Cell populations and related methods

Abstract

This invention relates to populations of immature neutrophils, methods of making and genetically engineering them, and their uses in therapy.

Description

[0001] CELL POPULATIONS AND RELATED METHODS

[0002] TECHNICAL FIELD

[0003] This invention relates to populations of immature neutrophils, methods of making and genetically engineering them, and their uses in therapy.

[0004] BACKGROUND

[0005] Neutrophils are the most abundant and potent antimicrobial effector cells in humans. They combat bacterial and fungal pathogens using an arsenal of antimicrobial responses, including phagocytosis, reactive oxygen species (ROS) production, degranulation and release of extracellular chromatin decorated with antimicrobial peptides, termed neutrophil extracellular traps (NETs) [1]. Patients with congenital neutropenia, who have abnormally low circulating neutrophils counts, succumb to severe bacterial and fungal infections [2, 3]. In contrast, excessive or dysregulated neutrophil activity promotes pathology in sepsis [4] and malaria [5, 6], as well as in non-infectious diseases such as cancer [7], autoimmunity [8] and cardiovascular disease [9]. Regulation of neutrophil development and function is therefore essential for health.

[0006] Neutrophils develop in the bone marrow from stem cells, such as CD34-expressing (CD34+) granulocyte-monocyte progenitors (GMPs)

[0010] . This process, termed granulopoiesis, produces an estimated 1011neutrophils daily in healthy individuals

[0011] . Development from the committed proliferative myeloblast to release of mature neutrophils into circulation takes approximately 14 days

[0012] , during which progenitors gradually acquire cytoplasmic granules, lobulated nuclei and ROS producing enzymes such as NADPH oxidase (NOX2) and myeloperoxidase (MPO). Granulopoiesis is driven by the growth factors granulocyte colony stimulating factor (GCSF) [13, 14] and granulocyte-macrophage colony-stimulating factor (GM-CSF) [15, 16]. In addition to promoting differentiation in the bone marrow, GCSF promotes mobilisation of mature and immature neutrophils out of the bone marrow and into the circulation, where it also extends their lifespan [17, 18].

[0007] Neutrophils were traditionally considered to be homogenous cells. This view has recently been challenged, with reports of various activation and differentiation states, both in healthy individuals

[0019] and during inflammation [7, 11]. One of the main determinants of neutrophil phenotype is maturity. During inflammation, elevated GCSF production promotes the release of immature neutrophils from the bone marrow. This phenomenon has been observed for decades in clinical settings, where the presence of immature morphological features in circulating neutrophils is termed 'left shift'. Flow cytometry studies of patient blood can identify immature neutrophils by reduced expression of surface maturity markers such as CD10 and CD101, relative to mature neutrophils

[0020] . Strikingly, the number of circulating immature neutrophils is often associated with poor prognosis in a variety of autoimmune and inflammatory diseases such as systemic lupus erythematosus (SLE), COVID-19 and late-stage cancer [20-26]. Despite this strong association with severe disease, the function and inflammatory potential of immature neutrophils remains unclear.

[0008] GCSF administration is sufficient to mobilise immature neutrophils into the circulation; this was demonstrated in GCSF-treated allogeneic stem cell donors (GCSF-D)

[0027] . Importantly, these studies suggested functional differences between control and GCSF-D neutrophils, including elevated production of proinflammatory cytokines, reduced motility, and capacity to produce ROS, as well as reduced ability to suppress the fungal pathogen Candida albicans (C. albicans) [18, 27-29].

[0009] Naveh et al., medRxiv 2023.07.12.23292345 discloses immature and genetically tractable neutrophils cultured ex vivo from CD34+ stem cells (and in incorporated herein by reference in its entirety).

[0010] SUMMARY OF THE INVENTION

[0011] The inventors have developed a novel culture method that produces a high yield of immature neutrophils. The immature neutrophils of the invention resemble native neutrophils in nuclear morphology, surface marker expression and neutrophil effector functions, such as reactive oxygen species (ROS) production, bacterial killing, phagocytosis, and chemotaxis. The immature neutrophils of the invention differ from native immature neutrophils (GCSF-D) based on their lack of expression of both CD16 (FcyRIII) and CXCR2 (interleukin-18 (IL-18) receptor beta). The immature neutrophils of the invention can be produced in high numbers, are useful in therapeutic contexts, and are amenable to genome editing to increase their activity. The immature neutrophils of the invention can also advantageously be stored for long periods of time because they maintain their function post-freezing / cryopreservation.

[0012] The invention provides a population of immature neutrophils, wherein: at least about 60% of the cells in the population express detectable levels of CD66b, at least about 60% of the cells in the population express detectable levels of CDllb, fewer than about 20% of the cells in the population express detectable levels of CD10, fewer than about 20% of the cells in the population express detectable levels of CD16, and fewer than about 20% of the cells in the population express detectable levels of CXCR2.

[0013] The invention also provides:

[0014] - a method of producing a population of immature neutrophils, comprising (a) culturing haematopoietic progenitor cells in the presence of interleukin-3 (IL-3), stem cell factor (SCF) and FMS-like tyrosine kinase 3 (Flt-3) Ligand (Flt3-L) and in the absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or granulocyte colony stimulating factor (G-CSF), (b) culturing the cells produced in step (a) in the presence of IL-3, SCF, Flt3-L and GM-CSF, (c) culturing the cells produced in step (b) in the presence of GM-CSF and G-CSF and (d) culturing the cells produced in step (c) in the presence of G-CSF; a population of immature neutrophils genetically engineered to express one or more activating receptors and / or to reduce expression of one or more inhibitory receptors; a method of producing a population of genetically engineered immature neutrophils of the invention, comprising (i) genetically engineering haematopoietic progenitor cells and (ii) culturing the genetically engineered haematopoietic progenitor cells under conditions which promote their differentiation to immature neutrophils;

[0015] - a cryopreserved population of the invention;

[0016] - a thawed population of the invention; a pharmaceutical composition comprising a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention or a thawed population of the invention and a pharmaceutically or physiologically acceptable diluent and / or carrier; a method of treating or preventing an infection in a subject in need thereof, comprising administering to the subject a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention; a method of treating or preventing cancer in a subject in need thereof, comprising administering to the subject a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention; and a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention for use in a method of treating or preventing an infection or cancer.

[0017] BRIEF DESCRIPTION OF THE FIGURES

[0018] All references to "native" neutrophils in the Figure legends and Examples relate to native, mature neutrophils isolated from peripheral blood.

[0019] Figure 1: Optimisation of neutrophil culture protocol. A: Representative Wright Giemsa- stained cytospin images of neutrophil differentiation steps, with cytokine protocol. Size bar=20 micron. B: Extrapolated fold expansion of cultured total cell count over 17 days of differentiation, n=7-ll. C-E: Surface marker expression of human stem and progenitor cell (HSPC) marker CD34 (C) and granulocyte markers CD66b (D) and CDllb (E) over days 5- 17 of differentiation, n=3-6.

[0020] Figure 2: Flow cytometry analysis of surface markers on native, GCSF-D and cultured neutrophils. A-B: Representative histogram and quantification of forward scatter (FSC) (A) and side scatter (SSC) (B). C: Representative scatter dot plots displaying CD101 and CD10 expression on native, GCSF-D and cultured neutrophils. D-H: Representative histograms and mean fluorescent intensity (MFI) quantifications of CD101 (D), CD10 (E), CD66b (F), CD15 (G) and CDllb (H). Histograms colour coded as: native (red), GCSF-D (blue) and cultured (black) neutrophils. Data were analysed by one-way ANOVA with Tukey's multiple comparisons displayed on graph, n=3-5, * = p<0.05, ** = p< 0.01, *** = p< 0.001.

[0021] Figure 3: Comparison of effector functions in cultured and native neutrophils. A-B: Detection of ROS with luminol (A) and APF (B) in native or cultured neutrophils stimulated with lOOnM PMA . Left: A representative kinetic plot of the respiratory burst in individual donors; right: area under the curve (AUC) quantification, n=3. C: Representative images of NETs induced with lOOnM PMA and C. albicans (MOI=5, 4h), stained with SYTO green and SYTOX orange. D: Quantification of average percent NETs per field of 10X view in response to PMA induction (n=2-4). E: Quantification of average NET frequency per 10X field of view, n=3 donors. F: Exocytosis of NE in response to stimulation with 25 pg / ml opsonised zymosan (OZ) for 1 hour, quantified by ELISA, n=3-5. G-H: IL-6 (G) and IL-8 (H) cytokine release from native and cultured neutrophils stimulated overnight with lOOng / mL LPS or 5pM R-848, quantified by ELISA, n=3-5. I: Viability of opsonised C. albicans after incubation with native or cultured neutrophils for 2.5h at MOI 2.5 (left) or 5 (right), n=3. J: Viability of opsonised S. aureus JE2 after incubation with native or cultured neutrophils at MOI=5, over 240 mins (all non-significant, n=3). Error bars indicate mean ± standard deviation. Data were analysed by two tailed students t test, * = p<0.05, ** = p< 0.01, *** = p< 0.001.

[0022] Figure 4: Mass spectrometry comparison of cultured and native neutrophil proteomes. A: Experimental design for TMT proteomic analysis. B: Stacked bar chart and heat map displaying significantly enriched proteins (red), significantly under-represented (blue) and unaltered proteins (grey), using native neutrophils as the baseline for comparison. Differentially expressed (DE) proteins were displayed using heatmap visualisation of Log2 Fold Change (Log2FC) values of both significantly enriched and underrepresented proteins. C: Volcano plot displaying total and differentially expressed proteins (P-value threshold of 0.05, absolute Log2FC threshold of 1.00). D: Top 8 unique Gene Ontology (GO) terms resulting from gene list enrichment analysis of enriched (red) and under- represented (blue) proteins using the GO Biological Pathway module. E: Representative Seahorse metabolic flux analyser mitochondrial stress test of native and cultured neutrophils treated with oligomycin, FCCP and rotenone / antimycin A. F-H: Oxygen consumption rate (OCR) of native and cultured neutrophils measuring basal respiration (F), spare respiratory capacity (G) and ATP production (H). Error bars indicate mean ± standard deviation (n=3 differentiations), two tailed students T test (***= P<0.001, ****= P<0.001). I: Normalised abundances of key granule proteins, n=3 differentiations, * = p<0.05, **=p<0.001.

[0023] Figure 5: Genome editing of cultured neutrophils. A-B: CRISPR / Cas9 mediated deletion of P2M (A) and CDllb (B) demonstrated by flow cytometric quantification of the percentage of cells expressing targeted protein (left) and representative histograms of differentiated neutrophils (right) at day 17 of differentiation, n=3. Data were analysed by two tailed students t test, * = p<0.05, ** = p< 0.01, *** = p< 0.001, ****=p<0.0001.

[0024] Figure 6: Quantification of cell death in cultured neutrophils. A: Representative scatter plots of heat treated (positive control) and untreated cultured neutrophils at days 17, 19 and 21 of differentiation. Cells were stained with propidium iodide (PI) and Annexin V-APC. B: Representative gating strategy of native and cultured cells. Native cells were gated on a singlet, appropriately sized population where monocytes and eosinophils were gated out using CD14 and IL-5R expression respectively. Cultured neutrophils were gated on a singlet, appropriately sized, live population using granulocyte markers CD66b and CD15.

[0025] Figure 7: Native and GCSF-D neutrophil staining properties and purity. A: Representative scatter plots of neutrophils gated on size, CD45, CD15 and CD66b expression. B-C: CD101 (B) and CD10 (C) expression in peripheral blood neutrophils from native (red), GCSF-D (blue) and cultured neutrophils (black), with fluorescence minus one (FMO) controls for cultured neutrophils and peripheral blood neutrophils displayed in grey. D: Representative images of NETs induced by 10 uM A23187, stained with SYOTO Green and SYTOX Orange.

[0026] Figure 8: Cultured neutrophils differ in mitochondrial and granule protein expression. A: Representative sorting gates for native and cultured neutrophils before TMT mass spectrometry. B: Top 8 unique Reactome 2022 pathway terms of enriched (red) and under- represented (blue) genes using the GO Biological Pathway module. C: Normalised protein abundances of key TCA cycle and respiratory electron transport proteins, n=3 differentiations. D-E: Enriched (D) and under-represented (E) proteins processed using the STRING database using a medium confidence setting to produce protein-protein interaction networks. Clusters are shown in different colours: E) ribosomal proteins in red, mitochondrial proteins in blue and granule proteins in green and in F) innate immune system and degranulation in red, mRNA processing in blue and chromatin organisation in green. Figure 9: CD66b expression and viability is unchanged by CRISPR / Cas9 mediated knockout in cultured neutrophils. A: Percentage of PI positive cells in Scr vs [32M (left) and CDllb (right) KO cells respectively by flow cytometric surface marker staining on day 7 of culture. B: Percentage of CD66b expressing cells in [32M (left) and CDllb (right) CRISPR / Cas9 KO cells, by flow cytometric surface marker staining on day 17 of culture.

[0027] Figure 10: Flow cytometry analysis of surface markers on mature neutrophils from circulating blood (native), immature, bone marrow-mobilised neutrophils (GCSF-D) and immature neutrophils of the invention (cultured). A-D: Percentage in positive flow cytometry gate, n=3-4 donors. Data were analysed by one-way ANOVA with Tukey's multiple comparisons displayed on graph,, * = p<0.05, ** = p< 0.01, *** = p< 0.001.

[0028] Figure 11: Secretion of TNF alpha by immature neutrophils of the invention, in response to the TLR8 agonist R848, measured by ELISA (n=3 donors per group).

[0029] Figure 12: Elevated mitochondrial metabolism in cultured neutrophils is a sign of immaturity. Mitochondrial oxygen consumption rate (OCR) of native neutrophils (Native) and cultured immature neutrophils of the invention (Cultured) measuring basal respiration (A), spare respiratory capacity (B) and ATP production (C) in metabolic flux analyser (Seahorse XF). Error bars indicate mean ± standard deviation (n=3 blood or apheresis cone donors), two tailed students T test (***= P<0.001, ****= P<0.001).

[0030] Figure 13: Comparison of viability of native neutrophils (native) and immature neutrophils of the invention (cultured). Apoptotic cells were quantified by measuring Annexin V positivity in a flow cytometry assay. N=3 blood or apheresis cone donors.

