of cells are administered to the subject.

[0195] For treatment, the subject may be administered once, twice, three times, four times, five times, or six times a week. Alternatively, the subject may be administered daily (e.g., once or twice a day). In other embodiments, the subject may be administered once a week or every other week. Preferably, the dosage is once a week. Those skilled in the art will understand that the dosage can be adjusted based on the needs of the subject and the efficacy of the drug. For example, if the effect of the drug is high, the dosage can be reduced.

[0196] In a preferred embodiment, for treatment, the subject is administered at least 2×10 9 cells or at least 2×10 10 cells once a week (e.g., once a week). Preferably, for treatment, the subject is administered at least 1×10 11 or at least 2×10 11 cells once a week.

[0197] The treatment period can be varied based on the subject's response to the treatment and / or the type and / or severity of the cancer or infection. For example, for treatment, the subject may be administered for at least 1 or 2 weeks. Preferably, for treatment, the subject may be administered for at least 3 or 4 weeks. In a preferred embodiment, for treatment, the subject is administered for at least 5 or 6 weeks, preferably at least 7 or 8 weeks.

[0198] In a preferred embodiment, for treatment, the subject is administered with at least 2×10 { 9 cells for 4 to 8 weeks, and the cells are administered once a week. Preferably, for treatment, the subject is administered with at least 2×10 9 cells (preferably at least 2×10 10or 2 × 10 11 The cells are administered for 8 weeks, once a week.

[0199] Administration may be by any suitable technique or route, including but not limited to intravenous injection, intra-arterial injection, intraperitoneal injection, injection into the tumor resection cavity, intrathecal injection, or a combination thereof. Preferably, the drug may be administered intravenously.

[0200] Leukocytosis growth factors may be administered together with the agents of the present invention. Administration may be sequential or simultaneous (preferably simultaneous). Suitable leukocytosis growth factors include granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone, serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, fetal bovine serum (FBS), retinoic acid, lipopolysaccharide (LPS), IFN-gamma, IFN-beta, or combinations thereof. Preferably, the leukocytosis growth factor includes IFN-gamma and GM-CSF. Preferably, the leukocytosis growth factor includes TNF-alpha. Preferably, the leukocytosis growth factor may include granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone, serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, and fetal bovine serum (FBS). Preferably, the leukocytosis growth factor may include granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone, serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, fetal bovine serum (FBS), retinoic acid, lipopolysaccharide (LPS), IFN-gamma, and IFN-beta. Specific examples of those mentioned above include, but are not limited to, salgramostim under the LEUKINE® brand, filgrastim under the NEUPOGEN® brand, and 5PEG-filgrastim under the NEULAST A® brand.

[0201] In a preferred embodiment, granulocyte-producing cells may be administered together with granulocyte colony-stimulating factor, growth hormone, serotonin, and interleukins (for example, sequentially or simultaneously, preferably simultaneously).

[0202] In some embodiments, the granulocyte-producing cells or pharmaceutical compositions of the present invention may be used in combination with other therapies, for example, existing cancer or infection therapies, such as radiotherapy, chemotherapy, and / or immunotherapy.

[0203] For example, the granulocyte-generating cells or pharmaceutical compositions of the present invention may be used in combination with cell-engaging therapies, such as T-cell-engaging therapies. Examples of such therapies that may be used in combination with the granulocyte-generating cells or pharmaceutical compositions of the present invention include those selected from the group consisting of bispecific T-cell engagers (BiTE), checkpoint inhibitory T-cell engagers (CiTE), simultaneous multiple-interaction T-cell engagers (SMiTE), triplicate killer engagers (TRiKE), and BiTE-expressing CAR-T cells (CART.BiTE cells). In particular, the finding that the granulocyte-generating cells or pharmaceutical compositions of the present invention can increase the expression of co-stimulatory molecules such as 4-1BB and OX40 by immune cells suggests that they may be advantageously used in combination with T-cell-engaging therapies, such as single / bispecific 4-1BB agonists or TAA / 4-1BB bispecific T-cell engagers.

[0204] Prior to administration, a matching step may exist between the agent of the present invention (e.g., granulocyte-producing cells or pharmaceutical composition of the present invention) and the subject to be treated. Matching may be based on donor-derived data from which the granulocyte-producing cells originate, and similar data obtained from the subject to be treated. Matching can be achieved based on blood type, similarity of human leukocyte antigen (HLA) type, or a combination thereof.

[0205] Treatment methods A second aspect of the present invention provides a method of therapy comprising amplifying a nongranulocyte-mediated therapeutic immune response, comprising providing granulocyte-producing cells to a subject in need of such therapy.

[0206] The granulocyte-producing cells provided may be cells according to any of the embodiments described herein. Preferably, the granulocyte-producing cells may also be provided by the pharmaceutical composition of the present invention.

[0207] Preferably, such subjects may be patients with cancer. Suitable patients may have any form of cancer, including those further described in this disclosure. For example, a patient may have pancreatic cancer.

[0208] Preferably, such patients may have an infection. Preferred patients may have any form of infection, including those further described in this disclosure. As merely an example, patients may have a viral infection.

[0209] Pharmaceutical manufacturing A third aspect of the present invention provides granulocyte-producing cells for use in the manufacture of drugs for use in amplifying nongranulocyte-mediated therapeutic immune responses.

[0210] The granulocyte-producing cells used in such production may be cells according to any of the embodiments described herein. A drug produced according to this aspect of the present invention may be a pharmaceutical composition of the present invention.

[0211] Cancers that are treated All medical uses, therapeutic methods, or pharmaceutical compositions of the present invention can be used in the treatment of cancer. Cancer can be treated by killing cancer cells or by therapeutically reducing the activity of cancer cells in other ways. This may result from the activity of non-granulocyte cells that provide therapeutically effective immunity, or it may result from cancer-killing activity in some of the granulocytes produced during the differentiation of granulocyte-producing cells used in the medical uses, therapeutic methods, or pharmaceutical compositions of the present invention.

[0212] In preferred embodiments, the cancer is a solid tumor carcinoma. The term “solid tumor carcinoma” refers to an abnormal malignant mass of tissue that does not contain cysts or fluid inclusions. Examples of solid tumor carcinomas include carcinomas, sarcomas, and lymphomas.

[0213] Solid tumors can be carcinomas. Carcinomas may be selected from one or more of the following: adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, or combinations thereof. Carcinomas may also be selected from epithelial neoplasms, squamous cell neoplasms, squamous cell carcinoma, basal cell neoplasms, basal cell carcinoma, transitional cell carcinoma, adenocarcinoma (e.g., adenocarcinoma unspecified (NOS), plastic gastritis, VIP-producing tumors, cholangiocarcinoma, hepatocellular carcinoma NOS, adenoid cystic carcinoma, renal cell carcinoma, Gravitz tumor), cutaneous adnexal neoplasms, mucoepidermal neoplasms, cystic mucinous and serous neoplasms, ductal lobular and medullary neoplasms, acinar cell neoplasms, or compound epithelial neoplasms.

[0214] Alternatively, solid tumor carcinomas may be sarcomas. Sarcomas may be selected from Askin tumors, staphyloid sarcomas, chondrosarcomas, Ewing's tumors, malignant hemangioendotheliomas, malignant Schwann cell tumors, osteosarcomas, or soft tissue sarcomas (including alveolar soft tissue sarcomas, angiosarcomas, phyllodes cystic sarcomas, dermatofibrosarcomas protuberans (DFSP), tendonoid tumors, fibroplastic round cell tumors, epithelioid sarcomas, exostosal chondrosarcomas, exostosal osteosarcomas, fibrosarcomas, gastrointestinal stromal tumors (GISTs), hemangiopericytomas, angiosarcomas, Kaposi's sarcoma, leiomyosarcomas, liposarcomas, lymphangiosarcomas, malignant fibrous histiocytomas, undifferentiated pleomorphic sarcomas, malignant peripheral nerve schwannomas (MPNSTs), neurofibrosarcomas, rhabdomyosarcomas, and synovial sarcomas). Alternatively, the solid tumor may be a lymphoma, such as a B-cell lymphoma, T-cell lymphoma, NK-cell lymphoma, or Hodgkin lymphoma.

[0215] In preferred embodiments, the medical use, treatment method, or pharmaceutical composition of the present invention is for use in treating one or more of the following: pancreatic cancer, liver cancer, esophageal cancer, gastric cancer, cervical cancer, ovarian cancer, lung cancer, bladder cancer, kidney cancer, brain cancer, prostate cancer, myeloma, non-Hodgkin lymphoma (NHL), laryngeal cancer, uterine cancer, or breast cancer.

[0216] If the medical use, treatment method, or pharmaceutical composition of the present invention is intended for use in treating pancreatic cancer, then pancreatic cancer may be a solid tumor cancer of the pancreas, such as pancreatic adenocarcinoma (e.g., pancreatic ductal adenocarcinoma).

[0217] Infections that can be treated All medical uses, therapeutic methods, or pharmaceutical compositions of the present invention can be used to treat infectious diseases. Such infectious diseases can be treated by killing infectious pathogens (e.g., cell-infecting pathogens) or by otherwise therapeutically reducing their activity, or by killing cells infected with infectious pathogens or by otherwise therapeutically reducing their activity.

[0218] As used herein, “infected cell” refers to a cell infected with an intracellular infectious agent. The intracellular infectious agent may be a pathogen, and therefore the cell is a “pathogen-infected cell.” In preferred embodiments, the cell may be infected with intracellular bacteria or a virus, preferably a virus.

[0219] In preferred embodiments, the infection to be treated is caused by Gram-negative or Gram-positive bacteria. Preferably, the infectant is a Gram-positive bacterium, such as a bacterium of the genus Staphylococcus.

[0220] Preferably, the infections to be treated include Staphylococcus species, multidrug-resistant Gram-negative bacteria (MRDGN bacteria), vancomycin-resistant Enterococcus (VRE), Mycobacterium species, carbapenem-resistant Enterobacteriaceae (CRE) enterobacteria, Acinetobacter species, Actinomyces species, Propionibacterium species, Anaplasma species, Bacillus species, Arcanobacterium species, Bacteroides species, Bartonella species, Brucella species, Yersinia species, Burkholderia species, Campylobacter species, Streptococcus species, Haemophilus species, Clostridium species, Corynebacterium species, Echinococcus species, and Ehrlichia species. It is caused by bacteria selected from one or more of the following species: Enterococcus, Rickettsia, Fusobacterium, Neisseria, Klebsiella, Helicobacter, Escherichia, Kingella, Legionella, Listeria, Borrelia, Mycoplasma, Chlamydia, Nocardia, Pasteurella, Bordetella, Prevotella, Chlamydophila, Coxiella, Salmonella, Group A Streptococcus, Shigella, Staphylococcus, Treponema, Vibrio, Francisella, Pseudomonas, and Ureaplasma.

[0221] In a suitable embodiment, the bacteria are resistant to 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 (for example, O157:H7, O111 and O104:H4), Kingella kingae, Legionella pneumophila, Listeria monocytogenes, Burkholderia pseudomallei, Neisseria meningitidis, Mycoplasma pneumoniae, Mycoplasma genitalium, Chlamydia trachomatis, Bordetella pertussis, Streptococcus pneumoniae, Chlamydophila psittaci, Coxiella burnetii, Treponema pallidum, ClostridiumSelected from one or more of the following: tetani, Chlamydophila pneumoniae, Vibrio cholera, Mycobacterium tuberculosis, Salmonella enterica subsp. enterica, serovartyphi, Ureaplasma urealyticum, and Francisella tularensis. Preferably, Mycobacterium tuberculosis.

[0222] Preferably, in a preferred embodiment, the bacteria are selected from one or more of the following: 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) enterobacteria.

[0223] Preferably, the infection to be treated is caused by a virus selected from one or more families selected from the adenoviridae, picornaviridae, herpesviridae, coronavirusidae, hepadnaviridae, flaviviridae, retroviridae, orthomyxoviridae, paramyxoviridae, papovaviridae, polyomavirus, rhabdoviridae, togaviridae, and bunyaviridae.

[0224] In preferred embodiments, the viruses include HIV-1 (human immunodeficiency virus), HIV-2, Junin virus, BK virus, Machupo virus, Sabia 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 (HHV-6), human herpesvirus 7 (HHV-7), enterovirus (e.g., EV71), coxsackie A virus, Sin Nombre virus, Heartland virus, hantavirus, 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) One or more of the following may be selected: 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, smallpox, smallpox, Venezuelan encephalitis virus, guanalitovirus, West Nile virus, yellow fever virus, and Zika virus.

[0225] Preferably, the infection to be treated is caused by fungi selected from one or more of the following: Aspergillus, Piedraia, Blastomyces, Candida, Fonsecaea, Coccidioides, Cryptococcus, Cryptosporidium, Geotrichum, Histoplasma, Microsporidia, Paracoccidioides, Pneumocystis, Sporothrix, Trichophyton, Epidermophyton, Hortaea, Malassezia, Trichosporon, and Mucorales.

[0226] In preferred embodiments, the pathogen is a fungus selected from one or more of the following: 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.

[0227] The macroparasite is selected from one or more of the species Angiostrongylus, Entamoeba, Anisakis, Ascaris, Babesia, Balantidium, Baylisascaris, Blastocystis, Capillaria, Trypanosoma, Clonorchis, Ancylostoma, Cyclospora, Taenia, Desmodesmus, Dientamoeba, Dracunculus, Enterobius, Fasciola, Filarioidea superfamily, Giardia, Gnathostoma, Necator, Hymenolepis, Isospora, Leptospira, Wuchereria, Rhinosporidium, Brugia, Plasmodium, Onchocerca, Opisthorchis, Paragonimus, Naegleria, Schistosoma, Strongyloides, Toxocara, Toxoplasma, Trichinella, Trichomonas, and Trichuris.

[0228] In a preferred embodiment, the macroparasites are 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, Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi Onchocerca One or more of the following are selected: volvulus, Opisthorchis viverrini, Opisthorchis felineus, Naegleria fowleri, Strongyloides stercoralis, Toxoplasma gondii, Trichinella spiralis, Trichuris trichiura, and Trichomonas vaginalis.

[0229] In a preferred embodiment, the infectant is an antibiotic-resistant bacterium (e.g., MRSA), preferably a multi-antibiotic-resistant bacterium. Antibiotic-resistant bacteria may be resistant to beta-lactam antibiotics such as methicillin.

[0230] Antibiotic resistance can be evaluated using any technique known in the art, such as the Kirby-Baure method, the Stokes method, Etest, and / or agar and culture medium dilution methods for determining the minimum inhibitory concentration (MIC).

[0231] In a preferred embodiment, the bacteria are resistant to one or more of the following: penicillin, penicillinase-resistant penicillin, cephalosporins, beta-lactamase inhibitors, tetracyclines and combinations thereof, or pharmaceutically acceptable salts thereof.

[0232] In preferred embodiments, bacteria are resistant to one or more of the following: vancomycin, naphicillin, oxacillin, teicoplanin, penicillin, methicillin, flucloxacillin, dicloxacillin, cefazolin, cephalothin, cephalexin, cefuroxime, clindamycin, cefazolin, amoxicillin / clavulanate, ampicillin / sulbactam, lincomycin, erythromycin, trimethoprim, sulfamethoxazole, daptomycin, linezolid, rifampin, ciprofloxacin, gentamicin, tetracycline, doxycycline, minosylcin, tigecycline and combinations thereof, or pharmaceutically acceptable salts thereof. In preferred embodiments, bacteria may be resistant to vancomycin and / or teicoplanin, or pharmaceutically acceptable salts thereof.

[0233] Multi-antibiotic resistant bacteria are resistant to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different antibiotics (e.g., chemical antibiotics).

[0234] In preferred embodiments, granulocytes produced during the differentiation of preferred granulocyte-producing cells kill infected organisms by phagocytosis. For example, in preferred embodiments, granulocytes produced during the differentiation of preferred granulocyte-producing cells kill viruses by phagocytosis of virus-infected cells. In preferred embodiments, granulocytes produced during the differentiation of preferred granulocyte-producing cells kill bacteria by phagocytosis of bacteria-infected cells. In preferred embodiments, granulocytes produced during the differentiation of preferred granulocyte-producing cells kill infected organisms by releasing one or more factors that kill infected organisms. For example, in preferred embodiments, granulocytes produced during the differentiation of preferred granulocyte-producing cells kill viruses by releasing one or more factors that kill viruses. In preferred embodiments, granulocytes produced during the differentiation of preferred granulocyte-producing cells kill bacteria by releasing one or more factors that kill bacteria. In some embodiments, granulocytes produced during the differentiation of preferred granulocyte-producing cells kill infected organisms in combination of the above.

[0235] Granulocyte-producing cells capable of generating granulocytes with desirable cell-destroying activity.

[0236] Preferably, granulocyte-producing cells for use in various embodiments of the present invention may be able to differentiate to produce granulocytes having cell-destructive activity that may further contribute to a therapeutic immune response. In particular, such cells may produce granulocytes that can kill cancer cells, infected cells, or cell infectants.

[0237] The inventors have developed several methods for identifying granulocytes that possess such cell-destroying activity.

[0238] For example, granulocyte-producing cells for use according to the present invention may have the ability to differentiate and produce granulocytes that have the ability to kill at least 5% of cancer cells in a cancer death assay, and the cancer death assay is a. Mixing granulocytes with cancer cells to form a mixture, b. Incubating the mixture, c. To measure the percentage of cancer cells that died in the mixture. Includes.

[0239] Preferably, in one embodiment of this type, the percentage of cancer cells killed in the mixture is the maximum percentage of cancer cells killed within 48 hours after the mixture is formed. The granulocytes thus produced may have the ability to kill at least 10%, 20%, 30%, 40%, 50%, 51.5%, 60%, 70%, or 80% of cancer cells in a cancer killing assay.

[0240] In preferred embodiments, the assay mixture comprises granulocytes versus cancer cells in a 1:1, 5:1, or 10:1 ratio.

[0241] Preferably, the cancer cells used in such assays are HeLa or PANC-1 cancer cells.

[0242] Those skilled in the art will recognize many suitable cancer cell death assays that can be used to evaluate the ability to kill cancer cells. As just one example, in a preferred embodiment, the cancer cell death assay is performed using the ACEA Biosciences xCELLigence RTCA DP Analyzer system (registered trademark) according to the manufacturer's instructions, as follows: a. Place 6,000 cancer cells at the bottom of a 16-well plate. b. Proliferate the cells until they reach confluence, which is determined by the plateau (i.e., the "normalization point") of the Cell Index (CI) value. c. Add 60,000 granulocytes (i.e., a ratio of 10 granulocytes per cancer cell), and incubate at 37°C. d. The percentage of dead cancer cells is the maximum percentage of cancer cells that have died within 48 hours after granulocyte addition, calculated using the following formula: ((Cell Index エフェクターなし -CellIndex エフェクター) / Cell Index エフェクターなし ) × 100.

[0243] In one embodiment, the cancer death assay is performed using a luciferase cytotoxicity assay as follows: a. Place cancer cells at the bottom of a plate (for example, a 96-well plate), b. Effector cells such as granulocyte-producing cells or granulocytes differentiated from granulocyte-producing cells are added to cancer cells (for example, after 17-24 hours, in an effector cell to cancer cell ratio of 10:1 or 20:1), to form a mixture. c. Incubate the mixture (e.g., in a 5% CO2 atmosphere at 37°C for 48 hours), d. After incubation, add a luciferase substrate (e.g., luciferin, preferably 5-fluoroluciferin) to the mixture (e.g., incubate at room temperature until the luminescence signal stabilizes (e.g., 7-10 minutes)), e. Measure the luminescence signal and determine the percentage of dead cancer cells.

[0244] The luciferase substrate can be added in any suitable concentration range, such as 1 to 1000 μM, for example, 10 to 500 μM or 100 to 400 μM.

[0245] In a preferred embodiment, the cancer death assay is performed using a luciferase cytotoxicity assay as follows: a. 1.5 × 10 4 Place individual cancer cells at the bottom of a 96-well plate. b. Effector cells, such as granulocyte-producing cells or granulocytes differentiated from granulocyte-producing cells, are added to cancer cells after 17-24 hours in an effector cell to cancer cell ratio of 10:1 or 20:1 to form a mixture. c. Incubate the mixture in a 5% CO2 atmosphere at 37°C for 48 hours. d. After incubation, add 100 μl of ONEglo® reagent to the mixture and incubate at room temperature until the luminescence signal stabilizes (e.g., 7-10 minutes). e. Measure the luminescence signal and determine the percentage of dead cancer cells.

[0246] The percentage of dead cancer cells can be calculated using the following formula: 100 - ((Corrected sample emission background) / (Corrected target-only emission background) × 100).

[0247] In this case, “sample” may be the above mixture containing effector cells and cancer cells, while “target only” may refer to a sample containing cancer cells but not effector cells. Those skilled in the art will understand that, for comparability, “sample” and “target only” may be exposed to the same steps, e.g., incubation. “Background” correction may be achieved by a normalization technique, for example, by subtracting any luminescence signals observed in the “culture medium only” sample. Preferably, “background” correction may be achieved by subtracting the luminescence of the culture medium only from the luminescence values ​​of the “sample” or “target only.”

[0248] Granulocyte-producing cells suitable for use in various embodiments of the present invention may have the ability to differentiate to produce granulocytes characterized by the following: a. Increased expression of one or more of the following compared to a reference standard derived from neutrophils unsuitable for cancer treatment: GM2A, CTSG, CAP37, ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, and PSMB2, and / or b. Reduced expression of ANXA1 and / or PPP3CB compared to a reference standard derived from neutrophils unsuitable for cancer treatment.

[0249] In a preferred embodiment, granulocyte-producing cells suitable for use in various aspects of the present invention may be characterized in that the granulocytes produced during the differentiation of the granulocyte-producing cells have a positively charged cell surface.

