Method of producing macrophages

Abstract

The present invention relates to a method of producing macrophages. The macrophages are produced from monocytes harvested from a subject by incubating said monocytes in a medium comprising a Colony Stimulating Factor 1 Receptor (CSF-1R) agonist. Before monocytes are harvested, the subject is treated with G-CSF. The macrophages produced by the method are capable of being polarised to a pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype. The invention also relates to methods of treating inflammatory diseases comprising administering the macrophages produced by the method to a subject.

Description

[0001] METHOD OF PRODUCING MACROPHAGES

[0002] Field of the Invention

[0003] The present invention relates to a method of producing macrophages. The macrophages are produced from monocytes harvested from a subject by incubating said monocytes in M-CSF, or another CSF-1R agonist such as IL-34. Before monocytes are harvested, the subject is treated with G-CSF. The invention also relates to methods of treating inflammatory diseases comprising administering the macrophages produced by the method to a subject.

[0004] Background to the Invention

[0005] Macrophage cell therapies are being utilised in various indications. To manufacture macrophages, cluster differentiation (CD) 14 positive monocytes can be isolated from patients and matured into macrophages in vitro. In vitro maturation is dependent on the addition of growth factors such as Granulocyte-macrophage colony stimulating factor (GM-CSF) or macrophage colony stimulating factor (M-CSF). Whereas the resulting macrophages from such maturation are unpolarised macrophages (also sometimes referred to as MO macrophages), the growth factor used for maturation influences the phenotype of the final macrophage, where GM- CSF promotes an inflammatory phenotype and M-CSF promotes an anti-inflammatory phenotype (1, 2). For autologous macrophage cell therapy that is envisioned for use in the oncology space, an inflammatory phenotype is favourable. Therefore, macrophages for such cell therapies are likely to be manufactured using GM-CSF (10). More recently, anti-inflammatory macrophages have been shown to be beneficial in inflammatory indications. To manufacture these, M-CSF is utilised (3).

[0006] Despite favourable mechanisms to shape the phenotype of macrophage cell therapies to ensure required potency, a major manufacturing limitation remains. Unlike NK or T cell therapies, monocytes and macrophages do not proliferate. Thus, cell loss during manufacture of macrophages from monocytes for cell therapy can limit the dosing regime in patients. A potential solution would be to perform multiple leukapheresis procedures in a patient (from which circulating monocytes can be isolated), and thus produce more starting material. However, this is often not feasible in a patient due to patient comfort, safety and / or compliance with multiple leukapheresis procedures.

[0007] To avoid multiple leukapheresis procedures, an alternative solution could be to administer Granulocyte colony stimulating factor (G-CSF) to patients prior to leukapheresis (also known as cell mobilisation). This results in the egress of all cells within the bone marrow, which in turn results in a higher proportion of monocytes within the circulation that can be isolated for macrophage maturation via GMP -manufacturing procedures.

[0008] G-CSF is a cytokine that disrupts the bone marrow niche via two independent mechanisms: 1) by promoting the release of proteases that cleave peptides that anchor bone marrow stem and progenitor cells to the bone marrow stroma and 2) by disrupting the interaction between CXCR4+ cells and CXCL12, a chemokine involved in cell tethering (4). Once cleaved, cells expressing CXCR4 are released from the bone marrow, regardless of maturation state. Monocyte maturation in the bone marrow is a complex interplay between CXCR4 and CCR2 (5). Simplified, immature monocytes are a transient population of CXCR4+CCR2- monocytes tethered to the bone marrow stroma in a CXCR4 dependent manner. As they mature, they lose CXCR4 expression and upregulate CCR2 which facilitates their emigration from the bone marrow (6, 7). Therefore, mobilised monocytes have a heterogeneous expression of CCR2 and are generally younger and likely to have a lower phagocytic ability than that non-mobilised monocytes. Moreover, the CSF-1R is known to be upregulated upon monocyte and macrophage maturation (13), hence more immature monocytes would be expected to be less responsive to CSF-1R agonists. It was therefore suspected that such monocytes might not be able to be differentiated to fully functional macrophages using M-CSF, IL-34, or indeed any agonist of the CSF-1R, and that they may result in macrophages that have a reduced phagocytic capability compared to that of macrophages generated from non-mobilised monocytes.

[0009] However, the inventors surprisingly found that such manufacturing of macrophages using M- CSF or IL-34 is feasible and procures functional macrophages. The inventors have found that agonists of the CSF-1R can promote the differentiation of G-CSF mobilised monocytes into functional macrophages.

[0010] Of note, while mobilization followed by use of GM-CSF to differentiate macrophages from mobilised monocytes has been described (10), the functionality of the resulting macrophages (e.g. their phagocytic capability and / or their ability to further polarize to a pro-restorative “M2- like” phenotype) was not demonstrated.

[0011] Summary of the Invention

[0012] Macrophage therapies have potential applicability to various fields of medicine. In a therapeutic context it is preferable for macrophages to be autologous to the subject to whom they are administered to avoid an immunological reaction to the therapy. Autologous macrophages may be made by harvesting monocytes from a subject, and differentiating them to macrophages in vitro. As monocytes and macrophages do not proliferate, production of sufficient therapeutic macrophages is a limiting factor for macrophage-based therapies.

[0013] While G-CSF is known to promote egress of monocytes from the bone marrow, and therefore enhance the harvest of monocytes from a single apheresis, all previous macrophages produced from mobilised monocytes have been inflammatory in character, and are therefore not at all applicable to the treatment of inflammatory conditions. The inventors have for the first time shown that G-CSF may be used to mobilise monocytes that can be differentiated into functional macrophages that are useful for treatment of inflammatory conditions.

[0014] WO2019 / 118888A1, W02022 / 047119A1 and W02012 / 062930 each describe therapeutic macrophages. Importantly, none of the macrophages described in WO2019 / 118888A1, W02022 / 047119A1 and WO2012 / 062930 are produced from monocytes extracted from a subject following a step of mobilisation with G-CSF. Therefore, none of these documents provide any indication that anti-inflammatory macrophages may be produced from monocytes harvested from a subject following a G-CSF mobilisation step.

[0015] WO2019 / 175595 describes use of autologous isolated non-engineered human macrophages in combination with another agent in the treatment of liver disease, wherein the other agent may be G-CSF. However, G-CSF is used in a completely different way in the context of WO2019 / 175595 compared to the present invention. In the present invention, the subject is treated with G-CSF before monocytes are extracted from the subject by apheresis, thereby increasing the number of monocytes which may be harvested from the subject in order to produce therapeutic macrophages. In contrast, in WO2019 / 175595, G-CSF is administered together with the therapeutic macrophages which have already been produced. The use of G-CSF in WO2019 / 175595 therefore does not have the technical effect of increasing the yield of monocytes and therefore autologous therapeutic macrophages which may be produced from a single apheresis. Accordingly, WO2019 / 175595 also does not suggest that monocytes mobilised using G-CSF may then be differentiated into macrophages using M-CSF.

[0016] Chihara et al., 2010 describes the differentiation of monocytes into macrophages using M-CSF and IL-34 (30). In contrast to the present invention, the monocytes used in Chihara et al. are not mobilised. Hence this document does not provide any evidence that CSF-1R agonists could be used to successfully differentiate mobilised monocytes into functional macrophages, which would be expected to have reduced expression of CSF-1R.

[0017] Boulakirba et al., 2018 describes the differentiation of monocytes into macrophages using M- CSF and IL-34 (31). However, the monocytes used are not mobilised, so this document also provides no indication that CSF-1R agonists could be used to successfully differentiate mobilised monocytes into functional macrophages.

[0018] A method of producing macrophages having a pro-inflammatory phenotype from monocytes has been described10. Said method comprises a step of treating the subject with G-CSF prior to the extraction of monocytes by apheresis. However, the monocytes were differentiated into macrophages using GM-CSF, rather than M-CSF or indeed any agonists of the CSF-1R. GM- CSF is known to induce macrophage properties associated with a pro-inflammatory phenotypen'12. Accordingly, there is no indication in the art that it would be possible to produce macrophages from G-CSF mobilised monocytes which are not pro-inflammatory, or indeed that G-CSF mobilised monocytes may be differentiated into macrophages using M-CSF or indeed any agonists of the CSF-1R, all the more so functional macrophages.

[0019] The inventors have shown, for the first time, that functional macrophages may differentiated in vitro from monocytes using M-CSF and / or IL-34, wherein the monocytes have been harvested from a subject, following mobilisation with G-CSF (and in particular, Filgrastim, a recombinant G-CSF). The G-CSF mobilisation step has the advantage of increasing the yield of monocytes possible from a single apheresis, reducing the need for repeated apheresis and therefore minimising the exposure of subjects to the risks associated with repeated apheresis. While the risk of complications from a single apheresis procedure is minimal, the impact on the subject becomes more significant with repeated procedures. For example, repeated apheresis procedures is not desirable, both for reasons of patient comfort, and also due to potential long-term negative consequences such as bone demineralization and cataract formation14'15. As described herein, a single apheresis may provide enough monocytes to produce sufficient therapeutic macrophages to complete an entire course of treatment with at least 4 doses of 1 x 108macrophages.

[0020] Based on understanding of existing practice, and as demonstrated by the data in the Examples, treating a subject with G-CSF causes cells expressing CXCR4 (such as monocytes) to be released from the bone marrow, regardless of maturation state. Therefore, mobilisation with G- CSF would be expected to increase the number and / or proportion of immature monocytes in the bloodstream. This is supported by the data in Figure 2, showing that populations of mobilised monocytes have a more heterogenous expression of CCR2 and a greater proportion of CD14+CCR2LOW cells, indicating an increased proportion of immature cells. Immature cells with reduced CCR2 expression are associated with reduced phagocytic capacity. It was therefore suspected that such monocytes might not be able to be differentiated to fully functional macrophages using M-CSF or other agonists of the CSF-1R and result in macrophages that have an equivalent phagocytic capability to that of macrophages generated from non-mobilised monocytes. However, the inventors surprisingly found that such manufacturing of macrophages results in macrophages with therapeutic functionality, including equivalent phagocytic capacity to macrophages produced from non-mobilised monocytes. The inventors have also confirmed that the macrophages produced by the methods of the invention can be polarised to a pro-restorative phenotype, for example using IL- 10. In fact, the macrophages produced by the methods of the invention have at least the same potential to be polarised to a pro-restorative phenotype as macrophages generated from non-mobilised monocytes.

[0021] Furthermore, the inventors have found that macrophages produced by the methods of the invention may also be engineered to express a payload such as IL-10 and / or MMP9. The inventors have found that macrophages produced by the methods of the invention, which are subsequently engineered to express IL- 10 and / or MMP9, can secrete the IL- 10 and / or MMP9 at the same level as macrophages generated from non-mobilised monocytes which have been engineered in the same way. Surprisingly, macrophages produced by the methods of the invention which have undergone a further step of being engineered to express IL- 10 may even secrete IL-10 at a greater level than macrophages generated from non-mobilised monocytes which have been engineered in the same way.

[0022] Moreover, prior to the present invention, it was understood that monocytes upregulate CSFR1, the receptor for M-CSF and IL-34, during maturation13. Therefore, it was also suspected that a population of mobilised monocytes would be less responsive to M-CSF or other agonists of the CSF-1R, and so it would not be possible to differentiate immature monocytes using M-CSF or other agonists of the CSF-1R (instead of e.g. GM-CSF) in order to produce macrophages which are not pro-inflammatory. The inventors are the first to show the feasibility of differentiating mobilised monocytes in medium comprising M-CSF and / or IL-34, suggesting the utility of agonists of the CSF-1R in general.

[0023] It is particularly surprising that the inventors have found that both IL-34 and M-CSF can be used to differentiate mobilised monocytes into mature macrophages, and that said macrophages have similar viability, phenotype and functionality, both in terms of polarisation potential and phagocytic capability. When the invention was devised, it was known in the art that IL-34 and M-CSF have different bioactivities, which was suggested to result in macrophages with different surface phenotypes and functionalities (e.g. differences in chemokine secretion) and different polarisation capabilities (see e.g. 30, 31). Accordingly, it would not be expected that both IL-34 and M-CSF are capable of producing functional, viable, mature macrophages, given the known differences between the activities of these molecules. In particular, it would not be expected that both IL-34 and M-CSF differentiated macrophages could both be polarised to a pro-regenerative phenotype, and that the resultant macrophages would be comparable in phenotype and function. Furthermore, it is particularly surprising that IL-34 can be used to differentiate mobilised monocytes into mature macrophages that may be polarised to an anti-inflammatory, prorestorative and / or anti-fibrotic phenotype, because previous work suggested that IL-34- differentiated mobilised monocytes may have different polarisation potential (see e.g. 30, 31).

[0024] Accordingly, it can be seen that the inventors have provided an improved method of producing functional monocyte-derived macrophages which may be polarised to an anti-inflammatory, prorestorative and / or anti-fibrotic phenotype, and which are capable of phagocytosis.

[0025] One or more aspects or embodiments of the claimed invention aim to solve one or more of the above-mentioned problems.

[0026] The invention provides a method of producing macrophages, comprising:

[0027] (a) treating a subject with G-CSF;

[0028] (b) obtaining leukocytes from the subject by leukapheresis following the treatment with G- CSF; (c) isolating monocytes from the leukocytes obtained by the leukapheresis; and

[0029] (d) incubating the monocytes in a medium comprising M-CSF to produce macrophages.

[0030] The invention also provides a method of producing macrophages wherein the method comprises incubating monocytes obtained from a subject in a medium comprising M-CSF to produce macrophages, wherein the monocytes used were previously isolated from leukocytes obtained from the subject through leukapheresis, and wherein prior to the leukapheresis, the subject was treated with G-CSF.