[0031] Figure 14: Comparison of neutrophil progenitor proliferation rates, starting from equivalent numbers of stem cells from either individual donors (blue), or 2 or more pooled donors. Differentiation was induced on day 3 (D3) post isolation and committed neutrophil progenitors were enumerated on day 10 (D10) of differentiation.

[0032] Figure 15: Cultured, immature neutrophils of the invention and native neutrophils were frozen at -80C with either Cryostor (CS) or Stem Cell Banker (SCB) freezing reagent. Cells were thawed 24h hours later and functionality was assessed by measuring ROS production in response to stimulation with phorbol ester (PMA). A: Pre-freezing and post-thaw ROS production of cultured, immature neutrophils of the invention (cultured). B, C: Pre-freezing and post-thaw ROS production of native neutrophils from two donors.

[0033] Figure 16: Gene editing of immature neutrophils of the invention. A: FACS plots showing surface expression of Siglec 9 on cultured neutrophils (day 17) treated with control (scramble, red) or siglec9 CRISPR / Cas9 gRNA, in two biological replicates, B: Combined mean fluorescent intensities (MFI) of siglec9 staining of the two experiments shown in A. C: ROS production of control and Siglec 9 knockdown cultured neutrophils, stimulated with the chemical inducer PMA (top) and opsonized C. albicans (bottom).

[0034] Figure 17: Lentiviral overexpression of CD16 (FcyRIIIb), the receptor for antibody- opsonised microbes, in cultured, immature neutrophils of the invention. Control vector= GFP only, CD16 vector= GFP+CD16. MFI= mean fluorescence intensity of surface CD16, quantified by flow cytometry. N=3 differentiations, from pooled apheresis donors.

[0035] DETAILED DESCRIPTION

[0036] General disclosure

[0037] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0038] The present invention is described with respect to particular embodiments and with reference to certain Figures but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein.

[0039] The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying Figures. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "some embodiments" or a "preferred embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment(s) is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in some embodiments" or "in a preferred embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may do so. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment.

[0040] In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a population" includes two or more populations, reference to "a marker" includes two or more such markers, reference to "a cell" includes two or more cells, reference to "a subject" refers to two or more subjects, reference to "a method" includes two or more methods and the like.

[0041] Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0042] "About" as used herein when referring to a measurable value such as a percentage or an amount and the like, is meant to encompass variations of ± 20 % or ± 10 %, more preferably ± 5 %, even more preferably ± 1 %, and still more preferably ± 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Any statement herein including the term "about" includes the same feature without the term. For instance, a population having at least "about" 60% of the cells in the population expressing detectable levels of CD66b includes a population having at least 60% of the cells in the population expressing detectable levels of CD66b.

[0043] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other components, integers, or steps.

[0044] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples, and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0045] In all instances herein, the term "native" is interchangeable with "naturally occurring".

[0046] In all instances herein, the term "genetically engineered" or the like is interchangeable with "genetically modified".

[0047] Immature neutrophils of the invention Population

[0048] The invention provides a population of immature neutrophils. This population is referred to herein as "a / the population" or "a / the population of the invention".

[0049] The population typically comprises at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% immature neutrophils. Neutrophils, and especially, immature neutrophils may be defined as discussed below. The identity of the cells making up the remainder of the population is not particularly limited. The cells making up the remainder of the population can be a single cell type or can be a mixture of different cell types. The cells making up the remainder of the population are typically the haematopoietic progenitor cells from which the immature neutrophils are derived. The cells making up the remainder of the population may be other immune cell, such as macrophages. These cells and methods for making immature neutrophils from them are discussed in more detail below.

[0050] The population may comprise any number of cells, such as at least about 5 x 105cells. The population preferably comprises at least about 1 x 106, at least about 2 x 106, at least about 2.5 x 106, at least about 5 x 106, at least about 1 x 107, at least about 2 x 107, at least about 5 x 107, at least about 1 x 108or at least about 2 x 108cells. In some instances, the population comprises at least about 1.0 x 107, at least about 1.0 x 108, at least about 1.0 x 109, at least about 1.0 x 1010, at least about 1.0 x 1011or at least about 1.0 x 1012cells or even more cells.

[0051] The population is preferably a mammalian population. The population may be a human, dog, cat, primate, horse, murine, rat, rodent, bovine, murine, porcine, or ovine population. The immature neutrophils are preferably mammalian. The immature neutrophils may be human, dog, cat, primate, horse, murine, rat, rodent, bovine, murine, porcine, or ovine immature neutrophils. The immature neutrophils are most preferably human.

[0052] The population is typically produced by cell culture. The immature neutrophils of the invention are typically produced from haematopoietic progenitor cells. The immature neutrophils may be produced using the method of the invention. The haematopoietic progenitor cells can be stem cells, haematopoietic stem cells, peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cell (iPSCs), or an immortalized cell line. The haematopoietic progenitor cells may be derived from one or more donors. Preferably the haematopoietic progenitor cells are CD34+. For instance, the haematopoietic progenitor cells can be CD34+ cells derived from donors, CD34+ stem cells, CD34+ PBMCs or iPSCs. CD34+ haematopoietic progenitor cells can be derived from stem cells, haematopoietic stem cells, peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cell (iPSCs), or an immortalized cell line.

[0053] The population of the invention may be isolated, substantially isolated, purified, or substantially purified. A population is isolated or purified if it is completely free of any other components, such as culture medium. A population is substantially isolated or substantially purified if it is mixed with carriers or diluents, such as a culture medium or a pharmaceutical composition, which will not interfere with its intended use. Other carriers and diluents are discussed in more detail below.

[0054] Immature neutrophils

[0055] The population of the invention comprises immature neutrophils. Neutrophils can be identified using known methods. Typical methods of identifying neutrophils include their nuclear morphology, surface marker expression (as described below) and some neutrophil effector functions, such as reactive oxygen species (ROS) production, bacterial killing, phagocytosis, and chemotaxis. Neutrophils display multi-lobbed nuclei which can be identified using routine microscopy techniques. Increased ROS production by neutrophils, especially in response to stimuli, can be measured using routine methods including those described in the Examples. Assays are also available for bacterial killing, phagocytosis, and chemotaxis.

[0056] At least about 60% of the cells in the population express detectable levels of CD66b (carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8)). CD66b is a granulocyte marker. Preferably, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% of the cells in the population express detectable levels of CD66b.

[0057] At least about 60% of the cells in the population express detectable levels of CDllb (integrin alpha M (ITGAM)). CDllb is also a granulocyte marker. Preferably, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% of the cells in the population express detectable levels of CDllb. Fewer than about 20% of the cells in the population express detectable levels of CD10 (membrane metallo-endopeptidase (MME)). CD10 is a marker of neutrophil maturity. Preferably, fewer than about 15%, fewer than about 10%, fewer than about 5%, fewer than about 2% or fewer than about 1% of the cells in the population express detectable levels of CD10.

[0058] Fewer than about 20% of the cells in the population express detectable levels of CD16 (FcyRIII). A low expression level of CD16 distinguishes the immature neutrophils of the invention from native immature neutrophils (GCSF-D). Preferably, fewer than about 15%, fewer than about 10%, fewer than about 5%, fewer than about 2% or fewer than about 1% of the cells in the population express detectable levels of CD16.

[0059] Fewer than about 20% of the cells in the population express detectable levels of CXCR2 (interleukin-18 (IL-18) receptor beta). A low expression level of CXCR2 distinguishes the immature neutrophils of the invention from native immature neutrophils (GCSF-D).

[0060] Preferably, fewer than about 15%, fewer than about 10%, fewer than about 5%, fewer than about 2% or fewer than about 1% of the cells in the population express detectable levels of CXCR2.

[0061] Preferably, at least about 60% of the cells in the population express detectable levels of CD66b, at least about 80% of the cells in the population express detectable levels of CDllb, fewer than about 10% of the cells in the population express detectable levels of CD10, fewer than about 10% of the cells in the population express detectable levels of CD16, and fewer than about 10% of the cells in the population express detectable levels of CXCR2.

[0062] Preferably, at least about 80% of the cells in the population express detectable levels of CD66b, at least about 80% of the cells in the population express detectable levels of CDllb, fewer than about 5% of the cells in the population express detectable levels of CD10, fewer than about 5% of the cells in the population express detectable levels of CD16, and fewer than about 10% of the cells in the population express detectable levels of CXCR2.

[0063] Preferably, fewer than about 40% of the cells in the population express detectable levels of CD101 (immunoglobulin superfamily, member 2 (IGSF2)). Preferably, fewer than about 35%, fewer than about 30%, fewer than about 20%, fewer than about 15%, fewer than about 10%, fewer than about 5%, fewer than about 2% or fewer than about 1% of the cells in the population express detectable levels of CD101.

[0064] Fewer than about 20% of the cells in the population express detectable levels of CD34. A low expression level of CD34 may distinguish the immature neutrophils of the invention from the haematopoietic progenitor cells from which they were produced. Preferably, fewer than about 15%, fewer than about 10%, fewer than about 5%, fewer than about 2% or fewer than about 1% of the cells in the population express detectable levels of CD34. Preferably, the cells in the population do not express detectable levels of CD34.

[0065] At least about 60% of the cells in the population express detectable levels of CD33 (Siglec- 3). Preferably, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% of the cells in the population express detectable levels of CD33.

[0066] Preferably, at least about 60% of the cells in the population express detectable levels of CD66b, at least about 60% of the cells in the population express detectable levels of CDllb, fewer than about 20% of the cells in the population express detectable levels of CD10, fewer than about 20% of the cells in the population express detectable levels of CD16, fewer than about 20% of the cells in the population express detectable levels of CXCR2, and fewer than about 20%, 15%, 10%, 5%, 2% or 1% of the cells in the population express detectable levels of CD34 and / or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% of the cells in the population express detectable levels of CD33. Preferably, at least about 60% of the cells in the population express detectable levels of CD66b, at least about 60% of the cells in the population express detectable levels of CDllb, fewer than about 20% of the cells in the population express detectable levels of CD10, fewer than about 20% of the cells in the population express detectable levels of CD16, fewer than about 20% of the cells in the population express detectable levels of CXCR2, and the cells in the population do not express detectable levels of CD34 and / or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% of the cells in the population express detectable levels of CD33.

[0067] Preferably, at least about 60% of the cells in the population express detectable levels of CD66b, at least about 80% of the cells in the population express detectable levels of CDllb, fewer than about 10% of the cells in the population express detectable levels of CD10, fewer than about 10% of the cells in the population express detectable levels of CD16, fewer than about 10% of the cells in the population express detectable levels of CXCR.2, and fewer than about 20%, 15%, 10%, 5%, 2% or 1% of the cells in the population express detectable levels of CD34 and / or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% of the cells in the population express detectable levels of CD33. Preferably, at least about 60% of the cells in the population express detectable levels of CD66b, at least about 80% of the cells in the population express detectable levels of CDllb, fewer than about 10% of the cells in the population express detectable levels of CD10, fewer than about 10% of the cells in the population express detectable levels of CD16, fewer than about 10% of the cells in the population express detectable levels of CXCR2, and the cells in the population do not express detectable levels of CD34 and / or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% of the cells in the population express detectable levels of CD33.

[0068] Preferably, at least about 80% of the cells in the population express detectable levels of CD66b, at least about 80% of the cells in the population express detectable levels of CDllb, fewer than about 5% of the cells in the population express detectable levels of CD10, fewer than about 5% of the cells in the population express detectable levels of CD16, fewer than about 10% of the cells in the population express detectable levels of CXCR2, and fewer than about 20%, 15%, 10%, 5%, 2% or 1% of the cells in the population express detectable levels of CD34 and / or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% of the cells in the population express detectable levels of CD33. Preferably, at least about 80% of the cells in the population express detectable levels of CD66b, at least about 80% of the cells in the population express detectable levels of CDllb, fewer than about 5% of the cells in the population express detectable levels of CD10, fewer than about 5% of the cells in the population express detectable levels of CD16, fewer than about 10% of the cells in the population express detectable levels of CXCR2, and the cells in the population do not express detectable levels of CD34 and / or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% of the cells in the population express detectable levels of CD33.

[0069] In the context of the present invention, a "detectable level" may comprise a detectable level of the mRNA encoding the relevant marker, e.g. CD66b, and / or the marker protein, e.g. the CD66b protein. Preferably, a "detectable level" comprises a detectable level of the protein, e.g. the CD66b protein. A "detectable level" may comprise a detectable level of the mRNA encoding the relevant marker, e.g. CD66b, and a detectable level of the marker protein, e.g. the CD66b protein. A "detectable level" may comprise a detectable activity of the relevant marker, e.g. CD66b (which is involved in cell adhesion, cell migration, and pathogen binding). In all instances herein, the term "level" is interchangeable with "amount", "activity" or "amount and activity".

[0070] The presence of mRNA and / or protein may be detected using any routine method in the art. Such methods include immunofluorescence, immunohistochemistry, western blotting, quantitative polymerase chain reaction (qPCR), reporter assays, enzyme-linked immunosorbent assay (ELISA), microscopy, flow cytometry, enzymatic staining, dye incorporation, chemiluminescent oxygen detection reagents, fluorometry, fluorescence in situ hybridization (FISH) and an alkaline phosphatase assay. The skilled person is capable of detecting the activity of any particular marker using known assays. Suitable assays are described in the Examples. The markers are preferably detected using flow cytometry.

[0071] Secreted factors The population of the invention preferably secretes a detectable level of one or more of (a) interleukin-6 (IL-6), (b) IL-8 and (c) tumour necrosis factor (TNF) alpha, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c). The secretion of these factors can be measured using routine assays, including the ones described in the Examples. The secretion of IL-6 may be stimulated using one or more factors, such as live or heat-killed microbes (bacteria and fungi), isolated component thereof, lipopolysaccharide (LPS) and / or R-848. The secretion of IL-8 may be stimulated using one or more factors, such as lipopolysaccharide (LPS). The secretion of TNF alpha may be stimulated using one or more factors, such as R-848.