[0250] Granulocyte-producing cells suitable for use in various embodiments of the present invention may also be specified in relation to the expression profiles of the granulocytes they can produce.

[0251] In a preferred embodiment, granulocyte-producing cells may be able to differentiate to produce granulocytes characterized by: a. Increased expression of one or more of GM2A, CTSG, CAP37, ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, ATM, IKBKB, BCAP31, TAPBP, PERM, PLEC, ACSL1, RAC1, and PSMB2 compared to a reference standard derived from granulocytes that do not have the ability to kill cancer cells, or infected organisms, or cells infected with infected organisms, and / or b. Reduced expression of ANXA1 and / or PPP3CB compared to a reference standard derived from granulocytes that lack the ability to kill cancer cells, or infected organisms, or cells infected with infected organisms.

[0252] Representative gene sequences for use in such embodiments of the present invention are shown in the sequence listings in international patent applications PCT / GB2020 / 053197 (published as WO2021 / 116711) and PCT / GB2020 / 053199 (published as WO2021 / 116713), and the appropriate Ensembl accession numbers, and their relevant disclosures, particularly those relevant to identifying sequence listings and sequences suitable for use in this embodiment of the present invention, are incorporated herein by reference.

[0253] Determining whether granulocytes have increased the expression of one or more of 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 done by measuring the expression of the above markers. The measurement of expression may be carried out by any means well known to those skilled in the art. The term “measuring” as used in relation to the expression of one or more genes in this invention encompasses measuring both negative (e.g., no expression) and positive (e.g., expression) of expression. In a preferred embodiment, expression is positive.

[0254] In some embodiments, expression may be measured using high-throughput techniques. For example, expression may be measured at the level of transcription (e.g., transcriptome techniques) or translation (e.g., proteomics techniques). Alternatively, the present invention may employ the use of genomics to detect, for example, the presence or absence of single nucleotide polymorphisms (SNPs), promoter sequences, gene copy number (e.g., replication), and / or enhancers or other relevant genetic features, preferably those that determine the expression level of one or more genes of the present invention. High-throughput techniques can be used to rapidly analyze the whole genome, proteome, and transcriptome to provide data including the expression levels of all genes, polypeptides, and transcripts within a cell. Proteomics is a technique for analyzing the proteome of a cell (e.g., at a specific point in time). The proteome differs in different cell types. Generally, proteomics is performed by gel-based techniques, including mass spectrometry, including tandem mass spectrometry, and differential gel electrophoresis. Proteomics can be used to detect polypeptides expressed in specific cell types and generate proteomic profiles that enable the identification of those specific cell types.

[0255] In preferred embodiments, the mRNA of the target gene can be detected and quantified, for example, by Northern blotting or by quantitative reverse transcription PCR (RT-PCR). Single-cell gene expression analysis can also be performed using a commercially available system (e.g., Fluidigm Dynamic Array). Alternatively, gene expression levels can be determined by analyzing polypeptide levels using Western blotting techniques, such as ELISA-based assays.

[0256] Therefore, in a preferred embodiment, the gene expression level is determined by measuring the mRNA / cDNA level of the gene of the present invention, such as by RNA sequencing (RNA-Seq).

[0257] In a preferred embodiment, the gene expression level is determined by measuring the polypeptide level produced by the gene of the present invention by mass spectrometry, for example, liquid chromatography and mass spectrometry (LC-MS / MS).

[0258] In a preferred embodiment, granulocytes (or stem cells) for treating cancer can be detected using an enzyme-linked immunosorbent assay (ELISA) or a Luminex assay (commercially available from R&D Systems, USA).

[0259] Therefore, in a preferred embodiment, expression measurement involves measuring and / or comparing the expression levels of one or more polypeptides 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 in granulocytes.

[0260] In a preferred embodiment, expression measurement involves measuring and / or comparing the amount produced by granulocytes of one or more polypeptides 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.

[0261] In a preferred embodiment, expression measurement involves measuring and / or comparing the expression levels of one or more polypeptides 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 in stem cells.

[0262] In a preferred embodiment, expression measurement involves measuring and / or comparing the amount produced by stem cells of one or more polypeptides 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.

[0263] In a preferred embodiment, expression measurements are compared to a genome-wide association study (e.g., a reference standard from a reference population, e.g., from a suitable or unsuitable donor, or from suitable or unsuitable granulocytes, or from a subject suitable or unsuitable for treatment with granulocyte-generating cells according to the present invention, or from a subject at or not at risk of cancer, or a combination thereof).

[0264] A suitable method for establishing a baseline or reference value for comparing expression levels is the prior art, known to those skilled in the art.

[0265] As used herein with respect to the expression of one or more genes in this invention, the term "increased" may refer to an expression level that is statistically significantly increased compared to a reference standard. Such genes may be considered upregulated.

[0266] In preferred embodiments, increased expression means an expression of 1x, 1.25x, or more than approximately 10x compared to a reference standard. In some embodiments, increased expression means an expression of at least approximately 1.1x, 1.2x, 1.25x, 1.5x, 1.75x, 2x, 4x, 5x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 50x, 75x, 100x, 150x, 200x, or at least more than approximately 300x compared to a reference standard.

[0267] As used herein with respect to the expression of one or more genes in this invention, the term "reduced" may refer to an expression level that is statistically significantly reduced compared to a reference standard. Such genes may be considered downregulated.

[0268] In preferred embodiments, a reduction in expression means an expression of less than 1x, 1 / 1.25, or about 1 / 10 compared to the reference standard. In some embodiments, a reduction in expression means an expression of less than at least about 1 / 1.1, 1 / 1.2, 1 / 1.25, 1 / 1.5, 1 / 1.75, 1 / 2, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 50, 1 / 75, 1 / 100, 1 / 150, 1 / 200, or at least about 1 / 300 compared to the reference standard.

[0269] The difference in magnification change may be an absolute numerical value of the expression level in the sample (e.g., CPM: parts per million) or Log2CPM (a standard measure in the field). Preferably, the magnification change is a Log2 magnification change. In a preferred embodiment, the 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 preferred embodiment, a Log2 change is a decrease of 0.1 or greater, 0.2 or greater, 0.3 or greater, 0.4 or greater, 0.5 or greater, 0.6 or greater, 0.7 or greater, 0.8 or greater, 0.9 or greater, 1.0 or greater, 1.1 or greater, 1.2 or greater, or 1.3 or greater. A decrease may be indicated by the presence of a "-" sign before the value.

[0270] In a preferred embodiment, the magnification change is measured and / or determined, for example, by RNA sequencing (RNA-Seq) as a whole.

[0271] With respect to the expression of one or more genes in this invention, the terms “unchanged” or “same” may refer to an expression level that is not statistically significant to a reference standard. Preferably, the same expression level as the reference standard.

[0272] Expression levels may be averages, such as mean expression levels. In preferred embodiments, statistical significance is determined using two-way ANOVA, for example, where n is at least 3 and the data are mean. + / - is shown as the standard error of the mean.

[0273] In preferred embodiments, the method of the present invention includes measuring the expression of the gene combination described herein.

[0274] When used in reference to genes described herein, the term “one or more” 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 genes. Preferably, the term “one or more” means all genes. Similarly, when used in reference to polypeptides described herein, the term “one or more” 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 polypeptides. Preferably, the term “one or more” means all polypeptides.

[0275] The expression of one or more of the following genes may correlate with the ability of granulocytes to kill cancer cells: ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PPP3CB, ANXA1, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2. Therefore, these genes may be referred to herein as genes associated with the ability to kill cancer cells. Therefore, the term "one or more genes associated with the ability to kill cancer cells" (and similar terms) can be synonymous with (and thus replace) 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." Therefore, the term (and similar terms) “one or more polypeptides associated with the ability to kill cancer cells” can be synonymous with (and thus replace) 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.”

[0276] In preferred embodiments, 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 may be increased in granulocytes capable of killing cancer cells compared to granulocytes not capable of killing cancer cells. Alternatively, in preferred embodiments, the expression of ANXA1 and / or PPP3CB may be decreased in granulocytes capable of killing cancer cells compared to granulocytes not capable of killing cancer cells.

[0277] In preferred embodiments, the expression of S100A9 and / or S100A8 may be increased in the granulocytes of the present invention compared to a reference standard derived from granulocytes that do not possess the ability to kill cancer cells.

[0278] The expression of one or more of the following genes is correlated with the ability of granulocytes to kill infected or infected cells: ITGB1, CYBB, SYK, DOCK8, COMP, ATG7, SLC2A1, GZMK, CTSG, ATM, IKBKB, BCAP31, TAPBP, PPP3CB, ANXA1, PERM, PLEC, ACSL1, RAC1, GM2A, CAP37, and PSMB2. Therefore, these genes are referred herein as genes associated with the ability to kill infected or infected cells. Therefore, the term (and similar terms) “one or more genes associated with the ability to kill an infectant or cells infected with an infectant” may be synonymous with (and thus replace) 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.”

[0279] In preferred embodiments, 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 in granulocytes capable of killing infected or infected cells compared to granulocytes that do not have the ability to kill infected or infected cells. Alternatively, or further, in preferred embodiments, the expression of ANXA1 and / or PPP3CB is decreased in granulocytes capable of killing infected or infected cells compared to granulocytes that do not have the ability to kill infected or infected cells.

[0280] In preferred embodiments, the method of the present invention may further include measuring the expression of one or more genes selected from S100A9 and S100A8. In preferred embodiments, the expression of S100A9 and / or S100A8 may be increased in the granulocytes of the present invention compared to a reference standard derived from granulocytes that do not have the ability to kill infected organisms or cells infected with infected organisms.

[0281] The expression levels of one or more genes of the present invention may be compared to a reference standard. The comparison may be carried out by any suitable technique known to those skilled in the art, for example, bioinformatics techniques. The expression levels of the genes described herein are appropriately determined in the above reference standard.

[0282] The reference standard may be a proteome profile (indicating the amount of polypeptides expressed by granulocytes), a transcriptome profile (indicating the amount of gene expression by granulocytes, for example, measured by RNA produced by the granulocytes), 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., replication), and / or enhancers or other relevant genetic features, preferably those that determine the expression level of one or more genes of the present invention. Those skilled in the art will understand that both proteome profiles and transcriptome profiles are measures of gene expression and will use the appropriate reference standard depending on the technique used to measure gene expression according to the present invention. For example, if proteomics is used in the implementation of the present invention, those skilled in the art will use a reference standard that is a proteome profile; if transcriptomics is used in the implementation of the present invention, those skilled in the art will use a reference standard that is a transcriptome profile; and if genomics is used in the implementation of the present invention, those skilled in the art will use a reference standard that is a genomic profile. A reference standard may refer to a database (e.g., a genome database), which may contain data from one or more sources, such as one or more subjects and / or cells.

[0283] The reference standard is preferably a reference standard of granulocytes that do not have the ability to kill cancer cells (e.g., a transcriptome or proteome profile of granulocytes unsuitable for cancer treatment). Such a reference standard may be derived from subjects without cancer (healthy subjects) or from subjects with cancer. Preferably, such a reference standard is derived from subjects without cancer.

[0284] In preferred embodiments, 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 compared to a reference standard derived from granulocytes that do not have the ability to kill cancer cells. In preferred embodiments, the expression of ANXA1 and / or PPP3CB is decreased compared to a reference standard derived from granulocytes that do not have the ability to kill cancer cells. In preferred embodiments, 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 compared to a reference standard derived from granulocytes that do not have the ability to kill cancer cells, and the expression of ANXA1 and / or PPP3CB is decreased compared to a reference standard derived from granulocytes that do not have the ability to kill cancer cells.

[0285] The reference standard may be a reference standard of granulocytes suitable for cancer treatment (e.g., a transcriptome or proteome profile of granulocytes suitable for cancer treatment). In a preferred 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 as that of a reference standard derived from granulocytes capable of killing cancer cells. In a preferred embodiment, the expression of ANXA1 and / or PPP3CB is decreased or the same as that of a reference standard derived from granulocytes capable of killing cancer cells.

[0286] In some embodiments, the present invention may include the use of a reference standard of granulocytes that does not have the ability to kill cancer cells, and a reference standard of granulocytes that has the ability to kill cancer cells.

[0287] The reference standard is preferably a reference standard of granulocytes that do not have the ability to kill the infected organism or cells infected with the infected organism (e.g., a transcriptome or proteome profile of granulocytes that do not have the ability to kill the infected organism or cells infected with the infected organism).

[0288] In preferred embodiments, 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 compared to a reference standard derived from granulocytes that lack the ability to kill infected cells or cells infected with infected cells. In preferred embodiments, the expression of ANXA1 and / or PPP3CB is decreased compared to a reference standard derived from granulocytes that lack the ability to kill infected cells or cells infected with infected cells. In preferred embodiments, 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 compared to a reference standard derived from granulocytes that lack the ability to kill infected or infected cells, and the expression of ANXA1 and / or PPP3CB is decreased compared to a reference standard derived from granulocytes that lack the ability to kill infected or infected cells.

[0289] The reference standard may be a reference standard of granulocytes capable of killing infected organisms or cells infected with infected organisms (e.g., a transcriptome or proteome profile of granulocytes suitable for the treatment of an infection). In a preferred 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 as that of a reference standard derived from granulocytes capable of killing infected organisms or cells infected with infected organisms. In a preferred embodiment, the expression of ANXA1 and / or PPP3CB is decreased or the same as that of a reference standard derived from granulocytes capable of killing infected organisms or cells infected with infected organisms.

[0290] In some embodiments, the present invention may include the use of a granulocyte reference standard that does not have the ability to kill infected organisms or cells infected with infected organisms, and a granulocyte reference standard that has the ability to kill infected organisms or cells infected with infected organisms.

[0291] In a preferred embodiment, granulocyte-producing cells suitable for use in various aspects of the present invention can produce granulocytes having a positively charged cell surface.

[0292] The inventors believe that the surface charge of granulocyte cells may correlate with their suitability for treating cancer and / or infectious diseases, and that granulocytes with a more positive charge (or a lower negative charge) (e.g., neutrophils) are more suitable for treating cancer and / or more effective in treating cancer and / or more suitable for treating infectious diseases and / or more effective in treating infectious diseases. The level of cell surface charge can be determined by comparison with a reference standard, preferably derived from granulocytes that do not have the ability to kill cancer cells and / or cells that have infected an infectious agent.

[0293] In a preferred embodiment, granulocyte-producing cells may be considered suitable for use in various aspects of the present invention if they can differentiate into granulocytes having a positively charged (or less negatively charged) cell surface. The cell surface charge can be determined using any suitable technique known in the art. In a preferred embodiment, the cell surface charge is determined using electrophoresis. Electrophoretic mobility assays may be those described in “Cell Electrophoresis” edited by Johann Bauer (ISBN 0-8493-8918-6, published by CRC Press, Inc.), the entirety of which is incorporated herein. In another embodiment, the cell surface charge can be determined using negatively and / or positively charged means. In a preferred embodiment, granulocytes have a positive cell surface charge if a negatively charged means, rather than a positively charged means, can bind to them. In a preferred embodiment, granulocytes have a negative cell surface charge if a positively charged means, rather than a negatively charged means, can bind to them. Such negatively and / or positively charged means can also be used to measure the concentration of granulocyte cells in a sample. The positively charged means may be positively charged particles, nanoprobes or nanoparticles, or cation exchange media. Suitable nanoparticles can be prepared by conjugating superparamagnetic iron(II,III)(Fe3O4) nanoparticles (NP) with (3-aminopropyl)triethoxysilane (APTES) to form a thin layer of silicon dioxide (SiO2) shell on the surface of the NP during the reaction with tetraethyl orthosilicate (TEOS) and ammonium hydroxide (NH4OH). Fluorescein isothiocyanate (FITC) can be embedded in the SiO2 shell, thereby exposing the Si-bonded hydroxyl group (SiO2-OH) and creating a negative surface charge. Branched poly(ethyleneimine) (PEI) molecules can be used not only to cover the SiO2-OH group in a non-covalent manner but also to expose additional positively charged amine groups.Therefore, in a preferred embodiment, negatively charged nanoparticles are prepared by conjugating Fe3O4 nanoparticles with APTES to form a thin layer of SiO2 shell on the nanoparticle surface during the reaction with tetraethyl orthosilicate (TEOS) and ammonium hydroxide (NH4OH), embedding FITC in the SiO2 shell and thereby exposing SiO2-OH groups (creating a negative surface charge). In another embodiment, positively charged nanoparticles are prepared by contacting negatively charged nanoparticles (as described herein) with PEI molecules (for example, to expose additional positively charged amine groups). In a preferred embodiment, the negatively charged means (e.g., nanoparticles) may have a negative surface charge of at least -5mV, -10mV, -20mV, -30mV, or -40mV. Preferably, the negatively charged means (e.g., nanoparticles) have a negative surface charge of at least -35mV. In a preferred embodiment, the positively charged means (e.g., nanoparticles) have at least... + 5mV, + 10mV, + 20mV, + 30mV, or + It may have a positive surface charge of 40 mV. Preferably, the positively charged means (e.g., nanoparticles) are at least + It has a positive surface charge of 35 mV. The surface charge of the positively or negatively charged means (e.g., nanoparticles) may refer to the surface zeta potential of the positively or negatively charged means (e.g., nanoparticles). The surface zeta potential may be measured with a dynamic light scattering particle size analyzer (e.g., Zetasizer Nano-ZS90, Malvern, UK).

[0294] Cells that have the ability to kill cancer cells In a preferred embodiment, granulocyte-producing cells suitable for use in various aspects of the present invention can be differentiated to produce granulocytes having the ability to kill cancer cells.

[0295] The "ability to kill cancer cells" can be determined by mixing cells (e.g., granulocytes such as neutrophils) with cancer cells and measuring the viability of the cancer cells (e.g., after incubation). If the cancer cells can no longer survive (i.e., are dead), the cells demonstrate the ability to kill cancer cells. In a preferred embodiment, the ability to kill cancer cells is determined using the cancer cell killing activity (CKA) assay described herein.

[0296] In a preferred embodiment, the CKA assay is performed a. To form a test sample by bringing cancer cells into contact with granulocytes (preferably in a granulocyte-to-cancer cell ratio of 10:1), b. Incubating the test sample, c. To measure the percentage of dead cancer cells in the test sample. Includes.

[0297] In a preferred embodiment, the CKA assay is performed a. Mixing granulocytes with cancer cells to obtain a mixture (preferably in a granulocyte-to-cancer cell ratio of 10:1), b. Incubating the mixture, c. To measure the percentage of cancer cells that died in the mixture. Includes.

[0298] As used herein, the term “mixing” means mixing one or more components together in any order, whether sequentially or simultaneously. In preferred embodiments, “mixing” means bringing a first component into contact with a second component (e.g., granulocytes and cancer cells).

[0299] Cancer cells for use in the assay may be one or more selected from pancreatic cancer cell lines, liver cancer cell lines, esophageal cancer cell lines, gastric cancer cell lines, cervical cancer cell lines, ovarian cancer cell lines, lung cancer cell lines, bladder cancer cell lines, kidney cancer cell lines, brain cancer cell lines, prostate cancer cell lines, myeloma cell lines, non-Hodgkin lymphoma (NHL) cell lines, laryngeal cancer cell lines, uterine cancer cell lines, or breast cancer cell lines. Suitable cell lines are commercially available from the American Type Culture Collection United Kingdom (UK), Guernsey, Ireland, Jersey and Liechtenstein, and LGC Standards (Queens Road, Teddington, Middlesex, TW11 0LY, UK). For example, pancreatic cell lines include Capan-2, ATCC HTB-80; Panc 10.05, ATCC CRL-2547; CFPAC-1, ATCC CRL-1918; HPAF-II, ATCC CRL-1997; SW 1990, ATCC CRL-2172; BxPC-3, ATCC CRL-1687; AsPC-1, ATCC CRL-1682; ATCC(registered trademark) TCP-1026(trademark); 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 It may be one or more of CRL-1469 or ATCC(registered trademark) TCP-2060(trademark). Preferably, the cancer cell line is a pancreatic cancer cell line such as PANC-1. In a preferred embodiment, the cancer cell line is a cervical cancer cell line such as HeLa cells.

[0300] The incubation step may be performed for between 1 hour and 100 hours. Preferably, the incubation step may be performed for between 5 hours and 75 hours, for example, between 10 hours and 20 hours. The incubation step may be performed for between 6 hours and 6 days. Preferably, the incubation step may be performed for between 6 hours and 2 days, for example, between 12 hours and 36 hours, for example, between 16 hours and 24 hours. In a preferred embodiment, the incubation step is performed for 24 hours. In another embodiment, the incubation step is performed for 48 hours. The incubation step may be performed at any temperature suitable for cell proliferation and survival, for example, between 35°C and 42°C, preferably at 37 or 39°C. Preferably, the incubation step is performed at 37°C or 39°C for 24 hours. Preferably, the incubation step is performed at 30°C to 40°C (for example, 37°C) for 16 to 24 hours.

[0301] The percentage of dead cancer cells can be measured by reference to the total number of starting cancer cells. The number of dead cancer cells can be measured using any suitable means, for example, by viability staining (e.g., trypan blue staining) and microscopy, or by other automated means, such as a cell electron detection device, such as the RT-CES® system available from ACEA Biosciences, Inc. (11585 Sorrento Valley Rd., Suite 103, San Diego, CA 92121, USA). In some embodiments, the percentage of dead cancer cells may be determined within 24 hours (e.g., of incubation of cancer cell lines and granulocytes). The percentage of dead cancer cells is preferably the maximum number of cancer cells that would have died if the method of the present invention had been carried out. The percentage of dead cancer cells in the mixture may be the maximum percentage of cancer cells that would have died within 48 hours after the formation of the mixture.