[0031] The invention also provides a method of treating an inflammatory condition in a subject, comprising administering the macrophages produced according to the methods described herein to the subject. The invention also provides a method of treating an inflammatory condition in a subject, comprising:

[0032] (a) treating a subject with G-CSF;

[0033] (b) obtaining leukocytes from the subject by leukapheresis following the treatment with G- CSF;

[0034] (c) isolating monocytes from the leukocytes obtained by the leukapheresis;

[0035] (d) incubating the monocytes in a medium comprising M-CSF to produce macrophages; and

[0036] (e) administering the macrophages to the subject.

[0037] The invention also provides a macrophage for use in such methods. Suitably, the invention also provides a macrophage for use in a method of treating an inflammatory condition in a subject, wherein the method comprises administering the macrophages to the subject, wherein the macrophages have been produced by:

[0038] (a) treating a subject with G-CSF;

[0039] (b) obtaining leukocytes from the subject by leukapheresis following the treatment with G- CSF;

[0040] (c) isolating monocytes from the leukocytes obtained by the leukapheresis; and

[0041] (d) incubating the monocytes in a medium comprising M-CSF to produce macrophages.

[0042] Alternatively, the invention provides a macrophage for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was administered with G-CSF, subsequently leukocytes were obtained from the subject by leukapheresis, and monocytes were isolated from the leukocytes, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising M-CSF to produce said macrophage

[0043] In a further aspect, the invention also provides a macrophage and G-CSF for use in a method of treating an inflammatory condition in a subject, wherein the method comprises

[0044] (a) treating a subject with G-CSF;

[0045] (b) obtaining leukocytes from the subject by leukapheresis following the treatment with G- CSF;

[0046] (c) isolating monocytes from the leukocytes obtained by the leukapheresis;

[0047] (d) incubating the monocytes in a medium comprising M-CSF to produce macrophages; and (e) administering the macrophages to the subject.

[0048] The invention also provides a macrophage and G-CSF for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was administered with G-CSF, subsequently leukocytes were obtained from the subject by leukapheresis, and monocytes were isolated from the leukocytes, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising M-CSF to produce said macrophage.

[0049] In a preferred embodiment, the invention provides a method of producing macrophages, comprising:

[0050] (a) treating a subject with Filgrastim, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0051] (b) obtaining leukocytes from the subject by leukapheresis less than 24 hours after the last dose of Filgrastim was administered;

[0052] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells; and

[0053] (d) incubating the monocytes in a medium comprising lOOng / mL M-CSF for 5-7 days to produce macrophages.

[0054] In another preferred embodiment, the invention provides a method of producing macrophages wherein the method comprises incubating monocytes obtained from the subject in a medium comprising lOOng / mL M-CSF for 5-7 days to produce macrophages, wherein the monocytes used were previously isolated from leukocytes obtained from the subject through leukapheresis, and wherein prior to the leukapheresis, the subject was treated with Filgrastim, which was administered subcutaneously at a dose of l Opg / kg / day for 4 or 5 consecutive days.

[0055] As described herein, the method comprises a step of isolating monocytes from the leukapheresis product. The monocytes may be isolated by any appropriate method. Appropriate methods are known to the person skilled in the art. In preferred embodiments, the monocytes are isolated by selection of CD14+ cells. Indeed, various methods are known in the art of isolating CD14+ cells, such as immunomagnetic separation17'20.

[0056] In a preferred embodiment, the invention also provides a method of producing macrophages, wherein the method comprises incubating monocytes obtained from the subject in a medium comprising lOOng / mL M-CSF for 5-7 days to produce macrophages, wherein the monocytes used were previously isolated from leukocytes obtained from the subject through leukapheresis, and wherein prior to the leukapheresis, , the subject was treated with Filgrastim, which was administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days, and wherein leukapheresis occurred less than 24 hours after the final dose of Filgrastim. A dose of lOpg of Filgrastim is equal to 1 million units. Suitably, in preferred embodiments, the subject is treated with 1 million units of Filgrastim / kg / day.

[0057] In a further preferred embodiment, the invention provides a method of producing macrophages, wherein the method comprises incubating monocytes isolated from a subject in-vitro in a medium comprising M-CSF at a sufficient concentration to differentiate the monocytes to macrophages, wherein the monocytes were isolated from a leukapheresis product following a leukapheresis process that has been performed on the subject, and wherein prior to the leukapheresis the subject has been treated with recombinant G-CSF, preferably Filgrastim. The skilled person would be able to select a concentration of M-CSF which would be sufficient to differentiate monocytes into macrophages. The concentration of M-CSF may depend on the concentration at which the monocytes are incubated in the medium. In some embodiments, the monocytes are incubated in the medium comprising M-CSF at a cell concentration of 4xlO6 / mL or 2xlO6 / mL. It has been demonstrated that various concentrations of M-CSF may potentially be used to successfully differentiate monocytes into macrophages, therefore the skilled person would be able to determine an appropriate protocol and a sufficient concentration of M-CSF to use in the medium21'24. In some embodiments, the concentration of the M-CSF in the medium is 10-150 ng / ml, optionally between about 10 ng / ml to about 100 ng / ml. More preferably, the concentration of M-CSF in the medium is between about 50 ng / ml to about 100 ng / ml. According to preferred embodiments, the concentration of the M-CSF in the medium is lOOng / mL or about lOOng / mL, optionally wherein the monocytes were incubated in the medium comprising M-CSF for 5-7 days (preferably 5 days). According to some embodiments, the monocytes were isolated from the leukapheresis product by isolating CD14+ leukocytes. According to some embodiments, the recombinant G-CSF is Filgrastim and prior to the leukapheresis the subject has been treated with Filgrastim at a dose of lOpg / kg / day for 4 or 5 consecutive days, and wherein leukapheresis occurred less than 24 hours after the final dose of Filgrastim.

[0058] In further preferred embodiments, the invention also provides a method of treating an inflammatory condition, wherein the method comprises

[0059] (a) treating a subject with Filgrastim, which is administered subcutaneously at a dose of lOpg / kg / day for 4 consecutive days;

[0060] (b) obtaining leukocytes from the subject by leukapheresis less than 24 hours after the last dose of Filgrastim was administered;

[0061] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells;

[0062] (d) incubating the monocytes in a medium comprising lOOng / mL M-CSF for 5-7 days to produce macrophages; and

[0063] (e) administering the macrophages to the subject in 4 doses of 1 x 108macrophages with 6 weeks between dose, wherein the macrophages are autologous to the subject.

[0064] The invention also provides a macrophage for use in such methods. Suitably, the invention also provides a macrophage for use in a method of treating an inflammatory condition in a subject, wherein the method comprises administering the macrophages to the subject in 4 doses of 1 x 108macrophages with 6 weeks between dose, wherein the macrophages are autologous to the subject, wherein the macrophages have been produced by:

[0065] (a) treating the subject with Filgrastim, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0066] (b) obtaining leukocytes from the subject by leukapheresis less than 24 hours after the last dose of Filgrastim was administered;

[0067] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells; and

[0068] (d) incubating the monocytes in a medium comprising lOOng / mL M-CSF for 5-7 days to produce macrophages. Alternatively, the invention provides a macrophage for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was previously treated with Filgrastim, which was administered subcutaneously at a dose of lOpg / kg / day for 5 consecutive days, and subsequently leukocytes were obtained from the subject by leukapheresis less than 24 hours after the last dose of Filgrastim was administered, monocytes were then isolated from the leukocytes obtained by the leukapheresis, optionally by selection for CD14+ cells, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising lOOng / mL M-CSF for 6 days to produce said macrophage.

[0069] In a further aspect, the invention also provides a macrophage and G-CSF for use in a method of treating an inflammatory condition in a subject, wherein the method comprises

[0070] (a) treating the subject with Filgrastim, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0071] (b) obtaining leukocytes from the subject by leukapheresis less than 24 hours after the last dose of Filgrastim was administered;

[0072] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells;

[0073] (d) incubating the monocytes in a medium comprising lOOng / mL M-CSF for 5-7 days to produce macrophages; and

[0074] (e) administering the macrophages to the subject in 4 doses of 1 x 108macrophages with 6 weeks between dose, wherein the macrophages are autologous to the subject.

[0075] In the methods of treatment and the macrophages for use in such methods, it is preferred that all the macrophages administered to the subject are autologous to the subject, and have been prepared from monocytes harvested from a single leukapheresis.

[0076] It should be noted that the step of administering G-CSF to the subject, followed by harvesting cells by apheresis, are not themselves capable of curing a disease present in the subject or returning them to a state of health. Notably, two European multicentre trials have failed to demonstrate that G-CSF increases survival or liver function in patients with liver cirrhosis (the REALISTIC study) or improves transplant-free survival in patients with ACLF (the GRAFT study)25'27. Moreover, the methods of the invention preferably harvest leukocytes by leukapheresis (rather than harvesting whole blood, for example), which is not considered to be therapeutic in patients with liver disease, such as liver cirrhosis. Furthermore, these steps are routine procedures requiring minimal specific training and medical expertise required from the person applying said steps. Such steps do not in themselves carry significant health risks or involve a substantial physical intervention on the body. G-CSF is routinely administered to patients, and its effects are typically transient and self-limiting16. Complications from single apheresis procedures are rare, and adverse effects are usually mild and easily treated14.

[0077] The invention also provides a method of producing macrophages, comprising:

[0078] (a) treating a subject with G-CSF;

[0079] (b) obtaining leukocytes from the subject by leukapheresis following the treatment with G- CSF;

[0080] (c) isolating monocytes from the leukocytes obtained by the leukapheresis; and (d) incubating the monocytes in a medium comprising a Colony Stimulating Factor 1 Receptor (CSF-1R) agonist to produce macrophages.

[0081] The invention also provides a method of producing macrophages wherein the method comprises incubating monocytes obtained from a subject in a medium comprising a CSF-1R agonist to produce macrophages, wherein the monocytes used were previously isolated from leukocytes obtained from the subject through leukapheresis, and wherein prior to the leukapheresis, the subject was treated with G-CSF.

[0082] The invention also provides a method of treating an inflammatory condition in a subject, comprising administering the macrophages produced according to the methods described herein to the subject.

[0083] The invention also provides a method of treating an inflammatory condition in a subject, comprising:

[0084] (a) treating a subject with G-CSF;

[0085] (b) obtaining leukocytes from the subject by leukapheresis following the treatment with G- CSF;

[0086] (c) isolating monocytes from the leukocytes obtained by the leukapheresis;

[0087] (d) incubating the monocytes in a medium comprising a CSF-1R agonist to produce macrophages; and

[0088] (e) administering the macrophages to the subject.

[0089] The invention also provides a macrophage for use in such methods. Suitably, the invention also provides a macrophage for use in a method of treating an inflammatory condition in a subject, wherein the method comprises administering the macrophages to the subject, wherein the macrophages have been produced by:

[0090] (a) treating a subject with G-CSF;

[0091] (b) obtaining leukocytes from the subject by leukapheresis following the treatment with G- CSF;

[0092] (c) isolating monocytes from the leukocytes obtained by the leukapheresis; and

[0093] (d) incubating the monocytes in a medium comprising a CSF-1R agonist to produce macrophages.

[0094] Alternatively, the invention provides a macrophage for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was administered with G-CSF, subsequently leukocytes were obtained from the subject by leukapheresis, and monocytes were isolated from the leukocytes, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising a CSF-1R agonist to produce said macrophage

[0095] In a further aspect, the invention also provides a macrophage and G-CSF for use in a method of treating an inflammatory condition in a subject, wherein the method comprises (a) treating a subject with G-CSF;

[0096] (b) obtaining leukocytes from the subject by leukapheresis following the treatment with G- CSF;

[0097] (c) isolating monocytes from the leukocytes obtained by the leukapheresis;

[0098] (d) incubating the monocytes in a medium comprising a CSF-1R agonist to produce macrophages; and

[0099] (e) administering the macrophages to the subject.

[0100] The invention also provides a macrophage and G-CSF for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was administered with G-CSF, subsequently leukocytes were obtained from the subject by leukapheresis, and monocytes were isolated from the leukocytes, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising a CSF-1R agonist to produce said macrophage.

[0101] In a preferred embodiment, the invention provides a method of producing macrophages, comprising:

[0102] (a) treating a subject with Filgrastim, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0103] (b) obtaining leukocytes from the subject by leukapheresis up to 24 hours after the last dose of Filgrastim was administered;

[0104] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells; and

[0105] (d) incubating the monocytes in a medium comprising 100-200ng / ml of the CSF-1R agonist for 5-7 days to produce macrophages.

[0106] In another preferred embodiment, the invention provides a method of producing macrophages wherein the method comprises incubating monocytes obtained from the subject in a medium comprising 100-200ng / ml of the CSF-1R agonist, for 5-7 days to produce macrophages, wherein the monocytes used were previously isolated from leukocytes obtained from the subject through leukapheresis, and wherein prior to the leukapheresis, the subject was treated with Filgrastim, which was administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days.

[0107] In a preferred embodiment, the invention also provides a method of producing macrophages, wherein the method comprises incubating monocytes obtained from the subject in a medium comprising 100-200ng / ml of the CSF-1R agonist for 5-7 days to produce macrophages, wherein the monocytes used were previously isolated from leukocytes obtained from the subject through leukapheresis, and wherein prior to the leukapheresis, the subject was treated with Filgrastim, which was administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days, and wherein leukapheresis occurred up to 24 hours after the final dose of Filgrastim. A dose of lOpg of Filgrastim is equal to 1 million units. Suitably, in preferred embodiments, the subject is treated with 1 million units of Filgrastim / kg / day.