[0072] Additional phenotypes

[0073] The population of the invention preferably has an increased capacity to produce reactive oxygen species (ROS), for instance in response to stimulation. The population of the invention preferably produces increased amounts of reactive oxygen species (ROS) in response to stimulation. The increased capacity and / or increased amount is typically compared with native, mature neutrophils and / or native, immature neutrophils. ROS production or capacity may be increased by any amount, such as by at least about 20%, at least about 50%, at least about 75%, at least about 90%, at least about a factor of 2, such as least about a factor of 5, at least about a factor of 10, at least about a factor of 50, at least about a factor of 100, at least about a factor of 500, as least about a factor of 1000, at least about a factor of 5000, at least about a factor of 10,000 or more. As discussed in more detail below, the population of the invention preferably also retains its increased capacity to produce ROS after freezing and thawing. ROS production can be measured as described in the Examples. Suitable factors which stimulate ROS production include, but are not limited to, live or heat-killed microbes (such as bacteria or fungi), calcium ionophores, zymosan, phorbol myristate acetate (PMA and aminophenyl fluorescein (APF).

[0074] The population of the invention is preferably capable of phagocytosis and / or chemotaxis. This can be done using routine methods. Phagocytosis of fluorescently labelled microbes or beads is measured by flow cytometry or by microscopy. Chemotaxis in response to a chemokine is measured in a transwell migration assay.

[0075] The population of the invention is preferably capable of killing or reducing the growth of one or more pathogenic agents. The one or more pathogenic agents may be selected from those described below. The one or more pathogenic agents preferably comprise Candida albicans and Staphylococcus aureus. Assays for measuring the ability of the population to kill or reduce the growth of pathogens are known and described in the Examples.

[0076] The population of the invention preferably demonstrates increased metabolic activity compared with native, mature neutrophils. Increased metabolic activity can be measured as one or more of (a) increased basal respiration, (b) increased spare respiratory capacity and (c) increased ATP production, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c). These can be measured using routine methods and as described in the Examples. Metabolic activity and one or more of (a)-(c) may be increased by any amount, such as by at least about 20%, at least about 50%, at least about 75%, at least about 90%, at least about a factor of 2, such as least about a factor of 5, at least about a factor of 10, at least about a factor of 50, at least about a factor of 100, at least about a factor of 500, as least about a factor of 1000, at least about a factor of 5000, at least about a factor of 10,000 or more.

[0077] The population of the invention preferably expresses detectable levels of one or more granule proteins. These can be measured as described in the Examples. The one or more granule proteins are preferably selected from MPO, ELAINE, NCF1B, LF and MMP9.

[0078] Preferably, at least about 20% of the cells in the population, such as at least about 30%, at least about 40%, at least about 50%, at least about 60% or at least about 70% of the cells in the population, remain viable in culture after about 48 hours. This means they remain viable in culture after about 48 hours after the method of the invention is conducted. Viability can be measured as described in the Examples, such as based on Annexin V positivity in a flow cytometry assay.

[0079] Genetically engineered immature neutrophils

[0080] At least some of the cells in the population of the invention are preferably genetically engineered to express or overexpress one or more activating receptors and / or to reduce expression of one or more inhibitory receptors. The cells may be genetically engineered in any of the ways discussed below. This population is referred to herein as "a / the genetically engineered population" or "a / the genetically engineered population of the invention".

[0081] Preferably, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the cells in the genetically engineered population are genetically engineered. This can be tested by measuring the number of cells comprising the exogenous polynucleotide that is configured to express or overexpress and / or reduce expression of the relevant factor(s). Changes in expression following genetical engineering can also be measured by flow cytometry or Western blot

[0082] Immature neutrophil of the invention

[0083] The invention also provides an immature neutrophil which expresses detectable levels of CD66b and CDllb and does not express detectable levels of CD10, CD16, and CXCR.2. The invention also provides a genetically engineered immature neutrophil which expresses detectable levels of CD66b and CDllb, does not express detectable levels of CD10, CD16, and CXCR.2 and is genetically engineered to express or overexpress one or more activating receptors and / or to reduce expression of one or more inhibitory receptors. The immature neutrophil may express an undetectable level of CD10, CD16, and CXCR.2.

[0084] The invention also provides a population of two or more immature neutrophils or genetically engineered immature neutrophils of the invention. Any of the embodiments discussed above and below with reference to the population of the invention and the genetically modified population of the invention equally apply to the immature neutrophil of the invention and populations thereof.

[0085] Method of producing immature neutrophils

[0086] The invention also provides a method of producing a population of the invention. The population of the invention may have any of the phenotypes discussed above. This method is referred to herein as "a / the method" or "a / the method of the invention".

[0087] The method comprises steps (a)-(d). Standard culture conditions are known in the art and are described in the Examples.

[0088] Step (a)

[0089] Step (a) comprises culturing haematopoietic progenitor cells in the presences of interleukin- 3 (IL-3), stem cell factor (SCF) and FMS-like tyrosine kinase 3 (Flt-3) Ligand (Flt3-L) and in the absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or granulocyte colony stimulating factor (G-CSF).

[0090] The haematopoietic progenitor cells may be any of those described above, including stem cells, haematopoietic stem cells, peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cell (iPSCs), or an immortalized cell line. The haematopoietic progenitor cells may be derived from induced pluripotent stem cell (iPSCs. The haematopoietic progenitor cells are preferably CD34+. The haematopoietic progenitor cells are preferably derived from one or more donors. The haematopoietic progenitor cells are preferably isolated from whole blood or apheresis waste. The haematopoietic progenitor cells are preferably isolated from one or more apheresis cones. The haematopoietic progenitor cells are more preferably isolated from two or more donors or two or more apheresis cones from two or more donors. The haematopoietic progenitor cells are preferably isolated from whole blood or apheresis waste from two or more donors. The invention may use any number of donors and / or apheresis cones, such as 1, 2, 3, 4, 5, 6 or more.

[0091] Step (a) preferably further comprises culturing the haematopoietic progenitor cells in the absence of IL-6 and thrombopoietin (TPO). The haematopoietic progenitor cells may be cultured for any amount of time. The haematopoietic progenitor cells may be cultured for from about 1 day to about 5 days, such as for about 1 day, about 2 days, about 3 days, about 4 days or about 5 days. Step (a) preferably comprises culturing the haematopoietic progenitor cells for about 3 days.

[0092] Any amounts of IL-3, SCF and Flt3-L may be used, such as concentrations of from about lOng / mL to about 500ng / mL. The concentration of IL-3 is preferably about lOng / mL. The concentration of SCF is preferably about 50ng / mL. The concentration of Flt-3-L is preferably about 50ng / mL. The concentration of IL-3 is preferably about lOng / mL, the concentration of SCF is preferably about 50ng / mL, and the concentration of Flt-3-L is preferably about 50ng / mL.

[0093] Step (b)

[0094] Step (b) comprises culturing the cells produced in step (a) in the presence of IL-3, SCF, Flt3-L and GM-CSF.

[0095] The cells produced in step (a) may be cultured for any amount of time. The cells produced in step (a) may be cultured for from about 1 day to about 5 days, such as for about 1 day, about 2 days, about 3 days, about 4 days or about 5 days. Step (b) preferably comprises culturing the cells for about 4 days.

[0096] Any amounts of IL-3, SCF, Flt3-L and GM-CSF may be used, such as concentrations of from about lOng / mL to about 500ng / mL. The concentration of IL-3 is preferably about lOng / mL. The concentration of SCF is preferably about 50ng / mL. The concentration of Flt-3-L is preferably about 50ng / mL. The concentration of GM-CSF is preferably about lOng / mL. The concentration of IL-3 is preferably about lOng / mL, the concentration of SCF is preferably about 50ng / mL, the concentration of Flt-3-L is preferably about 50ng / mL, and the concentration of GM-CSF is preferably about lOng / mL.

[0097] Step (c)

[0098] Step (c) comprises culturing the cells produced in step (b) in the presence of GM-CSF and G-CSF.

[0099] The cells produced in step (b) may be cultured for any amount of time. The cells produced in step (b) may be cultured for from about 1 day to about 5 days, such as for about 1 day, about 2 days, about 3 days, about 4 day or about 5 days. Step (c) preferably comprises culturing the cells for about 3 days.

[0100] Any amounts of GM-CSF and G-CSF may be used, such as concentrations of from about lOng / mL to about 500ng / mL. The concentration of GM-CSF is preferably about lOng / mL. The concentration of G-CSF is preferably about lOng / mL. The concentration of GM-CSF is preferably about lOng / mL and the concentration of G-CSF is preferably about lOng / mL.

[0101] Step (d)

[0102] Step (d) comprises culturing the cells produced in step (c) in the presence of G-CSF.

[0103] The cells produced in step (c) may be cultured for any amount of time. The cells produced in step (c) may be cultured for from about 1 day to about 10 days, such as for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about, about 9 days or about 10 days. Step (d) preferably comprises culturing the cells for about 7 days.

[0104] Any amount of G-CSF may be used, such as a concentration of from about lOng / mL to about 500ng / mL. The concentration of G-CSF is preferably about lOng / mL.

[0105] Preferred method

[0106] The method preferably comprises:

[0107] (a) culturing haematopoietic progenitor cells for about 3 days in the presence of IL-3 at a concentration of about lOng / mL, SCF at a concentration of about 50ng / mL and Flt3-L at a concentration of about 50ng / mL and in the absence of GM-CSF, G-CSF, IL-6 and TPO,

[0108] (b) culturing the cells produced in step (a) for about 4 days in the presence of IL-3 at a concentration of about lOng / mL, SCF at a concentration of about 50ng / mL, Flt3-L at a concentration of about 50ng / mL and GM-CSF at a concentration of about lOng / mL,

[0109] (c) culturing the cells produced in step (b) for about 3 days in the presence of GM-CSF at a concentration of about lOng / mL and G-CSF at a concentration of about lOng / mL and

[0110] (d) culturing the cells produced in step (c) for about 7 days in the presence of G-CSF at a concentration of about lOng / mL.

[0111] Optional pre-step

[0112] The invention preferably comprises before step (a) isolating CD34+ haematopoietic progenitor cells from one or more donors, such as 1, 2, 3, 4, 5, 6 or more donors. The invention preferably comprises before step (a) isolating CD34+ haematopoietic progenitor cells from two or more donors, such as 2, 3, 4, 5, 6 or more donors.

[0113] The invention preferably comprises before step (a) isolating CD34+ haematopoietic progenitor cells from one or more apheresis cones, such as 1, 2, 3, 4, 5, 6 or more apheresis cones. The invention preferably comprises before step (a) isolating CD34+ haematopoietic progenitor cells from two or more apheresis cones from two or more donors, such as 2, 3, 4, 5, 6 or more apheresis cones from 2, 3, 4, 5, 6 or more donors .

[0114] Optional step (e)

[0115] The method preferably further comprises (e) isolating immature neutrophils based on the expression of CD66b. This can be done using known methods and as described in the Examples.

[0116] Genetically engineered immature neutrophils

[0117] The invention provides a population of genetically engineered immature neutrophils. This population is also referred to herein "a / the genetically engineered population" or "a / the genetically engineered population of the invention".

[0118] The genetically engineered population typically comprises at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% immature neutrophils. Neutrophils, and especially, immature neutrophils may be defined as discussed above. The immature neutrophils may have any of the phenotypes discussed above. The remainder of the cells in the genetically engineered population may be the same as described above for the population of the invention.

[0119] The genetically engineered population of the invention may comprise native immature neutrophils or immature neutrophils produced using any other method. The immature neutrophils in the genetically engineered population of the invention may be produced using the method of the invention.

[0120] Preferably, at least about 60% of the cells in the genetically engineered population express detectable levels of CD66b. Preferably, at least about 60% of the cells in the genetically engineered population express detectable levels of CDllb. Preferably, fewer than about 20% of the cells in the genetically engineered population express detectable levels of CD10. Preferably, at least about 60% of the cells in the genetically engineered population express detectable levels of CD66b, at least about 60% of the cells in the genetically engineered population express detectable levels of CDllb, and fewer than about 20% of the cells in the genetically engineered population express detectable levels of CD10.

[0121] Preferably, fewer than about 40% of the cells in the genetically engineered population express detectable levels of CD101. Preferably, at least about 60% of the cells in the genetically engineered population express detectable levels of CD66b, at least about 60% of the cells in the genetically engineered population express detectable levels of CDllb, fewer than about 20% of the cells in the genetically engineered population express detectable levels of CD10, , fewer than about 40% of the cells in the genetically engineered population express detectable levels of CD101.

[0122] Preferably, fewer than about 20% of the cells in the genetically engineered population express detectable levels of CD16. Preferably, fewer than about 20% of the cells in the genetically engineered population express detectable levels of CXCR2. Preferably, at least about 60% of the cells in the genetically engineered population express detectable levels of CD66b, at least about 60% of the cells in the genetically engineered population express detectable levels of CDllb, fewer than about 20% of the cells in the genetically engineered population express detectable levels of CD10, fewer than about 20% of the cells in the genetically engineered population express detectable levels of CD16, and fewer than about 20% of the cells in the genetically engineered population express detectable levels of CXCR2. Preferably, at least about 60% of the cells in the genetically engineered population express detectable levels of CD66b, at least about 60% of the cells in the genetically engineered population express detectable levels of CDllb, fewer than about 20% of the cells in the genetically engineered population express detectable levels of CD10, and fewer than about 20% of the cells in the genetically engineered population express detectable levels of CXCR2.

[0123] Preferably, fewer than about 20% of the cells in the genetically engineered population express detectable levels of CD34. Preferably, at least about 60% of the cells in the genetically engineered population express detectable levels of CD33.

[0124] For CD66b, CDllb, CD10, CD101, CD16, CXCR2, CD34 and CD33, the genetically engineered population of the invention may have any of the %s discussed above for the population of the invention.

[0125] The phenotype of the cells may change from those discussed above based on the genetic engineering. For instance, the cells may have the marker expression pattern of the population of the invention described above except greater than 20% of the cells express detectable levels of CD16 as a result of the genetic engineering.

[0126] In some embodiments, the immature neutrophils themselves are genetically engineered. In alternative embodiments, haematopoietic progenitor cells are genetically engineered and then differentiated to immature neutrophils. Genetically engineered haematopoietic progenitor cells may be differentiated to immature neutrophils using any method, including the method of the invention. The genetically engineered population of the invention is preferably produced using the genetic engineering method of the invention.