[0302] A granulocyte-to-cancer cell ratio of at least 1:1, 5:1, or 10:1 may be used. Preferably, a granulocyte-to-cancer cell ratio of 5:1 is used. More preferably, a granulocyte-to-cancer cell ratio of 10:1 is used.

[0303] The number of dead cancer cells can also be measured using the ACEA Biosciences xCELLigence RTCA DP Analyzer system (registered trademark). The xCELLigence system is a real-time cell analyzer that enables label-free, dynamic, and continuous monitoring of changes in cell phenotype by measuring electrical impedance. Such measurements can be performed as detailed in Example 11. The system is commercially available from ACEA Biosciences, 6779 Mesa Ridge Road #100, San Diego, CA 92121, USA.

[0304] In a preferred embodiment, the CKA assay is performed using the ACEA Biosciences xCELLigence RTCA DP Analyzer System® in accordance with the manufacturer's instructions, as follows: e. Place 6000 cancer cells at the bottom of a 16-well plate. The cells are grown until they reach confluence, which is determined by the plateau (i.e., the "normalization point") of the f.Cell Index (CI) value. g. Add 60,000 granulocytes (i.e., a ratio of 10 granulocytes per cancer cell), and incubate at 37°C. h. The percentage of dead cancer cells is the maximum percentage of cancer cells that died within 48 hours after granulocyte addition, calculated using the following formula: ((Cell Index エフェクターなし -CellIndex エフェクター ) / Cell Index エフェクターなし ) × 100.

[0305] The maximum percentage of dead cancer cells may be referred to as "CKA%" in this specification. Preferably, the cancer cells are PANC-1 cells, which are commercially available from American Type Culture Collection United Kingdom (UK), Guernsey, Ireland, Jersey and Liechtenstein, LGC Standards (Queens Road, Teddington, Middlesex, TW11 0LY, UK) and have catalog number ATCC CRL-1469.

[0306] In a particularly preferred embodiment, granulocytes having the ability to kill cancer cells kill less than 15% of non-cancer cells in the "Non-Cancer Cell Killing Activity (NCKA) assay" described herein. Preferably, granulocytes kill less than 10% (e.g., less than 5% or less than 1%) of non-cancer cells in the "Non-Cancer Cell Killing Activity (NCKA) assay" described herein.

[0307] The "Non-Cancer Killing Activity (NCKA) Assay" or "NCKA Assay" can be performed using the ACEA Biosciences xCELLigence RTCA DP Analyzer System®, following the manufacturer's instructions, as follows: a. Place 6,000 non-cancerous cells at the bottom of a 16-well plate. b. Proliferate the cells until they reach confluence, which is determined by the plateau (i.e., the "normalization point") of the Cell Index (CI) value. c. Add 60,000 granulocytes (i.e., a ratio of 10 granulocytes to 1 non-cancer cell), and incubate at 37°C. d. The percentage of dead non-cancerous cells is the maximum percentage of non-cancerous cells that have died within 48 hours after granulocyte addition, calculated using the following formula: ((Cell Index エフェクターなし -CellIndex エフェクター ) / Cell Index エフェクターなし ) × 100.

[0308] 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 catalog number ATCC® CRL-10783®. In another embodiment, the non-cancerous cells are liver cells (e.g., primary non-transplantable liver tissue cells).

[0309] In a preferred embodiment, granulocytes may be considered "cancer cell-killing granulocytes" if they kill at least 5% of cancer cells in the method described herein. Granulocytes may be considered "cancer cell-killing granulocytes" if they kill at least 10%, 20%, 30%, 40%, 50%, or 51.5% of existing cancer cells. In a preferred embodiment, granulocytes may be considered "cancer cell-killing granulocytes" if they kill at least 60% of existing cancer cells. In a preferred embodiment, granulocytes may be considered "cancer cell-killing granulocytes" if they kill at least 70% of existing cancer cells. For example, granulocytes may be considered "cancer cell-killing granulocytes" if they kill at least 80% or 90% of existing cancer cells. In a particularly preferred embodiment, granulocytes may be considered "cancer cell-killing granulocytes" if they kill at least 51.5% of existing cancer cells. References to granulocyte-producing cells “having the ability to kill cancer cells” in this specification may be interpreted as referring to granulocyte-producing cells that can differentiate into granulocytes having the ability to kill cancer cells, consistent with the above definition.

[0310] In contrast, granulocytes that "lack the ability to kill cancer cells" or "cannot kill cancer cells" may be granulocytes that cannot kill at least 5% of cancer cells in the method described herein. Granulocytes that "lack the ability to kill cancer cells" or "cannot kill cancer cells" may be granulocytes that cannot kill at least 10%, 20%, 30%, 40%, 50%, or 51.5% of the cancer cells present. In a preferred embodiment, granulocytes that "lack the ability to kill cancer cells" or "cannot kill cancer cells" may be granulocytes that cannot kill at least 60% of the cancer cells present. In a preferred embodiment, granulocytes that "lack the ability to kill cancer cells" or "cannot kill cancer cells" may be granulocytes that cannot kill at least 70% of the cancer cells present. For example, granulocytes that "lack the ability to kill cancer cells" or "cannot kill cancer cells" may be granulocytes that cannot kill at least 80% or 90% of the cancer cells present. In a preferred embodiment, granulocytes that "lack the ability to kill cancer cells" or "cannot kill cancer cells" may be granulocytes that cannot kill at least 51.5% of the cancer cells present. Similarly, a reference to granulocyte-producing cells that "lack the ability to kill cancer cells" or "cannot kill cancer cells" may be interpreted as referring to granulocyte-producing cells that do not differentiate into granulocytes capable of killing cancer cells and / or differentiate into granulocytes that "lack the ability to kill cancer cells" or "cannot kill cancer cells."

[0311] Cells that have the "ability to kill pathogens or cells infected with pathogens" In a preferred embodiment, granulocyte-producing cells suitable for use in various aspects of the present invention can be differentiated to produce granulocytes having the ability to kill pathogens or cells infected with pathogens.

[0312] The “ability to kill infected organisms or cells infected with infected organisms” may be determined by mixing cells (e.g., granulocytes such as neutrophils) with infected organisms or cells infected with infected organisms and measuring the viability of the infected organisms or cells infected with infected organisms (e.g., after incubation). If the infected organisms or cells infected with infected organisms can no longer survive (i.e., have died), the cells exhibit the ability to kill infected organisms or cells infected with infected organisms. In a preferred embodiment, the ability to kill infected organisms or cells infected with infected organisms is determined using the Infection Killing Activity (IKA) assay described herein.

[0313] In a preferred embodiment, the IKA assay is performed a. Forming a test sample by bringing an infected body or cells infected with an infected body into contact with granulocytes, b. Incubating the test sample, c. To measure the percentage of dead infected organisms or cells infected with infected organisms in the test sample. Includes.

[0314] In a preferred embodiment, the IKA assay is performed a. Mixing granulocytes with infected organisms or cells infected with infected organisms to obtain a mixture, b. Incubating the mixture, c. To measure the percentage of dead infected organisms or cells infected with infected organisms in the mixture. Includes.

[0315] The incubation step or contact between granulocytes and infected organisms / infected cells may take place between 1 hour and 100 hours. Preferably, the incubation step or contact between granulocytes and infected organisms / infected cells may take place between 5 hours and 75 hours, for example between 10 hours and 20 hours. The incubation step or contact between granulocytes and infected organisms / infected cells may take place between 6 hours and 6 days. Preferably, the incubation step or contact between granulocytes and infected organisms / infected cells may take place between 6 hours and 2 days, for example between 12 hours and 36 hours, for example between 16 hours and 24 hours. In a preferred embodiment, the incubation step is performed for 24 hours. In another embodiment, the incubation step or contact between granulocytes and infected organisms / infected cells is performed for 48 hours. The incubation step or contact between granulocytes and infected cells may be carried out at any temperature suitable for cell proliferation and survival, for example, between 35°C and 42°C, preferably at 37°C or 39°C. Preferably, the incubation step or contact step between granulocytes and infected cells is carried out at 37°C or 39°C for 24 hours. Preferably, the incubation step or contact between granulocytes and infected cells is carried out at 30-40°C (e.g., 37°C) for 16-24 hours.

[0316] The conditions described above may be particularly suitable when incubating / contacting granulocytes with cells infected by an infectious agent.

[0317] The incubation step or contact between granulocytes and infected cells may be performed between 30 minutes and 24 hours (e.g., before assessing the mortality percentage). Preferably, the incubation step or contact between granulocytes and infected cells may be performed between 1 and 3 hours, for example, 2 hours. In other words, the assessment of mortality percentage may be determined after 2 hours of contact / incubation. The incubation step or contact between granulocytes and infected cells may be performed at any temperature suitable for cell proliferation and survival, for example, between 35°C and 42°C, preferably at 37°C.

[0318] The conditions described above may be particularly suitable when incubating / contacting granulocytes with infectious agents such as bacteria.

[0319] In preferred embodiments, the contact or incubation step is performed in solution. In other words, the infected organism or cells infected with the infected organism may grow in solution (i.e., they do not adhere to / grow on a surface such as the surface of a plate).

[0320] Preferably, if the infectious agent is bacteria, the contact or incubation step is performed in solution. In contrast, if the method uses cells infected with the infectious agent, it is preferable that the cells grow on or adhere to a surface, such as the surface of a plate.

[0321] In a preferred embodiment, the contact or incubation step is carried out under stirring at a speed of 100 to 250 rpm, for example, 120 rpm.

[0322] In preferred embodiments, when the method uses cells infected with an infected organism, the method of the present invention may include the use of a granulocyte-to-cell ratio of at least 1:1, 5:1, or 10:1. Preferably, the method includes the use of a granulocyte-to-cell ratio of 5:1. More preferably, the method includes the use of a granulocyte-to-cell ratio of 10:1.

[0323] The percentage of dead cells can be measured by reference to the total number of starting 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 by other automated means, such as a cell electron detection device, such as the RT-CES® system available from ACEA Biosciences, Inc. (11585 Sorrento Valley Rd., Suite 103, San Diego, CA 92121, USA). In some embodiments, the percentage of dead cells may be determined within 24 hours (e.g., of cell and granulocyte incubation). The percentage of dead cells is preferably the maximum number of dead cells when the method of the present invention is carried out.

[0324] The number of dead cells can also be measured using the ACEA Biosciences xCELLigence RTCA DP Analyzer system (registered trademark). The xCELLigence system is a real-time cell analyzer that enables label-free, dynamic, and continuous monitoring of changes in cell phenotype by measuring electrical impedance. Such measurements can be performed as detailed in the examples. The system is commercially available from ACEA Biosciences, 6779 Mesa Ridge Road #100, San Diego, CA 92121, USA.

[0325] In preferred embodiments, when the infectant is bacteria, a granulocyte-to-colony-forming unit ratio of at least 1:10, 1:5, 1:3, or 1:2 may be used. Preferably, a granulocyte-to-colony-forming unit ratio of 1:2 is used. More preferably, a granulocyte-to-colony-forming unit ratio of 1:1 is used.

[0326] In preferred embodiments, the ability to kill infected organisms or cells infected with infected organisms is determined using the MRSA assay described herein.

[0327] In a preferred embodiment, the MRSA assay is performed a. Mixing granulocytes with MRSA cells to form a mixture, b. Incubating the mixture, c. To measure the percentage of dead MRSA cells in the mixture. Includes.

[0328] The "MRSA assay" can be performed as follows: a. 1 x 10⁻¹⁰ MRSA strain USA300 in RPMI 1640 7 100 μl of CFU / ml solution, 1 × 10 7 Mix with 100 μl of a solution containing granulocytes / ml, b. Incubate the mixture at 37°C while shaking at 120 rpm. c. Collect a sample at 2 hours (diluted with sterile RPMI if necessary) and plate it onto triptych soy agar, d. Incubate the plated sample at 37°C for 24 hours, e. Counting bacterial colonies, f. To quantify the total CFU content, g.Formula((CFU content エフェクターなし -CFU content エフェクター ) / CFU content エフェクターなし Use ) × 100 to calculate the percentage of dead MRSA cells based on the CFU content in steps a and f.

[0329] In particularly preferred embodiments, the term “having the ability to kill infected or infected cells” as used herein further means that granulocytes kill less than 15% of healthy (uninfected) cells in the “healthy (uninfected) cell assay” described herein. Preferably, granulocytes kill less than 10% (e.g., less than 5% or less than 1%) of healthy (uninfected) cells in the “healthy (uninfected) cell assay” described herein.

[0330] The "healthy (uninfected) cell assay" can be performed using the ACEA Biosciences xCELLigence RTCA DP Analyzer system (registered trademark) according to the manufacturer's instructions, as follows: a. Place 6,000 healthy (uninfected) cells at the bottom of a 16-well plate. b. Proliferate the cells until they reach confluence, which is determined by the plateau (i.e., the "normalization point") of the Cell Index (CI) value. c. Add 60,000 granulocytes (i.e., a ratio of 10 granulocytes per non-pathogenic infected cell), and incubate at 37°C. d. The percentage of dead healthy (uninfected) cells is the maximum percentage of non-pathogenic infected cells that have died within 48 hours after granulocyte addition, calculated using the following formula: ((Cell Index エフェクターなし -CellIndex エフェクター ) / Cell Index エフェクターなし ) × 100.

[0331] Preferably, the 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 catalog number ATCC® CRL-10783®. In another embodiment, the healthy (uninfected) cells are liver cells (e.g., primary non-transplantable liver tissue cells).

[0332] In a preferred embodiment, a granulocyte may be considered an "infectious or infectious cell-killing granulocyte" if it kills at least 5% of the infectious agents or cells infected with infectious agents in the method described herein. A granulocyte may be considered an "infectious or infectious cell-killing granulocyte" if it kills at least 10%, 20%, 30%, 40%, or 50% of the infectious agents or cells infected with infectious agents that are present. In a preferred embodiment, a granulocyte may be considered an "infectious or infectious cell-killing granulocyte" if it kills at least 60% of the infectious agents or cells infected with infectious agents that are present. In a preferred embodiment, a granulocyte may be considered an "infectious or infectious cell-killing granulocyte" if it kills at least 70% of the infectious agents or cells infected with infectious agents that are present. Preferably, granulocytes may be considered "granulocytes capable of killing infected organisms or infected cells" if they kill at least 80% or 90% of the infected organisms or cells infected with infected organisms that are present. In a particularly preferred embodiment, granulocytes may be considered "granulocytes capable of killing infected organisms or cells infected with infected organisms" if they kill more than 41.23% of the infected organisms or cells infected with infected organisms that are present.

[0333] In contrast, granulocytes that "lack the ability to kill infected organisms or cells infected with infected organisms" or "cannot kill infected organisms or cells infected with infected organisms" may be granulocytes that cannot kill at least 5% of infected organisms or cells infected with infected organisms in the methods described herein. Granulocytes that "lack the ability to kill infected organisms or cells infected with infected organisms" or "cannot kill infected organisms or cells infected with infected organisms" may be granulocytes that cannot kill at least 10%, 20%, 30%, 40%, or 50% of the infected organisms or cells infected with infected organisms that are present. In a preferred embodiment, granulocytes that "lack the ability to kill infected organisms or cells infected with infected organisms" or "cannot kill infected organisms or cells infected with infected organisms" are granulocytes that cannot kill at least 60% of the infected organisms or cells infected with infected organisms that are present. In a preferred embodiment, granulocytes that "lack the ability to kill infected organisms or cells infected with infected organisms" or "cannot kill infected organisms or cells infected with infected organisms" are granulocytes that cannot kill at least 70% of the infected organisms or cells infected with infected organisms that are present. Preferably, granulocytes that "lack the ability to kill infected organisms or cells infected with infected organisms" or "cannot kill infected organisms or cells infected with infected organisms" are granulocytes that cannot kill at least 80% or 90% of the infected organisms or cells infected with infected organisms that are present. In a particularly preferred embodiment, granulocytes that "lack the ability to kill infected organisms or cells infected with infected organisms" or "cannot kill infected organisms or cells infected with infected organisms" are granulocytes that cannot kill more than 41.23% of the infected organisms or cells infected with infected organisms that are present.Similarly, a reference to granulocyte-producing cells that "lack the ability to kill infected organisms or cells infected with infected organisms" or "cannot kill infected organisms or cells infected with infected organisms" refers to granulocyte-producing cells that do not differentiate into granulocytes that have the ability to kill infected organisms or cells infected with infected organisms, and / or differentiate into granulocytes that "lack the ability to kill infected organisms or cells infected with infected organisms" or "cannot kill infected organisms or cells infected with infected organisms."

[0334] The infectant may refer to bacteria, fungi, viruses, macroparasites (e.g., helminths), or a combination thereof. Preferably, the infectant is a bacterium or a virus. For example, in a preferred embodiment, the infectant is a bacterium. In another embodiment, the infectant is a virus. Preferably, the infectant is a pathogen.

[0335] Cells that can express chemokines Granulocyte-producing cells that can be used in the embodiments of the present invention described herein include those capable of producing granulocytes that can express a desired chemokine. As just one example, the inventors have shown that granulocyte-producing cells suitable for use in various embodiments of the present invention can differentiate and produce granulocytes that secrete CXCL10.

[0336] Cells that can express co-stimulatory receptor ligands Granulocyte-producing cells that can be used in the embodiments of the present invention described herein include those capable of producing granulocytes that can express favorable ligands for costimulatory receptors. As just one example, the inventors have shown that granulocyte-producing cells suitable for use in various embodiments of the present invention can differentiate and produce granulocytes that express costimulatory receptor ligands, such as 4-1BBL and OX40L.

[0337] Methods for generating granulocyte-producing cells (or populations of such cells) that can be used therapeutically, and therapeutically useful granulocyte-producing cells In one embodiment, the present invention relates to a method for preparing granulocyte-producing cells for therapeutic use, ·below: · G-CSF, GM-CSF, · IL-3 and TNF The present invention provides a method for generating a population of granulocyte-producing cells by culturing a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells, including the presence of a certain substance.

[0338] Such a method may optionally further include the steps of purifying the population of granulocyte-producing cells that have been generated, and / or formulating the population of cells for medical use.

[0339] In one embodiment, the present invention provides a population of granulocyte-producing cells prepared for therapeutic use by the method of the first embodiment of the present invention.

[0340] The granulocyte-producing cells generated by the method of the present invention may be optionally recovered after generation. For the purposes of this disclosure, “recovery” of cells may be interpreted to include cell suspension, cell isolation, or cell separation.

[0341] The granulocyte-producing cells produced by the method of the present invention may optionally be cryopreserved after production. Granulocytes such as neutrophils do not respond well to cryopreservation, and it is known that the level of viable cells remaining after thawing of the frozen cell population is low. In contrast, the granulocyte-producing cells of the present invention are well-suited to cryopreservation, and a high level of viable cells is obtained after the freeze-thaw process. Therefore, the granulocyte-producing cell population of the present invention offers significant advantages compared to mature granulocyte cells in applications where it is desirable to cryopreserve cells before use for therapeutic purposes.

[0342] The granulocyte-producing cells produced by the method of the present invention may optionally be formulated for medical use after production. Methods suitable for formulating cell populations for therapeutic use are well known to those skilled in the art and can optionally be used to formulate the granulocyte-producing cell population of the present invention to produce the pharmaceutical composition of the present invention.

[0343] The characteristics of the granulocyte-producing cells that are generated, and the characteristics of the progenitor cell populations that can be used in such a method, will be considered in more detail elsewhere in this specification.

[0344] Optionally, cell culture conditions that promote progenitor cell differentiation may further include the presence of at least one cytokine selected from the group consisting of SCF and TPO.

[0345] The following paragraphs detail useful embodiments of methods for generating granulocyte-producing cells or populations of such cells. These include useful embodiments of progenitor cells that can be used as starting materials, granulocyte-producing cells that can be generated by the method, and cell culture conditions that can be utilized.

[0346] The cell culture conditions used in the method of the first aspect of the present invention to promote differentiation may include Iskov-modified Dulbecco's medium (IMDM) as the cell culture medium. In a preferred embodiment, IMDM is a medium containing high glucose, glutamine, HEPES, sodium pyruvate, and optionally phenol red.

[0347] This method utilizes cytokine granulocyte colony-stimulating factor (G-CSF) as an adjuvant.

[0348] Preferably, G-CSF is provided at a concentration of 0.013 μg / mL or higher. For example, G-CSF may be provided at concentrations of 0.016 μg / mL or higher, 0.02 μg / mL or higher, 0.03 μg / mL or higher, or 0.065 μg / mL or higher.

[0349] Preferably, G-CSF is provided at a concentration of 0.65 μg / mL or less. For example, G-CSF may be provided at concentrations of 0.52 μg / mL or less, 0.39 μg / mL or less, or 0.26 μg / mL or less.

[0350] Preferably, G-CSF is provided at concentrations of approximately 0.013 μg / mL to 0.65 μg / mL, 0.016 μg / mL to 0.52 μg / mL, 0.02 μg / mL to 0.39 μg / mL, 0.03 μg / mL to 0.26 μg / mL, or 0.065 μg / mL to 0.195 μg / mL. In a preferred embodiment, G-CSF is provided at a concentration of approximately 0.13 μg / mL. In fact, in a preferred embodiment, G-CSF is provided at a concentration of 0.13 μg / mL.

[0351] Examples of suitable forms of G-CSF that can be used in this manner include products produced by Peprotech and GMP products produced by BioLegend, details of which are described herein.

[0352] This method utilizes cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) as an adjuvant.

[0353] Preferably, GM-CSF is provided at a concentration of 0.001 μg / mL or higher. For example, GM-CSF may be provided at concentrations of 0.00125 μg / mL or higher, 0.00167 μg / mL or higher, 0.0025 μg / mL or higher, or 0.005 μg / mL or higher.