[0108] In a further preferred embodiment, the invention provides a method of producing macrophages, wherein the method comprises incubating monocytes isolated from a subject in-vitro in a medium comprising the CSF-1R agonist at a sufficient concentration to differentiate the monocytes to macrophages, wherein the monocytes were isolated from a leukapheresis product following a leukapheresis process that has been performed on the subject, and wherein prior to the leukapheresis the subject has been treated with recombinant G-CSF, preferably Filgrastim.

[0109] The skilled person would be able to select a concentration of the CSF-1R agonist which would be sufficient to differentiate monocytes into macrophages. The concentration of the CSF-1R agonist may depend on the concentration at which the monocytes are incubated in the medium. In some embodiments, the monocytes are incubated in the medium comprising the CSF-1R agonist at a cell concentration of 4xlO6 / mL or 2xlO6 / mL. In preferred embodiments, the monocytes are incubated in the medium comprising the CSF-1R agonist at a cell concentration of 4xlO6 / mL.

[0110] It has been demonstrated in the Examples of the application that various concentrations of CSF- 1R agonists such as M-CSF and IL-34 may potentially be used to successfully differentiate monocytes into macrophages, therefore the skilled person would be able to determine an appropriate protocol and a sufficient concentration of the CSF-1R agonist to use in the medium. In some embodiments, the concentration of the CSF-1R agonist in the medium is 10-500 ng / ml, optionally between about 10 ng / ml to about 100 ng / ml. In other embodiments, the concentration of M-CSF in the medium is between about 50 ng / ml to about 200 ng / ml. According to preferred embodiments, the concentration of the M-CSF in the medium is between lOOng / ml and 200ng / ml, lOOng / ml or 200ng / ml, optionally wherein the monocytes were incubated in the medium comprising the CSF-1R agonist for 5-7 days (preferably 5 days).

[0111] In some embodiments, the CSF-1R agonist is selected from the group consisting of: an agonist antibody, a small molecule agonist, a protein agonist, a peptide agonist and a combination thereof.

[0112] In some embodiments, the protein agonist is a growth factor or a cytokine. In some embodiments, the growth factor or cytokine is selected from the group consisting of: M-CSF, IL- 34 and a combination thereof.

[0113] According to some embodiments, the CSF-1R agonist is M-CSF and the M-CSF concentration in the medium used to differentiate monocytes into macrophages is about 100 ng / ml, optionally at a target density of 2 xl06 / cm2monocytes. According to other embodiments, the CSF-1R agonist is IL-34 and the IL-34 concentration in the medium used to differentiate monocytes into macrophages is about 200 ng / ml (or about at 50ng / mL per IxlO6monocytes).

[0114] In further preferred embodiments, the invention also provides a method of treating an inflammatory condition, wherein the method comprises

[0115] (a) treating a subject with Filgrastim, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0116] (b) obtaining leukocytes from the subject by leukapheresis up to 24 hours after the last dose of Filgrastim was administered;

[0117] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells;

[0118] (d) incubating the monocytes in a medium comprising 100-200ng / mL of a CSF-1R agonist for 5-7 days to produce macrophages; and (e) administering the macrophages to the subject in 1 to 4 doses of about 1 x 108macrophages with 6 weeks between dose, wherein the macrophages are autologous to the subject.

[0119] The invention also provides a macrophage for use in such methods. Suitably, the invention also provides a macrophage for use in a method of treating an inflammatory condition in a subject, optionally liver cirrhosis, preferably End Stage Liver Disease, wherein the method comprises administering the macrophages to the subject in 1 to 4 doses of about 1 x 108macrophages with 6 weeks between dose, wherein the macrophages are autologous to the subject, wherein the macrophages have been produced by:

[0120] (a) treating the subject with Filgrastim, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0121] (b) obtaining leukocytes from the subject by leukapheresis up to 24 hours after the last dose of Filgrastim was administered;

[0122] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells; and

[0123] (d) incubating the monocytes in a medium comprising 100-200ng / mL of a CSF-1R agonist for 5-7 days to produce macrophages. According to some embodiments, incubating the monocytes is with about 100 ng / ml of M-CSF1 or about 200 ng / ml of IL-34.

[0124] Alternatively, the invention provides a macrophage for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was previously treated with Filgrastim, which was administered subcutaneously at a dose of l Opg / kg / day for 4 or 5 consecutive days, and subsequently leukocytes were obtained from the subject by leukapheresis up to 24 hours after the last dose of Filgrastim was administered, monocytes were then isolated from the leukocytes obtained by the leukapheresis, optionally by selection for CD14+ cells, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising 100-200ng / mL of a CSF-1R agonist for 5 or 6 days to produce said macrophage. According to some embodiment, incubating the monocytes is with about 100 ng / ml of M-CSF1 or about 200 ng / ml of IL-34. According to some embodiments, the inflammatory condition if an inflammatory liver condition, optionally liver cirrhosis, preferably End Stage Liver Disease.

[0125] In a further aspect, the invention also provides a macrophage and G-CSF for use in a method of treating an inflammatory condition in a subject, wherein the method comprises

[0126] (a) treating the subject with G-CSF, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0127] (b) obtaining leukocytes from the subject by leukapheresis up to 24 hours after the last dose of G-CSF was administered;

[0128] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells;

[0129] (d) incubating the monocytes in a medium comprising 100-200ng / mL of a CSF-1R agonist for 5-7 days to produce macrophages; and (e) administering the macrophages to the subject in 1 to 4 doses of about 1 x 108macrophages with 6 weeks between doses, wherein the macrophages are autologous to the subject.

[0130] According to some embodiments, the invention also provides a macrophage and Filgrastim for use in a method of treating an inflammatory condition in a subject, wherein the method comprises

[0131] (a) treating the subject with Filgrastim, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0132] (b) obtaining leukocytes from the subject by leukapheresis up to 24 hours after the last dose of Filgrastim was administered;

[0133] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells;

[0134] (d) incubating the monocytes in a medium comprising 100-200ng / mL of a CSF-1R agonist for 5-7 days to produce macrophages; and

[0135] (e) administering the macrophages to the subject in 1 to 4 doses of about 1 x 108macrophages with 6 weeks between dose, wherein the macrophages are autologous to the subject.

[0136] In some aspects of the invention, the CSF-1R agonist is IL-34. In some aspects of the invention the monocytes are incubated in a medium which comprises IL-34. According to some embodiments, the CSF-1R agonist is M-CSF. According to some embodiments, the CSF-1R agonist is selected from the group consisting of: M-CSF, IL-34 and a combination thereof. In some aspects of the invention, the medium comprises more than one CSF-1R agonist. In some embodiments, the monocytes are incubated in a medium which comprises both M-CSF and IL- 34, optionally wherein the medium may further comprise one or more further CSF-1R agonists. In some embodiments, the monocytes are incubated in a medium which comprises M-CSF at a concentration of at least or about lOOng / ml, and IL 34 at a concentration of at least or about 200ng / ml.

[0137] In a preferred embodiment, the invention provides a method of producing macrophages, comprising:

[0138] (a) treating a subject with Filgrastim, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0139] (b) obtaining leukocytes from the subject by leukapheresis up to 24 hours after the last dose of Filgrastim was administered;

[0140] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells; and

[0141] (d) incubating the monocytes in a medium comprising at least 200ng / ml IL-34 for 5-7 days to produce macrophages.

[0142] In another preferred embodiment, the invention provides a method of producing macrophages wherein the method comprises incubating monocytes obtained from the subject in a medium comprising at least 200ng / ml IL-34, for 5-7 days to produce macrophages, wherein the monocytes used were previously isolated from leukocytes obtained from the subject through leukapheresis, and wherein prior to the leukapheresis, the subject was treated with Filgrastim, which was administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days.

[0143] In a preferred embodiment, the invention also provides a method of producing macrophages, wherein the method comprises incubating monocytes obtained from the subject in a medium comprising at least 200ng / ml IL-34 for 5-7 days to produce macrophages, wherein the monocytes used were previously isolated from leukocytes obtained from the subject through leukapheresis, and wherein prior to the leukapheresis, the subject was treated with Filgrastim, which was administered subcutaneously at a dose of l Opg / kg / day for 4 or 5 consecutive days, and wherein leukapheresis occurred up to 24 hours after the final dose of Filgrastim.

[0144] In a further preferred embodiment, the invention provides a method of producing macrophages, wherein the method comprises incubating monocytes isolated from a subject in-vitro in a medium comprising IL-34 at a sufficient concentration to differentiate the monocytes to macrophages, wherein the monocytes were isolated from a leukapheresis product following a leukapheresis process that has been performed on the subject, and wherein prior to the leukapheresis the subject has been treated with recombinant G-CSF, preferably Filgrastim.

[0145] The skilled person would be able to select a concentration of IL-34 which would be sufficient to differentiate monocytes into macrophages. The concentration of IL-34 may depend on the concentration at which the monocytes are incubated in the medium. In preferred embodiments, the monocytes are incubated in the medium comprising the IL-34 at a cell concentration of 4xl06cells / mL. In some embodiments, the monocytes are incubated in the medium comprising 200ng / ml of IL-34 and at a cell concentration of 4xl06cells / mL. In preferred embodiments, the medium comprises IL-34 at a concentration of at least 50ng IL-34 per 1 x 106cells. In preferred embodiments, the medium comprises IL-34 at a concentration of at least 200ng / ml, or a concentration of 200-3 OOng / ml, and the monocytes are present in the medium at a concentration of 4 x 106cells / ml, optionally wherein the monocytes were incubated in the medium comprising the IL-34 for 5-7 days (preferably 5 days).

[0146] In further preferred embodiments, the invention also provides a method of treating an inflammatory condition, wherein the method comprises

[0147] (a) treating a subject with Filgrastim, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0148] (b) obtaining leukocytes from the subject by leukapheresis up to 24 hours after the last dose of Filgrastim was administered;

[0149] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells;

[0150] (d) incubating the monocytes in a medium comprising at least 200ng / ml of IL-34, preferably wherein the monocytes are present in the medium at a concentration of 4 x 106cells / ml, for 5-7 days to produce macrophages; and

[0151] (e) administering the macrophages to the subject in 1 to 4 doses of about 1 x 108macrophages with 6 weeks between dose, wherein the macrophages are autologous to the subject. The invention also provides a macrophage for use in such methods. Suitably, the invention also provides a macrophage for use in a method of treating an inflammatory condition in a subject, wherein the method comprises administering the macrophages to the subject in 1 to 4 doses of about 1 x 108macrophages with 6 weeks between dose, wherein the macrophages are autologous to the subject, wherein the macrophages have been produced by:

[0152] (a) treating the subject with Filgrastim, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0153] (b) obtaining leukocytes from the subject by leukapheresis up to 24 hours after the last dose of Filgrastim was administered;

[0154] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells; and

[0155] (d) incubating the monocytes in a medium comprising at least 200ng / ml of IL-34for 5-7 days to produce macrophages.

[0156] Alternatively, the invention provides a macrophage for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was previously treated with Filgrastim, which was administered subcutaneously at a dose of l Opg / kg / day for 5 consecutive days, and subsequently leukocytes were obtained from the subject by leukapheresis up to 24 hours after the last dose of Filgrastim was administered, monocytes were then isolated from the leukocytes obtained by the leukapheresis, optionally by selection for CD14+ cells, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising at least 200ng / ml of IL-34, optionally wherein the monocytes are present in the medium at a concentration of 4 x 106cells / ml for 5 or 6 days to produce said macrophage.

[0157] Alternatively, the invention provides a macrophage for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was previously treated with Filgrastim, which was administered subcutaneously at a dose of lOpg / kg / day for 5 consecutive days, and subsequently leukocytes were obtained from the subject by leukapheresis up to 24 hours after the last dose of Filgrastim was administered, monocytes were then isolated from the leukocytes obtained by the leukapheresis, optionally by selection for CD14+ cells, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising at least 200ng / ml of IL-34 and / or at least lOOng / ml of M- CSF for 5 or 6 days to produce said macrophage.

[0158] In a further aspect, the invention also provides a macrophage and recombinant G-CSF for use in a method of treating an inflammatory condition in a subject, wherein the method comprises

[0159] (a) treating the subject with recombinant G-CSF, which is administered subcutaneously at a dose of lOpg / kg / day for 4 or 5 consecutive days;

[0160] (b) obtaining leukocytes from the subject by leukapheresis up to 24 hours after the last dose of the recombinant G-CSF was administered;

[0161] (c) isolating monocytes from the leukocytes obtained by the leukapheresis, optionally by separation of CD 14+ cells; (d) incubating the monocytes in a medium comprising at least 200ng / ml of IL-34 and / or at least lOOng / ml of M-CSF for 5-7 days to produce macrophages; and

[0162] (e) administering the macrophages to the subject in 1 to 4 doses of about 1 x 108macrophages with 6 weeks between dose, wherein the macrophages are autologous to the subject. According to preferred embodiments, the recombinant G-CSF is Filgrastim.

[0163] Any of the methods described herein may comprise a step of isolating monocytes from the leukapheresis product. The monocytes may be isolated by any appropriate method. Appropriate methods are known to the person skilled in the art. In preferred embodiments, the monocytes are isolated by selection of CD14+ cells. Indeed, various methods are known in the art of isolating CD14+ cells, such as immunomagnetic separation17'20.

[0164] Detailed Description of the Invention

[0165] The following definitions are provided.

[0166] ‘Mobilisation’ as used herein refers to a step of treating a subject with G-CSF. Mobilisation with G-CSF promotes the egress of cells from the bone marrow, which results in a greater number and proportion of monocytes in the peripheral circulation. Specifically, G-CSF causes cells expressing CXCR4 (such as monocytes) to be released from the bone marrow, regardless of maturation state. Therefore, mobilisation with G-CSF would be expected to increase the number and / or proportion of immature monocytes. Mobilisation is typically performed with recombinant G-CSF, such as, but not limited to, Filgrastim.