[0127] The genetically engineered population may comprise any number of cells, such as at least about 5 x 105cells. The genetically engineered population preferably comprises at least about 1 x 106, at least about 2 x 106, at least about 2.5 x 106, at least about 5 x 106, at least about 1 x 107, at least about 2 x 107, at least about 5 x 107, at least about 1 x 108or at least about 2 x 108cells. In some instances, the genetically engineered population comprises at least about 1.0 x 107, at least about 1.0 x 108, at least about 1.0 x 109, at least about 1.0 x IO10, at least about 1.0 x 1011or at least about 1.0 x 1012cells or even more.

[0128] In the context of the present invention, "genetically engineered" means that the cell or population of cells has been genetically modified to express or overexpress and / or reduce expression of the relevant factor(s), such as the one or more activating receptors and / or one or more inhibitory receptors. The genetically engineered cell or population preferably expresses a detectable level of one or more activating receptors. The genetically engineered cell or population preferably comprises a reduced expression of one or more inhibitory receptors.

[0129] Typically, "genetically engineered" means that the genetic material of the cell or population of cells has been altered to express or overexpress the relevant factor(s) and / or reduce expression of the relevant factor(s), such as one or more activating receptors and / or one or more inhibitory receptors. When "genetically engineered", the cell or population of cells typically comprises an exogenous polynucleotide that is configured to express or overexpress and / or reduce expression of the relevant factor(s), such as the one or more activating receptors and / or one or more inhibitory receptors. A skilled person is capable of identifying such exogenous polynucleotides in the cell or population of cells. The exogenous polynucleotide may comprise any of the control sequences discussed below and / or may be part of any of the vectors discussed below.

[0130] Prior to genetic engineering, the cell or population preferably does not express a detectable level of the one or more activating receptors. Prior to genetic engineering, the cell or population may express a detectable level of the one or more activating receptors. The detectable level may be low because of the cell or population's phenotype or epigenetics or because the factor is degraded or secreted from the cell. The cell or population may be genetically engineered to overexpress the relevant factor(s), such as the one or more activating receptors.

[0131] Prior to genetic engineering, the cell or population preferably expresses a detectable level of the one or more inhibitory receptors. The cell or population may be genetically engineered to under express the relevant factor(s), such as the one or more inhibitory receptors.

[0132] In the context of the invention, "genetically engineered to overexpress" means the cell or population of cells has been genetically modified to express an increased level of the relevant factor(s), such as the one or more activating receptors. The increased level may be an increased amount and / or an increased activity. The increased level is typically an increased detectable level. The increased level is typically compared with the level in the cell or population before the genetic engineering is conducted. The increased level is typically compared with the level in a cell or a population that has not undergone the genetic engineering, such as a corresponding, unmodified cell or population.

[0133] The marker, such as the one or more activating receptors, may be overexpressed by any amount. The marker, such as the one or more activating receptors, may be overexpressed by at least about 5%, such as least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, as least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%. The marker, such as the such as the one or more activating receptors, may be overexpressed by at least about a factor of 2, such as least about a factor of 5, at least about a factor of 10, at least about a factor of 50, at least about a factor of 100, at least about a factor of 500, as least about a factor of 1000, at least about a factor of 5000, at least about a factor of 10,000 or more.

[0134] The cell or population may be genetically modified to express a level of the marker, such as the such as the one or more activating receptors, that is increased by at least about 5%, such as least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, as least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%. The cell or population may be genetically modified to express a level of the marker, such as the such as the one or more activating receptors, that is increased by at least about a factor of 2, such as least about a factor of 5, at least about a factor of 10, at least about a factor of 50, at least about a factor of 100, at least about a factor of 500, as least about a factor of 1000, at least about a factor of 5000, at least about a factor of 10,000 or more.

[0135] In the context of the invention, "genetically engineered to reduce expression of" or "genetically engineered to under express" means the cell or population of cells has been genetically modified to express a decreased level of the relevant factor(s), such as the one or more inhibitory receptors. The decreased level may be an decreased amount and / or an decreased activity. The decreased level is typically an decreased detectable level. The decreased level is typically compared with the level in the cell or population before the genetic engineering is conducted. The decreased level is typically compared with the level in a cell or a population that has not undergone the genetic engineering, such as a corresponding, unmodified cell or population.

[0136] The expression of the marker may be reduced or the marker may be under expressed by any amount. The expression of the marker, such as the one or more inhibitory receptors, may be reduced or the marker may be under expressed by at least about 5%, such as least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, as least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%.

[0137] The cell or population may be genetically modified to express a level of the marker, such as the one or more inhibitory receptors, that is decreased by at least about 5%, such as least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, as least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%.

[0138] In the context of the invention, a cell or population is genetically engineered to express, overexpress, under express or reduce expression of a relevant factor(s), such as the one or more activating receptors and / or one or more inhibitory receptors, at one or more membranes. The one or more membranes may be selected from the cell membrane and intracellular membranes including, but not limited to, cell nucleus membranes, mitochondrial membranes, Golgi apparatus membranes, endoplasmic reticulum (ER) membranes, lysosomal membranes, vacuole membranes and granule membranes.

[0139] The immature neutrophils are genetically engineered to express or overexpress one or more activating receptors and / or to reduce expression of one or more inhibitory receptors. The immature neutrophils may be genetically engineered in any of the ways discussed above or below. Preferably, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the cells in the genetically engineered population are genetically engineered. This can be tested by measuring the number of cells comprising the exogenous polynucleotide that is configured to express or overexpress and / or reduce expression of the relevant factor(s).

[0140] The genetically engineered population is preferably a mammalian population. The genetically engineered population may be a human, dog, cat, primate, horse, murine, rat, rodent, bovine, murine, porcine, or ovine population. The genetically engineered immature neutrophils are preferably mammalian. The genetically engineered immature neutrophils be human, dog, cat, primate, horse, murine, rat, rodent, bovine, murine, porcine, or ovine genetically engineered immature neutrophils. The genetically engineered immature neutrophils are most preferably human.

[0141] The genetically engineered population of the invention may be isolated, substantially isolated, purified, or substantially purified. A population is isolated or purified if it is completely free of any other components, such as culture medium. A population is substantially isolated or substantially purified if it is mixed with carriers or diluents, such as a culture medium or a pharmaceutical composition, which will not interfere with its intended use. Other carriers and diluents are discussed in more detail below. The genetically engineered population is genetically engineered to express or overexpress one or more activating receptors and / or to reduce expression of one or more inhibitory receptors. The genetically engineered population may be genetically engineered to express or overexpress one or more activating receptors. The genetically engineered population may be genetically engineered to reduce expression of one or more inhibitory receptors. The genetically engineered population may be genetically engineered to express or overexpress one or more activating receptors and to reduce expression of one or more inhibitory receptors. Expression, overexpression, reduced expression and under expression are described above.

[0142] The genetically engineered population may be genetically engineered to express or overexpress any number of activating receptors, such as 1, 2, 3, 4, 5, 6 or more activating receptors. The one or more activating receptors are preferably selected from CD16, CD10, CDllb, CD15, CD18, CD32a / c, CD62L, CD64, CD66b, CD66d, CD85h, CD89, CD172b, C177, CD300b, CD300c, CD354, PILRB, SIRPB2, CEACAM4, Siglec-14, and 02 integrin. The one or more activating receptors preferably comprises or consists of CD16.

[0143] Methods of expressing or overexpressing exogenous polynucleotides in cells are known in the art. The genetic engineering of cells can be carried out according to standard cloning and expression techniques, which are known in the art (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0144] Cells may be genetically engineered by introducing a vector and / or polynucleotide into the cells. The cells may be transfected with the vector and / or polynucleotide. The term "transfection" and its various forms may be used to describe non-virus-mediated nucleic acid transfer. Non-viral delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. The preparation of lipid: nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0145] Alternatively, the cells may be transduced with the vector and / or polynucleotide. The vector is typically an expression vector. Various expression vectors can be employed to genetically engineer the cells. Both viral-based and non-viral expression vectors can be used in the invention. Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., 1997, Nat Genet. 15:345). For example, non-viral vectors useful for expression in mammalian (e.g., human) cells include pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C, (Invitrogen, San Diego, Calif.), MPS V vectors, and numerous other vectors known in the art for expressing other proteins and / or nucleotide sequences. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, 1995, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., 1992, Cell 68: 143.

[0146] Preferably, the vector is a retroviral, lentiviral, adenoviral, or adeno-associated viral vector. The vector is preferably a retroviral or lentiviral vector. The vector is preferably a lentiviral vector, such as pLV[Exp]-EGFP-SFFV

[0147] Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., 1986, Immunol. Rev. 89:49-68), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell typespecific, stage-specific, and / or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, the SSFV promoter, the EFl alpha promoter, the phosphoglycerate kinase (PGK) promoter and promoter-enhancer combinations known in the art.

[0148] The genetically engineered population may be genetically engineered to reduce expression of any number of inhibitory receptors, such as 1, 2, 3, 4, 5, 6 or more inhibitory receptors. The one or more inhibitory receptors are preferably selected from Siglec 9 (CD329), CD33, CD66a, CD85a, CD85d, CD85k, CD170, CD172A, CD300a, CD300f, CD305, PILRA, SIRL-1 and CLEC12A. The one or more inhibitory receptors preferably comprise or consist of Siglec 9 (CD329). Methods for reducing expression of, or under expressing, specific genes are known in the art. In this context, genetic engineering typically involves modifying endogenous genes using nucleases. These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome. The induced DSBs may be repaired through non- homologous end joining (NHEJ) or homology directed repair (HDR), resulting in targeted mutations or edits. HDR and NHEJ are described in Pardo et al, Cell Mol Life Sci, 66(6): 1039-1056 (2009) and Cox et al., Nature Medicine, 21(2): 121-131 (2015).

[0149] Suitable nucleases include, but are not limited to, (a) clustered regularly interspaced short palindromic repeats (CRISPR), such as CRISPR / Cas9, (b) zinc finger nucleases (ZFN), (c) transcription activator-like effector nucleases (TALEN), (d) meganucleases and (e) combinations thereof, such as (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a), (c) and (d); (b), (c) and (d); or (a), (b), (c) and (d). Suitable CRISPR nucleases in (a) include, but are not limited to, SpCas9 and SaCas9. Cpfl may also be used.

[0150] All of these methods are capable of modifying gene sequences to produce mutants, variants or fragments. All of these methods are also capable of deleting parts of or all of gene sequences.

[0151] These methods are well-known in the art. ZFN, TALEN and CRISPR / Cas-based methods for gene editing are described in Govindan and Ramalingam, J Cell Physiol, 9999: 1-13 (2016); Gaj et al., Trends in Biotechnology, 31(7): 397-405 (2013); Sander and Young, Nature Biotechnology, 32: 347-355 (2014); Hu et al., Cell Chem Biol, 23(l):57-73 (2016). Multiple genes may be modified at the same time. Alternatively, multiple genes may be modified sequentially.

[0152] The nucleases may be delivered to cells in any manner. For instance, the nucleases may be delivered as ribonucleoproteins (RNPs), as messenger RNA (mRNA), in viral delivery systems, such as adeno-associated viruses (AAV) or lentiviruses, or as part of a plasmid.

[0153] Nanoparticle delivery systems may be used to transfect the cell with the one or more constructs or nucleases. Such delivery systems include, but are not limited to, electroporation, lipid-based systems, liposomes, micelles, microvesicles, exosomes, and gene gun. With regards to nanoparticles that can deliver RNA, see, e.g., Alabi et al., Proc Natl Acad Sci U S A. 2013 Aug 6;110(32): 12881-6; Zhang et al., Adv Mater. 2013 Sep 6;25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar 13;13(3): 1059-64; Karagiannis et al., ACS Nano. 2012 Oct 23;6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug 28;6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun 3;7(6):389-93. Lipid Nanoparticles, Spherical Nucleic Acid (SNA™) constructs, nanoplexes and other nanoparticles (particularly gold nanoparticles) are also contemplated as a means for delivery of a construct or vector in accordance with the invention.

[0154] The cells are typically genetically engineered to express a marker or factor which ensures that only successfully genetically engineered cells remain viable. Thus, any non-genetically engineered cells are eliminated, thereby improving the purity of the resulting genetically engineered population.

[0155] Method of genetic engineering

[0156] The invention also provides a method of producing a genetically engineered population of the invention. The genetically engineered population may be any of those described above. This method is referred to herein as "a / the genetic engineering method" or"a / the genetic engineering method of the invention".

[0157] The genetic engineering method comprises steps (i) and (ii).

[0158] Step (i)

[0159] Step (i) comprises genetically engineering haematopoietic progenitor cells to express or overexpress one or more activating receptors and / or to reduce expression of one or more inhibitory receptors. Any of the embodiments discussed above with reference to the genetically engineered population of the invention apply to this step. The haematopoietic progenitor cells are preferably genetically engineered using a lentivirus to express or overexpress one or more activating receptors, such as CD16. The haematopoietic progenitor cells are preferably genetically engineered using CRISPR, such as CRISPR / Cas9, to reduce expression of or under express one or more inhibitory receptors, such as Siglec 9 (CD329).

[0160] The haematopoietic progenitor cells may be any of those described above, including stem cells, haematopoietic stem cells, peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cell (iPSCs), or an immortalized cell line. The haematopoietic progenitor cells are preferably CD34+. The haematopoietic progenitor cells are preferably derived from one or more donors. The haematopoietic progenitor cells are preferably isolated from one or more apheresis cones. The haematopoietic progenitor cells are more preferably isolated from two or more donors or two or more apheresis cones from two or more donors. Any of the embodiments discussed above with reference to the method of the invention equally apply to this step.

[0161] Step (ii)

[0162] Step (ii) comprises culturing the genetically engineered haematopoietic progenitor cells under conditions which promote their differentiation to immature neutrophils. Any method may be used to do this, including the methods described in Kuhikar et al., Stem Cell Research, 2021. 50: p. 102150 and Cordero et al., bioRxiv 2023.06.17.545406. Step (ii) preferably comprises conducting a method of the invention. Any of the embodiments of the method of the invention discussed above apply to step (ii).

[0163] Cryopreserved and thawed populations

[0164] The invention also provides a cryopreserved population of immature neutrophils. This population is referred to herein as "a / the cryopreserved population" or "a / the cryopreserved population of the invention".

[0165] The cryopreserved population may comprise a population of the invention or a genetically modified population of the invention. Any of the embodiments discussed above equally apply to the cryopreserved population of the invention.