[0354] Preferably, GM-CSF is provided at a concentration of 0.05 μg / mL or less. For example, GM-CSF may be provided at concentrations of 0.04 μg / mL or less, 0.03 μg / mL or less, or 0.02 μg / mL or less.

[0355] Preferably, GM-CSF is provided at concentrations of approximately 0.001 μg / mL to 0.05 μg / mL, 0.125 μg / mL to 0.04 μg / mL, 0.00167 μg / mL to 0.03 μg / mL, 0.0025 μg / mL to 0.02 μg / mL, or 0.005 μg / mL to 0.015 μg / mL. In a preferred embodiment, GM-CSF is provided at a concentration of approximately 0.01 μg / mL. In fact, in a preferred embodiment, GM-CSF is provided at a concentration of 0.01 μg / mL.

[0356] Examples of suitable forms of GM-CSF that can be used in this manner include products produced by Peprotech and BioTechne, as well as GMP products produced by BioTechne, details of which are described herein.

[0357] This method utilizes the cytokine interleukin-3 (IL-3) as an adjunct.

[0358] Preferably, IL-3 is provided at a concentration of 0.013 μg / mL or higher. For example, IL-3 may be provided at concentrations of 0.016 μg / mL or higher, 0.02 μg / mL or higher, 0.03 μg / mL or higher, or 0.065 μg / mL or higher.

[0359] Preferably, IL-3 is provided at a concentration of 0.65 μg / mL or less. For example, IL-3 may be provided at concentrations of 0.52 μg / mL or less, 0.39 μg / mL or less, or 0.26 μg / mL or less.

[0360] Preferably, IL-3 is provided at concentrations of approximately 0.013 μg / mL to 0.65 μg / mL, 0.016 μg / mL to 0.52 μg / mL, 0.02 μg / mL to 0.39 μg / mL, 0.03 μg / mL to 0.26 μg / mL, or 0.065 μg / mL to 0.195 μg / mL. In a preferred embodiment, IL-3 is provided at a concentration of approximately 0.13 μg / mL. In fact, in a preferred embodiment, IL-3 is provided at a concentration of 0.13 μg / mL.

[0361] Examples of suitable forms of IL-3 that can be used in this manner include products manufactured by PeproTech and GMP products manufactured by PeproTech or BioTechne, details of which are described herein.

[0362] In preferred embodiments, GM-CSF and IL-3 are provided to cells for a period of 12 to 72 hours, preferably 48 hours, during the cell culture conditions. For example, GM-CSF and IL-3 may be provided to cells for the last 48 hours of the period the cells are in culture. GM-CSF and IL-3 may be provided to cells on days 4 and 5 of the cell culture conditions that promote the differentiation of progenitor cells. GM-CSF and IL-3 may be provided to cells on days 3 and 4 of the cell culture conditions that promote the differentiation of progenitor cells.

[0363] This method utilizes tumor necrosis factor (TNF) as an adjuvant. The terms TNF and TNF-alpha are used synonymously in this specification.

[0364] Preferably, TNF is provided at a concentration of 0.0001 μg / mL or higher. For example, TNF may be provided at concentrations of 0.000125 μg / mL or higher, 0.000167 μg / mL or higher, 0.00025 μg / mL or higher, or 0.0005 μg / mL or higher.

[0365] Preferably, TNF is provided at a concentration of 0.005 μg / mL or less. For example, TNF may be provided at a concentration of 0.004 μg / mL or less, 0.003 μg / mL or less, or 0.002 μg / mL or less.

[0366] Preferably, TNF is provided at concentrations of approximately 0.0001 μg / mL to 0.005 μg / mL, 0.000125 μg / mL to 0.004 μg / mL, 0.000167 μg / mL to 0.003 μg / mL, 0.00025 μg / mL to 0.002 μg / mL, or 0.0005 μg / mL to 0.0015 μg / mL. In a preferred embodiment, TNF is provided at a concentration of approximately 0.001 μg / mL. In fact, in a preferred embodiment, TNF is provided at a concentration of 0.001 μg / mL.

[0367] Examples of suitable forms of TNF that can be used in this manner include products produced by PeproTech and GMP products produced by BioTechne, details of which are described herein.

[0368] In a preferred embodiment, TNF is provided to the cells for a period of 12 to 36 hours, preferably 24 hours, during the cell culture conditions. For example, TNF may be provided to the cells for the last 24 hours of the period the cells are in culture. TNF may be provided to the cells on day 4 to 5 of the cell culture conditions that promote the differentiation of progenitor cells. TNF may be provided to the cells on day 5 of the cell culture conditions that promote the differentiation of progenitor cells. TNF may be provided to the cells on day 4 of the cell culture conditions that promote the differentiation of progenitor cells.

[0369] This method may optionally utilize cytokine stem cell factors (SCFs) as adjuncts.

[0370] Preferably, SCF is provided at a concentration of 0.013 μg / mL or higher. For example, SCF may be provided at concentrations of 0.016 μg / mL or higher, 0.02 μg / mL or higher, 0.03 μg / mL or higher, or 0.065 μg / mL or higher.

[0371] Preferably, SCF is provided at a concentration of 0.65 μg / mL or less. For example, SCF may be provided at concentrations of 0.52 μg / mL or less, 0.39 μg / mL or less, or 0.26 μg / mL or less.

[0372] Preferably, SCF is provided at concentrations of approximately 0.013 μg / mL to 0.65 μg / mL, 0.016 μg / mL to 0.52 μg / mL, 0.02 μg / mL to 0.39 μg / mL, 0.03 μg / mL to 0.26 μg / mL, or 0.065 μg / mL to 0.195 μg / mL. In a preferred embodiment, SCF is provided at a concentration of approximately 0.13 μg / mL. In fact, in a preferred embodiment, SCF is provided at a concentration of 0.13 μg / mL.

[0373] Examples of suitable forms of SCF that can be used in this manner include products produced by Peprotech and GMP products produced by Peprotech or BioTechne, details of which are described herein.

[0374] This method may optionally utilize the cytokine thrombopoietin (TPO) as an adjunct.

[0375] Preferably, TPO is provided at a concentration of 0.013 μg / mL or higher. For example, TPO may be provided at concentrations of 0.016 μg / mL or higher, 0.02 μg / mL or higher, 0.03 μg / mL or higher, or 0.065 μg / mL or higher.

[0376] Preferably, TPO is provided at a concentration of 0.65 μg / mL or less. For example, TPO may be provided at concentrations of 0.52 μg / mL or less, 0.39 μg / mL or less, or 0.26 μg / mL or less.

[0377] Preferably, TPO is provided in concentrations of approximately 0.013 μg / mL to 0.65 μg / mL, 0.016 μg / mL to 0.52 μg / mL, 0.02 μg / mL to 0.39 μg / mL, 0.03 μg / mL to 0.26 μg / mL, or 0.065 μg / mL to 0.195 μg / mL. In a preferred embodiment, TPO is provided at a concentration of approximately 0.13 μg / mL. In fact, in a preferred embodiment, TPO is provided at a concentration of 0.13 μg / mL.

[0378] Examples of suitable forms of TPO that can be used in this manner include products produced by Peprotech and GMP products produced by BioTechne or Peprotech, details of which are provided herein.

[0379] In preferred embodiments, the cell culture conditions used when culturing a population of progenitor cells to generate granulocyte-producing cells further include the presence of at least one adjuvant selected from the group consisting of insulin transferrin selenium (ITS) and human serum albumin (HSA). In preferred embodiments, such cell culture conditions include the presence of both ITS and HSA. Preferably, both ITS and HSA are present in the differentiation medium of the present invention.

[0380] This method may appropriately utilize insulin as an adjuvant at a concentration between approximately 0.1 g / L and approximately 5 g / L, for example, at a concentration of approximately 1.0 g / L. These methods and cell culture media may appropriately utilize transferrin as an adjuvant at a concentration between approximately 0.01 g / L and approximately 2.5 g / L, for example, at a concentration of approximately 0.55 g / L. Preferably, such methods and cell culture media may utilize selenium as an adjuvant at a concentration between approximately 0.0001 g / L and approximately 0.003 g / L, for example, at a concentration of approximately 0.00067 g / L.

[0381] This method may optionally utilize HSA as an auxiliary agent.

[0382] Preferably, HSA may be provided in a concentration between 0.1% and 5%. For example, HSA provided as an adjuvant may be provided in a concentration of approximately 1%.

[0383] Preferably, the cell culture conditions or differentiation medium used in the method of the present invention to promote the differentiation of progenitor cells may include GM-CSF, G-CSF, SCF, TPO, IL-3, TNF, ITS, and HSA. The cell culture medium may optionally include IMDM supplemented with Glutamax.

[0384] Therefore, in a preferred embodiment, the cell culture conditions or differentiation medium used in the method of the present invention to promote the differentiation of progenitor cells may include approximately 0.01 μg / mL of GM-CSF, approximately 0.13 μg / mL of G-CSF, approximately 0.13 μg / mL of SCF, approximately 0.13 μg / mL of TPO, approximately 0.13 μg / mL of IL-3, approximately 0.001 μg / mL of TNF, 1 × ITS, and approximately 1% HSA. The cell culture medium may optionally include IMDM supplemented with Glutamax.

[0385] The method of the present invention may include culturing a population of progenitor cells for any appropriate period of time under cell culture conditions that promote the differentiation of progenitor cells. For example, progenitor cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days under conditions that produce a population of granulocyte-producing cells. The method according to the first aspect of the present invention may include culturing a population of progenitor cells for a period of 1 to 7 days under cell culture conditions that promote the differentiation of progenitor cells. For example, such a method may include culturing cells for a period of 4 to 7 days under the relevant conditions. In a preferred embodiment, such a method may include culturing cells for approximately 1 day, or approximately 2 days, or approximately 3 days, or approximately 4 days, or approximately 5 days, or approximately 6 days, or approximately 7 days. Progenitor cells may be cultured for 1 to 10 days, 2 to 9 days, 3 to 8 days, 4 to 7 days, or 5 to 6 days under conditions that produce a population of granulocyte-producing cells. Preferably, the progenitor cells are cultured for 4, 5, or 6 days under conditions that produce a population of granulocyte-producing cells. In a preferred embodiment, the progenitor cells are cultured for 5 days under conditions that produce a population of granulocyte-producing cells. In a preferred embodiment, the progenitor cells are cultured for 5 days under conditions that produce a population of granulocyte-producing cells. In a preferred embodiment, the progenitor cells are cultured for 6 days under conditions that produce a population of granulocyte-producing cells.

[0386] In a preferred embodiment of the method of the present invention, the progenitor cells are 1 cm 2Approximately 1 x 10 5 From 10x10 6 The cells may be cultured at an initial seeding density.

[0387] The method of the present invention may also include increasing the number of cells present in the culture so that the number of granulocyte-producing cells obtained by the method is greater than the number of progenitor cells present at the start of the method. In a preferred embodiment, the number of granulocyte-producing cells in the resulting population may increase by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 times compared to the number of progenitor cells present at the start of the method. The method described in the examples achieves a population of granulocyte-producing cells that is approximately 3.5 times larger than the initial population of progenitor cells.

[0388] In preferred embodiments, the method is carried out with respect to a population of progenitor cells generated by in vitro proliferation of a population of stem cells. Thus, such a method of the present invention may further include the step of culturing a population of stem cells under cell culture conditions that produce a population of progenitor cells.

[0389] In a preferred embodiment, the method further includes the step of culturing a population of stem cells under cell culture conditions that generate a population of progenitor cells, • The cell culture conditions for generating progenitor cells are: · SCF, • Flt-3 ligand, IL-3, · IL-6, and TPO This includes the presence of.

[0390] The number of progenitor cells generated by such methods may be significantly increased compared to the number of stem cells present at the start of the cell culture conditions. As just one example, such embodiments of the method of the present invention can achieve an increase in the number of progenitor cells of at least 50 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 300 times, or at least 350 times or more compared to the number of stem cells at the start of the cell culture conditions. The examples detail the protocol used by the inventors to achieve an increase of approximately 75 times in the number of progenitor cells compared to the initial stem cell population.

[0391] The present invention also provides a cell culture medium for use in the method of the present invention, comprising SCF, Flt-3 ligand, IL-3, IL-6, and TPO. The cell culture medium according to this aspect of the present invention may also be referred to as "expansion medium."

[0392] Therefore, the method for preparing cells for therapeutic use according to such embodiments of the present invention is a) · SCF, • Flt-3 ligand, IL-3, · IL-6, and TPO To generate a population of progenitor cells by culturing a population of stem cells under cell culture conditions that include the presence of, b) · G-CSF, GM-CSF, · IL-3 and TNF To culture a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells, including the presence of [specific element], to generate a population of granulocyte-producing cells, and optionally, c) To collect granulocyte-producing cells and It may include.

[0393] Representing the change in cell number from the initial population of stem cells to the population of granulocyte-generating cells generated, the total increase in cell number achieved by such a method of the present invention may be at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 300 times, at least 350 times, at least 400 times, at least 450 times, at least 500 times, at least 550 times, at least 600 times, at least 650 times, at least 700 times, at least 750 times, at least 800 times, at least 850 times, at least 900 times, at least 950 times, at least 1000 times, at least 1050 times, at least 1100 times, at least 1150 times, at least 1200 times, at least 1250 times, or at least 1300 times. Examples detail the protocols used by the inventors to achieve an increase of more than 250 times in the number of granulocyte-generating cells compared to the initial stem cell population.

[0394] The method according to such embodiments of the present invention may include a total incubation period of 10 to 25 days, for example, 11 to 20 days, for example, 12, 13, 14, 15, 6, 17, 18, or 19 days.

[0395] SCF may optionally be provided as an adjuvant in embodiments of the method of the present invention that include the step of generating a population of progenitor cells.

[0396] Preferably, SCF is provided at a concentration of 0.02 μg / mL or higher. For example, SCF may be provided at concentrations of 0.025 μg / mL or higher, 0.03 μg / mL or higher, 0.05 μg / mL or higher, or 0.1 μg / mL or higher.

[0397] Preferably, SCF is provided at a concentration of 1 μg / mL or less. For example, SCF may be provided at a concentration of 0.8 μg / mL or less, 0.6 μg / mL or less, or 0.4 μg / mL or less.

[0398] Preferably, SCF is provided at concentrations of approximately 0.02 μg / mL to 1 μg / mL, 0.025 μg / mL to 0.8 μg / mL, 0.03 μg / mL to 0.6 μg / mL, 0.05 μg / mL to 0.4 μg / mL, or 0.1 μg / mL to 0.3 μg / mL. In a preferred embodiment, SCF is provided at a concentration of approximately 0.2 μg / mL. In fact, in a preferred embodiment, SCF is provided at a concentration of 0.2 μg / mL.

[0399] The above-described form of SCF is also suitable for use in such embodiments.

[0400] Flt-3 ligand (F3L) may optionally be provided as an adjuvant in embodiments of the method of the present invention that include the step of generating a population of progenitor cells.

[0401] Preferably, F3L is provided at a concentration of 0.02 μg / mL or higher. For example, F3L may be provided at concentrations of 0.025 μg / mL or higher, 0.03 μg / mL or higher, 0.05 μg / mL or higher, or 0.1 μg / mL or higher.

[0402] Preferably, F3L is provided at a concentration of 1 μg / mL or less. For example, F3L may be provided at a concentration of 0.8 μg / mL or less, 0.6 μg / mL or less, or 0.4 μg / mL or less.

[0403] Preferably, F3L is provided at concentrations of approximately 0.02 μg / mL to 1 μg / mL, 0.025 μg / mL to 0.8 μg / mL, 0.03 μg / mL to 0.6 μg / mL, 0.05 μg / mL to 0.4 μg / mL, or 0.1 μg / mL to 0.3 μg / mL. In a preferred embodiment, F3L is provided at a concentration of approximately 0.2 μg / mL. In fact, in a preferred embodiment, F3L is provided at a concentration of 0.2 μg / mL.

[0404] Examples of suitable forms of F3L that can be used in this manner include products produced by Peprotech and GMP products produced by Peprotech or BioTechne, details of which are described herein.

[0405] IL-3 may optionally be provided as an adjuvant in embodiments of the method of the present invention that include the step of generating a population of progenitor cells.

[0406] Preferably, IL-3 is provided at a concentration of 0.0015 μg / mL or higher. For example, IL-3 may be provided at concentrations of 0.0019 μg / mL or higher, 0.0025 μg / mL or higher, 0.00375 μg / mL or higher, or 0.0075 μg / mL or higher.

[0407] Preferably, IL-3 is provided at a concentration of 0.075 μg / mL or less. For example, IL-3 may be provided at concentrations of 0.06 μg / mL or less, 0.045 μg / mL or less, or 0.03 μg / mL or less.

[0408] Preferably, IL-3 is provided at concentrations of approximately 0.0015 μg / mL to 0.075 μg / mL, 0.0019 μg / mL to 0.06 μg / mL, 0.0025 μg / mL to 0.045 μg / mL, 0.00375 μg / mL to 0.03 μg / mL, or 0.0075 μg / mL to 0.0225 μg / mL. In a preferred embodiment, IL-3 is provided at a concentration of approximately 0.015 μg / mL. In fact, in a preferred embodiment, IL-3 is provided at a concentration of 0.015 μg / mL.

[0409] The above-described IL-3 configuration is suitable for use in such embodiments.

[0410] Interleukin-6 (IL-6) may optionally be provided as an adjuvant in embodiments of the method of the present invention that include the step of generating a population of progenitor cells.

[0411] Preferably, IL-6 is provided at a concentration of 0.0015 μg / mL or higher. For example, IL-6 may be provided at concentrations of 0.0019 μg / mL or higher, 0.0025 μg / mL or higher, 0.00375 μg / mL or higher, or 0.0075 μg / mL or higher.

[0412] Preferably, IL-6 is provided at a concentration of 0.075 μg / mL or less. For example, IL-6 may be provided at concentrations of 0.06 μg / mL or less, 0.045 μg / mL or less, or 0.03 μg / mL or less.

[0413] Preferably, IL-6 is provided at concentrations of approximately 0.0015 μg / mL to 0.075 μg / mL, 0.0019 μg / mL to 0.06 μg / mL, 0.0025 μg / mL to 0.045 μg / mL, 0.00375 μg / mL to 0.03 μg / mL, or 0.0075 μg / mL to 0.0225 μg / mL. In a preferred embodiment, IL-6 is provided at a concentration of approximately 0.015 μg / mL. In fact, in a preferred embodiment, IL-6 is provided at a concentration of 0.015 μg / mL.

[0414] Examples of suitable forms of IL-6 that can be used in this manner include products manufactured by PeproTech and GMP products manufactured by PeproTech or BioTechne, details of which are described herein.

[0415] TPO may optionally be provided as an adjuvant in embodiments of the method of the present invention that include the step of generating a population of progenitor cells.

[0416] Preferably, TPO is provided at a concentration of 0.002 μg / mL or higher. For example, TPO may be provided at concentrations of 0.0025 μg / mL or higher, 0.003 μg / mL or higher, 0.005 μg / mL or higher, or 0.01 μg / mL or higher.

[0417] Preferably, TPO is provided at a concentration of 0.1 μg / mL or less. For example, TPO may be provided at a concentration of 0.08 μg / mL or less, 0.06 μg / mL or less, or 0.04 μg / mL or less.

[0418] Preferably, TPO is provided in concentrations of approximately 0.002 μg / mL to 0.1 μg / mL, 0.0025 μg / mL to 0.08 μg / mL, 0.003 μg / mL to 0.06 μg / mL, 0.005 μg / mL to 0.04 μg / mL, or 0.01 μg / mL to 0.03 μg / mL. In a preferred embodiment, TPO is provided at a concentration of approximately 0.02 μg / mL. In fact, in a preferred embodiment, TPO is provided at a concentration of 0.02 μg / mL.

[0419] The TPO forms described above are also suitable for use in these embodiments.

[0420] Preferably, the cell culture conditions used to promote the generation of progenitor cells in the method of the present invention may include SCF, and Flt-3 ligand, and IL-3, and IL-6, and TPO, and ITS, and HSA. The cell culture medium may optionally include IMDM with added Glutamax.

[0421] Therefore, in a preferred embodiment, the cell culture conditions that promote the generation of progenitor cells used in the method of the present invention may include approximately 0.2 μg / mL of SCF, approximately 0.2 μg / mL of Flt-3 ligand, approximately 0.015 μg / mL of IL-3, approximately 0.015 μg / mL of IL-6, approximately 0.02 μg / mL of TPO, 1 × ITS, and approximately 1% HSA. The cell culture medium may optionally include IMDM with added Glutamax.

[0422] Examples of stem cells that can be used in such methods of the present invention as starting materials for the generation of progenitor cells (and ultimately granulocyte-producing cells) include, but are not limited to, hematopoietic stem cells (HSCs). Further details of suitable stem cells and sources of stem cells are provided elsewhere herein and include (but are not limited to) umbilical cord blood and mobilized blood.

[0423] In preferred embodiments, the cell culture conditions used when culturing stem cells to generate progenitor cells further include the presence of at least one adjuvant selected from the group consisting of ITS and HSA. In preferred embodiments, such cell culture conditions include the presence of both ITS and HSA. Preferably, both ITS and HSA are present in the expanded medium of the present invention.

[0424] ITS may be provided as an adjuvant in embodiments of the method of the present invention, which includes the step of generating a population of progenitor cells.

[0425] Such embodiments of the method may appropriately utilize insulin as an adjuvant at a concentration between about 0.1 g / L and about 5 g / L, for example, at a concentration of about 1.0 g / L. These methods may appropriately utilize transferrin as an adjuvant at a concentration between about 0.01 g / L and about 2.5 g / L, for example, at a concentration of about 0.55 g / L. Preferably, such methods may utilize selenium as an adjuvant at a concentration between about 0.0001 g / L and about 0.003 g / L, for example, at a concentration of about 0.00067 g / L.