[0167] A ‘macrophage’ as used herein refers to a phagocytic cell which is responsible for detecting, engulfing and destroying pathogens and apoptotic cells, and which is produced through the differentiation of monocytes.

[0168] ‘Payload’ as used herein is at least one nucleic acid encoding a gene or genes of therapeutic interest (such as, but not limited to, DNA or RNA), which is / are introduced into a macrophage, optionally via transfection.

[0169] ‘Non-polarised macrophage’ or ‘unpolarised macrophage’ as used herein refers to a mature macrophage which has not received any further stimulation to induce particular functional capacity. Non-polarized macrophages may also refer to naive or non-activated macrophages or MO macrophages. The macrophages produced by the method of the invention may be nonpolarised, but are capable of being polarised to a pro-restorative, anti-inflammatory and / or anti- fibrotic phenotype.

[0170] ‘Mature macrophage’ refers to a macrophage which expresses mature cell surface markers, preferably CCR2-, CD14+, CD206+, CD163+ and 25F9+.

[0171] Macrophages may acquire various states, referred to as “polarisation”, which are usually, but simplistically, divided into two main extremes, “pro-inflammatory” (or classically-activated, “Ml”, “Ml-like”) and “pro-regenerative” (or “pre-restorative”, alternatively-activated, “M2”, “M2 -like”, anti-inflammatory or anti-fibrotic). However, macrophages may adopt a state between these extremes.

[0172] It is generally postulated that Ml macrophages are pro-inflammatory, whereas M2 macrophages are responsible for immunomodulation and wound-healing responses. However, it is increasingly clear that this binary classification does not address the more complex heterogeneity in vivo, where macrophages adopt distinct phenotypes and even switch between phenotypes in response to the myriad of stimuli to which they are exposed. These in vivo macrophage phenotypes are impossible to recapitulate exactly in tissue culture models, emphasizing the importance of the characterization of macrophages on the basis of function. Macrophages acquire a “pro- regenerative” state under the action of various factors in combination, including macrophage colony-stimulating factor (M-CSF), IL-4, IL-13, IL-10 and TGF-p. These macrophages mediate wound healing and tissue regeneration primarily.

[0173] Due to the complexity of macrophage biology, the categorisation to “Ml” and “M2” can be considered over simplified. For example, “M2” macrophages are through to in fact be a spectrum depending on their environment. Further sub-categorisation of the pro-regenerative M2-like state has therefore been attempted, such as the following sub-categorisation suggested by Gharavi, A.T et al., “The role of macrophage subtypes and exosomes in immunomodulation”, Cell Mol Biol Lett 27, 83 (2022). In this sub-categorisation, for example:

[0174] “M2a” cells are thought to be anti-inflammatory, profibrotic and to have roles in allergy and wound healing. Such cells are categorised by the expression of IL-10, 11-1R, IL-27a, CCL1, CCL17, CCL18, CCL22, CDl lb, CD45, CD206, YM1, RELMa, IGF1. DCIR, Stabilin 1, Factor XIII-A, Ly6C, TREM-2 and DC-SIGN. The M2a state may be acquired under the action of factors such as IL-4, IL- 13, IL- 10 and PPARg.

[0175] “M2b” cells are thought to be involved in activation of a T-helper 2 (Th2) type response, immune regulation and promoting tumour progression. Such cells are categorised by the expression of IL-6, TNF-a, CD86 and SPHK1. The M2b state may be acquired under the action of IL-lb or exposure to LPS.

[0176] “M2c” cells are associated with immunosuppression, phagocytosis, tissue repair and extracellular matrix remodelling. Such cells are categorised by the expression of IL-10, CXCL13, CD163, CD206, CXCR4, TGF-b and MerTK. The M2c state may be acquired under the action of IL- 10, glucocorticoids, IL-6, IL- 10, TNF-a and TLR stimulation.

[0177] “M2d” cells are associated with tumour progression, angiogenesis, and clearance of apoptotic tissue. Such cells are categorised by the expression of IL-10, VEGF and TGF-b. The M2d state may be acquired upon exposure to LPS.

[0178] The macrophage produced by the invention may express markers consistent with functional human monocyte derived macrophages, such as CD45, CD14, CD206, CD163, CD169 and 25F9.

[0179] ‘Treatment’ as used in the present invention means an intervention in a physiological condition which prevents, reduces, or removes the clinical symptoms associated with a given physiological condition in a subject.

[0180] A “therapeutically effective amount” of macrophages described in this specification, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression may be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays. Therapeutically effective amounts and dosage regimens can be determined empirically by testing in known in vitro or in vivo (e.g. animal model) systems.

[0181] By ‘subject’ or ‘individual’ or ‘animal’ or ‘patient’ is meant any subject, particularly a mammalian subject, preferably a human subject, for whom diagnosis, prognosis, or therapy is desired, except where the subject is defined as a ‘healthy subject’. Mammalian subjects include humans; domestic animals; farm animals; such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

[0182] ‘GMP-complianf as used herein means that the method complies with Good Manufacturing Practice principles and may be used interchangeably with ‘GMP-compatible’ and ‘GMP -graded’. By way of example a GMP-compliant medium has to be serum-free, antibiotic-free, animal substance free and xenoprotein-free. The WHO provides guidance on what is required for good manufacturing practice: “Chapter 1 : WHO good manufacturing practices: Main principles for pharmaceutical products". Quality Assurance of Pharmaceuticals: A compendium of guidelines and related materials - Good manufacturing practices and inspection. 2 (2nd updated ed.). WHO Press, pp. 17-18. ISBN 9789241547086.

[0183] It is to be noted that the term "a" or "an" entity refers to one or more of that entity.

[0184] ‘about’ means + / - 10% of the value given, + / - 9%, + / - 8%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1%, unless otherwise stated.

[0185] Mobilisation Step

[0186] In all aspects of the invention, the subject is treated with G-CSF prior to performing apheresis, preferably leukapheresis, on the same subject. The step of treatment with G-CSF is referred to herein as mobilisation. Suitably, mobilisation with G-CSF is performed before leukocytes are harvested from the subject using a leukapheresis process, and before monocytes are isolated from the leukocytes obtained following leukapheresis. The G-CSF mobilisation step and the leukapheresis are therefore performed on the same subject. In preferred embodiments, the subject on which the mobilisation and leukapheresis are performed is the same subject to whom the macrophages are subsequently administered. In such embodiments, the macrophages which are administered to the subject are autologous to the subject. In other embodiments, the macrophages may be allogeneic to the subject. In embodiments where the macrophages are allogeneic to the subject, the macrophages are preferably hypoimmunogenic.

[0187] In some embodiments, the G-CSF which is administered to the subject during the mobilisation step is recombinant G-CSF. In some embodiments, the recombinant G-CSF is selected from Lenograstim, Filgrastim, Pegfilgrastim and Lipegfilgrastim. In preferred embodiments, the G- CSF is Filgrastim. In some embodiments, the Filgrastim is selected from Zarzio, Granix, Nivestim, Releuko, Ratiograstim, Accofil, Tevagrastim and Neupogen. In preferred embodiments, the Filgrastim is Neupogen. In some embodiments, the G-CSF is administered intravenously or subcutaneously. Preferably, the G-CSF is administered subcutaneously.

[0188] In some embodiments, one or more doses of G-CSF are administered to the subject. In some embodiments, one, two, three, four or five doses of G-CSF are administered to the subject. In some embodiments, multiple (more than one) doses of G-CSF are administered to the subject. In preferred embodiments, 4 or 5 doses of G-CSF are administered to the subject. In a preferred embodiment, 5 doses of G-CSF are administered to the subject.

[0189] In preferred embodiments, the G-CSF, preferably Filgrastim, is administered at a dose of lOpg / kg / day. In preferred embodiments, the doses are administered to the subject on consecutive days. In preferred embodiments, the dose of G-CSF is administered to the subject on four consecutive days. In preferred embodiments, the dose of G-CSF administered to a patient is at a concentration of 60pg / ml. In some embodiments, the unit dose of G-CSF is 30pg G-CSF in a solution with a volume of 0.5mL. In a preferred embodiment, the G-CSF is at a concentration of 60pg / ml, and is administered to the subject at a dose of lOpg / kg / day for 4 or 5 consecutive days. In an especially preferred embodiment, the G-CSF is Filgrastim, is at a concentration of 60pg / ml, and is administered to the subject subcutaneously at a dose of I Opg / kg / day for 5 consecutive days. According to some embodiments, a dose of lOpg of Filgrastim is equal to 1 million units. Suitably, the subject is treated with 1 million units of Filgrastim / kg / day. In an especially preferred embodiment, the G-CSF is Filgrastim, is at a concentration of 60pg / ml, and is administered to the subject subcutaneously at a dose of 1 million units / kg / day for 5 consecutive days.

[0190] Harvesting of Monocytes

[0191] In all aspects of the invention, leukocytes (including monocytes) are harvested from the subject by apheresis, after the mobilisation step in which the subject is treated with G-CSF. Apheresis refers a process in which whole blood is removed from a subject, components of whole blood are extracted, and components which are not extracted may be returned to the subject’s circulation. Leukapheresis is a specific form of apheresis in which white blood cells (leukocytes) are harvested, and other components of the whole blood may be returned to the subject. Cells harvested following an apheresis procedure are referred to herein as an apheresis product. The leukocytes harvested following a leukapheresis procedure are referred to herein as a ‘leukapheresis product’. In preferred embodiments, the apheresis is leukapheresis. Accordingly, as used herein, the terms “apheresis” and “leukapheresis” may be used interchangeably. Similarly, the terms “apheresis product” and “leukapheresis product” may be used interchangeably. In preferred embodiments, following harvesting of leukocytes in a leukapheresis process, monocytes are further isolated from the leukapheresis product. According to some embodiments, the monocytes are isolated from the leukapheresis product up to 72 hours following the leukapheresis process. In preferred embodiments, the apheresis takes place less than 24 hours after the final dose of G-CSF is administered to the subject.

[0192] In preferred embodiments, the monocytes, from which all therapeutic macrophages for a course of treatment are prepared are generated, are harvested from the patient in a single leukapheresis. As used herein, “course of treatment” refers to the completion of the dose regimen of therapeutic macrophages, as described below. For example, the course of treatment may preferably comprise up to 4 doses of 1 x 108macrophages. In preferred embodiments, the apheresis therefore allows harvest of greater than 4 x 108monocytes, in order to produce at least 4 x 108therapeutic macrophages for administration to the subject. Accordingly, in preferred embodiments, a single apheresis is performed on any particular subject during one course of treatment. The step of mobilisation with G-CSF increases the proportion and / or of monocytes circulating in the peripheral blood. Accordingly, the step of mobilisation has the advantage of increasing the number of monocytes which may be harvested in a single leukapheresis, thus reducing the need for the subject to undergo repeated rounds of apheresis. Producing therapeutic macrophages as described herein therefore may reduce the adverse effects associated with repeated apheresis.

[0193] Monocytes harvested from the subject may be characterised, such as at the point of harvesting or on the same day as harvesting. The monocytes present in the apheresis product after harvesting from the subject by apheresis and after processing to isolate CD14+ cells, may be referred to herein as “day 0” monocytes. Day 0 monocytes obtained from a subject previously treated with G-CSF may be referred to as mobilised day 0 monocytes. Comparable day 0 monocytes obtained by apheresis from a subject who was not treated with G-CSF prior to apheresis, may be referred to herein as non-mobilised day 0 monocytes.

[0194] Monocytes may be identified by the expression of CD14 and CD45, for example by flow cytometry. As shown in the data in Figure 1, the apheresis product harvested from subjects after a step of G-CSF mobilisation exhibits comparable expression of CD14 on CD14+CD45+ cells at the population level compared to non-mobilised apheresis product. Suitably, the population of cells harvested by apheresis expresses the same or a similar level of CD14 on CD14+CD45+ cells compared to non-mobilised apheresis product. References to “same”, ’’similar” and “comparable”, for example with respect to expression of markers, refers to two or more measurements of marker expression which are not significantly different from each other. A statistical test may be applied to determine whether measurements are statistically significantly different from each other. References to “significant” in the context of comparison, for example with reference to a value being significantly higher, indicates that there is a statistically significant difference between the compared values. Appropriate statistical tests are known to those skilled in the art.

[0195] In some embodiments, the method of producing macrophages, and specifically the step of obtaining monocytes from the subject, comprises a step of selecting and or isolating monocytes sites by selecting CD14+ cells, from the product prior to differentiating the monocytes into macrophages. For example, the isolation of monocytes may be by cell sorting of a population labelled with anti-CD14 antibodies, such as by fluorescence activated or immunomagnetic cell sorting, as appropriate.

[0196] As described above, and as would be understood by the skilled person, the monocytes obtained from a subject following mobilisation with G-CSF may comprise a greater proportion of immature monocytes compared to a population of non-mobilised monocytes. For example, at a population level, a greater proportion of mobilised day 0 obtained from the subject, may exhibit a cell surface phenotype associated with immature monocytes, compared to non-mobilised day 0 monocytes. Mobilisation may increase the proportion of cells, expressing low levels of CCR2 on their surface. In particular, mobilisation may increase the proportion of CD14+, CCR2i0Wmonocytes. Suitably, at a population level, mobilised day 0 monocytes may have reduced expression of CCR2 compared to non-mobilised day 0 monocytes. Suitably, at a population level, mobilised day 0 monocytes may have an increased proportion of CCR2i0Wmonocytes compared to non-mobilised day 0 monocytes. At a population level, mobilised day 0 monocytes may have a greater proportion of CD14+ CCR2i0Wcells compared to non-mobilised day 0 monocytes. For example, at a population level, mobilised day 0 monocytes may have a proportion of CD14+ CCR2i0Wcells of at least 2.5%, at least 5% at least 7.5%, or at least 10%. In particular, the day zero mobilised monocytes may have a proportion of CD14+ CCR2i0Wcells of at least 5%.