[0166] The invention also provides a thawed population of immature neutrophils. This population is referred to herein as "a / the thawed population" or "a / the thawed population of the invention".

[0167] The thawed population may comprise a population of the invention or a genetically modified population of the invention. Any of the embodiments discussed above equally apply to the thawed population of the invention.

[0168] As shown in the Examples (and especially Figure 15), the population of the invention retains its ability to produce ROS post thawing.

[0169] The cryopreserved population may comprise a cryopreservation medium. Suitable cryopreservation media are known in the art. The cryopreservation medium is preferably CryoStor®CS10 (#07930, STEMCELLTM TECHNOLOGIES) or STEM-CELL BANKER® GMP Grade (ZENOGEN PHARMA).

[0170] The cryopreserved population may be cryopreserved according to known protocols, including the standard protocols used for the cryopreservation media discussed above. For instance, the population of the invention or the genetically engineered population of the invention may be incubated at about 4°C for about 10 minutes and then cryopreserved using a standard slow rate-controlled cooling using Nalgene® Mr. Frosty. Alternatively, the population of the invention or the genetically engineered population of the invention may be directly placed in about -80°C.

[0171] The thawed population may comprise a buffer or culture medium comprising the components of a cryopreservation medium. Suitable cryopreservation media are discussed above. A cryopreserved population may be thawed using standard protocols. The cryopreserved population may be thawed and diluted in a suitable buffer or culture medium. The buffer may be any of the pharmaceutically or physiologically acceptable diluents and / or carriers discussed below in relation to the pharmaceutical composition of the invention.

[0172] For instance, the cryopreserved population may be thawed in a 37°C water bath by gently shaking and removed from the bath when only a small ice crystal remains. The thawed population may then be diluted 1 in 10 by adding warm Hanks' Balanced Salt Solution (HBSS -Ca -Mg) in small increments (50 pL - 1 mL) and centrifuged gently at 300 g for 10 min. After the centrifugation, the cell pellet may be gently resuspended in warm HBSS.

[0173] The cryopreserved or thawed population may comprise any number of cells, such as any of the numbers of cells discussed above. The cryopreserved or thawed population may be isolated, substantially isolated, purified, or substantially purified as described above.

[0174] Pharmaceutical compositions

[0175] Also provided is a pharmaceutical composition comprising a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention or a thawed population of the invention and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0176] The carrier and / or diluent is generally selected to be suitable for the intended mode of administration and can include agents for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Typically, these carriers and / or diluents include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and / or buffered media.

[0177] Suitable further agents for inclusion in the pharmaceutical composition include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulphite, or sodium hydrogensulphite), buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as free serum albumin, gelatin, or immunoglobulins), colouring, flavouring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides, such as sodium or potassium chloride, or mannitol sorbitol), delivery vehicles, excipients and / or pharmaceutical adjuvants.

[0178] The carrier and / or diluent may be a parenteral, optionally intravenous vehicle. Suitable parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically acceptable thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates may be included. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. In some cases, one might include agents to adjust tonicity of the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in a pharmaceutical composition. For example, in many cases it is desirable that the composition is substantially isotonic. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present. The precise formulation will depend on the route of administration. Additional relevant principle, methods and components for pharmaceutical formulations are well known (see, e.g., Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22ndEdition).

[0179] A pharmaceutical composition of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled person, the route and / or mode of administration will vary depending upon the desired results. Routes of administration for pharmaceutical compositions of the invention include intravenous, intramuscular, intradermal, intraperitoneal, intrapleural, subcutaneous, intratumoural, spinal, intra-bone marrow or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumoural, intrapleural and intra- sternal injection and infusion. In some embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, administration is intrapleural or intraperitoneal. When parenteral administration is contemplated, the pharmaceutical compositions are usually in the form of a sterile, pyrogen-free, parenterally acceptable composition. A particularly suitable vehicle for parenteral injection is a sterile, isotonic solution, properly preserved. The pharmaceutical composition can be in the form of a lyophilizate, such as a lyophilized cake. Alternatively, the pharmaceutical composition of the invention can be administered by a nonparenteral route, such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0180] In some embodiments, the pharmaceutical composition is for subcutaneous administration. Typically, the pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (e.g., amino acids, such as methionine, and or saccharides such as sucrose), buffering agents and tonicifying agents. Alternatively, the pharmaceutical composition may be for intra-bone marrow administration.

[0181] Therapy

[0182] The invention also provides a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention for use in therapy. The invention also provides use of a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for use in therapy. The invention also provides a method of treating a subject in need thereof, comprising administering a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention to the subject.

[0183] The therapy may relate to improving the outcome of bone marrow transplant. The invention also provides a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention for use in treating or preventing an infection or cancer improving the outcome of bone marrow transplant. The invention also provides use of a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for improving the outcome of bone marrow transplant. The invention also provides a method of improving the outcome of bone marrow transplant in a subject, comprising administering a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention to the subject. The product of the invention may be administered before, during or after the bone marrow transplant.

[0184] The therapy may relate to treating or preventing an infection or cancer. The invention also provides a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention for use in treating or preventing an infection or cancer. The invention also provides use of a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating or preventing an infection or cancer. The invention also provides a method of treating or preventing an infection or cancer in a subject, comprising administering a population of the invention, a genetically engineered population of the invention, a cryopreserved population of the invention, a thawed population of the invention or a pharmaceutical composition of the invention to the subject.

[0185] The infection may be caused by any pathogenic agent. The pathogenic agent may be a bacterium, an archaeon, a fungus, or a virus. The infection is preferably antibiotic or antimycotic resistant.

[0186] The bacterium may be Gram negative or Gram positive. The Gram-positive bacterium is preferably from the genus Bacillus, Clostridium, Enterococcus, Mycobacterium, Staphylococcus or Streptococcus. The Gram-positive bacterium may be from the genus Pasteurella or Nocardia.

[0187] The Gram-negative bacterium is preferably from the genus Aggregatibacter, Bacteroides, Bartonella, Brucella, Campylobacter, Chylamidia, Enterbacter, Francisella, Haemophilus, Heliobacter, Klebsiella, Legionella, Moraxella, Neisseria, Porphyromonas, Pseudomonas, Salmonella, Serratia, Stenotrophomonas, Vibrio or Yersinia. The Gram-negative bacterium is preferably from the genus Escherichia or Pseudomonas.

[0188] The bacterium may be from the genus Borrelia, Chlamydophila, Listeria, Mycoplasma, Proteus, or Treponema. The bacterium is preferably Aggregatibacter actinomycetemcomitans, Bacillus anthracis, Bacillus licheniformis, Bacteroides fragilis, Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Campylobacter jejuni, Chlamydia trachomatis, Chlamydophila pneumoniae, Clostridium difficile, Clostridium perfringens, Enterobacter aerogenes, Enterococcus faecalis, Enterococcus faecium, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Legionella pneumophila, Listeria monocytogenes, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycoplasma genitalium, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Porphyromonas gingivalis, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enter ica, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Stenotrophomonas maltophilia, Streptococcus mutans, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Treponema pallidum, Vibrio cholera, Vibrio parahaemolyticus or Yersinia enterocolitica. Other specific examples of bacteria include, but are not limited, to Mycobacterium tuberculosis, Mycobacterium intracellilare, Mycobacterium kansaii, Mycobacterium gordonae, Streptococcus agalactiae, Streptococcus viridans group, Streptococcus faecalis, Streptococcus bovis, Streptococcus pneumoniae, Corynebacterium diptheriae, Erysipelothrix rhusiopathie, Clostridium tetani, Klebsiella pneumoniae, Pasteurella multocida, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pertenue and Actinomyces israelii.

[0189] The bacterium is preferably Escherichia coli, Pseudomonas aeroginosa, or Staphylococcus aureus.

[0190] The fungus is preferably from the genus Absidia, Acremonium, Aspergillus, Aureobasidium, Basidiobolus, Blastomyces, Blastoschizomyces, Candida, Cladosporium, Coccidioides, Cryptococcus, Cunninghamella, Curvularia, Debaryomyces, Exophiala, Exserohilum, Fonsecea, Fusarium, Geotrichum, Histoplasma, Issatchenkia, Kluyveromyces, Malezzesia, Mucor, Paracoccidioides, Paecilomyces, Penicillium, Pichia, Pneumocystis, Rhizomucor, Rhizopus, Rhodotorula, Saccharomyces, Scedosporium, Schizophyllum, Scopulariopsis, Sporothrix, Trichoderma, Trichophyton or Trichosporon. The fungus is preferably Aspergillus fumigatus, Aspergillus flavus, Aspergillus lentulus, Aspergillus terreus, Aspergillus nidulans, Aspergillus oryzae, Aspergillus niger, Candida albicans, Candida caribbica ( Candida fermentati), Candida dubliniensis, Candida famata (Debaryomyces hansenii), Candida fukuyamaensis (Candida xestobii or Candida carpophila), Candida guilliermondii, Candida kefyr (Kluyveromyces marxianus), Candida krusei (Issatchenkia orientalis), Candida metapsilosis, Candida orthopsilosis, Candida parapsilosis, Candida parapsilosis, Candida pelliculosa, Candida psychrophila, Candida rugosa, Candida smithsonii, Candida tropicalis, Candida utilis, Coccidioides immitis , Cryptococcus bacillisporus, Cryptococcus gattii, Cryptococcus grubii, Cryptococcus neoformans, Debaryomyces coudertii, Debaryomyces maramus, Debaryomyces nepalensis, Debaryomyces prosopidis, Debaryomyces robertsiae, Debaryomyces udenii, Histoplasma capsulatum, Kluyveromyces lactis, Pichia cecembensis, Rhodotorula araucariae, Rhodotorula babjevae, Rhodotorula dairensis, Rhodotorula diobovatum, Rhodotorula glutinis, Rhodotorula kratochvilovae, Rhodotorula paludigenum, Rhodotorula sphaerocarpum, Rhodotorula toruloides, Rhodotorula mucliaginosa, Saccharomyces 'sensu stricto', Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces cariocanus, Saccharomyces kudiavzevii, Saccharomyces mikatae, Saccharomyces paradioxus, Saccharomyces pastorianus, Saccharomyces uvarum, Saccharomyces cerevisiae or Tsuchiyaea wingfieldii.

[0191] The virus may belong to the family Retroviridae, such as human deficiency viruses, such as HIV-I (also referred to as HTLV- III), HIV-II, LAC, IDLV-III / LAV, HIV-III or other isolates such as HIV-LP, the family Picornaviridae, such as poliovirus, hepatitis A, enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses, the family Calciviridae, such as viruses that cause gastroenteritis, the family Togaviridae, such as equine encephalitis viruses and rubella viruses, the family Flaviviridae, such as dengue viruses, encephalitis viruses and yellow fever viruses, the family Coronaviridae, such as coronaviruses, including SARS-Cov-2 (COVID-19), the family Rhabdoviridae, such as vesicular stomata viruses and rabies viruses, the family Filoviridae, such as Ebola viruses, the family Paramyxoviridae, such as parainfluenza viruses, mumps viruses, measles virus and respiratory syncytial virus, the family Orthomyxoviridae, such as influenza viruses, the family Bungaviridae, such as Hataan viruses, bunga viruses, phleoboviruses and Nairo viruses, the family Arena viridae, such as hemorrhagic fever viruses, the family Reoviridae, such as reoviruses, orbiviruses and rotaviruses, the family Bimaviridae, the family Hepadnaviridae, such as hepatitis B virus, the family Parvoviridae, such as parvoviruses, the Papovaviridae, such as papilloma viruses and polyoma viruses, the family Adenoviridae, such as adenoviruses, the family Herpesviridae, such as herpes simplex virus (HSV) I and II, varicella zoster virus and pox viruses, or the family Iridoviridae, such as African swine fever virus). The virus may be an unclassified virus, such as the etiologic agents of Spongiform encephalopathies, the agent of delta hepatitis, the agents of non-A, non-B hepatitis (class 1 enterally transmitted; class 2 parenterally transmitted such as Hepatitis C), Norwalk and related viruses and astroviruses.

[0192] Preferably, the cancer is anal cancer, bile duct cancer (cholangiocarcinoma), bladder cancer, blood cancer, bone cancer, bowel cancer, brain tumours, breast cancer, colorectal cancer, cervical cancer, endocrine tumours, eye cancer (such as ocular melanoma), fallopian tube cancer, gall bladder cancer, head and / or neck cancer, Kaposi's sarcoma, kidney cancer, larynx cancer, leukaemia, liver cancer, lung cancer, lymph node cancer, lymphoma, melanoma, mesothelioma, myeloma, neuroendocrine tumours, ovarian cancer, oesophageal cancer, pancreatic cancer, penis cancer, primary peritoneal cancer, prostate cancer, Pseudomyxoma peritonei, skin cancer, small bowel cancer, soft tissue sarcoma, spinal cord tumours, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, trachea cancer, unknown primary cancer, vagina cancer, vulva cancer or endometrial cancer. The leukaemia is preferably acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic lymphocytic leukaemia, or chronic myeloid leukaemia. The lymphoma is preferably Hodgkin lymphoma or non-Hodgkin lymphoma. The cancer is preferably primary cancer or secondary cancer.

[0193] The administration route may be any of those discussed above. In preferred embodiments, administration is conducted by intravenous injection.

[0194] The therapy or method typically comprises administering a therapeutically effective amount or a prophylactically effective amount of the cells. A therapeutically effective amount is an amount which ameliorates one or more symptoms, such as all the symptoms, of the disease or disorder and / or abolishes one or more symptoms, such as all the symptoms, of the disease or disorder. The therapeutically effective amount preferably cures the disease or disorder. A prophylactically effective amount is an amount which prevents the onset of the disease or disorder and / or prevents the onset of one or more symptoms, such as all the symptoms, of the disease or disorder. The prophylactically effective amount preferably prevents the subject from developing the disease or disorder. Suitable amounts are discussed in more detail below.

[0195] Any of the populations or the pharmaceutical composition may be administered to a subject that displays symptoms of disease or disorder. Any of the populations or the pharmaceutical composition may be administered to a subject that is asymptomatic, i.e., does not display symptoms of disease or disorder. Any of the populations or the pharmaceutical composition may be administered when the subject's disease status is unknown, or the subject is expected not to have a disease or disorder. Any of the populations or the pharmaceutical composition may be administered to a subject that is predisposed, such as genetically predisposed, to developing the disease or disorder.