[0426] HSA may be provided as an adjuvant in embodiments of the present invention that include the step of generating a population of progenitor cells.

[0427] Preferably, HSA may be provided in a concentration between 0.1% and 5%. For example, HSA provided as an adjuvant may be provided in a concentration of approximately 1%.

[0428] In embodiments of the present invention's method for culturing stem cells to obtain progenitor cells, this may include increasing the number of cells present in the culture.

[0429] The method of the present invention may include culturing a population of stem cells for any appropriate period of time under cell culture conditions that generate a population of progenitor cells. For example, cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days under conditions that generate a population of progenitor cells. Preferably, cells are cultured for 8 or 9 days under conditions that generate a population of progenitor cells. Stem cells may be cultured for 1-15 days, 1-10 days, 2-14 days, 3-13 days, 4-12 days, 5-11 days, 6-10 days, 7-9 days, or 8-9 days under conditions that generate a population of progenitor cells. Preferably, stem cells such as HSCs are cultured for 8-9 days under conditions that generate a population of progenitor cells.

[0430] In a preferred embodiment of such a method of the present invention, stem cells are cultured for a period of 6 to 10 days under conditions that produce a population of progenitor cells. For example, such a method may include culturing the cells for a period of 7 to 8 days. In a preferred embodiment, such a method may include culturing the cells for approximately 6, or approximately 7, or approximately 8, or approximately 9, or approximately 10 days under cell culture conditions that produce a population of progenitor cells.

[0431] Therefore, the present invention's method for preparing cells for therapeutic use is (a) A population of stem cells is cultured for 6 to 10 days, or preferably 8 days, under cell culture conditions for generating progenitor cells that include the presence of SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, thereby generating a population of progenitor cells. (b) To obtain a population of granulocyte-producing cells by culturing a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells. It may include.

[0432] A preferred method of the present invention for preparing cells for therapeutic use is: (a) A population of stem cells is cultured for 6 to 10 days, or preferably 8 days, under cell culture conditions for generating progenitor cells that include the presence of IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, thereby generating a population of progenitor cells. (b) A population of progenitor cells is cultured for 1 to 6 days, preferably 5 days, under cell culture conditions that promote the differentiation of progenitor cells, including IMDM, G-CSF, GM-CSF, IL-3, and TNF, to obtain a population of granulocyte-producing cells. It may include.

[0433] Such a method of the present invention for preparing cells for therapeutic use is (a) A population of stem cells is cultured for 6 to 10 days, or preferably 8 days, under cell culture conditions for generating progenitor cells that include the presence of IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, thereby generating a population of progenitor cells. (b) To obtain a population of granulocyte-producing cells by culturing a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells. It may include.

[0434] For example, the present invention's method for preparing cells for therapeutic use is: (a) A population of stem cells is cultured for 6 to 10 days, or preferably 8 days, under cell culture conditions for generating progenitor cells that include the presence of IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, thereby generating a population of progenitor cells. (b) Culture a population of progenitor cells for 1 to 6 days, or preferably 5 days, under cell culture conditions that promote the differentiation of progenitor cells including IMDM, SCF, TPO, GCSF, ITS, and HSA, to obtain a population of granulocyte-producing cells. It may include.

[0435] Appropriately replenished cell culture medium may be replaced or replenished at any appropriate point in the culture of stem cells under conditions for generating progenitor cells. For example, cell culture medium may be replenished on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of stem cell culture. Preferably, cell culture medium is replenished on days 1 and 6 of stem cell culture. Cell culture medium may be replaced on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of stem cell culture. Preferably, cell culture medium is replaced on day 4 of stem cell culture.

[0436] Stem cells, such as HSCs, from which progenitor cells are generated, may be seeded at any appropriate cell density. For example, stem cells may be seeded at a density of 1 × 10⁶. 5 cells / mL~1×10 6 cells / mL, 2.5×10 5 cells / mL~1×10 6 cells / mL, 3×10 5 cells / mL~8×10 5 cells / mL or 4 × 10 5 cells / mL~6×10 5 cells / mL, preferably 5 × 10 5 Stem cells may be seeded at a density of cells / mL. 5 cells / cm 2 ~1 × 10 6 cells / cm 2 , 2.5×10 5 cells / cm 2 ~1 × 10 6 cells / cm 2 , 3 x 10 5 cells / cm 2 ~8×10 5 cells / cm 2 or 4 x 10 5 cells / cm 2 ~6×10 5 cells / cm 2 Preferably 5 × 10 5 cells / cm 2They may be seeded at a density of 5 × 10. In a preferred embodiment, stem cells (e.g., HSCs) are arranged as follows: 5 cells / mL and 5 × 10 5 cells / cm 2 It is sown at this density.

[0437] Cells may be seeded in any suitable culture vessel. For example, cells may be seeded in a G-Rex 6M or G-Rex 10M culture vessel. Cells may be transferred to a new culture vessel at any appropriate time. Cells may be successively transferred to cell culture vessels with increasing surface area. Such transfers may be performed on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the culture for generating progenitor cells. For example, stem cells (e.g., HSCs) may be transferred from a smaller G-Rex to a G-Rex 100M on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the culture for generating progenitor cells. For example, stem cells (e.g., HSCs) may be transferred to a G-Rex 100M or a larger cell culture vessel, such as a G-Rex 500M, on day 4 of expansion. In a preferred embodiment, progenitor cells may be transferred to a new culture vessel on day 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of culture conditions that promote the differentiation of progenitor cells into granulocyte-producing cells.

[0438] According to such embodiments, a preferred method for preparing cells for therapeutic use is: (a) 5 × 10 5 cells / mL and 5 × 10 5 cells / cm 2 This involves seeding stem cells (e.g., HSCs), (b) To obtain a population of progenitor cells by culturing cells for 8 days in a cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, wherein the cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA is replenished on days 1 and 6 of the culture, and the cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA is replaced on day 4 of the culture, (c) A population of progenitor cells is cultured for 5-6 days in a cell culture medium containing IMDM, SCF, TPO, GCSF, ITS, and HSA to obtain a population of granulocyte-producing cells, wherein the cell culture medium containing IMDM, SCF, TPO, GCSF, ITS, and HSA is replenished on the third day of differentiation, and a population of progenitor cells is cultured to obtain a population of granulocyte-producing cells. It may include.

[0439] A preferred method for preparing cells for therapeutic use is: (a) 5 × 10 5 cells / mL and 5 × 10 5 cells / cm 2 This involves seeding stem cells (e.g., HSCs), (b) To obtain a population of progenitor cells by culturing cells for 8 days in a cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA, wherein the cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA is replenished on days 1 and 6 of the culture, and the cell culture medium containing IMDM, SCF, FLT-3, TPO, IL-3, IL-6, ITS, and HSA is replaced on day 4 of the culture, (c) A population of progenitor cells is cultured for 5-6 days in a cell culture medium containing IMDM, SCF, TPO, G-CSF, ITS, and HSA to obtain a population of granulocyte-producing cells, wherein the cell culture medium containing IMDM, SCF, TPO, GCSF, ITS, HAS, GM-CSF, IL-3, and TNF is replenished on the third day of differentiation, and a population of progenitor cells is cultured to obtain a population of granulocyte-producing cells. It may include.

[0440] The inventors have also identified a method by which granulocyte-producing cells can be primed to amplify cellular properties that enhance their therapeutic utility. In particular, priming granulocyte-producing cells by such a method can amplify their cytotoxic activity in a manner that enhances their therapeutic utility.

[0441] A preferred method for priming granulocyte-producing cells for therapeutic use comprises culturing a population of granulocyte-producing cells in the presence of GM-CSF and optionally one or more cytokines selected from the group consisting of TNF, IFN-α, IFN-β, IL-15, and IL-18.

[0442] The method may also include a step of priming granulocyte-producing cells for therapeutic use by culturing a population of granulocyte-producing cells in the presence of GM-CSF and optionally one or more cytokines selected from the group consisting of TNF, IFN-α, IFN-β, IL-15, and IL-18.

[0443] A method comprising the step of priming granulocyte-producing cells may optionally further include the steps of purifying the resulting population of primed granulocyte-producing cells and / or formulating the population of primed cells for medical use. The population of primed granulocyte-producing cells may be as defined elsewhere in this disclosure (e.g., with respect to the biological activity of the primed cells or the expression of their specific markers).

[0444] GM-CSF may be used at concentrations of 1-1000 ng / mL, 2-500 ng / mL, 3-250 ng / mL, or 4-200 ng / mL in the cell culture conditions during the priming step. GM-CSF may also be used at concentrations of 5-150 ng / mL, for example, 10-130 ng / mL.

[0445] TNF may be used in the cell culture conditions of the priming step at concentrations of 0.001–10 ng / mL, 0.002–5 ng / mL, 0.003–2.5 ng / mL, or 0.004–2 ng / mL. TNF may also be used at concentrations of 0.005–1.5 ng / mL, for example, 0.01–1 ng / mL.

[0446] IFN-α may be used at concentrations of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, or 4-20 ng / mL in the cell culture conditions during the priming step. IFN-α may also be used at concentrations of 5-15 ng / mL, for example, 10 ng / mL.

[0447] IFN-β may be used at concentrations of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, or 4-20 ng / mL in the cell culture conditions during the priming step. IFN-β may also be used at concentrations of 5-15 ng / mL, for example, 10 ng / mL.

[0448] IL-15 may be used in the cell culture conditions of the priming step at concentrations of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, or 4-20 ng / mL. IL-15 may also be used at concentrations of 5-15 ng / mL, for example, 10 ng / mL.

[0449] IL-18 may be used in the cell culture conditions of the priming step at concentrations of 1-100 ng / mL, 2-50 ng / mL, 3-25 ng / mL, or 4-20 ng / mL. IL-18 may also be used at concentrations of 5-15 ng / mL, for example, 10 ng / mL.

[0450] IL-3 may be used in the cell culture conditions of the priming step at concentrations of 1-1000 ng / mL, 2-500 ng / mL, 3-250 ng / mL, or 4-200 ng / mL. IL-3 may also be used at concentrations of 5-150 ng / mL, for example, 10-130 ng / mL.

[0451] In a preferred embodiment, priming includes culturing a population of granulocyte-producing cells in the presence of GM-CSF at a concentration of approximately 130 ng / mL, and optionally one or more cytokines selected from the group consisting of TNF at a concentration of approximately 0.01 to 1.0 ng / mL, IFN-α at a concentration of approximately 10 ng / mL, IFN-β at a concentration of approximately 10 ng / mL, IL-15 at a concentration of approximately 10 ng / mL, IL-18 at a concentration of approximately 10 ng / mL, and IL-3 at a concentration of approximately 130 ng / mL.

[0452] In preferred embodiments, the primed cells may be cultured in the presence of GM-CSF, G-CSF, SCF, TPO, and IL-15. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 10 ng / mL, G-CSF at a concentration of approximately 130 ng / mL, SCF at a concentration of approximately 130 ng / mL, TPO at a concentration of approximately 130 ng / mL, and IL-15 at a concentration of approximately 10 ng / mL.

[0453] In preferred embodiments, the primed cells may be cultured in the presence of GM-CSF, G-CSF, SCF, TPO, and TNF. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 100 ng / mL, G-CSF at a concentration of approximately 130 ng / mL, SCF at a concentration of approximately 130 ng / mL, TPO at a concentration of approximately 130 ng / mL, and TNF at a concentration of approximately 10 ng / mL.

[0454] In a preferred embodiment, the primed cells may be cultured in the presence of GM-CSF and IL-3. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL and IL-3 at a concentration of approximately 130 ng / mL.

[0455] In a preferred embodiment, the primed cells may be cultured in the presence of GM-CSF and IL-15. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL and IL-15 at a concentration of approximately 10 ng / mL.

[0456] In a preferred embodiment, the primed cells may be cultured in the presence of GM-CSF and IL-18. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL and IL-18 at a concentration of approximately 10 ng / mL.

[0457] In a preferred embodiment, the primed cells may be cultured in the presence of GM-CSF and IL-16. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL and IL-16 at a concentration of approximately 10 ng / mL.

[0458] In a preferred embodiment, the primed cells may be cultured in the presence of GM-CSF and TNF. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL and TNF at a concentration of approximately 1 ng / mL.

[0459] In preferred embodiments, the primed cells may be cultured in the presence of GM-CSF, G-CSF, SCF, TPO, and IFN-α. For example, the cells may be cultured in the presence of GM-CSF at a concentration of approximately 130 ng / mL, G-CSF at a concentration of approximately 130 ng / mL, SCF at a concentration of approximately 130 ng / mL, TPO at a concentration of approximately 130 ng / mL, and IFN-α at a concentration of approximately 10 ng / mL.

[0460] The priming step can be continued for any appropriate period. For example, the priming step may be continued for 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, 72 hours, 78 hours, 84 hours, 90 hours, or 96 hours. The priming step may be continued for 1 to 96 hours, 2 to 90 hours, 3 to 84 hours, 6 to 78 hours, 12 to 72 hours, 18 to 54 hours, or 24 to 48 hours. Preferably, priming may include a culture incorporating the cytokines mentioned above for a period of 1, 2, or 3 days, for example, at the concentrations mentioned above. In particular, priming may include a culture incorporating the combination of priming cytokines mentioned for 2 days.

[0461] The priming step may be suitably incorporated at any appropriate stage of the method. However, priming is typically performed during the period when the progenitor cells are cultured under conditions that promote differentiation of the progenitor cells into granulocyte-producing cells. For example, priming may be initiated on day 1, day 2, day 3, day 4, or day 5 of culturing the progenitor cells under conditions that promote differentiation into granulocyte-producing cells.

[0462] Alternatively, in a preferred embodiment, the priming step may be performed after the granulocyte-producing cells have been generated and optionally after the granulocyte-producing cells have been collected. For example, priming may be performed before or after the cryopreservation of the population of granulocyte-producing cells according to the present invention.

[0463] As a mere example, if the priming step is performed over two days, priming may be performed on days 3 and 4 of culture conditions that promote differentiation of progenitor cells into granulocytes, on days 4 and 5 of such culture, or on days 5 and 6 of such culture. To avoid misunderstanding, any of the above priming protocols may be preferably performed on days 3 and 4, days 4 and 5, or days 5 and 6 of culture conditions that promote differentiation of progenitor cells into granulocytes.

[0464] The priming step developed by the present inventors does not appear to have a significant effect on the immunomodulatory capacity of the granulocyte population. Therefore, in embodiments where only the immunomodulatory activity of granulocytes is desired, it may be preferable to omit the priming step from the method of generating the granulocyte population.

[0465] Unless otherwise required by context, the following definitions are applicable to granulocyte-producing cells in any embodiment of the present invention or in any other context in which granulocyte-producing cells or populations of such cells are referred to.

[0466] For a cell to be considered "granulocyte-generating" in the terminology of this invention, it must be able to produce granulocytes (e.g., neutrophils) or granulocyte precursor cells of the granulocyte lineage. Indeed, a suitable granulocyte-generating cell can produce such cells. To avoid misunderstanding, granulocytes themselves should be considered "granulocyte-generating" for the purposes of this invention, but in many embodiments, granulocyte-generating cells are not granulocytes themselves, but cells capable of producing granulocytes. Preferably, granulocyte-generating cells in the context of this invention may be interpreted as excluding other cell lineages, for example, monocyte lineages and / or lymphocyte lineages.

[0467] In the context of the present invention, a suitable population of granulocyte-producing cells may be defined by reference to the expression of their different markers. Those skilled in the art will be well aware of suitable methods by which cells can be characterized and / or isolated and, if necessary, enriched based on the expression of a particular profile of cell surface markers.

[0468] The following definitions, based on appropriate marker expression profiles, can be used alone or in combination to identify appropriate populations of granulocyte-producing cells.

[0469] Unless otherwise specified (for example, in lists that state "or" or "and / or"), any reference in this disclosure to cells that are positive or negative for the expression of certain markers should be interpreted as meaning that such cells must have the listed expression (either positive or negative) of each of the markers mentioned. Thus, for example, a reference to a cell or population of cells as "CD15+CD66b+" should be interpreted as meaning that the cell is positive for the expression of both CD15 and CD66b, and that the population of such cells includes cells that are CD15+ and cells that are CD66b+.

[0470] This disclosure includes definitions of cell populations or subpopulations with respect to the enumerated expression (either positive or negative) of several specific markers.

[0471] In a preferred embodiment, such a definition can be interpreted as requiring that the population or subpopulation of interest include cells that are positive or negative (if required by the definition) with respect to the enumerated markers. For example, in the case of a population defined as being positive for the expression of a first marker, negative for the expression of a second marker, and positive for the expression of a third marker, this requirement may be satisfied by a population of cells that include cells that are positive for the first marker, and at the same time also include cells that are negative for the second marker, and further include cells that are positive for the third marker. In such an embodiment, the population or subpopulation of cells may be heterogeneous with respect to cells having the enumerated expressions (whether positive or negative). Preferably, the cells that each show the required expression for each of the enumerated markers may constitute the largest group of cells in such a population or subpopulation. Preferably, the cells that each show the required expression for each of the enumerated markers may constitute the majority of cells in such a population or subpopulation. Preferably, cells that each exhibit the required expression for each of the enumerated markers constitute at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in such population or subpopulation.

[0472] In one embodiment, in a given population or subpopulation, each cell in that population or subpopulation may express at least two, three, four, or five of the enumerated markers.

[0473] In a preferred embodiment, such a definition can be interpreted as requiring that the population or subpopulation of interest consist of cells that are positive or negative (if required by the definition) with respect to the enumerated markers. In such an embodiment, the population or subpopulation of cells is homogeneous with respect to cells having the enumerated expression (whether positive or negative).

[0474] In a preferred embodiment, the population of granulocyte-producing cells includes cells that are "Lin-" (i.e., negative for a cocktail of common leukocyte markers, defined for this purpose as negative expression of each of CD3, CD16, CD19, CD20, CD14, and CD56). For example, a preferred population of granulocyte-producing cells may contain at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% Lin- cells. As an example, a preferred population of granulocyte-producing cells may contain at least 90% Lin- cells. A preferred population of granulocyte-producing cells may contain approximately 95-99% Lin- cells. Preferably, the population of granulocyte-producing cells may contain approximately 97% Lin- cells.

[0475] Alternatively, or furthermore, a preferred population of granulocyte-producing cells includes CD34+ cells. For example, such a population of granulocyte-producing cells may contain less than 50%, less than 45%, less than 40%, or less than 35% CD34+ cells. As an example, such a population of granulocyte-producing cells may contain less than 30% CD34+ cells. In such embodiments, the proportion of CD34+ cells may be between approximately 5% and 25%. Preferably, the population of granulocyte-producing cells contains approximately 14% CD34+ cells.

[0476] Alternatively, or furthermore, a preferred population of granulocyte-producing cells includes CD38+ cells. For example, such a population of granulocyte-producing cells may include at least 10%, at least 15%, or at least 20% CD38+ cells. In such embodiments, the proportion of CD38+ cells may be between approximately 10% and 80%, for example, between approximately 10% and 30%. Preferably, the population of granulocyte-producing cells includes approximately 12% CD38+ cells.

[0477] Alternatively, or furthermore, a preferred population of granulocyte-producing cells includes cells having a hematopoietic stem cell (HSC) phenotype (defined for this purpose as Lin-CD34+CD38-CD45RA-CD90+). For example, such a population of granulocyte-producing cells may include cells having less than 5%, less than 4%, less than 3%, or less than 2% of the HSC phenotype. As an example, such a population of granulocyte-producing cells may include cells having less than 1% of the HSC phenotype. A preferred population of granulocyte-producing cells may include cells having approximately 0.01–0.15% of the HSC phenotype. Preferably, the population of granulocyte-producing cells includes cells having approximately 0.04% of the HSC phenotype.

[0478] Alternatively, or furthermore, a preferred population of granulocyte-producing cells may include cells having less than 1% of the long-term reconstituted hematopoietic stem cell (LT-HSC) phenotype (defined for this purpose as Lin-CD34+CD38-CD45RA-CD90+CD49f+). For example, such a population of granulocyte-producing cells may include cells having less than 5%, less than 4%, less than 3%, or less than 2% of the LT-HSC phenotype. As an example, such a population of granulocyte-producing cells may include cells having less than 1% of the LT-HSC phenotype. A preferred population of granulocyte-producing cells may include cells having approximately 0.01–0.05% of the LT-HSC phenotype. Preferably, the population of granulocyte-producing cells includes cells having approximately 0.02% of the LT-HSC phenotype.

[0479] Alternatively, or further, a preferred population of granulocyte-producing cells may include cells having the lymphoid priming pluripotent progenitor (LMPP) phenotype (defined for this purpose as Lin-CD34+CD38-CD45RA+). For example, such a population of granulocyte-producing cells may include cells having less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, or less than 25% of the LMPP phenotype. As an example, such a population of granulocyte-producing cells may include cells having less than 20% of the LMPP phenotype. A preferred population of granulocyte-producing cells may include cells having approximately 2-15% of the LMPP phenotype. Preferably, the population of granulocyte-producing cells includes cells having approximately 5% of the LMPP phenotype.

[0480] Alternatively, or furthermore, a preferred population of granulocyte-producing cells includes cells having a pluripotent progenitor (MPP) phenotype (defined for this purpose as Lin-CD34+CD38-CD45RA-). For example, such a population of granulocyte-producing cells may include cells having less than 30%, less than 25%, less than 20%, or less than 15% of the MPP phenotype. As an example, such a population of granulocyte-producing cells may include cells having less than 10% of the MPP phenotype. A preferred population of granulocyte-producing cells may include cells having approximately 1-6% of the MPP phenotype. Preferably, the population of granulocyte-producing cells includes cells having approximately 2% of the MPP phenotype.