[0197] Incubation of monocytes in medium comprising CSF-1R agonist

[0198] In all aspects of the invention, monocytes harvested from a subject by apheresis (preferably leukapheresis) are incubated in a medium comprising an agonist of Colony Stimulating Factor 1 Receptor (CSF-1R). CSF-1R may also be referred to as CD115, CSF-1 receptor, and Feline McDonough Sarcoma (FMS). CSF-1R is a receptor tyrosine kinase, and M-CSF and IL-34 are known to be ligands for this receptor30.

[0199] As used herein, “agonist” refers to a substance which binds a receptor, and activates signalling through the receptor. Suitably, a CSF-1 R agonist binds the receptor and activates signalling by tyrosine phosphorylation. In preferred embodiments, the agonist selectively binds CSF-1R. By “selectively” binding CSF-1R, the agent has a greater affinity for CSF-1R than other molecules, such as ligands or receptors. Accordingly, said agent has a greater tendency to form stable complexes with CSF-1R, or molecules with extensive sequence similarity thereto, than other molecules. In preferred embodiments, the agonist specifically binds the CSF-1R. By “specifically” binding CSF-1R, the agent does not form stable complexes with molecules other than CSF-1R (or fragments thereof), or molecules with extensive sequence similarity thereto. In preferred embodiments, the CSF-1R agonist activates signalling through the receptor to the same degree as M-CSF or IL-34. In some embodiments, the monocytes are incubated in a medium comprising one agonist of CSF-1R. In some embodiments, the monocytes are incubated in a medium comprising more than one agonist of CSF-1R. In some embodiments, the monocytes are incubated in a medium comprising 2 agonists of CSF-1R. Preferably, the medium does not comprise GM-CSF.

[0200] In some embodiments, the monocytes are incubated in the medium comprising the CSF-1R agonist for 5-7 days. In some embodiments, the monocytes are incubated in the medium comprising the CSF-1R agonist for 5 days.

[0201] In some embodiments, the CSF-1R agonist is present in the medium at a concentration of 10- 500ng / ml. In some embodiments, the CSF-1R agonist is present at a concentration of 5- 300ng / ml. In some embodiments, the CSF-1R agonist is present at a concentration of lOOng / ml, 200ng / ml, 300ng / ml, 400ng / ml or 500ng / ml. In some embodiments, the CSF-1R agonist is present at a concentration of at least lOOng / ml, at least 200ng / ml, at least 300ng / ml, at least 400ng / ml or at least 500ng / ml.

[0202] In some embodiments, the monocytes are present in the medium at a concentration of 4 x 106cells / ml. In other embodiments, the monocytes are present in the medium at a concentration of 2 x 106 cells / ml. In some embodiments, the CSF-1R agonist is selected from the group consisting of: an agonist antibody, a small molecule agonist, a protein agonist, a peptide agonist and a combination thereof. As used herein, a small molecule agonist refers to an agonist with a molecular weight of less than 1000 Daltons. For example, a full-length protein or antibody agonist would not be considered a small molecule agonist.

[0203] In some embodiments, the protein agonist is a growth factor or a cytokine. In some embodiments, the growth factor or cytokine is selected from the group consisting of: M-CSF, IL- 34 and a combination thereof.

[0204] In some embodiments, the agonist of CSF-1R is M-CSF. In some embodiments, the agonist of CSF-1R is IL-34. In some embodiments, the agonist is a peptide fragment of M-CSF or IL-34. In some embodiments, the agonist is a fusion peptide comprising M-CSF and / or IL-34 or a peptide fragment of M-CSF and / or IL-34. Preferred peptide fragments comprise the domain that binds CSF-1R. In some embodiments, the CSF-1R agonist is an agonist antibody. In some embodiments, the CSF-1R agonist is a small molecule agonist. In some embodiments, the medium comprises M-CSF. In some embodiments, the medium comprises IL-34. In some embodiments, the medium comprises both M-CSF and IL-34.

[0205] In some embodiments, the medium comprises M-CSF at a concentration of 10-150ng / ml and IL- 34 at a concentration of 50-300ng / ml. In preferred embodiments, the medium comprises M-CSF at a concentration of lOOng / ml, and IL-34 at a concentration of 200ng / ml. In preferred embodiments, the medium comprises M-CSF at a concentration of at least lOOng / ml, and IL-34 at a concentration of at least 200ng / ml.

[0206] Suitably the medium is suitable for generating macrophages from monocytes. Suitably the medium may be a T-cell medium. Suitably the medium may be selected from: X-Vivo 10, X- Vivo 15, TexMACS, AIMv, RPMI, DMEM, and DMEM / F12. Suitably the medium is TexMACS (Miltenyi). Suitably the medium is serum-free. Suitably the medium is xenoprotein- firee. Suitably the medium is GMP-compliant. In a preferred embodiment, the medium is TexMACS GMP medium.

[0207] In some embodiments, the medium may comprise one or more further growth factors. Suitable factors include growth factors, polysaccharides, cytokines and chemokines. Suitably therefore the factors are growth factors. Suitably, the one or more factors are GMP-compliant. Incubation of monocytes in medium comprising IL-34

[0208] In some aspects of the invention, monocytes harvested from a subject by apheresis (preferably leukapheresis) are incubated in a medium comprising IL-34. In some embodiments, the medium comprises IL-34 and one or more further CSF-1R agonists. Preferably, the medium does not comprise GM-CSF.

[0209] In some embodiments, the concentration of IL-34 in the medium is 50ng / mL,100ng / mL, 200ng / mL or 300ng / mL. In some embodiments, the concentration of IL-34 in the medium is at least 50ng / mL, at least lOOng / mL, at least 200ng / mL or at least 300ng / mL.

[0210] In preferred embodiments, the concentration of IL-34 in the medium is at least 200ng / mL. In especially preferred embodiments, the concentration of IL-34 in the medium is at least 200ng / mL and the monocytes are present in the medium at a concentration of 4 x 106cells / ml. In other words, the IL-34 was at a concentration of 50 ng I 1 x 106cells.

[0211] Suitably the medium is suitable for generating macrophages from monocytes. Suitably the medium may be a T-cell medium. Suitably the medium may be selected from: X-Vivo 10, X- Vivo 15, TexMACS, AIMv, RPMI, DMEM, and DMEM / F12. Suitably the medium is TexMACS (Miltenyi). Suitably the medium is serum-free. Suitably the medium is xenoprotein- firee. Suitably the medium is GMP-compliant. In a preferred embodiment, the medium is TexMACS GMP medium.

[0212] In some embodiments, the medium may comprise one or more further growth factors. Suitable factors include growth factors, polysaccharides, cytokines and chemokines. Suitably therefore the factors are growth factors. Suitably, the one or more factors are GMP-compliant.

[0213] Incubation of Monocytes in M-CSF

[0214] In aspects of the invention, monocytes harvested from a subject by apheresis (which are preferably isolated from leukocytes harvested by leukapheresis) are incubated in medium comprising M-CSF in order to differentiate the monocytes into macrophages. In a preferred embodiment, the incubation in medium comprising M-CSF produces mature, functional macrophages, as described in more detail below.

[0215] Monocytes are most commonly cultured with either M-CSF or GM-CSF in order to differentiate them into macrophages. Culturing monocytes with GM-CSF skews them towards an “inflammatory” phenotype, whereas culturing monocytes with M-CSF or another CSF-1R agonist such as IL-34, skews them towards a “pro-restorative” phenotype, or a phenotype whereby they may be easily polarised to a pro-restorative phenotype. Thus, as M-CSF or another CSF-1R agonist such as IL-34 is used as the growth factor to generate macrophages in any of the methods of the invention, it is preferred that the medium does not also comprise GM-CSF. Of note, as described herein above, macrophages differentiated using M-CSF or GM-CSF or any CSF-1R agonist, without further exposure to other factors, are considered non-polarised macrophages.

[0216] Suitably the macrophages are produced in vitro from the monocytes obtained from the subject by a culturing method lasting between 3 to 8 days optionally 4 to 8 days. Suitably the macrophages are produced in vitro from monocytes by a culturing method lasting between 3 to 7 days, notably 4 to 7 days, or 5 to 7 days. In some embodiments, the monocytes are incubated in the medium comprising M-CSF for 5-7 days. In a preferred embodiment, the macrophages are incubated in the medium comprising M-CSF for 5 days. In another embodiment, the macrophages are produced in vitro from monocytes by a culturing method that lasts 3-5 days, 4 or 5 days, or 7 days, known as a day 5 method or a day7 method, respectively. One example of an in vitro method of producing macrophages from monocytes is described in WO2019 / 175595. The ‘day5’ method is described in application number PCT / GB2021 / 051294 (the contents of which is herein incorporated by reference).

[0217] Suitably, the macrophages may be produced by a ‘day5’ method comprising:

[0218] (a) Culturing monocytes in medium for 3 - 5 or 4 - 5 days to produce macrophages, wherein the medium comprises M-CSF and optionally one or more further growth factors to stimulate macrophage production; wherein step (a) may take place entirely in the same medium.

[0219] In aspects of the invention, the medium comprises M-CSF. Suitably the medium contains M- CSF at a concentration of between 10 and 150ng / ml, such as 25-150ng / mL. In some embodiments, the concentration of the M-CSF in the medium is between about 10 ng / ml to about 100 ng / ml. More preferably, the concentration of M-CSF in the medium is between about 50 ng / ml to about 100 ng / ml. According to preferred embodiments, the concentration of the M-CSF in the medium is lOOng / mL or about lOOng / mL. In a preferred embodiment, the medium comprises lOOng / mL M-CSF. In some embodiments, the M-CSF is GMP -graded recombinant human macrophage colony-stimulating factor 1 (rhM-CSF-1; also known as ‘rh (recombinant human) CSF-1’). Suitably the medium contains 100 ng / mL GMP-graded recombinant human macrophage colony-stimulating factor 1 (rhM-CSF-1; also known as ‘rh (recombinant human) CSF-1’). In a preferred embodiment, the monocytes are incubated in a medium comprising lOOng / mL GMP-graded recombinant human macrophage colony-stimulating factor 1 for 5 days.

[0220] Suitably the medium is suitable for generating macrophages from monocytes. Suitably the medium may be a T-cell medium. Suitably the medium may be selected from: X-Vivo 10, X- Vivo 15, TexMACS, AIMv, RPMI, DMEM, and DMEM / F12. Suitably the medium is TexMACS (Miltenyi). Suitably the medium is serum-free. Suitably the medium is xenoprotein- firee. Suitably the medium is GMP-compliant. In a preferred embodiment, the medium is TexMACS GMP medium.

[0221] In some embodiments, the medium may comprise one or more further growth factors. Suitable factors include growth factors, polysaccharides, cytokines and chemokines. Suitably therefore the factors are growth factors. Suitably, the one or more factors are GMP-compliant.

[0222] Characteristics of Macrophages

[0223] In some embodiments, the monocytes may be incubated in M-CSF, or any agonist of CSF- 1R, until the macrophages produced are mature and functional. In preferred embodiments, the macrophages administered to the subject are mature and functional, and differentiation may be continued until the desired phenotype is acquired. “Functional “macrophages as referred to herein are capable of carrying out the normal functions of a macrophage, including phagocytosis and ability to be polarised upon exposure to polarising stimuli.

[0224] In some embodiments, the maturation state and / or functionality of the macrophages is assessed before the incubation in M-CSF, or the agonist of CSF-1R, is ended. Assessing macrophages maturation and / or functionality may comprise determining macrophage surface phenotype, such as by flow cytometry, and / or by functional assays, such as a phagocytosis assay as described in the Examples. If the macrophages do not exhibit a mature phenotype or are not functional, incubating in medium comprising M-CSF, or the agonist of CSF-1R, may be continued until the macrophages are mature and functional. In some embodiments, macrophage maturation and / or functionality may be determined by an at least 5-fold increase in CD206 and / or 25F9 expression compared to day 0 monocytes. In some embodiments, the macrophages produced by the method of the invention have at least 5-fold increased expression of CD206 and / or 25F9 compared to the monocytes harvested from the subject.

[0225] In some embodiments, the macrophages produced by the method of the invention have at least 10-fold increased expression of CD206 and / or 25F9 compared to the day 0 monocytes. In some embodiments, the macrophages produced by the method of the invention have at least 20-fold, at least 30-fold, at least 40-fold or at least 50-fold increased CD206 expression compared to the day 0- monocytes. In some embodiments, the macrophages produced by the method of the invention have at least 10-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold or at least 800-fold increased expression of 25F9 compared to the day 0 monocytes.

[0226] In some embodiments, the macrophage produced from mobilised monocytes has the same, similar, or comparable expression of CD 14, compared to macrophages produced from nonmobilised monocytes. The macrophage may have the same, similar or comparable expression of 25F9 compared to non-mobilised macrophages. Alternatively, the macrophage may have reduced expression of 25F9 compared to non-mobilised macrophages.