[0196] Administration of any of the populations or the pharmaceutical composition to the subject may reduce symptoms by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%, when compared to an untreated subject.

[0197] The subject may be a mammal. Optionally, the subject is a human, horse, dog, or cat. The subject is preferably human.

[0198] In embodiments where the subject is human, the subject may be a human adult or child. In the context of the present invention, an adult will be understood to be an at least 18-year- old human. A child will be understood to be a human less than 18 years old. In some embodiments, the adult is at least 60 years old.

[0199] The number of cells administered to the subject should take into account the route of administration, the disease or disorder being treated, the weight of the subject and / or the age of the subject. In general, from about 1 x 106to about 1 x 1011cells are administered to the subject. In one embodiment, from about 1 x 107to about 1 x 1010cells, or from about 1 x 108to about 1 x 109cells are administered to the subject.

[0200] In some embodiments, from about 1 mg / ml to about 100 mg / ml of any of the populations or the pharmaceutical composition may be administered to the subject. In some embodiments, from about 1 mg / ml to about 10 mg / ml of any of the populations or the pharmaceutical composition are administered to the subject. The invention may be used in combination with other means of, and substances for, treating disease or disorder. In some cases, any of the populations or the pharmaceutical composition may be administered simultaneously, sequentially, or separately with other substances which are intended for treating the disease or disorder or ameliorating the symptoms of the disease or disorder, or for providing pain relief. Any of the populations or the pharmaceutical composition may be used in combination with existing treatments for disease or disorder and may, for example, be simply mixed with such treatments. Thus the invention may be used to increase the efficacy of existing treatments for disease.

[0201] In some embodiments, the cells can be autologous with respect to the subject. For example, haematopoietic progenitor cells can be removed from the subject, differentiated to immature neutrophils and returned to the subject. Preferably, however, the cells are allogeneic with respect to the subject. In this embodiment, the cells may be matched to the subject.

[0202] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples, and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0203] EXAMPLES

[0204] Example 1 - Production and analysis of immature neutrophils

[0205] To address the knowledge gap in neutrophil development, we optimised an ex vivo protocol for differentiation of neutrophils from human hematopoietic stem and progenitor cells (HSPCs). Previous culture protocols have described neutrophil differentiation from diverse sources such as embryonic stem cells, induced pluripotent stem cells as well as bone marrow and peripheral blood HSPCs [30-41]; these studies report that cultured neutrophils resemble native neutrophils in nuclear morphology, surface marker expression and some neutrophil effector functions such as ROS production, bacterial killing, phagocytosis and chemotaxis. Based on these reports, we optimised the culture conditions to obtain a high yield of CD34+HSPC-derived neutrophils. We show that cultured neutrophils more closely resemble immature neutrophils than steady state circulating neutrophils, and that they are amenable to genome editing, enabling mechanistic studies of immature neutrophil function.

[0206] Materials and methods

[0207] CD34+stem cell isolation Peripheral blood mononuclear cells (PBMCs) were isolated from apheresis waste products (NHSBT, Filton, Bristol, UK) with ethical approval from NHS Research Ethics committee (REC 18 / EE / 0265). PBMCs were isolated by density centrifugation using Histopaque 1077 (Sigma Aldrich) as previously described [58, 59]. 8-10mL of blood from apheresis were mixed with 60pL citrate-dextrose solution (ACD, Sigma Aldrich). lOmL of Hanks' balanced salt solution (HBSS, Lonza) was added to samples and the mixture was layered over 25mL of room temperature (RT) Histopaque 1077 (Sigma Aldrich) then centrifuged at 400xg at RT for 35 minutes with no brake. The interface layer that contains PBMCs was collected and washed five times with HBSS supplemented with ACD (0.6% v / v, Sigma Aldrich). Finally, cells were resuspended in lOmL red cell lysis buffer (55mM NH4CI, 0.137mM EDTA, ImM KHCO3, pH 7.5 in water) and washed once with HBSS supplemented with ACD (0.6% v / v, Sigma Aldrich). CD34+cells were isolated from PBMCs using a Human CD34 MicroBead Kit (Miltenyi Biotec) and LS columns (Miltenyi Biotec) as outlined in the manufacturer's instructions.

[0208] Neutrophil differentiation

[0209] CD34+cells were cultured in IMDM (Biochrom, Source Biosciences, Cambridge UK) supplemented with 10% (v / v) fetal calf serum (FCS, Life Technologies) and 1% (v / v) penicillin / streptomycin (P / S, Sigma Aldrich), at 37°C in 5% CO2. Stem Cell Factor (SCF, 50ng / mL, Miltenyi Biotech), Flt-3 Ligand (50ng / mL, Miltenyi Biotech), Interleukin-3 (IL-3, lOng / mL, R&D Systems), GM-CSF (lOng / mL, Miltenyi Biotech) G-CSF (lOng / mL, Miltenyi Biotech) were introduced at the following times post CD34+isolation: IL-3, SCF and Flt3-L from day 0-3; GM-CSF, IL-3, SCF and Flt3-L from day 3-7; GM-CSF and G-CSF from day 7- 10 and G-CSF only from day 10-17. CD34+cells were initially plated at 0.1-0.2 x 106cells / mL on Day 0. A full media change was completed at Day 3 post CD34+isolation, after which point cultures were supplemented with additional media every 2-3 days to maintain a cell density of 0.5 x 106cells / mL. Unless stated otherwise all functional assays were completed between Day 17 and Day 19 of culture.

[0210] Native neutrophil isolation from peripheral blood

[0211] Blood samples from consented healthy donors at the University of Bristol were collected with ethical approval from NHS Research Ethics committee (REC 18 / EE / 0265), into EDTA tubes. Neutrophils were isolated using the EasySep™ direct human neutrophil isolation kit (STEMCELL Technologies) as per the manufacturer's instructions.

[0212] Multi-fluorophore flow cytometry analysis

[0213] Flow cytometry analysis was conducted using 5 x 105Day 17-19 cultured or GCSF-D neutrophils as previously described

[0020] . Briefly, samples were washed with PBS, resuspended in 0.1% Zombie Aqua live / dead stain in PBS (BioLegend) and incubated for 10 minutes in the dark at RT. Samples were then incubated in FC Block (BioLegend) diluted in flow buffer (5mM ETDA and 0.5% bovine serum albumin (BSA) in PBS) on ice for 5 minutes, after which a master mix of primary antibodies was added and incubated for 30 minutes on ice protected from light (at the concentrations indicated in Supplemetary Table S2).

[0214] Samples were washed twice with flow buffer and fixed using 2-4% paraformaldehyde for 20 minutes at RT. Cells were analysed using a BD X20 Fortessa flow cytometer within 7 days of sample preparation. Appropriate single fluorescence colour compensation controls were conducted in parallel using Invitrogen OneComp eBeads (Thermo Fisher Scientific). At least 10,000 events were recorded per sample, gated on a singlet, live population and data were processed in FlowJo software (version 9).

[0215] S. aureus killing assay

[0216] 2.5 x 106native neutrophils or Day 18 cultured neutrophils were resuspended in 500pL HBSS (Lonza) and combined with 5 x 105S. aureus JE2 strain bacteria in HBSS supplemented with 2mM CaCL2, 2mM MgCL2and 10% pooled human serum (Seqens, 21000P). Samples were incubated at 37°C with atmospheric CO2levels on a rotator. 50pL of each sample was taken immediately and thereafter every hour for 4 hours and streaked on agar plates. Agar plates were then incubated at 37°C overnight and then scored for colonies the following day. Killing efficiency was calculated as (number of colonies with neutrophils I number of colonies with serum only).

[0217] C. albicans killing assay

[0218] C. albicans CaSSl strain was grown overnight in Yeast Extract-Peptone-Dextrose (YPD) medium at 30°C, 200 RPM in 5% CO2for 16 hours. The next day, C. albicans concentration was determined by optical density, and yeast were sub-cultured in YPD with 0.05 pg / ml doxycycline for 3 hours at 30°C, 200 RPM in 5% CO2. Yeast were resuspended in RPMI-1640 and opsonised with 5% pooled human serum for 30 minutes before incubating with native or cultured neutrophils at a MOI of 2.5 or 5 at 37°C for 2.5 hours. Samples were treated with 0.1% Triton X-100 (Sigma Aldrich) to lyse neutrophils, washed three times with PBS, incubated with alamarBlueTM (Thermo Fisher Scientific) for 17 hours and fluorescence was measured using a FLUOstar Omega Microplate Reader (BMG Labtech) to quantify metabolically active C. albicans.

[0219] C. albicans NET assay

[0220] C. albicans CaSSl strain was grown overnight in YPD medium at 30°C, 200 RPM in 5% CO2for 16 hours. The next day, C. albicans concentration was determined by optical density, sub-cultured in YPD for 3 hours at 30°C, 200 RPM in 5% CO2. Fungi were resuspended in RPMI-1640 and opsonised with 10% pooled human serum for 30 minutes. Fungi were pelleted and plated at 0.5 x 106in a 24 well plate in 1ml in NETs media (RPMU640 supplemented with 0.025% HSA and lOmM HEPES). Fungi were incubated at 37 °C until pseudo hyphae formed. 1 x 105native or cultured neutrophils were added per well and incubated for 4 hours. Cells were stained with IpM of SYTO green to label all neutrophils and IpM SYTOX orange for NETs (Thermo Fisher Scientific) and imaged using an EVOS® FL Cell Imaging System (Thermo Fisher Scientific). NETs were quantified on ImageJ Fiji software.

[0221] Cytokine release

[0222] 1 x 105neutrophils were plated in 200pl in triplicate in RPMI-1640 with phenol red (GIBCO), 10% fetal bovine serum (FBS, Sigma) and 1% P / S (Biochrom) and stimulated with lOOng / mL bacterial lipopolysaccharide (LPS from E. coli O127:B8, Sigma Aldrich) or 5 pM resiquimod (Sigma Aldrich) overnight at 37° C in 5% CO2. IL-8 and IL-6 levels in the resulting supernatants were measured using Human IL8 / CXCL8 DuoSet ELISA and Human IL-6 DuoSet kits following the manufacturers protocol (both R&D Systems).

[0223] Zymosan degranulation assay

[0224] 1 x 105neutrophils were plated in duplicate, in 200pl RPMI-1640 (GIBCO) supplemented with 0.025% human serum albumin (HSA; Sigma-Aldrich) and lOmM HEPES (Sigma- Aldrich) and were stimulated with 25pg / mL opsonised Zymosan (Sigma Aldrich). After one hour, samples were centrifuged at 300xg, lOOpL of supernatant was removed from each well and the NE concentration measured using a human Neutrophil Elastase / ELA2 DuoSet ELISA kit (R&D Systems) as per the manufacturer's protocol.

[0225] APF reactive oxygen species production

[0226] 1 x 105neutrophils were plated in lOOul of ROS media (HBSS with lOmM HEPES and 0.025% HSA, both Sigma Aldrich) supplemented with lOpM APF (Thermo Fisher) in black, clear, flat-bottomed plates. Cells were incubated at 37°C, 5% CO2 for 45 minutes, centrifuged at 400xg for 5 minutes and media was aspirated and replaced with plain ROS media. Neutrophils were stimulated with lOOnM PMA (Sigma Aldrich), and fluorescence measured every 2.5 minutes for 4 hours using a BMG FLUOstar plate reader (emission: 490nm, excitation: 515nm).

[0227] TMT mass spectrometry

[0228] Day 18 cultured neutrophils and native neutrophils were isolated or cultured from 3 separate donors as described above. Sample pairs were donor-matched, with CD34+cells and native neutrophils isolated from the same fresh apheresis cone. Neutrophils were sorted using CD66b expression using a BD Influx Cell Sorter (BD Biosciences). Cells were immediately pelleted and lysed in supplemented RIPA buffer (EDTA 10 mM, 50 mmol / l TCEP (Sigma Aldrich), 2mM PMSF protease inhibitor, 1 / 50 v / v Protease Inhibitor Cocktail Set V (Calbiochem) and flash-frozen in liquid nitrogen for storage until later analysis. All samples were then thawed on ice, sonicated to fragment DNA, and measured for protein concentration with a Pierce® BCA Protein Assay Kit (Thermo Scientific, cat no 23227) according to the manufacturer's instructions. Due to TCEP being used during cell lysis, a reducing agent-compatible kit was used. 100 pg of each sample were then digested with trypsin and labelled with TMT reagents according to the manufacturer's protocol (Thermo Fisher Scientific). The resulting peptides were identified by nano LCMS / MS with a Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific). Raw files were analysed using Proteome Discoverer software v. 2 and cross-referenced against the human UniProt database (human). PD analysis was conducted for full trypsin digestion, removing all hits with more than one missed cleavage. All peptides were filtered to meet an FDR of 1%. Log? fold changes (Log2FC) were calculated between cultured and native neutrophils to identify differentially expressed proteins (|Log2FC| > 1, P value < 0.05). Log2FC volcano plots were generated using Microsoft Excel. Log?FC clustering analysis and the resulting heatmap visualisations were generated using R v. 4.2.2. Protein-protein interaction networks for differentially expressed proteins were generated using the STRING database and Cytoscape v. 3.9.1.

[0229] CRISPR / Cas9 gene editing

[0230] Nucleofection of ribonucleoproteins (RNP) was completed using the Nucleofector 4D (Lonza) using a P3 Primary Cell 4D-NucleofectorTM X Kit S (Lonza) and Invitrogen™ TrueCut™ Cas9 Protein v2 (Thermo Fisher Scientific) following the manufacturer's recommended protocols. CD34+cells were isolated and cultured as described above until Day 3 of culture. 0.3 x 106cells were transduced per reaction. 50 pmol of Cas9 was mixed with 125 pmol of gRNA (62.5pmol of two guides with the same gene target or a scrambled (SCR) gRNA control) per reaction and incubated at 25°C for 15 minutes to form RNPs, which were stored on ice for up to 4 hours. All guides were designed using the Synthego CRISPR Design Tool and produced by Synthego (Redwood City, USA). Supplemented Nucleofector Solution (SNS) was made up fresh at a 4.5: 1 ratio of nucleofector solution to supplement. 0.3 x 106Day 3 cells were spun down, washed in PBS, and resuspended in 20pL SNS. Cells were added to RNPs, mixed gently, and transferred to a nucleocuvette cassette. Cells were then electroporated using the manufacturers recommended Nucleofector 4D program EO-100. 80pL of prewarmed 37°C StemSpan was dripped gently into each reaction to dilute the SNS. Cells were replated in 2mL of StemSpan (Stem Cell Technologies) supplemented with 1% (v / v) P / S (Sigma Aldrich), SCF (50ng / mL, Miltenyi Biotech), Flt3-L (50ng / mL, Miltenyi Biotech), IL-3 (lOng / mL, R&D Systems), GM-CSF (lOng / mL, Miltenyi Biotech) and GCSF (lOng / mL, Miltenyi Biotech). Cells were allowed to recover for 48 hours, after which they were cultured from Day 5 as indicated in the neutrophil culture methodology section (Fig. 1A).