[0481] In a preferred embodiment, the population of granulocyte-producing cells may include more than 90% Lin- cells (e.g., approximately 97% Lin- cells), and / or less than 30% CD34+ cells (e.g., approximately 14% CD34+ cells), and / or more than 10% CD38+ cells (e.g., approximately 12% CD38+ cells), and / or less than 1% of cells having the above-defined HSC phenotype (e.g., approximately 0.04% of cells having the HSC phenotype), and / or less than 1% of cells having the above-defined LT-HSC phenotype (e.g., approximately 0.02% of cells having the LT-HSC phenotype), and / or less than 20% of cells having the above-defined LMPP phenotype (e.g., approximately 5% of cells having the LMPP phenotype), and / or less than 10% of cells having the above-defined MPP phenotype (e.g., approximately 2.5% of cells having the MPP phenotype).

[0482] In preferred embodiments, the population of granulocyte-producing cells may include more than 90% Lin- cells (e.g., approximately 97% Lin- cells), less than 30% CD34+ cells (e.g., approximately 14% CD34+ cells), more than 10% CD38+ cells (e.g., approximately 12% CD38+ cells), less than 1% of cells having the above-defined HSC phenotype (e.g., approximately 0.04% of cells having the HSC phenotype), less than 1% of cells having the above-defined LT-HSC phenotype (e.g., approximately 0.02% of cells having the LT-HSC phenotype), less than 20% of cells having the above-defined LMPP phenotype (e.g., approximately 5% of cells having the LMPP phenotype), and less than 10% of cells having the above-defined MPP phenotype (e.g., approximately 2.5% of cells having the MPP phenotype).

[0483] Alternatively, or furthermore, a preferred population of granulocyte-producing cells may include CD15- versus CD15+ cells in a ratio of approximately 1:1.

[0484] A suitable population of granulocyte-producing cells may contain approximately 25-75% or 35-60% CD15- cells. For example, a suitable population of granulocyte-producing cells may contain approximately 50% CD15- cells.

[0485] A suitable population of granulocyte-producing cells may contain approximately 30-70% or 40-65% CD15+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 50% CD15+ cells.

[0486] A preferred population of granulocyte-producing cells may contain approximately 5-25%, 5-20%, 7-18%, or 10-15% CD15+CD66b+ cells. For example, a preferred population of granulocyte-producing cells may contain approximately 12% CD15+CD66b+ cells.

[0487] A preferred population of granulocyte-producing cells may contain approximately less than 30% or less than 25% CD11b+ cells. For example, a preferred population of granulocyte-producing cells may contain approximately 10-25% or 15-25% CD11b+ cells, for example, approximately 19% CD11b+ cells.

[0488] A preferred population of granulocyte-producing cells may contain at least 30%, at least 35%, at least 40%, or at least 45% CD71+ cells. For example, a preferred population of granulocyte-producing cells may contain approximately 60% CD71+ cells.

[0489] A suitable population of granulocyte-producing cells may contain approximately 60-95% or 65-90% CD49d+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 75% CD49d+ cells.

[0490] A preferred population of granulocyte-producing cells may contain less than 5%, less than 4%, less than 3%, or less than 2% CD10+ cells. A preferred population of granulocyte-producing cells may contain approximately 0.03–2% CD10+ cells. For example, a preferred population of granulocyte-producing cells may contain approximately 0.5% CD10+ cells.

[0491] A suitable population of granulocyte-producing cells may contain approximately 1-120% or 2-15% CD177+ cells. A suitable population of granulocyte-producing cells may contain approximately 6% CD177+ cells.

[0492] A preferred population of granulocyte-producing cells may contain less than 20% or less than 15% CD62L+ cells. For example, a preferred population of granulocyte-producing cells may contain between approximately 2% and 15%, for example, approximately 8% CD62L+ cells.

[0493] A suitable population of granulocyte-producing cells may contain approximately 40-85% or 50-75% CD54+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 63% CD54+ cells.

[0494] A suitable population of granulocyte-producing cells may contain approximately 2-15% or approximately 5-10% CD63+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 7% CD63+ cells.

[0495] A suitable population of granulocyte-producing cells may contain approximately 70-90% or 75-85% CD18+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 80% CD18+ cells.

[0496] A suitable population of granulocyte-producing cells may contain approximately 35-55% HLA-DR+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 47% HLA-DR+ cells.

[0497] A suitable population of granulocyte-producing cells may contain approximately 6-8% CD115+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 5% CD115+ cells.

[0498] A suitable population of granulocyte-producing cells may contain approximately 5-30% CD40+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 15% CD40+ cells.

[0499] A suitable population of granulocyte-producing cells may contain approximately 5-30% CD64+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 15% CD64+ cells.

[0500] A suitable population of granulocyte-producing cells may contain approximately 20-55% CD32+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 40% CD32+ cells.

[0501] A suitable population of granulocyte-producing cells may contain approximately 4-9% CXCR2+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 6% CXCR2+ cells.

[0502] A suitable population of granulocyte-producing cells may contain approximately 0.04–1% CD16+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 0.25% CD16+ cells.

[0503] A suitable population of granulocyte-producing cells may contain approximately 2-15% CD14+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 8% CD14+ cells.

[0504] A suitable population of granulocyte-producing cells may contain approximately 0.5–4% CD68+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 1.5% CD68+ cells.

[0505] A suitable population of granulocyte-producing cells may contain approximately 2-18% CD206+ cells. For example, a suitable population of granulocyte-producing cells may contain approximately 10% CD206+ cells.

[0506] A suitable isolation population of granulocyte-producing cells is: • Over 90% of Lin- cells (for example, approximately 97% of Lin- cells), • Less than 30% CD34+ cells (for example, approximately 14% CD34+ cells), • Over 30% of CD38+ cells (for example, approximately 65% ​​of CD38+ cells), Cells with less than 1% HSC phenotype (for example, cells with approximately 0.04% HSC phenotype), • Cells with less than 1% LT-HSC phenotype (for example, cells with approximately 0.02% LT-HSC phenotype) • Cells with less than 20% LMPP phenotype (for example, cells with approximately 5% LMPP phenotype), and • Cells with less than 10% MPP phenotype (e.g., cells with approximately 2.5% MPP phenotype) Includes.

[0507] A suitable isolation population of granulocyte-producing cells is: • The first subgroup of cells that are CD15+CD64+CD18+CD49d+CD71+ • A second subgroup of cells that are CD15-CD11b+ / -CD18+CD49d+CD32+HLA-DR- • A third subgroup of cells that are CD15-CD11b-HLA-DR+CD18+CD49d+ and CD71+ Includes.

[0508] A preferred population of granulocyte-producing cells may further include a fourth subpopulation of cells that are CD15-CD11b+HLA-DR+.

[0509] It will be understood that if information on the markers expressed by these subpopulations of cells is obtained, one or more of these subpopulations can be easily isolated from the cell population according to the sixth aspect of the present invention.

[0510] A preferred subpopulation of granulocyte-producing cells that are CD15+CD64+CD18+CD49d+CD71+ may also be positive for one, two or more, or all of the markers selected from the group consisting of CD177, CD11b, CD71, CD66b, HLA-DR, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14. Such a preferred subpopulation of cells may also be negative for the markers CD16 and / or CD62L (in addition to the required or optional expression or absence of expression of the other markers mentioned above). Preferably, the population or subpopulation of cells is heterogeneous with respect to the enumerated marker profiles (which may preferably include any of the components referred to herein). Preferably, a population or subpopulation of cells according to this embodiment of the present invention is uniformly positive for CD15 and heterogeneous for other markers in the enumerated marker profile (which may preferably include any of the components referred to herein). Preferably, a population or subpopulation of cells is uniform in the enumerated marker profile (which may preferably include any of the components referred to herein).

[0511] A first subpopulation of cells expresses markers very similar to those expressed by committed neutrophil progenitor cells. However, the cells disclosed in this embodiment of the present invention are CD64+, and may also be CD16- and CD62L-. This is in contrast to neutrophil precursor cells that are CD64-CD16+ and CD62L+, which are observed in circulation and during homeostasis. The expression of CD64 by CD15+CD64+CD18+CD49d+CD71+ cells therefore provides a useful means of distinguishing the cells disclosed herein from those occurring in nature, as well as the lack of CD16 and / or CD62L expression. Cells, or populations of cells, that are CD15+CD64+CD18+CD49d+CD71+ and also CD16- and / or CD62L- can be distinguished as being produced by the method described herein, rather than being naturally occurring granulocyte-producing cells or populations of such cells.

[0512] The inventors have confirmed that cells from this subpopulation exhibit cytotoxic activity that makes them particularly effective for medical use. Indeed, such cells appear to constitute the main source of cytotoxic activity in the isolated population of granulocyte-producing cells described above. Therefore, such cells may be particularly useful in clinical situations where it is necessary to kill cells (e.g., cancer cells, infected cells, or cell-infecting pathogens) to achieve a therapeutic effect.

[0513] A first subpopulation of cells may express 4-1BBL and / or OX40L. These markers are ligands for T cells and NK cells, and their expression by these cells can indicate that the cells possess immunomodulatory activity. Similarly, a first subpopulation of cells may express CD38 and / or CD40 and / or CD54, as well as other costimulatory molecules associated with functional interactions with immune cells such as T cells. Therefore, such cells, or pharmaceutical compositions containing such cells, may be effective in biological or therapeutic applications that utilize the modulation of the activity of such nongranulocytic inflammatory cell types.

[0514] In addition to expressing markers indicating immunomodulatory capacity, this cell population also expresses molecules (particularly CD11b, CD18, Mac1, and CD32) that suggest they possess direct cytotoxic activity. This could make them suitable for applications where therapeutic elimination of cells, such as cancer cells or infected cells, is desired.

[0515] A preferred population of granulocyte-producing cells may include cells that are CD15-, CD11b+ / -, CD18+, CD49d+, CD32+, and HLA-DR-. A preferred subpopulation of cells that are CD15-CD11b+ / -CD18+CD49d+CD32+HLA-DR- may also be positive for one, two or more, or all of the markers selected from the group consisting of: CD177, CD11b, CD71, CD66b, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14. Preferably, the population or subpopulation of cells is heterogeneous with respect to the enumerated marker profiles (which may preferably include any of the components referred to herein). Preferably, the population or subpopulation of cells according to this embodiment of the present invention is uniformly negative for CD15 and HLA-DR and heterogeneous for other markers in the enumerated marker profile (which may preferably include any of the components referred to herein). Preferably, the population or subpopulation of cells is uniform in the enumerated marker profile (which may preferably include any of the components referred to herein).

[0516] A second subpopulation of cells expresses markers such as Mac-1 (including CD11b and CD18) and CD32, consistent with their high capacity for cytotoxic activity. Therefore, these cells may also be beneficial in medical or therapeutic applications requiring direct cytotoxic activity, such as the killing of cancer or infected cells. These cells may also express molecules such as 4-1BBL and / or OX40L, indicating immunomodulatory potential and suitability for use in biological or therapeutic applications requiring such activity. Cells in this group may also express CXCR2, which can be elevated by exposure to IL-3 during the methods according to the present invention, and is a marker that may contribute to increased chemotaxis (in response to drugs such as IL-8) and targeting of these cells to TMEs.

[0517] A preferred subpopulation of cells that are CD15-CD11b-HLA-DR+CD18+CD49d+ and CD71+ may also be positive for one, two or more, or all of the markers selected from the group consisting of: CD177, CD71, CD66b, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14. Preferably, the population or subpopulation of cells is heterogeneous with respect to the enumerated marker profile (which may preferably include any components referred to herein). Preferably, the population or subpopulation of cells according to this embodiment of the Invention is uniformly negative for CD15 and CD11b, uniformly positive for HLA-DR, and heterogeneous with respect to the other markers of the enumerated marker profile (which may preferably include any components referred to herein). Preferably, the population or subpopulation of cells is uniform with respect to the enumerated marker profile (which may preferably include any of the components referred to herein).

[0518] A third subpopulation of cells expresses markers indicating relatively low levels of differentiation. Consistently, these cells may also be CD34+. Cells in this group may also express markers such as 4-1BBL and / or OX40L and / or CD40 and / or CD54, indicating their suitability for use in applications requiring immunomodulation of nongranulocyte immune cells. While cells in this group do not express markers indicating direct cytotoxic activity, they may possess the ability to further differentiate and express markers such as CD11b and CD15, which would confer such activity. Therefore, these cells could be utilized in medical uses or therapeutic methods where in vivo signaling would induce such differentiation, resulting in the ability to kill harmful cell types.

[0519] A preferred subpopulation of granulocyte-producing cells that are CD15-CD11b+HLA-DR+ may also be positive for one, two or more, or all of the markers selected from the group consisting of: CD177, CD71, CD66b, CD115, CD49d, CD40, CD62L, CD54, CD18, CD34, CXCR4, CD64, CD32, CXCR2, CD38, Mac1, 4-1BBL, OX40L, PD-L1, and CD14. Preferably, the cell population or subpopulation is heterogeneous with respect to the enumerated marker profile (which may preferably include any of the components referred to herein). Preferably, the cell population or subpopulation according to this embodiment of the Invention is uniformly negative for CD15, uniformly positive for HLA-DR and CD11b, and heterogeneous with respect to the other markers of the enumerated marker profile (which may preferably include any of the components referred to herein). Preferably, the population or subpopulation of cells is uniform with respect to the enumerated marker profile (which may preferably include any of the components referred to herein).

[0520] These cells express markers similar to those expected to activate myeloid cells. These cells may further express markers such as CD14 and / or CD11b and / or CD206. They may be suitable for applications where either direct cytotoxicity or immunomodulatory activity is desired.

[0521] Suitable granulocyte-producing cells, or populations of granulocyte-producing cells, for use in the medical use of the therapeutic methods of the present invention may be CD64+, CD16-, and / or CD62L-. For example, granulocyte-producing cells may be CD64+. Granulocyte-producing cells may be CD64+ and CD16-. Granulocyte-producing cells may be CD64+ and CD62L-. Granulocyte-producing cells may be CD16- and CD62L-. Preferably, granulocyte-producing cells are CD64+, CD16-, and CD62L-. The expression of CD64, as well as the absence of CD16 and CD62L expression, by the granulocyte-producing cells of the present invention is in contrast to neutrophils, which are CD64-CD16+ and CD62L+, observed in circulation and during homeostasis. The expression of CD64, therefore, provides a useful means of distinguishing the granulocyte-producing cells disclosed herein from those naturally occurring, as is the absence of CD16 and / or CD62L expression. Granulocyte-producing cells that are CD64+, CD16-, and / or CD62L- can be distinguished as being produced by the method according to the present invention, rather than being naturally occurring granulocyte-producing cells or populations of such cells.

[0522] Therefore, in preferred embodiments, the medical use or therapeutic method of the present invention uses granulocyte-producing cells, or populations of such cells, which are CD64+ granulocyte-producing cells. Preferably, CD64+ granulocyte-producing cells are CD64+ and CD16- granulocyte-producing cells. CD64+ granulocyte-producing cells may be CD64+ and CD62L- granulocyte-producing cells. CD64+ granulocyte-producing cells may be CD64+, CD16- and CD62L- granulocyte-producing cells.

[0523] In another embodiment, the medical use or therapeutic method of the present invention utilizes granulocyte-producing cells, which are CD16-granulocyte-producing cells. CD16-granulocyte-producing cells may be CD16- and CD62L-granulocyte-producing cells.

[0524] In another embodiment, the medical use or therapeutic method of the present invention uses granulocyte-producing cells, which are CD62L-granulocyte-producing cells.

[0525] Source of granulocyte-producing cells Granulocyte-producing cells suitable for use in various embodiments of the present invention can be obtained from any suitable source. The granulocyte-producing cells may be homogeneous with respect to their intended recipient. They may be obtained from or derived from any suitable donor.

[0526] Any of the cells or populations of cells disclosed herein may be derived from mammals such as humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Preferably, the cells or populations of cells are of human origin. Therefore, the cells may be human cells, or the populations of cells may be populations of human cells. In particular, the granulocyte-producing cells or populations of granulocyte-producing cells disclosed herein may be derived from mammals such as humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Preferably, the granulocyte-producing cells or populations of granulocyte-producing cells are of human origin. Therefore, the granulocyte-producing cells may be human granulocyte-producing cells. The populations of granulocyte-producing cells may be populations of human granulocyte-producing cells.

[0527] Granulocyte-producing cells may be obtained from any suitable source. For example, granulocyte-producing cells may be obtained from a PBMC sample or a cord blood sample. PBMC samples or cord blood samples may be obtained from a donor. Preferably, granulocyte-producing cells may be obtained from a sample of αβT cell-depleted PBMCs. Granulocyte-producing cells may be obtained from hematopoietic stem cells or stem cells such as iPSCs (for example, they can be differentiated in vitro).

[0528] As used herein, the term “possible to obtain” also includes the term “obtainable.” In one embodiment, “possible to obtain” means “obtainable.”

[0529] As used herein, the term “donor” may also refer to a subject (preferably a human subject) from which a sample can be obtained (e.g., can be obtained). Any suitable sample from which granulocyte-producing cells and / or non-granulocyte immune cells can be obtained may be obtained from a donor. Donors may be selected based on one or more of the following characteristics: sex, age, medical history, and / or blood type. Donors may be selected if they are healthy. Donors may be selected if they do not have cancer and do not have infectious diseases. For example, a donor may be selected if they do not have cancer. A donor may be selected if they do not have infectious diseases. A donor may be selected if they are male. A donor may be selected if they are between 18 and 55 years old, preferably between 18 and 35 years old (more preferably between 18 and 24 years old). Preferably, the donor may be selected if the donor is male between 18 and 55 years of age, preferably between 18 and 35 years of age (more preferably between 18 and 24 years of age). In another embodiment, the donor may be selected if the donor is female. The donor may be selected if the donor is over 40 years of age. Preferably, the donor may be selected if the donor is female over 40 years of age.

[0530] Granulocyte-producing cells suitable for use in various embodiments of the present invention may be generated by in vitro differentiation of stem cells. As used herein, the term “stem cell” encompasses any cell capable of differentiating into granulocyte-producing cells (preferably capable of producing neutrophils). For example, the term “stem cell” may encompass totipotent, pluripotent, compound pluripotent, or unipotent cells. In preferred embodiments, the term “stem cell” encompasses hematopoietic stem cells, as well as precursor cells (e.g., differentiated from hematopoietic stem cells), which are capable of differentiating into granulocytes (preferably neutrophils). Preferably, the term “stem cell” as used herein does not encompass human embryonic stem cells.

[0531] Stem cells can be part of a stem cell culture.

[0532] "Stem cells" may be natural stem cells or induced stem cells. In preferred embodiments, natural stem cells may be cells of the hematopoietic pathway or equivalent cells. In preferred embodiments, granulocyte-producing cells are derived from induced pluripotent stem cells (iPSCs) or equivalent induced stem cells.

[0533] In preferred embodiments, iPSCs can be obtained from somatic cells, such as donor somatic cells. The generation of iPSCs is a well-known technique in the art. See Yu et al (2007), Science, 318:1917–1920, whose teachings are incorporated herein by reference.

[0534] In another embodiment, iPSCs can be obtained from stem cells (e.g., those obtained from a donor), such as hematopoietic pathway stem cells. Preferably, iPSCs can be obtained from hematopoietic stem cells or precursor cells as described herein.

[0535] In preferred embodiments, the stem cells are nuclear-transfer embryonic stem cells (NT-ESCs) or equivalent. In preferred embodiments, NT-ESCs can be obtained by injecting the nuclei of cells from a donor into egg cells from which the original nuclei have 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, the teachings of which are incorporated herein by reference.

[0536] Stem cells may be immortalized. Those skilled in the art are particularly familiar with immortalization techniques, including the introduction of viral genes that deregulate the cell cycle (e.g., adenovirus type 5 E1 gene) and the artificial expression of telomerase. Immortalization advantageously allows for the preparation of cell lines that can be stably cultured in vitro. Thus, in one embodiment, the present invention provides immortalized cell lines and stable stem cell cultures that can be obtained (e.g., obtained) from selected stem cells. Preferably, immortalized cell lines or stable stem cell cultures can be obtained (e.g., obtained) by the methods of the present invention.

[0537] The term "stable" as used in reference to stem cell cultures or cell lines means that the cell culture or cell line has been modified to be more suitable for in vitro cell culture than unmodified cells (i.e., cells obtained from a donor and directly subjected to in vitro cell culture). Therefore, the above-mentioned "stable" cell culture or cell line can withstand more replication (preferably for a longer period) compared to unmodified cells.

[0538] Methods to enhance the therapeutic activity of nongranulocyte immune cells A fifth aspect of the present invention provides a method for promoting the therapeutic activity of nongranulocyte immune cells, wherein the nongranulocyte immune cells are incubated together with granulocyte-producing cells.

[0539] The method according to this aspect of the present invention can be carried out in vitro or in vivo. Preferably, the method is carried out in vivo. The method according to this aspect of the present invention can be used to enhance the therapeutic activity of nongranulocyte immune cells before administering them to a patient as a therapeutic agent.

[0540] The method according to this embodiment of the present invention may be carried out with respect to any nongranulocyte immune cells. The method may be carried out with respect to host nongranulocyte immune cells. Preferably, the method is carried out with respect to NK cells.

[0541] An increase in therapeutic activity may be demonstrated by an increase in activation, according to any of the parameters further discussed herein.

[0542] A method using granulocyte-producing cells to improve immune cell cultures. In an eighth aspect, the present invention provides a method for increasing the viability of immune cells in culture, the method comprising culturing immune cells in the presence of a feeder layer of granulocyte-producing cells.