[0227] In some embodiments, the functionality of the macrophage may be assessed by determining its phagocytic capacity. Phagocytic capacity may be assessed by any method known in the art, such as by pHrodo assay, as described in the Examples. As noted above, mobilisation with G-CSF increases the proportion of immature monocytes with reduced CCR2 expression collected from subjects when performing apheresis. Immature cells with reduced CCR2 expression are associated with reduced phagocytic capacity. It was therefore suspected that mobilised monocytes will not give rise to macrophages that have an equivalent phagocytic capability to that of macrophages generated from non-mobilised monocytes. However, the inventors surprisingly found that such manufacturing of macrophages results in macrophages with therapeutic functionality, including equivalent phagocytic capacity to macrophages produced from non-mobilised monocytes. Accordingly, in some embodiments, the macrophages produced by the method of the invention have the same or a similar phagocytic capacity compared to nonmobilised macrophages (macrophages differentiated from monocytes in M-CSF, or the agonist of CSF-1R, wherein the monocytes were harvested from a subject not previously mobilised with G-CSF). In preferred embodiments, the macrophages produced by the method of the invention have at least an equivalent phagocytic capacity to non-mobilised macrophages. In some embodiments, the functionality of the macrophage may be assessed by determining whether it is capable of being polarised to a pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype, which may also be described as “M2”, or “M2-like”. As would be understood by the skilled person, there is a spectrum of macrophage phenotypes and classification of phenotypes in a binary M1 / M2 paradigm is an oversimplification. Nonetheless, the macrophage produced by the method of the invention is not an Ml, Ml-like, pro-inflammatory and / or pro-fibrotic macrophage. Instead, the macrophage produced by the method of the invention is capable of being polarised to an M2, M2 -like, pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype. In particular, the macrophage may be capable of being polarised to a pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype in response to IL-4, IL-10 and / or IL-13.

[0228] In some embodiments, the method further comprises incubating the macrophages in IL-4, IL-13 and / or M-CSF. Such a step may polarise the macrophage to a pro-restorative phenotype, as described above. Additionally, this step improves cryoresilience of the macrophage product. As used herein, the term “cryoresilience” (can also be referred to as “recovery”) refers to the survival rate of macrophages following cryopreservation, optionally as measured by the percentage of viable cells post cryogenesis out of the macrophages that have been cryopreserved. In some embodiments, the concentration of IL-4 and IL- 13 in the medium are 20ng / ml, the concentration of M-CSF is lOOng / ml, and the macrophages are at a concentration of 4 x 106cells / ml. In some embodiments, the cells are incubated overnight in the medium comprising IL-4, IL-13 and / or M-CSF.

[0229] In some embodiments, the macrophage may be polarised following engineering to overexpress polarising factors, such as IL- 10 (and optionally further MMP9). Such engineered cells are described in detail herein, and are capable of self-polarisation. In other words, the overexpression of the polarising factors by the engineered macrophage causes the cell to be polarised to exhibits a M2, M2 -like, pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype.

[0230] The macrophage produced according to the method of the invention is unpolarised. In some embodiments, the macrophage produced by the method of the invention exhibits a prorestorative, anti-inflammatory and / or anti-fibrotic properties, even before undergoing a further step of polarisation.

[0231] In some embodiments, macrophages which have been differentiated using IL-34 and subsequently polarised to a pro-restorative phenotype, have a comparable surface phenotype and function to macrophages differentiated using M-CSF and subsequently polarised to a prorestorative phenotype.

[0232] Whether a macrophage exhibits an M2, M2 -like, pro-restorative, anti-inflammatory and / or anti- fibrotic phenotype may be determined at least in part by examining surface marker expression. In some embodiments, a pro-restorative phenotype may be described using one or more of the following markers: an increase CD206 and / or CD163, a decrease in inflammatory markers such as CD86 and / or MHC class II (HLA-DR). These increases / decreases are as compared to nonpolarised (resting) or pro-inflammatory macrophages. In terms of secretion profile, these macrophages are expected not to express TNFa, IFNg and ILlb, normally associated with a pro- inflammatory and pro-fibrotic profile. In preferred embodiments, the engineered macrophage expresses CD206 at a 5-fold greater level than non-engineered, non-polarised cells, such as those described in WO2019 / 17559.

[0233] Accordingly, the macrophages produced by the method of the invention, or said macrophages following a further step of polarisation and / or engineering, preferably have an increased expression of CD 163 and CD206, and a reduced expression of HLA DR and CD86 compared to cells not polarised to a pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype. In further preferred embodiments, the macrophages produced by the method of the invention, or said macrophages following a further step of polarisation and / or engineering, do not secrete TNFa, IFNg, ILlb, IL-12p70 and / or IL-2. In preferred embodiments, the macrophages secrete less than 40pg / ml TNFa. In some embodiments, the macrophages secrete the same or similar levels of TNFa, IFNg, ILlb, IL-12p70 and / or IL-2 as non-engineered, non-polarised cells.

[0234] In some embodiments, the macrophages produced by the methods of the invention, following a further step of polarisation to a pro-restorative phenotype using IL- 10, have a reduced expression of HLA DR and CD86 relative to before the polarisation step. In some embodiments, following the further step of polarisation, the expression of HLA DR and CD86 on the macrophage is reduced by at least 50% relative to before the polarisation step. In some embodiments, following the further step of polarisation, the expression of HLA DR and CD86 on the macrophage is reduced by at least 75% relative to before the polarisation step. In some embodiments, following the further step of polarisation, the reduction of expression of HLA DR and CD86 on the macrophage produced according to the invention is the same as the reduction of expression of HLA DR and CD86 on a non-mobilised macrophage which has undergone the same polarisation step. In some embodiments, following the further step of polarisation, the reduction of expression of HLA DR and CD86 on the macrophage produced according to the invention is greater than the reduction of expression of HLA DR and CD86 on a non-mobilised macrophage which has undergone the same polarisation step. In some embodiments, following a further step of polarisation with IL-10, the macrophage produced by the method of the invention expresses HLA DR and CD86 at a lower level than a non-mobilised macrophage which has undergone the same further step of polarisation.

[0235] Administration of Therapeutic Macrophages

[0236] In accordance with aspects of the invention, the macrophages are administered to the subject. Administration of the macrophages to the subject may comprise delivering the engineered macrophage to a subject by systemic administration, suitably by systemic injection, preferably intravenously. Peripheral vein injection is favoured for liver conditions in order to avoid invasive procedures in cirrhotic patients, for example. Local injection, such as renal artery for kidney conditions may be better tolerated.

[0237] In accordance with aspects of the invention, the macrophages are administered to the subject. In preferred embodiments, the macrophages administered to the subject are autologous to the subject. In such embodiments, the subject who is treated with G-CSF and from whom the monocytes are harvested by apheresis is the same subject to whom the macrophages are administered. In other words, it is preferred that the macrophages administered to the subject are derived from monocytes obtained from the same subject.

[0238] In some embodiments, one or more doses of the therapeutic macrophages are administered to the subject. In a preferred embodiment, up to four doses of the therapeutic macrophages are administered to the subject. Suitably, one, two, three or four doses may be administered to the subject. In preferred embodiments, 4 doses are administered to the subject. In preferred embodiments, all doses of the engineered macrophages are derived from monocyte obtained from a single apheresis.

[0239] The therapeutic macrophages may preferably be formulated as a dispersion for infusion, such as intravenous infusion. In some embodiments, the macrophages may be administered at between 70 and 130 million macrophages (i.e. 7 x 107and 1.3 x 108macrophages) per dose. In preferred embodiments, the therapeutic macrophages are administered at a dose of 1 x 108macrophages. The engineered macrophages as administered to the subject may be formulated in a pharmaceutical composition additionally comprising one or more pharmaceutically acceptable excipients. In embodiments where multiple doses are administered to the subject, the doses are preferably administered at 6 week intervals. In a preferred embodiment, the engineered macrophages or pharmaceutical composition comprising the engineered macrophages are administered to the subject in 4 doses of 1 x 108macrophages with 6 weeks between doses (i.e. at 6 week intervals).

[0240] Suitably, the invention may also provide a pharmaceutical composition which may be used in any of the methods of the invention. The pharmaceutical composition may comprise the engineered macrophage and one or more pharmaceutically acceptable excipients. The pharmaceutical composition may have the following formulation of excipients: 33.13% saline, 0.17% human serum albumin, 66.7% Cryostor-10, such that the final concentration of DMSO may be 6.7%. The pharmaceutical composition may be frozen.

[0241] Methods of Treating Inflammatory Conditions The macrophage produced by the methods of the invention may be used in therapy, most notably cellular therapy, to a subject in need thereof. The subject may have a condition, disease or disorder that would benefit from the administration of the macrophages of the present invention.

[0242] The macrophages produced according to the method described herein may be useful an antiinflammatory, pro-restorative and / or anti-fibrotic phenotype. Alternatively, the macrophages produced by the methods described herein may be capable of being further polarised to an antiinflammatory, pro-restorative and / or anti-fibrotic phenotype. Accordingly, the macrophages produced by the method of the invention may be particularly applicable to the treatment of inflammatory conditions.

[0243] Suitably, such a condition, disease or disorder may have an inflammatory and / or fibrotic element. Such condition, disease or disorder may be acute or chronic, or acute-on-chronic. Such condition, disease or disorder may result in organ damage.

[0244] In some embodiments, the condition, disease or disorder in a subject is a chronic inflammatory condition with a fibrotic element. In some embodiments, the condition is chronic organ damage associated with chronic inflammation. In some embodiments, the condition is an acute inflammatory condition. In some embodiments, the condition is an acute-on-chronic inflammatory condition.

[0245] In some embodiments the condition may be Acute-on-chronic liver failure (ACLF). ACLF is a syndrome characterised by acute decompensation of chronic liver disease associated with organ failures and high short-term mortality. An excessive systemic inflammatory response seems to play a crucial role in the development of ACLF.

[0246] In some embodiments, the condition may be liver injury. In preferred embodiments, the liver injury is a chronic liver injury, optionally an inflammatory liver injury. In preferred embodiments, the inflammatory liver injury has a fibrotic element. In preferred embodiments, the condition is a chronic inflammatory liver injury with a fibrotic element, preferably liver cirrhosis.

[0247] Cirrhosis represents the end-stage of chronic liver injury and progressive fibrosis (scarring), irrespective of the underlying aetiology. It is characterised by severe liver fibrosis leading to architectural disruption, hepatocyte dysfunction and portal hypertension. Various aetiologies may lead to liver cirrhosis. Hepatic disorders having a fibrotic component which may lead to fibrosis include, but are not limited to, non-alcoholic fatty liver disease (NAFL) (e.g., nonalcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)) or alcoholic liver disease (e.g., alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH)).

[0248] The aetiologies leading to fibrosis may include, but are not limited to, steatotic liver disease (SLD), such as metabolic dysfunction-associated steatotic liver disease (MASLD), Metabolic- associated steatohepatitis (MASH) or Met-ALD. In some instances, the cause of fatty liver disease may be unknown, and may be termed cryptogenic SLD.

[0249] Metabolic dysfunction-associated steatotic liver disease refers to a non-alcoholic fatty liver disease, and therefore may also be known as NAFLD. Metabolic-associated steatohepatitis refers to a more severe form of MASLD, which may also be known as NASH. “Met-ALD” refers to individuals who have steatotic liver disease and who also drink alcohol. “Cryptogenic SLD” refers to SLD whose cause is unknown, such as in individuals who do not carry any known metabolic risk factors for SLD.

[0250] According to some embodiments, the aetiology of liver disease is selected from at least one of: (1) pure metabolic dysfunction-associated steatotic liver disease (MASLD) (i.e. not Met-ALD) , or (2) metabolic and alcohol related / associated liver disease (Met-ALD), or (3) HCV sustained virologic response (HCV SVR) with ongoing cirrhosis, or (4) alcohol-related liver disease (ALD or Met-ALD) only if patient is confirmed to not be drinking alcohol above Met-ALD limits (i.e. does not show alcohol misuse) and has a value of <25 mg / 1 in a PEth alcohol test. Patients with ‘pure’ ALD (i.e. not Met-ALD) only if they are confirmed to not be drinking alcohol above Met- ALD / alcohol misuse limits defined in this protocol AND have negative PEth test. Alcohol misuse (i.e. the threshold of alcohol consumption above which the aetiology of liver disease may be attributed to alcohol -related liver disease) is defined as an alcohol intake of greater than > 14 units / week or more than more than 2 units / day for females and 3 units / day for males. One unit is equivalent to 8 g of alcohol: a half-pint (-240 mL) of beer, 1 glass (125 mL) of wine or 1 (25 mL) measure of spirits.

[0251] Fibrotic diseases, disorders, and conditions can include mechanical trauma, biliary obstruction, autoimmune hepatitis, iron overload, Hepatitis B infection (HBV), and / or Hepatitis C infection (HCV). However, the engineered macrophages according to the present invention are able to treat cirrhosis irrespective of the underlying aetiology.

[0252] Cirrhosis may be either compensated or decompensated cirrhosis (also referred to herein as hepatic decompensation or HD). Decompensated cirrhosis is defined as an acute deterioration in liver function in a patient with cirrhosis and is characterised by symptoms such as, but not limited to, jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, variceal haemorrhage, gastrointestinal haemorrhage and any combination thereof. In other embodiments, the liver disease is decompensated cirrhosis. In some embodiments, the liver disease is compensated cirrhosis. In some embodiments, the patient has compensated cirrhosis and had at least one event (or exactly one event) of decompensated cirrhosis.

[0253] In some embodiments, the patient has a MELD score of between 10 and 18, or more preferably 10-16 or 12-18. In a preferred embodiment, the patient has a MELD 3.0 score of between 12-20, optionally between 12-18, optionally between 14-20.