[0231] Seahorse metabolic flux analysis

[0232] Seahorse XFe 96 calibration plates were hydrated in 200ul culture grade water in a non-CO? incubator overnight. Water was replaced with pre-warmed XF calibrant and incubated in a non- CO2 incubator for at least 45 minutes prior to calibration. 4 x 105cells were plated in 180pl Seahorse media (Seahorse XF DMEM medium, 5mM glucose, 2mM glutamine). 20pl of 10X oligomycin (final concentration 1.5pM) was added to port A, 22.2ul of FCCP (final concentration 500nM) in port B and 24.7ul of AA / RO into port C (final concentration IpM). The injection plate was overlayed on the calibration plate and inserted into the Seahorse XFe 96 analyser for calibration. The culture plate with seeded neutrophils was later exchanged after calibration. 3 basal reads were acquired before the first injection and reads were obtained every 6 minutes thereafter.

[0233] Statistical analysis

[0234] Data was organised and analysed using GraphPad Prism 8 software. Mean ± SD is plotted for a minimum of n=3 differentiations unless otherwise stated. Statistical analysis was completed where appropriate using student's t test when comparing two different samples, or one-way ANOVA where multiple samples were being examined, with asterisks on the graph represent the following: *P < 0.05, **P < 0.01, and ***P < 0.001.

[0235] Cytospin preparation and imaging

[0236] 1 x 105cells were removed from cultures at the indicated timepoints post CD34+isolation. Cells were washed with PBS and spun onto glass slides at lOOOxg for 5 minutes (Thermo Scientific Cytospin). Samples were then fixed in 100% methanol for 10 minutes and then stained with May Gruwald-Giesma stains as per the manufacturer's instructions (Merck).

[0237] Four fluorophore flow cytometry analysis

[0238] 1 x 105cells were removed on day 8 of cell culture and labelled with conjugated antibodies for 25 minutes at 4°C, using commercial nonspecific IgG controls for nonspecific staining. Propidium iodide labelling was used to identify the dead cell population. Samples were analysed using a MacsQuant flow cytometer (Miltenyi Biotec) and processed using FlowJo software (Version 9).

[0239] Table 1: Proteins uniquely expressed in cultured neutrophils and not native neutrophils.

[0240]

[0241] Table 2: Antibodies and fluorescent dyes used for flow cytometry

[0242] Table 3: Synthego guide RIMA for CRISPR-Cas9 gene editing

[0243] Results Optimisation of a neutrophil culture and differentiation protocol from haematopoietic stem cells We isolated CD34+HSPCs from apheresis cones using immunomagnetic selection and cultured these with multiple combinations of stem cell proliferation and expansion factors (stem cell factor (SCF), interleukin-3 (IL-3) and fms-like tyrosine kinase 3 ligand (Flt3-L)), followed by neutrophil differentiation factors (GM-CSF and G-CSF). We tested a range of reported differentiation conditions [30, 32, 34, 37, 40, 42-44] to generate our optimised differentiation protocol shown in Fig. 1A, which was selected based on superior yield, differentiation efficiency and viability of cells (Fig. 6A). This protocol resulted in an average 326 ± 248 fold expansion (n= 7-11, Fig. IB) with a gradual reduction and loss after day 5 of CD34 expression (Fig. 1C) and progressive increase after day 7 of granulocyte marker expression (CD66b and CDllb, Fig. ID and E respectively). Neutrophil nuclear lobulation and surface markers peaked at differentiation day 17 (Fig. 1A and C-E), yielding an average of 75.45% neutrophils co-expressing the markers CD66b and CD15 (n=4, Fig. 6B).

[0244] Cultured neutrophils phenotypically resemble GCSF-mobilised immature neutrophils

[0245] GCSF treated healthy donors (GCSF-D) are known to have a significant population of immature neutrophils circulating in peripheral blood

[0045] . We used flow cytometry to compare profiles of cultured, GCSF-D and steady state native neutrophils (gating strategy shown in Fig. 7A). GCSF-D and steady state native neutrophils had similar forward (FSC) and side scatter (SSC) (Fig. 2A-B), indicating similar size and granularity. Cultured neutrophils displayed similar FSC but reduced SSC indicating decreased granularity, possibly due to reduced abundance of cytoplasmic vesicles. As expected, GCSF-D neutrophils had significantly lower expression of maturity markers CD10 and CD101 compared to native neutrophils (Fig. 2C-E, Fig. 7B-C). Cultured neutrophils also had reduced abundance of maturity markers, indicating immature status. In contrast, we found similar levels of granulocyte markers in all three cell types (Fig. 2F-H), with a trend for increased CD66b in cultured neutrophils. In summary, based on their surface marker expression, neutrophils cultured from CD34+HSPCs phenocopy immature GSCF-D neutrophils.

[0246] Functional comparison of cultured and native neutrophils

[0247] Immature peripheral blood neutrophils are reported to have reduced oxidative burst, impaired capacity to kill C. albicans and increased cytokine production to toll-like receptor (TLR) agonists [27-29]. We compared effector responses in cultured neutrophils and steady state native neutrophils isolated from peripheral blood, to explore any possible functional differences. We observed no difference in total ROS production, in response to phorbol myristate acetate (PMA), a protein kinase C (PKC) agonist, when analysed by area under the curve (AUC) (Fig. 3A). The kinetic curve did however reveal subtle differences in the temporal response across donors (Fig. 3A), potentially indicating differences in in antioxidant response or NOX2 assembly. ROS detection with aminophenyl fluorescein (APF), a dye that detects peroxynitrites and MPO-catalysed hypochlorous acid, also showed comparable production of intracellular ROS (Fig. 3B). In summary, NOX2 and MPO activity are similar in cultured and native neutrophils.

[0248] Next, we measured the ability of cultured neutrophils to release NETs in response to the fungal pathogen C. albicans and the protein kinase C agonist phorbol-12-myristate-13- acetate (PMA). NETs were stained with SYTOX Orange dye, which labels extracellular DNA, while intact neutrophils were detected with SYTO Green. Cultured neutrophils did not engage in NET formation in response to PMA (Fig. 3C and D); however the response to C. albicans was equivalent to native neutrophils (Fig. 3C and E). These two stimuli are known to engage different NETosis pathways: while PMA is entirely NOX2-dependent, C. albicans is partially independent of the oxidase

[0046] . We therefore tested another NOX2-independent stimulus: the calcium ionophore A23187, which induced comparable levels of NETs in both native and cultured neutrophils (Fig 7D). In conclusion, cultured neutrophils can engage in NOX2-independent NETosis but are deficient in the NOX2-dependent pathway induced by PMA.

[0249] Next, we measured exocytosis of primary granules by quantifying neutrophil elastase (NE) release in response to stimulation with serum-opsonized zymosan (OZ), a fungal cell wall component. We observed a 27% reduction in extracellular NE release in stimulated cultured neutrophils compared to native ones (Fig. 3F), suggesting either reduced NE expression or decreased propensity to degranulate in response to OZ.

[0250] As reported for immature neutrophils from GCSF-D, production of proinflammatory cytokine interleukin-6 (IL-6) was significantly elevated in cultured neutrophils in response to both TLR4 agonist lipopolysaccharide (LPS) and TLR7 / 8 agonist resiquimod (R-848) (Fig. 3G). However, no difference in IL-8 production was detected in response to LPS (Fig. 3H).

[0251] Lastly, we measured the ability of cultured neutrophils to suppress proliferation of two important human pathogens. We observed that cultured neutrophils were able to kill C. albicans, however this ability was reduced compared to native neutrophils (approx. 50% reduction; Fig. 31). In contrast, cultured neutrophils were able to suppress growth of a clinical isolate of the gram-positive bacterium Staphylococcus aureus (S. aureus) at rates comparable to those observed with native neutrophils (Fig. 3J).

[0252] Cultured and native neutrophils have distinct proteomes

[0253] To investigate what underpins the functional differences described above, we analysed the proteomes of cultured and steady state native neutrophils, using tandem mass tag (TMT) mass spectrometry. Cultured and native neutrophils were obtained from the same donors, allowing for matched proteomic analysis. Prior to mass spectrometry, both native and cultured neutrophils were FACS sorted for CD66b+to eliminate contamination with precursors or other cell types (Fig. 4A, Fig. 8A). We applied a stringent false discovery rate (FDR) filtering step of < 1%, which led to identification of 2359 proteins. A large majority, 74% of detected proteins (1745 in total), were unchanged between cultured and native neutrophils (Fig. 4B). Enriched (red) and underrepresented (blue) proteins were defined by an absolute Iog2 fold change (Log2FC) of at least 1 and comparison p-value <0.05. Enriched and underrepresented proteins were altogether consistent among all donors as visualised by heatmap (Fig. 4B) and volcano plot (Fig. 4C) of all differentially expressed proteins. Despite the change in relative abundances of proteins, only 12 proteins were exclusively detected in cultured neutrophils and not in native cells (Table 1), although it remains unclear if these are functionally relevant.

[0254] To identify differentially regulated pathways, we conducted gene ontology (GO) term analysis on enriched and underrepresented protein sets (Figure 4D) with the entire human proteome set as the background. This identified 'cellular respiration' as the top enriched pathway in cultured cells, indicating an altered metabolic state. Similarly, both 'protein stabilisation' and 'nucleotide catabolic process' were among the top 6 enriched pathways, further suggesting that biosynthetic pathways are altered in cultured neutrophils, a finding consistent with the fact that enhanced mitochondrial respiration and biosynthesis are both more prominent in neutrophil precursors, compared to mature neutrophils

[0047] . On the other hand, multiple antimicrobial pathways were downregulated in cultured neutrophils, including 'antimicrobial humoral response', 'inflammatory response' and 'defence response to bacterium', potentially explaining the impaired fungal killing.

[0255] To further interrogate specific molecular pathways altered between cultured and native neutrophils, we also carried out Reactome pathway analysis. As with the GO analysis, the top enriched Reactome pathway in native neutrophils was 'TCA Cycle and respiratory electron transport' (Fig. 8B), again identifying an enrichment of mitochondrial proteins in immature cultured neutrophils. Moreover, we found that individual proteins important for the TCA cycle and respiratory electron transport were enriched (Fig. 8C). We confirmed enhanced mitochondrial activity in cultured neutrophils using the Seahorse metabolic flux analyser mitochondrial stress test, which showed increased basal oxygen consumption rate (OCR), spare respiratory capacity and mitochondrial ATP production (Fig. 4E-H). STRING analysis of the 225 overexpressed proteins also highlighted a cluster of diverse mitochondrial proteins (highlighted in blue, Fig. 8D). Mitochondrial respiration is a hallmark immature neutrophils circulating in inflammatory disease such as COVID-19 and cancer [20, 48].

[0256] Several granule proteins stood out as significantly reduced in cultured neutrophils, including matrix metalloprotease 9 (MMP9), cysteine rich secretory protein 3 (CRISP3), cyclic adenosine monophosphate (CAMP) (Fig. 4C and 41), supporting our findings on reduced SSC and reduced degranulation in cultured versus native cells. Reactome analysis also identified the 'innate immune response' and 'neutrophil degranulation' as the most underrepresented pathways in cultured neutrophils (Fig. 9B). Neutrophil degranulation was enriched in both overrepresented and underrepresented proteins, albeit more strongly in the underrepresented protein set (Fig. 8D and 8E), so we further investigated granule protein abundance. Indeed, comparison of the individual abundance of representative granule proteins highlighted significant reduction of core granule protein abundance, with the exception of MPO (Fig. 41). Collectively, these data argue for a significant perturbation of granule protein synthesis in cultured neutrophils, which is supportive of our suggestion that they have not reached complete maturity.

[0257] CRISPR / Cas9 genome editing of cultured neutrophils

[0258] Neutrophils are notoriously short lived and difficult to transfect, therefore the ability to culture them from CD34+stem cells offers a window of opportunity to explore gene editing

[0049] . We investigated whether cultured neutrophil precursors are amenable to genetic manipulation, prior to differentiation, as a tool for modifying gene expression in immature neutrophils. We targeted CD34+HSPCs (day 3 of culture) and used nucleofection to deliver ribonucleoproteins (RNPs) of Cas9 and guide RNA (gRNA). We used two different gRNAs, targeting p2microglobulin (p2M), a transmembrane protein expressed on all nucleated cells, and CDllb, an integrin expressed on myeloid cells. Nucleofection was efficient and did not require selection, with differentiated neutrophils demonstrating 92.5% and 88.1% loss of [32M (Fig. 5A) and CDllb (Fig. 5B) surface expression, respectively, at culture endpoint (day 17). We found no significant difference in viability of the gene-edited cells (Fig. 9A). Moreover, CD66b expression and therefore neutrophil differentiation was not affected (Fig. 9B), confirming that modification of precursors by CRISPR / Cas9 is well tolerated and can be used as a molecular tool to investigate immature neutrophils.