[0543] In a ninth aspect, the present invention provides a method for increasing the proliferation of immune cells in culture, the method comprising culturing immune cells in the presence of a feeder layer of granulocyte-producing cells.

[0544] The immune cells cultured by the method of the eighth or ninth aspect of the present invention may be selected from the group consisting of T cells and NK cells. In embodiments in which the cultured immune cells include T cells, the cells are CD8 + T cells, CD4 + The group may be selected from T cells, NK T cells, αβ T cells, γδ T cells, peripheral blood T cells, and tumor-infiltrating T cells.

[0545] The method according to the eighth aspect of the present invention may be suitable for use in culturing NK or NK T cells. The method according to the ninth aspect of the present invention may be suitable for use in culturing αβ T cells.

[0546] How to select the appropriate treatment regimen A sixth aspect of the present invention provides a method for selecting an appropriate treatment regimen for a patient. This method is: This includes identifying whether the patient has impaired nongranulocyte immune response, If the patient is identified as having impaired non-granulocyte immune response, treatment with granulocyte-producing cells is selected as the appropriate treatment. If the patient is identified as having no impairment in the non-granulocyte immune response, treatment with therapies other than granulocyte-producing cells will be selected.

[0547] Those skilled in the art will recognize many suitable methods for evaluating impairments (or other) in a patient's nongranulocyte immune response.

[0548] Such methods may be particularly relevant in patients suspected of having impaired nongranulocyte immune responses. Patients with impaired or suspected nongranulocyte immune responses may have or be receiving treatment for immunosuppressive diseases.

[0549] A seventh aspect of the present invention provides an alternative or additional method for selecting an appropriate treatment regimen for a patient. This method is This includes incubating the patient's nongranulocyte immune cells together with granulocyte-producing cells. If the activation of non-granulocyte immune cells in the patient increases in response to incubation, then treatment with granulocyte-producing cells is selected as the appropriate treatment. If the activation of non-granulocyte immune cells in the patient increases in response to incubation, treatment with therapies other than granulocyte-producing cells will be selected.

[0550] The activation of a patient's nongranulocyte cells can be assessed by any appropriate means, including, but not limited to, any appropriate indicators of activation, and any appropriate means, including indicators and means further discussed herein.

[0551] In a method according to any sixth or seventh aspect of the present invention, if treatment with granulocyte-producing cells is selected as an appropriate treatment, the treatment may be carried out using granulocyte-producing cells as considered in any aspect or embodiment of the present invention. Such cells may also be provided by the pharmaceutical composition of the present invention.

[0552] Screening method of the present invention The tenth, eleventh, twelfth, thirteenth, and fourteenth aspects of the present invention relate to a screening method for identifying granulocyte-producing cells suitable for therapeutic use. The tenth aspect provides a method for determining whether granulocyte-producing cells are suitable for use in the treatment of cancer by beneficially modulating the tumor microenvironment; the eleventh aspect provides a method for determining whether granulocyte-producing cells are suitable for use in the treatment of cancer by increasing the recruitment and / or activation of immune cells to tumors; the twelfth aspect provides a method for determining whether granulocyte-producing cells are suitable for use in the treatment of cancer by directly promoting the death of cancer cells; the thirteenth aspect provides a method for determining whether granulocyte-producing cells are suitable for use in the treatment of infectious diseases by directly promoting the death of cell-infecting pathogens or infected cells; and the fourteenth aspect provides a method for determining whether granulocyte-producing cells are suitable for use in the treatment of diseases by amplifying a therapeutic immune response.

[0553] In a preferred embodiment, the method according to the tenth aspect of the present invention may include evaluating the expression of inflammatory cytokines selected from the group consisting of IFN-γ and TNF.

[0554] Preferably, the method according to the 11th aspect of the present invention may include evaluating the expression of the chemokine CXL10.

[0555] In a preferred embodiment, the method according to the 11th aspect of the present invention may include evaluating the expression of a degranulation marker selected from the group consisting of CD107a, perforin, and granzymes.

[0556] Preferably, a method according to a twelfth aspect of the present invention may positively identify granulocyte-producing cells as suitable for use in the treatment of cancer by directly promoting the death of cancer cells if the rate of cancer cell death when incubated with granulocyte-producing cells or cells derived from granulocyte-producing cells is at least three times higher than the rate of non-cancer cell death.

[0557] A method according to a thirteenth aspect of the present invention may include positively identifying granulocyte-producing cells as suitable for use in the treatment of an infectious disease if the rate of death of a cell-infecting pathogen or infected cells incubated with granulocyte-producing cells or cells derived from granulocyte-producing cells is at least three times higher than the rate of death of uninfected cells.

[0558] A method according to a fourteenth aspect of the present invention may include identifying granulocyte-producing cells as suitable for therapeutic use if the activation of immune cells is increased in accordance with any of the considerations expressed herein. Granulocyte-producing cells may be incubated with any form of immune cells. For example, granulocyte-producing cells may be incubated with non-granulocyte cells. The immune cells may be derived from an individual in need of treatment.

[0559] If a screening method according to any of these embodiments of the present invention identifies granulocyte-producing cells as suitable for therapeutic use, the method may further include identifying the donor from which the granulocyte-producing cells were collected or derived as a donor capable of providing therapeutically effective granulocyte-producing cells. Alternatively, the method may further include obtaining stem cells from the donor from which the granulocyte-producing cells were collected or derived. The stem cells may be naturally occurring cells such as hematopoietic stem cells, or they may be artificial stem cells such as iPSCs. Such stem cells may be preserved. Such stem cells can be used, for example, to generate more therapeutically effective granulocyte-producing stem cells for incorporation into the pharmaceutical composition of the present invention.

[0560] The present invention will now be further described with reference to the following examples. [Examples]

[0561] Materials and methods Co-culture of PBMCs with IMANp or PDAC patient donor neutrophils: PBMCs from healthy donors were cultured with granulocyte-producing cells (referred to as "IMANp" by the inventors, as indicated in the figure) (n=4) or blood-derived neutrophils from pancreatic ductal adenocarcinoma (PDAC) donors (n=1) in various ratios (2:1, 1:1, or 0.5:1 granulocyte-producing cells:PBMCs, or 1:1 or 0.5:1 donor neutrophils:PBMCs). For identification and proliferation analysis, PBMCs were labeled with cell-tracking far-infrared (CTFR) dyes before co-culture. Co-culture was performed in or without anti-CD3 stimulation (OKT3, 1 μg / ml). After 72 hours, proliferation and / or activation of various T cell and NK cell populations were investigated by flow cytometry.

[0562] Digestion of biopsy samples from PDAC patients and co-culture with IMANp: Biopsy samples from PDAC patients were digested using Miltenyi's Human Tumor Isolation Kit (130-095-929) according to the manufacturer's instructions. Briefly, tumor biopsy samples were cut into small fragments (2-4 mm) and transferred to gentleMACS C tubes containing 4.7 mL of RPME, 200 μl of enzyme H, 20 μl of enzyme R, and 25 μl of enzyme A. The C tubes were then tightly sealed and mounted upside down into the sleeve of the gentleMACS Dissociator (Miltenyi). The program for hard tumors (37C_h_TDK_3) was selected and executed. After the program finished, the C tubes were removed, the samples were taken out, passed through a 70 μm cell strainer, and then washed in complete medium. For identification purposes, digested tumor cells were labeled with cell-tracking far-infrared (CTFR) dyes before co-culture. Next, CTFR-labeled tumor digests were co-cultured with IMANp (n=2) in a 2:1 ratio (tumor digest:IMANp). Co-culture was performed in or without anti-CD3 stimulation (OKT3, 1 μg / ml). After 72 hours, the activation of various T cell and NK cell populations was investigated by flow cytometry.

[0563] Flow cytometry: For identification and proliferation analysis, PBMCs were labeled with cell-tracking far-infrared (CTFR) dyes before co-culture. Granulocyte-producing cells (IMANp) or patient-donor neutrophils were not labeled before co-culture. After 72 hours of culture, cells were washed in PBS and incubated for 20 minutes with a viability / dead stain (Fixable Viability Dye eFluor 780, 1:500 dilution) and an FcγR block (Human TruStain FcX, 1:50 dilution). Cells were then washed in flow cytometry buffer and surface-stained with antibodies specific to CD3 (OKT3), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (HCD56), CD107a (H4A3), 4-1BB (4B4-1), and OX40 (Ber-ACT35). In other experiments, granulocytes generated during the differentiation of granulocyte-producing cells (designated here as "IMAN") were surface-stained for the expression of 4-1BBL (5F4) and OX40L (11C3.1). All antibodies were used at a 1:50 dilution, and staining was performed at 50 μl / sample. After surface staining, cells were fixed using 100 μl of 1×BD CellFix and acquired using MACSQuant 16 (Miltenyi). Data were analyzed using FlowLogic software. Analysis of stained populations was performed by gatedting with single live cells.

[0564] Quantitative determination of cytokines / chemokines in cell culture supernatant: The cell culture supernatant was 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 1-1] [Table 1-2]

[0565] Example 1 Co-culture with IMANp granulocyte-producing cells is associated with blood-derived CD8 + Increases T cell activation Methods: PBMCs from healthy donors were cultured with granulocyte-producing cells (n=4) or blood-derived neutrophils from PDAC donors (n=1) in various ratios shown. Co-culture was performed in or without anti-CD3 stimulation (OKT3, 1 μg / ml). After 72 hours, CD8 + T cell activation was investigated by flow cytometry. Before co-culturing, PBMCs were labeled with a cell-tracking far-infrared (CTFR) dye, and CD8 + Cells are called living cells, singlets, and CTFRs. + CD3 + CD8 + The cells were gated as follows. CD8 cell activation was investigated by measuring the expression of degranulation markers such as CD107a on the cell surface, as well as co-stimulatory molecules such as 4-1BB and OX40.

[0566] Results: The results were (Figure 1A) unstimulated and (Figure 1B) anti-CD3 stimulated CD8 + This shows the expression percentages of CD107a, 4-1BB, and OX40 on T cells.

[0567] (Figure 1A) Co-culture with granulocyte-producing cells, rather than patient-donor neutrophils, is in the absence of TCR stimulation for CD8 + As indicated by the increased expression of CD107a, 4-1BB, and OX40 on T cells, blood-derived CD8 +Stimulation with anti-CD3 increased T cell activation. (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 enhanced in the presence of granulocyte-producing cells but not in the presence of patient-donor neutrophils. These results indicate that the presence of granulocyte-producing cells enhances activation, as indicated by degranulation (CD107a) and the expression of further activation markers (costimulatory molecules OX40 and 4-1BB) of activated CD8 T cells. OX40 and 4-1BB are costimulatory markers expressed on activated T cells. The linkage of these costimulatory receptors on activated CD8 T cells should increase the effector function of these cells (e.g., increased cytotoxicity and IFN-γ production).

[0568] Furthermore, the data suggest that granulocyte-producing cells provide signal 2 (co-stimulation) and / or signal 3 (cytokine stimulation) for T cell activation.

[0569] This data also suggests that granulocyte-generating cells may be usable in combination therapy with T cell engagers, such as single / bispecific 4-1BB agonists or TAA / 4-1BB bispecific T cell engagers.

[0570] Example 2 Co-culture with granulocyte-producing cells is used for blood-derived CD4 + Increases T cell activation Methods: PBMCs from healthy donors were cultured with granulocyte-producing cells (n=4) or blood-derived neutrophils from PDAC donors (n=1) in various ratios shown. Co-culture was performed in or without anti-CD3 stimulation (OKT3, 1 μg / ml). After 72 hours, CD4 + T cell activation was investigated using flow cytometry. Before co-culturing, PBMCs were labeled with a cell-tracking far-infrared (CTFR) dye, and CD4 + Cells are called living cells, singlets, and CTFRs. + CD3 + CD4 +The cells were gated. CD4 cell activation was investigated by measuring the expression of co-stimulatory molecules (particularly 4-1BB and OX40) on the cell surface.

[0571] Results: The results were (Figure 2A) unstimulated and (Figure 2B) anti-CD3 stimulated CD4 + This shows the expression percentages of 4-1BB and OX40 on T cells. (Figure 2A) Co-culture with granulocyte-producing cells, rather than patient-donor neutrophils, shows CD4 expression in the absence of TCR stimulation. + As indicated by the increased expression of 4-1BB and OX40 on T cells, blood-derived CD4 + Stimulation with anti-CD3 increased T cell activation (Figure 2B). Stimulation with anti-CD3 increased the expression of 4-1BB and OX40 on CD4 T cells, and the expression of these activation markers was further enhanced in the presence of granulocyte-producing cells but not in the presence of patient-donor neutrophils. These results indicate that the presence of granulocyte-producing cells enhances CD4 T cell activation (OX40 and 4-1BB). OX40 and 4-1BB are costimulatory markers expressed on activated T cells. The binding of these costimulatory receptors on CD4 T cells should increase the effector function of these cells (e.g., increase cytokine production).

[0572] The data suggest that granulocyte-producing cells provide signal 2 (co-stimulation) and / or signal 3 (cytokine stimulation) for T cell activation.

[0573] Example 3 Co-culturing with granulocyte-producing cells enhances the proliferation and accumulation of αβ T cells. Methods: PBMCs from healthy donors were cultured in a 1:1 ratio with granulocyte-producing cells (n=4) or blood-derived neutrophils from a PDAC donor (n=1). Co-culture was performed in the presence of anti-CD3 stimulation (OKT3, 1 μg / ml). After 72 hours, CD4 + and CD8 +T cell proliferation was investigated by flow cytometry. Before co-culturing, PBMCs were labeled with a cell-tracking far-infrared (CTFR) dye (Invitrogen, C34572), and T cells were identified as live cells, singlets, and CTFR-labeled cells. + CD3 + CD8 + or CD3 + CD4 + The cells were gated as follows. Proliferating cells were identified as having a decreased median fluorescence intensity (MFI) of CTFR, which occurs when cells divide and the dye is diluted.

[0574] Results: The result was (Figure 3A) CD4 after 72 hours of incubation. + and CD8 + Figure 3B shows the percentage of T cell proliferation and the absolute number of both cell types. Co-culturing with granulocyte-producing cells, rather than patient-donor neutrophils, enhanced αβ T cell proliferation (indicated by increased proliferation of anti-CD3 stimulated CD4 and CD8 T cells) and the accumulation of such cells (indicated by increased absolute number of CD4 and CD8 T cells present after 72 hours of culture). Granulocyte-producing cells can amplify the proliferation resulting from the TCR of αβ T cells and increase the accumulation of immune cells.

[0575] The data suggest that granulocyte-producing cells provide signal 2 (co-stimulation) and / or signal 3 (cytokine stimulation) for T cell activation.

[0576] Example 4 Co-culturing with granulocyte-producing cells promotes the survival of blood-derived NK cells and NKT cells. Methods: PBMCs from healthy donors were cultured with granulocyte-producing cells (n=4) or blood-derived neutrophils from PDAC donors (n=1) in various ratios as shown. After 72 hours, the absolute number of NK and NKT cells was quantified by flow cytometry. Before co-culture, PBMCs were labeled with cell-tracking far-infrared (CTFR) dyes, and live cells, singlets, and CTFRs were identified. + They were gated as follows: NK cells CD3 - CD56 +Gate as and NKT cells CD 3 +CD56 + It was gated as such.

[0577] Results: The results show the absolute number of NK cells (Figure 4A) and NKT cells (Figure 4B) in both PBMC donors. In both donors, co-culture with granulocyte-producing cells, rather than with neutrophils derived from PDAC patients, promoted the survival of NK and NKT cells, as indicated by the absolute numbers.

[0578] The results indicate that granulocyte-producing cells have a favorable effect on the survival of immune cells, as exemplified by their effect on the survival of NK cells and NKT cells. This further suggests that medical uses or therapeutic methods using granulocyte-producing cells may be used in combination with NK cell therapy, for example, as feeder cells for NK cell therapy generation, or in combination with NK cell therapy to support NK cell therapy function in vivo.

[0579] Example 5 Co-culturing with granulocyte-producing cells promotes the activation of blood-derived NK cells and NKT cells. Methods: PBMCs from healthy donors were cultured with granulocyte-producing cells (n=4) or blood-derived neutrophils from PDAC donors (n=1) in various ratios as shown. After 72 hours, NK cell and NKT cell activation was investigated by flow cytometry. Prior to co-culture, PBMCs were labeled with cell-tracking far-infrared (CTFR) dyes, and live cells, singlets, and CTFRs were identified. + They were gated as follows: NK cells CD3 - CD56 + Gate as and CD3 + CD56 + The cells were gated. Activation of NK cells and NKT cells was investigated by measuring the expression of degranulation markers such as CD107a on the cell surface, as well as co-stimulatory molecules such as 4-1BB and OX40.

[0580] Results: The results show the expression percentages of CD107a, 4-1BB, and OX40 on NK cells (Figure 5A) and NKT cells (Figure 5B). Co-culture with granulocyte-producing cells, rather than patient donor neutrophils, increased the activation of blood-derived NK and NKT cells, as indicated by the increased expression of CD107a, 4-1BB, and OX40 on NK cells (Figure 5A) and NKT cells (Figure 5B). These results indicate that the presence of granulocyte-producing cells enhances the degranulation (CD107a) and activation (OX40 and 4-1BB) of NK and NKT cells.

[0581] The results indicate that granulocyte-producing cells have a favorable effect on the activation of NK cells and NKT cells. This suggests that therapies using granulocyte-producing cells can be used in combination with NK cell therapy, for example, as feeder cells for NK cell therapy generation, or in combination with NK cell therapy to support in vivo NK cell therapy function.

[0582] Example 6 Co-culturing with granulocyte-producing cells enhances the activation of tumor-infiltrating leukocytes (CD8, CD4, and NK cells). Methods: Tumor digestive tissue ± granulocyte-producing cells (n=2) from a PDAC patient (n=1) were cultured. After 72 hours, the activation of tumor-infiltrating αβ T cells and NK cells was investigated by flow cytometry. Before co-culture, digestive tumor cells were labeled with cell-tracking far-infrared (CTFR) dyes, and live cells, singlets, and CTFR-labeled cells were identified. + It was gated as such. Next, the group of effectors, CD3 + CD8 + CD3 + CD4 + , or CD3 - CD56 + TIL activation was investigated by measuring the expression of degranulation markers such as CD107a on the cell surface, as well as co-stimulatory molecules such as 4-1BB and / or OX40, as shown.

[0583] Results: The results show the multiplier changes in the expression of indicated activation markers on tumor-infiltrating (Figure 6A) CD8 T cells, (Figure 6B) NK cells, and (Figure 6C) CD4 T cells. Data are shown as the multiplier of increase in expression compared to the condition of tumor digestion only. (Figures 6A and 6B) The data show that co-culture with granulocyte-producing cells increased the activation of tumor-infiltrating leukocytes (exemplified by CD8 and NK cells), as indicated by the increased expression of CD107a and 4-1BB on CD8 T cells and NK cells. This demonstrates the ability of granulocyte-producing cells to promote degranulation of cytotoxic effector cells in TME and to enhance their activation by increasing 4-1BB expression. (Figure 6C) The data show that co-culture with granulocyte-producing cells increased the activation of tumor-infiltrating CD4, as indicated by the increased expression of 4-1BB and OX40 costimulatory receptors on tumor-infiltrating CD4 T cells. The linkage of these costimulatory receptors increases the effector function of T cells, e.g., increases cytokine production.

[0584] Example 7 Co-culturing with granulocyte-producing cells increases cytokine production by PBMCs. Methods: PBMCs from healthy donors (n=2) were cultured with granulocyte-producing cells (n=4) or blood-derived neutrophils from PDAC donors (n=1) in various ratios as shown. Co-culture was performed in the presence of anti-CD3 stimulation (OKT3, 1 μg / ml). After 72 hours, the supernatant was collected, and the concentration of secreted cytokines, e.g., IFN-γ, was measured by quantitative sandwich ELISA (ab174443) according to the manufacturer's instructions.

[0585] Results: The results show the concentration of IFN-γ detected in the supernatant (Figure 7). Co-culture with granulocyte-producing cells, rather than patient-donor neutrophils, increased IFN-γ production by PBMCs. This data, as indicated by the increased IFN-γ production by PBMCs, suggests that granulocyte-producing cells enhance T-cell effector function for potent anti-tumor immunity.

[0586] The data suggest that granulocyte-producing cells provide signal 2 (co-stimulation) and / or signal 3 (cytokine stimulation) for T cell activation. Furthermore, they show that granulocyte-producing cells do not result in unregulated T cell activation, which is important regarding the safety of medical use or therapeutic methods.

[0587] 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 activation.

[0588] Example 8 Co-culturing with granulocyte-producing cells increases cytokine production by tumor-infiltrating lymphocytes (TILs). Methods: Tumor digests ± granulocyte-producing cells (n=2) from a PDAC patient (n=1) were cultured. Co-culture was performed in the presence of anti-CD3 stimulation (OKT3, 1 μg / ml). After 72 hours, the supernatant was collected, and the concentration of secreted cytokines, e.g., IFN-γ, was measured by quantitative sandwich ELISA (ab174443) according to the manufacturer's instructions.

[0589] Results: (Figure 8) The results show the concentration of IFN-γ detected in the cell culture supernatant. Co-culture with granulocyte-producing cells increased IFN-γ production by TILs. This data indicates that granulocyte-producing cells enhance the T cell effector function for potent antitumor immunity, as shown by the increased IFN-γ production by TILs.

[0590] The data suggest that granulocyte-producing cells provide signal 2 (co-stimulation) and / or signal 3 (cytokine stimulation) for T cell activation.