[0254] In some embodiments, the liver disease is one in which cirrhosis is caused by damage to hepatocytes, for example, is a hepatocyte-derived disease, such as those diseases of viral origin (including treated (sustained viral response) hepatitis C (HCV), hepatitis B), damage through alcoholism (alcohol related liver disease (ALD)), or non-alcoholic fatty liver disease (NAFLD), including Non-alcoholic steatohepatitis (NASH) (including NASH resulting from diabetes or obesity), cryptogenic cirrhosis, hemochromatosis or alpha- 1 -antitrypsin deficiency. In some embodiments, the underlying aetiology has been removed (for example, a patient suffering from damage through alcoholism is no longer drinking, or a patient suffering from damage through HCV no longer has HCV etc.). In some embodiments, the patient with liver disease is at risk of end stage renal disease.

[0255] In some embodiments, the liver disease is steatotic liver disease (SLD). In some embodiments, the steatotic liver disease is metabolic dysfunction-associated steatotic liver disease (MASLD), is Met-ALD or cryptogenic SLD. In some embodiments, the metabolic dysfunction-associated steatotic liver disease (MASLD) is Metabolic-associated steatohepatitis (MASH).

[0256] Cirrhosis may lead to acute-on-chronic liver failure (ACLF). In some embodiments, the liver disease is ACLF. ACLF is a distinct condition from hepatic decompensation. Hepatic decompensation is characterised by the development of ascites, hepatic encephalopathy, gastrointestinal haemorrhage, or any combination of these conditions in patients with liver cirrhosis. ACLF in contrast is associated with organ failures and carries high short-term mortality in excess of 15% at 28 days. Three major features characterise this syndrome: ACLF occurs in the context of intense systemic inflammation; ACLF frequently develops in close temporal relationship with pro-inflammatory precipitating events (e.g. infections or alcoholic hepatitis); and ACLF is associated with single- or multiple-organ failure. In some embodiments, the engineered macrophage is used in the treatment of cirrhosis in a subject with ACLF.

[0257] A diseased patient suitable for a treatment or use in accordance with any aspect or embodiment of the invention may be a patient with a relevant disease and severity.

[0258] In some embodiments, the subject has undergone their first hepatic decompensation event. As used herein, a “first hepatic decompensation event” or a “first decompensation event” refers to a decompensation event that is not preceded by an earlier decompensation event in the preceding six months. For example, the first decompensation event may not be preceded by an earlier decompensation event in the preceding 12 months. In certain embodiments, the first decompensation event is not preceded by any earlier decompensation event in the subject and in such embodiments, the subject has never previously suffered an earlier decompensation event before the first decompensation event.

[0259] In other embodiments, the subject has intractable ascites (also referred to herein as Medically Refractory Ascites) which began (second large volume paracentesis) within six months prior to treating with the engineered macrophages. As used herein, the term “Refractory ascites” or “Medically refractory ascites” refers to ascites that cannot be mobilised or that recurs such that it requires repeated (> 2) large volume paracentesis, LVP (i.e. therapeutic, not diagnostic) at least once per 8 weeks, in which case the onset date of the refractory ascites is defined as the date of the second LVP. As used herein, the term “intractable ascites” which requires repeated (>1) large volume paracentesis (therapeutic, not diagnostic) despite best medical attempts to control ascites by the use of diuretics and dietary sodium restriction. As used herein, the term “Recurrent ascites” refers to an ascites that recurs (i.e. a second large volume paracentesis) within a six month period.

[0260] In preferred embodiments, the subject has been hospitalised following their first hepatic decompensation event. In other embodiments, the subject has undergone a hepatic decompensation event not preceded by an earlier decompensation event in the preceding months. The subject may exhibit, or more preferably may have recovered from one or more clinical signs of hepatic decompensation selected from the list consisting of one or more clinical signs of hepatic decompensation selected from the group consisting of ascites, hepatic encephalopathy, variceal bleed, hepatorenal syndrom e / acute kidney injury (HRS / AKI), Spontaneous Bacterial Peritonitis (SBP) or a combination thereof. The cells, compositions and methods of the invention are expected to be particularly effective in treating patients that have been hospitalised following their first hepatic decompensation event. Furthermore, the data provided in the examples demonstrate that the cells, compositions and methods of the invention are suitable for treating these particular patients, which have severe disease that is difficult to treat.

[0261] Hospitalisation following a hepatic decompensation event is a measure of disease severity and provides a specific clinical situation. Certain symptoms of hepatic decompensation are similar to those of less severe liver cirrhosis, but when a patient is hospitalised following their first hepatic decompensation event, or their first hepatic decompensation event requiring hospitalisation in six months, this indicates that their diseases is severe enough to particularly benefit from the present invention. Accordingly, in certain embodiments, the invention provides cells and compositions for use in a method of treating a patient exhibiting one or more clinical signs of hepatic decompensation selected from the list consisting of ascites, hepatic encephalopathy, variceal bleed, hepatorenal syndrom e / acute kidney injury (HRS / AKI), Spontaneous Bacterial Peritonitis (SBP) or a combination thereof, where the one or more clinical signs require hospital admission. Hospitalisation refers to admission to a hospital for treatment. Hospitalisation thus normally requires that the patient stays in the hospital for at least 24 hours. Hospitalisation is a measure of disease severity, because symptoms cannot be managed outside of the hospital setting.

[0262] Hospitalisation is a recognised measure of disease severity and patient status in the context of liver cirrhosis (Balcar et al., United European Gastroenterol J. 2021; 9(4): 427-437.)

[0263] In some embodiments, the invention provides engineered macrophages and compositions comprising the same for use in a method of treating a patient diagnosed as having liver cirrhosis; optionally wherein the patient has an aetiology of liver disease of: (1) pure metabolic dysfunction- associated steatotic liver disease (MASLD) or metabolic and alcohol related / associated liver disease (Met-ALD), or (2) HCV sustained virologic response (HCV SVR) with ongoing cirrhosis, or (3) alcohol -related liver disease (ALD or Met-ALD) only if patient is confirmed to not be drinking alcohol at or above alcohol misuse levels and has a value of <25 mg / 1 in a PEth alcohol test; wherein the patient:

[0264] (a) has been hospitalised for the first time in six months as an inpatient for a major hepatic decompensation event defined as either ascites, hepatic encephalopathy (HE), a variceal bleed, hepatorenal syndrom e / acute kidney injury (HRS / AKI), Spontaneous Bacterial Peritonitis (SBP) or a combination thereof; or

[0265] (b) has intractable ascites (also referred to herein as Medically Refractory Ascites) which began (second large volume paracentesis) within six months prior to treating with the engineered macrophages.

[0266] Phosphatidylethanol (PEth) is a serum biomarker that can detect alcohol use within the last 28 days with excellent sensitivity and specificity. In preferred embodiments, the subject is administered with the engineered macrophages only if they have been determined to be clinically stable following their qualifying event, preferably within 6 weeks of the qualifying event, and providing that they have remained clinically stable prior to administration.

[0267] As used herein, “clinically stable” (also referred to herein as “recompensated”) is defined as either or both of:

[0268] (i) a decrease in Model for End Stage Liver Disease 3.0 score in 2 separate MELD score assessments (within one point or more of each other) taken within 2 weeks of each other; and

[0269] (ii) the subject being assessed by a physician, including documented review of MELD, as sufficiently stable to safely undergo cell mobilisation (with G-CSF, such as Filgrastim), to be subjected to apheresis and / or to be able to be treated with the engineered macrophages, without substantial risk to patient safety.

[0270] In some embodiments, the method comprises assessing the subject for indicators that treatment would be particularly beneficial to the subject, or the subject to be treated has been assessed for such indicators. In preferred embodiments, the subject has one or more of, or preferably all of, the following characteristics: no known contraindications for treatment with G-CSF (and in particular Filgrastim), no known contraindications for undergoing apheresis (and in particular leukapheresis); has been hospitalised as an inpatient for the first time for a hepatic decompensation event (qualifying event) defined by the presence of ascites, hepatic encephalopathy or variceal bleed, preferably within the six months prior to treatment or apheresis, or has been admitted as an outpatient for intractable ascites, preferably within the six months prior to treatment or apheresis; a MELD 3.0 score of 12-20 as measured within 2 weeks of the qualifying event requiring hospitalisation or as measured at admission for an outpatient subject; an aetiology of liver disease of pure metabolic dysfunction-associated steatoic liver disease (MASLD), or metabolic and alcohol related / associated liver disease (MetALD), or ‘pure’ ALD (i.e. not Met-ALD) without alcohol misuse and with a negative PEth test prior to administration of the engineered macrophages, or HCV sustained virologic response (HCV SVR) with ongoing cirrhosis; and diagnosis of liver cirrhosis based on at least one of (i) clinical and radiological features that correlate with a diagnosis of cirrhosis, (ii) a liver stiffness score as measured with transient elastography of greater than 15kPa, or (iii) a previous liver biopsy confirming histological features of cirrhosis. According to some embodiments, if the subject has intractable ascites then the intractable ascites is considered as the qualifying event.

[0271] Sustained virologic response refers to an absence of virus in serum for an extended period following a treatment program. Therefore, sustained virologic response may be determined by measuring viral load e.g. by quantifying viral RNA, in blood or serum over a period of time, such as 12 weeks, 3 months or 6 months following the completion of treatment. In some embodiments, sustained virologic response in a subject is defined as a viral load of less than 25 international units of RNA per millilitre of blood for 12 weeks, or as measured at 12 weeks, following the completion of treatment.

[0272] In some embodiments, the method comprises assessing the subject for co-morbidities and / or indicators of increased risk prior to treatment. In preferred embodiments, the subject does not have, or is not known to have and / or suspected to have, any combination of the following co-morbidities or indicators of increased risk at the start of treatment: liver cirrhosis due to any viral hepatitidies with the exception of HCV SVR; liver cirrhosis due to autoimmune and cholestatic aetiologies including, but not limited to, primary biliary cholangitis and primary sclerosing cholangitis; any current organ failure requiring more than outpatient supportive care which is not associated with the hepatic decompensation event (qualifying event); known splenomegaly, optionally wherein the largest dimension of the subject’s spleen is greater than or equal to 16cm; thrombocytopaenia, optionally wherein the thrombocytopaenia is defined by a platelet count of less than 50 x 109platelets / L; a history of liver or other organ transplantation, such as having undergone at least one previous liver or organ transplant; acute-on-chronic liver failure (ACLF); recent sepsis with positive microbial cultures or as defined by a doctor, unless stable and is at least 5 weeks after having completed full course of IV antibiotics; known infection with human immunodeficiency virus (HIV) or human T-Lymphotrophic Virus 1; pulmonary embolism ; hepatocellular carcinoma (HCC) or active malignant disease (with the exception of non-melanoma skin cancer, cervical carcinoma in situ, or superficial bladder cancer) within the 5 years preceding the start of treatment; co-hepatic morbidities such as hepatic hydrothorax (refractory to medical management) or portal vein thrombosis; chronic renal impairment, wherein the subject may require dialysis; unresolved acute kidney injury (AKI); acute heart failure; chronic heart failure; porto-pulmonary hypertension; severe chronic lung disease such as chronic obstructive pulmonary disease (COPD) or interstitial lung disease where forced expiratory volume in one second (FEV1) is less than 50% or 60%, and / or forced vital capacity (FVC) is less than 60%; hepatopulmonary syndrome; a history of chronic albumin treatment, or current chronic albumin treatment; significant untreated or unstable psychiatric disease; undergone a Transjugular intrahepatic portosystemic shunt (TIPS) procedure in the preceding 6 months; as judged by the Investigator, any evidence of intercurrent illness that is either life threatening or of clinical significance such that it might limit compliance with study procedures; current or planned use of immunomodulators or immunosuppressive medication (with the exception of low doses of corticosteroids up to 10 mg / kg / day prednisone or equivalent are permitted, or inhaled steroids to manage asthma, which are allowed); received a gene or cell therapy at any time; a known hypersensitivity to dimethyl sulfoxide (DMSO); is pregnant or breast-feeding or plans to become pregnant over the next year, or of childbearing potential and unwilling to comply with contraceptive requirements; has current or recurrent alcohol misuse in the three months prior to screening; and / or is taking non-medically supervised drugs of abuse that are judged (by the Investigator) to be a high risk to the participants acute health or which makes the participant likely to be non-compliant with follow-up . Additionally, the subject preferably is not judged by the investigator as unlikely to comply with study procedures, restrictions, and requirements. In preferred embodiments, the subject does not have any of the aforementioned co-morbidities or indicators of increased risk. In other embodiments, the subject does not have any evidence of intercurrent illness that is life threatening and / or has clinical significance to the subject’s liver cirrhosis.

[0273] In a preferred embodiment, the subject does not have any of the aforementioned co-morbidities or indicators of increased risk, the subject has undergone a first hepatic decompensation event which required hospitalisation, and has undergone an additional or subsequent hepatic decompensation event, and is treated following the additional or subsequent decompensation event.

[0274] Forced expiratory lung volume (FEV1) refers to the volume of air exhaled by an individual during a forced exhale, expressed as a percentage of a reference value. Forced vital capacity (FVC) refers to the maximum volume of air exhaled by an individual during a forced exhale, following a complete inhale. The reference value may be based on the measurement of forced expiratory volume in a population of healthy individuals i.e. an individual without a restrictive lung disease. As used herein, references to FEV1 or FVC expressed as a percentage are with respect to a reference value that would be expected for a healthy individual.

[0275] Thrombocytopaenia refers to the subject having a low platelet count, such as a platelet count lower than a reference sample taken from a healthy individual. A subject may be considered as having thrombocytopaenia if they have a platelet count lower than a given reference point, such as lower than 150,000 platelets / mm3, lower than 100,000 pl atelets / mm3or lower than 50,000 platelets / mm3. In particular embodiments, the subject may be considered to have thrombocytopaenia if they have a platelet count of less than 50,000 platelets / mm3.