[0259] Discussion

[0260] Many basic questions in neutrophil biology remain unanswered, despite the importance of these cells in antimicrobial defence and inflammatory disease. This is in part because, neutrophil research is hampered by a lack of tools for genetic manipulation and by the short lifespan of neutrophils isolated from peripheral blood. One important outstanding question is the existence of neutrophil heterogeneity in disease conditions and whether neutrophils produced during inflammation differ from those produced at steady state. Studies using single cell RNA sequencing are providing compelling evidence for the presence of multiple neutrophil states

[0019] . Unsurprisingly for a cell with a short circulating lifespan (1-5 days in circulation) [1], neutrophil maturity is emerging as a major factor in determining phenotype and function. Building on previous published work in the field, we developed an improved method for ex vivo culture and genetic manipulation of neutrophils. Our optimised protocol uses CD34+stem cells isolated from waste apheresis cones, rather than the more restricted and costly embryonic or induced pluripotent stem cells, or HSPCs isolated from cord blood or bone marrow [31, 32, 37-40]. Compared to previous reports [30, 32, 34, 37, 40, 42-44], our protocol offers the combined advantages of 1) a shortened 17-day differentiation, 2) high levels of purity and 3) high yield. Most recently, Kuhikar et al. described neutrophil differentiation from apheresis cones, showing an average 72.4-fold expansion, with 57.37% of neutrophils expressing CD66b and 70.48% expressing CD15

[0050] . Our protocol achieves an average 326-fold expansion with 75.45% of neutrophils co-expressing CD66b and CD15. The yields obtained using this culture method are robust, we observed there was significant variation in yield between the apheresis blood donors. We currently do not know the reason for this but assume it is natural biological variation in the ability of donor CD34+stem cells to proliferate under our standardised conditions.

[0261] Quantification of expression of the well-established maturity markers CD10 and CD101 demonstrated that cultured neutrophils phenotypically resemble immature neutrophils mobilised by GCSF administration. Similarly to GCSF-D neutrophils

[0018] , cultured neutrophils demonstrated reduced C. albicans killing and overproduction of IL-6, compared to steady state native cells. The overproduction of IL-6 may be a potential pathogenic mechanism in diseases where immature neutrophils are implicated. Indeed, secretion of IL- 6 by immature neutrophils has been implicated in autoinflammatory diseases such as chronic graft versus host disease and adult-onset Still's disease [51, 52].

[0262] Cultured neutrophils produce an efficient NOX2 oxidative burst, as previously shown [32, 33, 36, 40, 44, 53], and have comparable levels of MPO activity to native cells. Interestingly MPO was enriched in the proteome of cultured cells, reflecting reports of elevated MPO expression in immature CD10" neutrophils circulating in myocardial infarction patients

[0054] .

[0263] Our proteomic dataset represents a useful resource for the field and reveals important developmental differences between native and cultured neutrophils. In contrast to MPO, we found many other granule proteins to be significantly reduced in the proteome of cultured neutrophils. This paucity of cytoplasmic granule proteins was observed for primary (NE), secondary (lactoferrin) and tertiary (MMP9 and CR.ISP3) granules, and may explain the defect in C. albicans killing. Degranulation has been the least studied antimicrobial response in cultured neutrophils, with only Dick et al. reporting reduced primary granule exocytosis in CD34+ex vivo differentiated versus native neutrophils

[0039] , in line with our finding of reduced NE release. It is likely that both findings are explained by perturbations in granule synthesis rather than reduced function of exocytosis machinery. The decrease in granule protein abundance and the maintenance of mitochondrial metabolism are both indicative of incomplete maturation of cultured neutrophils. This is reminiscent of in vitro erythroid culture from CD34+HSPCs, where the majority of differentiated cells are immature erythrocytes (reticulocytes), which subsequently mature in circulation [55, 56]. This also means that care is needed when exploring patient phenotypes with such culture models as they may not reproduce all aspects of a disease, in instances where disease consequences manifest in terminally differentiated neutrophils.

[0264] Ex vivo culture presents opportunities for genome editing that are not possible in neutrophils isolated from peripheral blood. Similar to previous knockout

[0049] and overexpression efforts

[0038] , we show that CRISPR / Cas9 can be used to modulate gene expression in cultured neutrophils. Nucleofection of ribonucleoprotein negates the need for using plasmid constructs, which we have found can impact neutrophil differentiation. This represents an important advance in the manipulation of cultured neutrophils that will facilitate moving away from use of imperfect immortalised neutrophil-like cell lines and mouse models, which do not fully recapitulate human neutrophil phenotypes and functions

[0057] . This technique also provides a model system for investigating the molecular bases of neutropenia and neutrophil immunodeficiencies caused by germline mutations.

[0265] Conclusion

[0266] Neutrophils produced using our described culture method are reminiscent of immature, bone marrow-mobilised neutrophils. Use of ex vivo differentiation from donor or patient CD34+cells, coupled with genome editing, are useful new tools for increasing our understanding of neutrophil development.

[0267] Example 2 - Further analysis of immature neutrophils

[0268] Immature neutrophils were produced and tested as described in Example 1. Marker expression was compared with immature, bone marrow-mobilised neutrophils (GCSF-D) and cultured / manufactured mature neutrophils. TNF was quantified by TNF DuoSet ELISA (R8iD Systems). The results are shown in Figures 10 and 11. Figure 10 defines the immature neutrophils of the invention.

[0269] Example 3 - Mitochondrial metabolism of the immature neutrophils

[0270] Immature neutrophils of the invention were produced as described in Examples 1 and 2.

[0271] Mitochondrial metabolism was measured in native mature neutrophils (Native) and cultured, immature neutrophils of the invention (Cultured), using Seahorse metabolic flux analyser. The results are shown in Figure 12 and demonstrate that immature cultured neutrophils have active mitochondria, which consume oxygen at basal (left panel) and challenged states (middle panel, oligomycin treatment to inhibit ATP synthase). Unlike mature native neutrophils, immature neutrophils generate ATP in mitochondria (right panel). Example 4 - Viability of the immature neutrophils

[0272] Immature neutrophils of the invention were produced as described in Examples 1 and 2.

[0273] Viability of native mature neutrophils (native) and cultured, immature neutrophils of the invention (cultured) measured using Annexin V staining to detect phosphatidylserine (apoptosis marker) at 0 hours and 48 hours. The results are shown in Figure 13 and demonstrate that cultured neutrophils have an extended lifespan compared to native neutrophils,

[0274] Example 5 - Enhancing yield by pooling CD34 cells from 2 apheresis cones

[0275] The proliferation rates of neutrophil progenitors were measured during the production method described in Example 1. Differentiation was induced on day 3 (D3) post isolation and committed neutrophil progenitors were enumerated on day 10 (DIO) of differentiation. The results are shown in Figure 14. And demonstrate that pooling stem cells from multiple heterologous apheresis cones leads to enhanced proliferation and yield of neutrophil progenitors.

[0276] Example 6 - Freezing and thawing immature neutrophils

[0277] Immature neutrophils of the invention were produced as described in Examples 1 and 2.

[0278] Cultured, immature neutrophils of the invention and native mature neutrophils were frozen either in CryoStor®CS10 (#07930, STEMCELL™ TECHNOLOGIES) or STEM-CELL BANKER® GMP Grade (ZENOGEN PHARMA) according to manufacturers' protocols. In brief, a cell suspension was gently pelleted by centrifugation either at 400 g for 5 min at RT (CryoStor®) or at 2000 rpm for 5 min at 4°C (STEM-CELL BANKER), and then the supernatant was carefully removed. Next, the cell pellet was carefully resuspended in either cold CryoStor®CS10 or STEM-CELL BANKER® at 10xl06cells / mL and transferred to cryovials. For CryoStor®CS10 the vials were incubated at 4°C for 10 min and cryopreserved using a standard slow rate-controlled cooling using Nalgene® Mr. Frosty. For STEM-CELL BANKER® cells were directly placed in -80°C.

[0279] The cells were thawed according to the same protocol for both reagents. In brief, the cells were guickly thawed in a 37°C water bath by gently shaking the vial and removed from the bath when only a small ice crystal remained. Next, the cells were diluted 1 in 10 by adding warm Hanks' Balanced Salt Solution (HBSS -Ca -Mg) in small increments (50 pL - 1 mL) and centrifuged gently at 300 g for 10 min. After the centrifugation, the cell pellet was gently resuspended in warm HBSS, and the cells were directly used for assays.

[0280] The results are shown in Figure 15. Neutrophil activity was tested by measuring ROS production using the luminol assay, in response to activation by PMA, in cultured neutrophils (blue) or native neutrophils from two different donors (red and green). ROS production was measured either before cryopreservation (dark lines) or after thawing (light lines). The data demonstrate that cultured neutrophils maintain function post-freezing, while native neutrophils do not.

[0281] Example 7: Genetic engineering of cultured neutrophils to enhance function

[0282] Immature neutrophils were gene edited using CRISPR / Cas9 to deplete expression of the inhibitory receptor Siglec 9 (CD329) as described in Example 1. The results are shown in Figure 16. A: surface expression of Siglec 9, determined by flow cytometry in cells treated with a Siglec 9-targeting CRISPR gRNA (blue) and a control scrambled gRNA (Ctrl, red ) in cultured neutrophils derived from stem cells isolated from two independent donors. Histograms show depletion of Siglec 9 expression. B: guantification of median fluorescence intensity (MFI) from A. C: Siglec 9 knockdown cells demonstrate elevated ROS production in response to PMA and C. albicans, in donor 1 (left and donor 2 (right).

[0283] Example 8: CD16 overexpression

[0284] Immature neutrophils were genetically engineered to overexpress CD16, using lentiviral delivery of the CD16 cDNA, using the vector pLV[Exp]-EGFP-SFFV, at day 10 of the differentiation protocol. Surface expression of CD16 was guantified at day 17 of the differentiation protocol using flow cytometry. The results are shown in Figure 17 and demonstrate that cultured neutrophils are amenable to overexpression experiments.

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Claims

CLAIMS1. A population of immature neutrophils, wherein: at least about 60% of the cells in the population express detectable levels of CD66b, at least about 60% of the cells in the population express detectable levels of CDllb, fewer than about 20% of the cells in the population express detectable levels of CD10, fewer than about 20% of the cells in the population express detectable levels of CD16, and fewer than about 20% of the cells in the population express detectable levels of CXCR2.

2. A population according to claim 1, wherein: at least about 60% of the cells in the population express detectable levels of CD66b, at least about 80% of the cells in the population express detectable levels of CDllb, fewer than about 10% of the cells in the population express detectable levels of CD10, fewer than about 10% of the cells in the population express detectable levels of CD16, and fewer than about 10% of the cells in the population express detectable levels of CXCR2.

3. A population according to claim 1 or 2, wherein fewer than about 20% of the cells in the population express detectable levels of CD34.

4. A population according to any one of the preceding claims, wherein the population secretes a detectable level of one or more of interleukin-6 (IL-6), IL-8 and tumour necrosis factor (TNF) alpha.

5. A population according to any one of the preceding claims, wherein at least some of the cells in the population are genetically engineered to express one or more activating receptors and / or to reduce expression of one or more inhibitory receptors.

6. A method of producing a population of immature neutrophils, comprising (a) culturing haematopoietic progenitor cells in the presence of interleukin-3 (IL-3), stem cell factor (SCF) and FMS-like tyrosine kinase 3 (Flt-3) Ligand (Flt3-L) and in the absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or granulocyte colony stimulating factor (G-CSF), (b) culturing the cells produced in step (a) in the presence of IL-3, SCF, Flt3-L and GM-CSF, (c) culturing the cells produced in step (b) in the presence of GM-CSF and G-CSF and (d) culturing the cells produced in step (c) in the presence of G-CSF.

7. A method according to claim 6, wherein the method further comprises (e) isolating immature neutrophils based on the expression of CD66b.

8. A method according to claim 6 or 7, wherein step (a) comprises culturing the haematopoietic progenitor cells for about 3 days, step (b) comprises culturing the cells for about 4 days, step (c) comprises culturing the cells for about 3 days, and / or step (d) comprises culturing the cells for about 7 days.

9. A method according to any one of claims 6-8, wherein the concentration of IL-3 is about lOng / mL, the concentration of SCF is about 50ng / mL, the concentration of Flt-3-L is about 50ng / mL, the concentration of GM-CSF is about lOng / mL, and / or the concentration of G-CSF is about lOng / mL.

10. A method according to any one of claims 6-9, wherein the haematopoietic progenitor cells are stem cells, haematopoietic stem cells, peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cell (iPSCs), or an immortalized cell line.

11. A method according to any one of claims 6-10, wherein the haematopoietic progenitor cells are CD34+.

12. A population of immature neutrophils genetically engineered to express one or more activating receptors and / or to reduce expression of one or more inhibitory receptors.

13. A population according to claim 12, wherein the one or more activating receptors are selected from CD16, CD10, CDllb, CD15, CD18, CD32a / c, CD62L, CD64, CD66b, CD66d, CD85h, CD89, CD172b, C177, CD300b, CD300c, CD354, PILRB, SIRPB2, CEACAM4, Siglec-14, and [32 integrin.

14. A population according to claim 12 or 13, wherein the one or more inhibitory receptors are selected from Siglec 9 (CD329), CD33, CD66a, CD85a, CD85d, CD85k, CD170, CD172A, CD300a, CD300f, CD305, PILRA, SIRL-1 and CLEC12A.

15. A population according to any one of claims 12-14, wherein the population is as defined in any one of claims 1-4.

16. A population according to any one of claims 1-5 and 12-15, wherein the population comprises at least about 5 x 105cells.

17. A method of producing a population of genetically engineered immature neutrophils according to any one of claims 12-15, comprising (i) genetically engineering haematopoietic progenitor cells and (ii) culturing the genetically engineered haematopoietic progenitor cells under conditions which promote their differentiation to immature neutrophils.

18. A method according to claim 17, wherein step (ii) comprises conducting a method according to any one of claims 6-11.

19. A cryopreserved population according to any one of claims 1-5 and 12-15.

20. A thawed population according to any one of claims 1-5 and 12-15.

21. A pharmaceutical composition comprising a population according to any one of claims 1- 5, 12-15 and 19-20 and a pharmaceutically or physiologically acceptable diluent and / or carrier.

22. A method of treating or preventing an infection in a subject in need thereof, comprising administering to the subject a population according to any one of claims 1-5, 12-15 and 19-20 or a pharmaceutical composition according to claim 21.

23. A method according to claim 22, wherein the infection is caused by a bacterium, an archaeon, a fungus, or a virus.

24. A method of treating or preventing cancer in a subject in need thereof, comprising administering to the subject a population according to any one of claims 1-5, 12-15 and 19-20 or a pharmaceutical composition according to claim 21.

25. A population according to any one of claims 1-5, 12-15 and 19-20 or a pharmaceutical composition according to claim 21 for use in a method of treating or preventing an infection or cancer.

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