[0591] Example 9 Granulocyte-producing cells promote the recruitment of immune cells into the tumor microenvironment. Methods: Fresh tumor biopsies (RCCs) ± granulocyte-producing cells from patients were encapsulated in a tumor-on-chip model and co-cultured with matching donor PBMCs. (Figure 9) PBMC recruitment to microtumors was measured daily for 3 days using live-cell imaging.

[0592] Results: The results show the multiplier of immune cell infiltration into the microtumor at the indicated time point compared to the tumor on day 0 only (Figure 9). These data suggest that granulocyte-producing cells are immunomodulatory due to their ability to recruit immune cells into the tumor microenvironment.

[0593] Example 10 Granulocyte-producing cells promote enhanced tumor death. Methods: Fresh tumor biopsies (RCCs) ± granulocyte-producing cells from patients were embedded in a tumor-on-chip model and co-cultured with matching donor PBMCs. (Figure 10) The cytotoxicity of tumor cells was measured daily for 3 days using live-cell imaging.

[0594] Results: The results show the percentage of tumor death at the indicated time points (Figure 10). These data suggest that granulocyte-producing cells increased tumor cell death.

[0595] It will be recognized that the killing of tumor cells is a key objective of anticancer therapy. Therefore, the increased tumor-killing activity observed in therapy with granulocyte-producing cells clearly demonstrates that the medical uses, therapeutic methods, and pharmaceutical compositions of the present invention can exert therapeutic anticancer activity. As demonstrated in previous examples, this is achieved by amplifying the immune response, particularly the effect of non-granulocyte cells in the immune response.

[0596] Example 11 Granulocytes, which are generated during the differentiation of granulocyte-producing cells, promote the recruitment of immune cells into the tumor microenvironment through the secretion of chemokines. Methods: Granulocyte-producing cells were differentiated, and the resulting granulocytes ("IMAN") (n=4) were stimulated with ±IFN-α, IFN-β, or TNF (all 10 ng / ml) for 24 hours. The data show the concentrations of chemokines such as CXCL10 in the cell culture supernatant, quantified by LEGENDplex according to the manufacturer's instructions.

[0597] Results: The results show that granulocytes generated during the differentiation of granulocyte-producing cells release CXCL10 when activated by various cytokines (Figure 11). These data suggest that therapy using granulocyte-producing cells may play a further role in promoting immune cell recruitment into the tumor microenvironment by generating granulocytes capable of releasing chemokines such as CXCL10. CXCL10 is related to CXCR3 + It is known to be a potent chemotactic for T cells and NK cells.

[0598] These data indicate that granulocytes, generated during the differentiation of granulocyte-producing cells, can be activated through many different pathways. The data suggest that granulocyte-producing cell therapy, particularly the granulocytes generated as a result of such therapy, may be combined with single / bispecific antibodies that activate innate immune cells. For example, combinations with anti-CD40 mAbs or anti-CD40 / TAA bispecific antibodies for granulocyte activation and tumor targeting.

[0599] Example 12 Granulocytes derived from granulocyte-producing cells express ligands for T cell and NK cell costimulatory receptors. Methods: Granulocytes (n=3) generated during the differentiation of granulocyte-producing cells suitable for use in the medical use or therapeutic method of the present invention were analyzed by flow cytometry for the expression of T cell and NK cell costimulatory receptors, 4-1BBL, and OX40L.

[0600] Results: The results show the expression percentages of 4-1BBL and OX40L on granulocytes derived from granulocyte-producing cells of three individual donors (Figure 12). These data suggest that granulocyte-producing cells enhance T cell and NK cell effector function through co-stimulation, as indicated by the expression of both 4-1BBL and OX40L.

[0601] Materials and methods Preparation of a population of granulocyte-producing cells (IMANp) The preparation of a population of granulocyte-producing cells (referred to as "IMANp" by the inventors, as shown in the figure) from hematopoietic stem cells (HSCs) consists of three main steps after the recovery of donor leukocytes: - Isolation and cryopreservation of CD34+ cells from donor leukocyte apheresis. - Isolated CD34+ cells (E0-E8) are expanded for 9 days to generate intermediate progenitor cells. On day 9 (E8D0), the expansion medium is replaced with differentiation medium. - Intermediate / undifferentiated progenitor cells (D0-D5) are differentiated for 5 days into a heterogeneous mixture of cells, mainly granulocyte-producing progenitor cells called IMANp. -Optional freezing and saving of IMANp.

[0602] The materials used to prepare IMANp are as follows: [Table 2] [Table 3]

[0603] Preparation of culture medium Cytokines are reconstituted in cell culture-grade water containing 5% HSA, and aliquots are stored at -80°C before being added to the culture medium.

[0604] Expanded culture medium CD34 +HSCs were expanded in expanded medium containing ISKOFF-modified Dulbecco's medium (IMDM) containing cytokines including SCF, FLT-3, TPO, IL3, and IL6, as well as ITS and HAS, at the following concentrations. [Table 4]

[0605] Differentiation medium Cells are differentiated in a differentiation medium containing IMDM, SCF, TPO, GCSF, ITS, and HSA at the following concentrations. [Table 5]

[0606] CD34 + Expansion of HSC Donor CD34 HSCs were thawed in a 37°C water bath on day 0 (E0) and transferred to thawing medium consisting of IMDM and 1% HSA. Cell count and viability were measured immediately after thawing for all donor samples. Subsequently, cells were measured at 5e5 / mL and 5e5 / cm³. 2 So, with a volume of 10 mL per well, 10 cm³ 2 Seeds were seeded in expanded medium in G-Rex 6M or G-Rex 10M having a surface area of ​​[specified surface area].

[0607] On day 1 (E1), samples were collected for flow cytometry analysis to characterize cell number, viability, and phenotypic characteristics using a panel of progenitor cells and neutrophils. The wells were supplemented with 40 mL of expansion medium to increase the volume to 4 mL / cm2. On E2 and E3, the cells were left in G-Rex to continue expansion.

[0608] Cells seeded from 1 G-Rex 6M or G-Rex 10M were transferred to a G-Rex with a larger surface area, e.g., 1 G-Rex 100M. The G-Rex was carefully removed from the incubator, and the expansion medium was removed to 15 mL per well. After resuspending the cells in the remaining volume by swirling, samples were taken for flow cytometry analysis to characterize cell number, viability, and phenotype using a panel phenotype of progenitor cells, neutrophils, and mature neutrophils. Subsequently, the cells were transferred to G-Rex 100M, and 85 mL of fresh expansion medium was added to the G-Rex. The cells were left as they were in E5, and in E6, any samples may be taken for flow cytometry analysis to characterize cell number, viability, and phenotype using a panel phenotype of progenitor cells, neutrophils, mature neutrophils, and off-target bone marrow and lymphoid systems. Furthermore, 100 mL of expansion medium was supplied to each well and left for 48 hours. In E7, the cells were left as they were. In E8, the G-Rex were 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 swirling, and samples were taken for flow cytometry analysis to characterize cell number, viability, and phenotypic characteristics using a panel of progenitor cells, neutrophils, mature neutrophils, and off-target bone marrow and lymphoid cells. Subsequently, the medium was changed to initiate the differentiation process.

[0609] Differentiation of intermediate progenitor cells On day 9 of the manufacturing process (E8D0), the expansion medium was removed, leaving 100 mL per well, then 400 mL of fresh differentiation medium was added to each well, and the G-Rex was returned to the incubator. Days D1 and D2 were left as is. On D3, any samples may be taken for cell count, viability, and flow staining of progenitor cells, neutrophils, mature neutrophils, and off-target bone marrow and lymphoid panels. Furthermore, on D3, the volume of differentiation medium per well was doubled to a total volume of 1 L per well. The cells were left as is until D4 to differentiate, and cell harvesting was performed on D5.

[0610] The magnification of cells (E0 stem cells → E8D0 progenitor cells → E8D5 granulocyte-producing cells) achieved using this exemplary method of the present invention is as follows: [Table 6]

[0611] IMANp recovery, a heterogeneous mixture of progenitor cells, mainly granulocyte-generating progenitor cells. On day 14 of the manufacturing process (E8D5), G-Rex was carefully removed from the incubator, and the culture medium was aspirated to 100 mL. The cells were resuspended by swirling and transferred to a sterile centrifuge tube. Samples may be taken for cell count, viability, and staining of progenitor cells, neutrophils, mature neutrophils, and off-target bone marrow and lymphoid panels. The cells were washed by centrifugation at 350 g for 10 minutes, and the used culture medium was aspirated and removed. The cells were then resuspended in cryoformulation medium (CS10) containing 10% DMSO at the required density at a cell concentration of less than 100 E6 cells / mL. The obtained samples were divided into cryogenic storage containers (bags and vials) and immediately frozen. The samples were then stored in a gas-phase liquid nitrogen freezer (≤-130°C). After 24 hours of storage, the cryovials were removed for post-thaw analysis, and flow cytometry analysis was performed to evaluate cell viability, cell recovery rate, and phenotypic characterization using progenitor cells, neutrophils, mature neutrophils, and off-target bone marrow and lymphoid panels.

[0612] IMANp Characterization The population of granulocyte-producing cells was characterized with respect to the following panel of markers. [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12-1] [Table 12-2]

[0613] Results of characterization of granulocyte-producing cell populations Figures 12-16 show the results of characterization of a population of granulocyte-producing cells produced by the method of the present invention.

[0614] As described above, in relation to the sixth aspect of the present invention, four subgroups of granulocyte-producing cells were identified within the overall population of granulocyte-producing cells.

[0615] Figure 12 shows the relative proportions of these subpopulations within the granulocyte-producing cell population generated by the exemplary method of the present invention. v0.2 is the method of the present invention without a priming step, while v0.3a-c include any priming step as follows: v0.3a (priming with GM-CSF the day before the last day of culture, and priming with TNF on the last day of culture), v0.3b (priming with both GM-CSF and IL-3 the day before the last day of culture), and v0.3c (priming with both GM-CSF and IL-3 the day before the last day of culture, and priming with TNF on the last day of culture).

[0616] In each of these conditions, the first subgroup represented the largest proportion of the total, the third subgroup represented the second largest proportion, and the second subgroup represented the third largest proportion of the total granulocyte-producing cell population. The fourth subgroup represented the smallest subgroup generated by each protocol and was hardly present in the population of granulocyte-producing cells generated using the v0.2 (no priming) protocol.

[0617] Figure 13 further characterizes the first subpopulation of granulocyte-producing cells (the population according to the seventh aspect of the present invention) with respect to the expression of various markers.

[0618] Figure 14 further characterizes a second subpopulation of granulocyte-producing cells (the population according to the eighth aspect of the present invention) with respect to the expression of various markers.

[0619] Figure 15 further characterizes a third subgroup of granulocyte-producing cells (the group according to the ninth aspect of the present invention) with respect to the expression of various markers.

[0620] Figure 16 further characterizes a fourth subgroup of granulocyte-producing cells (the subgroup according to the 10th aspect of the present invention) with respect to the expression of various markers.

[0621] Any priming step for a population of granulocyte-producing cells (IMANp) IMANp may be primed with additional cytokines after thawing to enhance its cytotoxicity. [Table 13]

[0622] After thawing E8D5 cells, IMANp was cultured for 48 hours in the presence of GM-CSF (10-130 ng / mL) alone, or in combination with TNFα (0.01-1 ng / mL), IFNα (10 ng / mL), IFNβ (10 ng / mL), IL-3 (130 ng / mL), IL-15 (10 ng / mL), or IL-18 (10 ng / mL) in the presence of GM-CSF (10-130 ng / mL).

[0623] Any priming during IMANp differentiation IMANp may be primed during the differentiation stage to enhance cytotoxicity.

[0624] The donor HSCs were thawed and enlarged as described above.

[0625] After proliferation (E8D0), HSCs are differentiated for up to 6 days (E8D0-E8D6). Between D3-D4, D4-D5, or D5-D6, cells are stimulated in either 1% or 2% HSA using GM-CSF (10-130 ng / mL) alone or in combination with TNFα (0.01-1 ng / mL), IFNα (10 ng / mL), IFNβ (10 ng / mL), IL-3 (130 ng / mL), IL-15 (10 ng / mL), or IL-18 (10 ng / mL).

Claims

1. Granulocyte-producing cells, or populations of such cells, for use in modulating therapeutic immune responses.

2. The aforementioned cells are CD62L － The granulocyte-producing cells for use according to claim 1, or a population of such cells.

3. The granulocyte-producing cells or a population of such cells have a marker expression profile: CD10 － , CD11b － , CD16 － , CD62L － , CD66b － , CD177 － , CD15 ＋ , CD38 ＋ , CD49d ＋ , CD54 ＋ , CD63 ＋ The granulocyte-producing cells or a population of such cells for use according to claim 1 or claim 2, having the above

4. The granulocyte-producing cells have the following marker expression profile: CD11b ｈｉ CD15 ＋ CD66b ＋ CD177 ＋ CD18 ｈｉ CD16 － CD34 － CD38 － CD49d － A granulocyte-producing cell for use according to claim 1 or claim 2, or a population of such cells.

5. The granulocyte-producing cells have a marker expression profile of CD34. ＋／－ CD38 ＋／－ CD15 ＋／－ CD49d ＋ CD18 ＋ CD66b － CD177 － CD16 － A granulocyte-producing cell for use according to claim 1 or claim 2, or a population of such cells.

6. The granulocyte-producing cells or populations of such cells have a marker expression profile: CD10 － CD11b － CD16 － CD62L － CD66b － CD177 － CD15 ＋ CD38 ＋ CD49d ＋ CD54 ＋ CD63 ＋ A granulocyte-producing cell for use according to claim 1 or claim 2, or a population of such cells.

7. The granulocyte-producing cells or populations of such cells have the marker expression profile: CD11b ｈｉ CD15 ＋ CD66b ＋ CD177 ＋ CD18 ｈｉ CD16 － CD34 － CD38 － CD49d － A granulocyte-producing cell for use according to claim 1 or claim 2, or a population of such cells.

8. The granulocyte-producing cells or populations of such cells have a marker expression profile: CD34 ＋／－ CD38 ＋／－ CD15 ＋／－ CD49d ＋ CD18 ＋ CD66b － CD177 － CD16 － A granulocyte-producing cell for use according to claim 1 or claim 2, or a population of such cells.

9. Granulocyte-producing cells, or populations of such cells, for use according to any of the prior claims, for amplifying a therapeutic immune response.

10. Granulocyte-producing cells, or populations of such cells, for use according to any of the prior claims, for modulating a nongranulocyte immune response.

11. The granulocyte-producing cells described above can differentiate into granulocytes having the ability to kill cancer cells, as described in any of the prior claims, for use, or a population of such cells.

12. The therapeutic immune response is a host therapeutic immune response, as described in any of the prior claims, of granulocyte-producing cells for use, or a population of such cells.

13. The granulocyte-producing cells are of the same kind as the granulocyte-producing cells for use described in any of the prior claims, or a population of such cells.

14. The granulocyte-producing cells can be differentiated to produce cells that secrete CXCL10 and / or express ligands of costimulatory molecules selected from the group consisting of 4-1BBL and OX40L, as described in any of the prior claims, for use, or a population of such cells.

15. Granulocyte-producing cells, or populations of such cells, for use in the treatment of cancer, as described in any of the prior claims.

16. The granulocyte-producing cells for use according to claim 10, or a population of such cells, are selected from the group consisting of pancreatic cancer, liver cancer, esophageal cancer, gastric cancer, cervical cancer, ovarian cancer, lung cancer, bladder cancer, kidney cancer, brain cancer, prostate cancer, myeloma, non-Hodgkin lymphoma (NHL), laryngeal cancer, uterine cancer, and breast cancer.

17. Granulocyte-producing cells, or populations of such cells, for use in the treatment of infectious diseases, as described in any of the prior claims.

18. The infectious disease is selected from the group consisting of viral infections, bacterial infections, and fungal infections, and is a granulocyte-producing cell for use according to claim 12, or a population of such cells.

19. Granulocyte-producing cells, or populations of such cells, for use according to any of the prior claims, for use in amplifying a nongranulocyte therapeutic immune response by increasing the activation of nongranulocyte immune cells.

20. The activation increases the expression by the immune cells of a degranulation marker selected from the group consisting of CD107a, perforin, and granzyme, as described in claim 14, for use by granulocyte-producing cells, or a population of such cells.

21. The activation increases the expression by the immune cells of a costimulatory molecule selected from the group consisting of 4-1BB, OX40, CD27, CD28, ICOS, HVEM, LIGHT, CD40L, DR3, GITR, CD30, TIM1, CD2, and CD226, as described in claim 14 or claim 15, granulocyte-producing cells for use, or a population of such cells.

22. Granulocyte-producing cells, or populations of such cells, for use according to any of the prior claims, for use in amplifying a therapeutic immune response by increasing T cell activation.

23. The aforementioned T cells are CD8 ＋ T cells, CD4 ＋ Granulocyte-producing cells for use according to claim 17, or a population of such cells, selected from the group consisting of T cells, NK T cells, αβ T cells, γδ T cells, peripheral blood T cells, and tumor-infiltrating T cells.

24. Granulocyte-producing cells, or populations of such cells, for use according to any of the prior claims, for use in amplifying a therapeutic immune response by increasing the activation of NK cells.

25. Granulocyte-producing cells, or populations of such cells, for use according to any of the prior claims, for use in amplifying a therapeutic immune response by increasing the activation of PBMCs.

26. Granulocyte-producing cells, or populations of such cells, for use according to any of the prior claims, for use in amplifying a therapeutic immune response by increasing TIL activation.

27. Granulocyte-producing cells, or populations of such cells, for use according to any of the prior claims, for use in amplifying a therapeutic immune response by increasing the tumor cell-killing activity of immune cells.

28. Granulocyte-producing cells, or populations of such cells, for use according to any of the prior claims, for use in amplifying a therapeutic immune response by increasing the proliferation of immune cells.

29. Granulocyte-producing cells, or populations of such cells, for use according to any of the prior claims, for use in combination with further cellular immunotherapy.

30. A method for promoting the therapeutic activity of nongranulocyte immune cells, comprising incubating nongranulocyte immune cells with granulocyte-producing cells described in any of the prior claims, or a population of such cells.

31. A pharmaceutical composition comprising an enriched population of granulocyte-producing cells according to any one of claims 1 to 29.

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

33. A method for selecting an appropriate treatment regimen for a patient, wherein the method is: - This includes incubating the patient's nongranulocyte immune cells together with granulocyte-producing cells. If the activity of the non-granulocyte immune cells in the patient increases in response to the incubation, then treatment with granulocyte-producing cells is selected as the appropriate treatment. The method wherein, if the activity of the patient's non-granulocyte immune cells increases in response to the incubation, treatment with a therapy other than granulocyte-producing cells is selected.

34. A method for increasing the survival of immune cells in culture, comprising culturing the immune cells in the presence of a feeder layer of granulocyte-producing cells. A method for increasing the proliferation of immune cells in culture, comprising culturing the immune cells in the presence of a feeder layer of granulocyte-producing cells.

35. A method for determining whether granulocyte-producing cells are suitable for use in the treatment of cancer by beneficially modulating the tumor microenvironment, - To evaluate whether the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, can express inflammatory cytokines, and / or - To evaluate whether the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, can stimulate the expression of inflammatory cytokines by non-granulocyte immune cells, Based on this evaluation, we will identify whether granulocyte-producing cells are suitable for use in cancer treatment by beneficially modulating the tumor microenvironment. The method, including the method described above.

36. A method for determining whether granulocyte-producing cells are suitable for use in the treatment of cancer by increasing the recruitment of immune cells to tumors and / or activation of immune cells, - To evaluate whether the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, can express chemokines associated with promoting cell transport, and / or - To evaluate whether the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, can stimulate the expression of degranulation markers by non-granulocyte immune cells, Based on this evaluation, we will determine whether granulocyte-producing cells are suitable for use in the treatment of cancer by increasing the recruitment of immune cells to tumors and / or immune cell activation. The method, including the method described above.

37. A method for determining whether granulocyte-producing cells are suitable for use in the treatment of cancer by directly promoting the death of cancer cells, - Incubating the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, together with cells of a cancer cell line, - To evaluate whether the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, can increase the death of the cancer cell line to a greater extent than the death of non-cancer cells, Based on this evaluation, we will determine whether granulocyte-producing cells are suitable for use in cancer treatment by directly promoting the death of cancer cells. The method, including the method described above.

38. A method for determining whether granulocyte-producing cells are suitable for use in treating infectious diseases by directly promoting the death of cell-infecting pathogens or infected cells, - Incubating the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, together with a sample of a cell-infecting pathogen or infected cells, - To evaluate whether the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, can increase the death of cell-infecting pathogens or infected cells, Based on this evaluation, we will determine whether granulocyte-producing cells are suitable for use in treating infections by directly promoting the death of cell-infecting pathogens or infected cells. The method, including the method described above.

39. A method for determining whether granulocyte-producing cells are suitable for use in therapy by amplifying a therapeutic immune response, - Incubating the granulocyte-producing cells, or cells derived from the granulocyte-producing cells, together with immune cells, - To evaluate whether the granulocyte-producing cells can increase the activation of the immune cells, Based on this evaluation, we will determine whether granulocyte-producing cells are suitable for use in therapy by amplifying therapeutic immune responses. The method, including the method described above.

40. A method for preparing granulocyte-producing cells for therapeutic use, ·below: G-CSF, GM-CSF, IL-3 and TNF The method comprising culturing a population of progenitor cells under cell culture conditions that promote the differentiation of progenitor cells, including the presence of a certain substance, to generate a population of granulocyte-producing cells.

41. A population of granulocyte-producing cells prepared for therapeutic use by the method of claim 40.

42. An immortalized cell line or stable stem cell culture that can be obtained from selected stem cells (for example, obtained from selected stem cells).

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