[0276] In accordance with the methods described herein, the subject’s MELD score may be determined. The MELD score may be MELD, MELD 3.0 or MELD-Na, for example. MELD scores use three components; bilirubin (BIL), creatinine (CRE) and international normalized ratio (INR) which are measured from the patient, and used in a formula to generate a MELD score. Multiple variations on MELD scores are known in the art, as described for example in Kim et al., 2021.

[0277] MELD may be calculated as follows:

[0278] MELD= 3.78 X ln[Bilirubin (mg / dL)] + 9.57 X ln[Creatinine (mg / dL)] + 11.2 X [INR] + 6.43

[0279] MELD-Na uses the MELD score as calculated according to the preceding formula, and the serum sodium concentration (referred to as “Na”), where the serum sodium concentration (Na) is bound between 125 and 137 mmol / L, as defined by the Organ Procurement and Transplantation Network29.

[0280] MELD-Na may be calculated as follows:

[0281] MELD-Na = MELD + 1.32 x (137-Na) -[0.033 x MELD x (137-Na)]

[0282] In preferred embodiments, the MELD 3.0 score of the subject is determined within 2 weeks of the qualifying hepatic decompensation event requiring hospitalisation or as measured at admission for an outpatient subject. In a preferred embodiment, the subject has a MELD 3.0 score of between 10 and 20, or more preferably 10-16,12-18 or 12-20. In a most preferred embodiment, the subject has a MELD 3.0 score of 12-20.

[0283] MELD 3.0 may be calculated as follows: MELD 3.0 = 1.33 (if female subject) + [4.56 x In (bilirubin)] + [0.82 x (137 -Na)] - [0.24 x (137 - Na)xIn(bilirubin)] + [9.09 x In(INR)] + [11.14 x In(creatinine)] + [1.85 x (3.5 - albumin)] - [1.83 x (3.5 - albumin) x In(creatinine)] + 6.

[0284] The number produced by the formula for calculating MELD 3.0 is rounded to the nearest integer.

[0285] In some embodiments, the macrophage is autologous or allogenic to the subject. In all aspects of the invention, the macrophage is derived from a monocyte obtained from a subject. In preferred embodiments, mobilisation and apheresis are performed are the same subject to whom the macrophages are subsequently administered. In such embodiments, the macrophages which are administered to the subject are autologous to the subject. In other embodiments, the macrophages may be allogeneic to the subject. In embodiments where the macrophages are allogeneic to the subject, the macrophages are preferably hypoimmunogenic.

[0286] Engineered Macrophages

[0287] The macrophages produced by the methods of the invention are suitable for engineering, particularly for engineering to express IL-10 and / or MMP9. As described in Example 8 and shown in Figure 9, macrophages produced by the methods of the invention which are engineered to express IL-10 and MMP9 may secrete IL-10 and MMP9 at concentrations at least as great, if not greater than, non-mobilised macrophages which have been engineered to express IL-10 and MMP9 in the same way.

[0288] Accordingly, the method of producing macrophages may further comprise a step of engineering, in order to produce an engineered macrophage. In some embodiments, the macrophage is engineered to express IL- 10 and MMP9. The macrophages may be engineered to express these proteins by inclusion of exogenous coding sequences. Said exogenous coding sequences may be extrachromosomal or integrated into the cell’s genome. An engineered macrophage expresses the exogenous coding sequence(s) for IL- 10 and MMP9.

[0289] An ‘engineered macrophage’ is a macrophage that has been engineered to express a particular payload, such as IL-10 and / or MMP9. In particular, the expression is above endogenous levels, such that the engineered macrophages express e.g. IL- 10 and / or MMP9 at greater levels than non-engineered cells. The macrophages may be engineered to express these proteins by inclusion of exogenous coding sequences. In some embodiments, the macrophage secretes IL- 10 at a culture supernatant concentration of at least 10,000pg / ml when cultured in vitro at a cell concentration of 4xl06 / m. In some embodiments, the macrophage secretes MMP9 at a culture supernatant concentration of at least 200ng / ml when cultured in vitro at a cell concentration of 4xl06 / ml.

[0290] MMP9 (Matrix metallopeptidase 9) is a matrix metalloprotease, a type IV collagenase. MMP9 is also known as 92kDa type IV collagenase, 92 kDa gelatinase or gelatinase B (GELB). Matrix metalloproteases (MMPs), also known as matrix metalloproteinases or matrixins are a family of peptidases, collectively capable of cleaving all components of the extracellular matrix (ECM). MMPs are also able to process bioactive mediators, such as growth factors, cytokines, chemokines, and cell-surface receptors. 25 mammalian MMPs have been identified, with varying roles in the maintenance of the ECM and processes of tissue repair, and both inhibitory and stimulatory roles in fibrosis.

[0291] In some embodiments, the engineered macrophages carry one or more exogenous sequence that can turn on endogenous expression of IL- 10 and / or MMP9. Any macrophage that has been genetically modified by any means through an exogenous sequence (i.e., a sequence that is not a part of the natural macrophage genome) is an engineered macrophage according to the invention. Engineered Macrophages The step of engineering may polarise the macrophage to an M2, M2 -like, anti-inflammatory, pro-restorative and / anti-fibrotic phenotype. The engineered macrophage may be engineered to overexpress IL- 10. In certain embodiments, the macrophage secretes IL- 10 at a culture supernatant concentration of at least 10,000pg / ml when cultured in vitro at a cell concentration of 4xl06 / ml. As described herein, engineered macrophages overexpressing IL- 10 alone demonstrate a significant ability to recruit monocytes, anti-inflammatory secretome and to convert unpolarised or pro-inflammatory macrophages to a pro-restorative phenotype. The examples demonstrate that such a macrophage is useful in therapy, in particular for treating liver cirrhosis.

[0292] It is preferred that the macrophage for use in therapy is engineered ex vivo and delivered to the patient.

[0293] However, in some embodiments, the macrophage may be engineered in vivo. In some embodiments, the macrophage is engineered in vivo via the administration to a subject of a preparation of exogenous coding sequence(s) for IL- 10 and MMP9 suitable for transfecting a macrophage. The preparation may include any of the exogenous coding sequences discussed herein. Suitable delivery vehicles for in vivo engineering may include targeting molecules for macrophages by virtue of their cell surface markers. Should an in vivo transfection of macrophages be envisaged, localized application of the preparation may be more effective, such as localized injection of the preparation to liver or kidneys, or nebulization to the lungs. Such treatment may be prepared as nanoparticles to assist macrophage targeting.

[0294] In some embodiments, the method polarizes the host monocytes / macrophages to a prorestorative phenotype. In some embodiments, the method polarizes unpolarized host macrophages to an anti-inflammatory and / or pro-restorative phenotype. In some embodiments, the method polarizes inflammatory host macrophages to an anti-inflammatory and / or prorestorative phenotype. In some embodiments, the method comprises electroporation. In some embodiments, the macrophage is contacted with an anti-inflammatory treatment after electroporation.

[0295] The macrophages produced by, or for use in accordance with, the methods described herein may be engineered to overexpress IL- 10 and IL-4; IL- 10 and MMP12; IL-4, IL- 13 and MMP9; or IL- 4, IL-13 and MMP12.

[0296] Engineered macrophages for use according to any aspect of the invention may have particular structural and advantageous functional properties, as described herein.

[0297] In preferred embodiments, when exposed to non-engineered macrophages, the engineered or polarised macrophages polarise non-engineered macrophages to a pro-restorative phenotype. When administered to a subject, the engineered macrophages may polarise host macrophages (such as endogenous monocyte derived macrophages that migrate into the liver) to a prorestorative phenotype. In some embodiments, the engineered macrophage may convert unpolarised host macrophages to a pro-restorative macrophage. In some embodiments, the engineered macrophage may convert pro-inflammatory host macrophages to a pro-restorative macrophage. In some embodiments, conditioned medium from engineered macrophages may convert non-engineered macrophages to a pro-restorative phenotype. Accordingly, the engineered macrophages may convert non-engineered macrophages to a pro-restorative phenotype in vitro or in vivo. Conversion to a pro-restorative phenotype may increase the expression of CD206 and CD 163, and decrease the expression of CD86 and HLA-DR on the cell surface. In some embodiments the engineered macrophage has an at least two-fold reduced expression of CD86 compared to non-engineered, non-polarised cells. In some embodiments the engineered macrophage has an at least two-fold reduced expression of HLA-DR compared to nonengineered, non-polarised cells. In some embodiments the engineered macrophage has an at least 1000-fold increased secretion of IL-10 compared to non-engineered, non-polarised cells. In some embodiments the engineered macrophage has an at least 10-fold increased secretion of MMP3 compared to non-engineered, non-polarised cells. In some embodiments the engineered macrophage has an at least 20-fold increased secretion of MMP10 compared to non-engineered, non-polarised cells.

[0298] In some embodiments the macrophage secretes IL- 10 at a culture supernatant concentration of at least 10,000pg / ml when cultured in vitro at a cell concentration of 4xl06 / ml. In some embodiments the macrophage secretes MMP9 at a culture supernatant concentration of at least 200ng / ml when cultured in vitro at a cell concentration of 4xl06 / ml. In some embodiments the engineered macrophage has an at least 5-fold increased expression of CD206 compared to monocytes. In some embodiments engineered macrophage has an at least 5-fold increased expression of 25F9 compared to monocytes. In some embodiments the engineered macrophage has an at least ten-fold reduced expression of CD80 compared to non-engineered cells. In some embodiments the macrophage of the invention secretes TNF-a at a culture supernatant concentration of at least 40pg / ml when cultured in vitro at a cell concentration of 4xl06 / ml. In some embodiments the engineered macrophage has the same phagocytic ability as a nonengineered, non-polarised cell.

[0299] In one embodiment, the engineered macrophage comprises one or more exogenous coding sequences for IL- 10 and MMP9. Said exogenous coding sequence may be any suitable nucleic acid sequence. Said exogenous coding sequence may be present in the cytoplasm or nucleus as an extrachromosomal nucleic acid or integrated into the macrophage genome. Said exogenous coding sequence may encode IL- 10 and MMP9, or a plurality of exogenous coding sequences may each encode IL- 10 or MMP9.

[0300] In some embodiments, endogenous IL- 10 and / or MMP9 gene expression can be stimulated by genetic engineering. For example, gene editing techniques such as CRISPR can be used to turn on and off the endogenous genes that encode IL- 10 and or MMP9, generating an engineered macrophage that expresses IL-10 and / or MMP9 under conditions in which it would not otherwise express these proteins. This may be done by altering the promoter sequence, for example.

[0301] Natural non-engineered macrophages are capable of expressing IL-10 and / or MMP9 under relevant physiological conditions, but generally natural macrophages do not secrete significant levels of IL-10. However, “engineered macrophages” as described herein refer to macrophages wherein the expression level of IL-10 in particular has been raised to supra-physiological levels, thereby improving the anti-inflammatory properties of the therapeutic macrophages. As used herein, the inventors describe this as “overexpression” of IL-10. In order to overexpress the IL- 10, the macrophage may be engineered such that it possesses additional or exogenous coding sequences for IL- 10. In some embodiments, the cell is transfected with mRNA encoding IL- 10 / MMP9.

[0302] A cell which overexpresses IL- 10 and / or MMP9 comprises coding sequences expressing IL- 10 and / or MMP9 at a higher level than in non-engineered cells. As described above, overexpression may be achieved through the introduction of exogenous nucleic acid encoding IL- 10 and / or MMP9 such as mRNA, or genetic modification which stimulates expression of IL-10 and / or MMP9 from endogenous coding sequences. An engineered macrophage which overexpresses IL- 10 and / or MMP9 may not necessarily secrete a greater amount of IL-10 and / or MMP9 than a non-engineered macrophage.

[0303] In other embodiments, the macrophage may be engineered to turn on endogenous genes that encode IL-10 and / or MMP9. In any case, the macrophages of the disclosure have been modified through alterations in IL- 10 and / or MMP9 expression levels by any means and thus are referred to as engineered macrophages.

[0304] In over-expressing IL-10, the natural activity levels of the MMPs, including MMP9, fall.

[0305] Therefore, the engineered macrophage is provided with an additional or exogenous MMP coding sequence to maintain at least "physiological" levels of MMP9 expression, or levels just above physiological. In some embodiments, MMP9 expression is modified to rescue, restore or return the macrophages into expressing levels of MMP9 comparable or increased when compared to macrophages not transfected with IL- 10. Thus, despite the fact that the engineered macrophage is engineered with additional / exogenous MMP9 coding sequence, the engineered macrophage demonstrates MMP9 expression levels similar or slightly above wild-type / natural expression. Slightly above may mean an enhancement of expression over natural expression levels of between 1.2 and 1.5 times, such as 1.2, 1.3, 1.4 or 1.5 times the natural level.

[0306] The macrophage may be engineered to express both IL- 10 and MMP9. This expression may be driven from the endogenous genes in some embodiments. In other embodiments, the macrophage is engineered to contain exogenous coding sequence(s) for IL- 10 and MMP9. It may be preferred that the macrophage over-expresses IL-10. It may be preferred that the macrophage over-expresses MMP9. Alternatively, the macrophage may be engineered such that there is over-expression of both IL-10 and MMP-9. Expression levels of IL-10 and / or MMP9 are increased when compared to a non-transfected macrophage. Expression levels of MMP9 are increased when compared to a macrophage transfected with IL- 10 alone.

[0307] In some embodiments, the baseline macrophages (i.e., pre-engineered or natural) are referred to as unpolarised human monocyte-derived macrophages, also terms “resting” macrophages.

[0308] As used herein, over-expression relates to the artificial expression of a gene in increased quantity.

[0309] As referred to herein, expression levels of the proteins may be quantified at between 16 to 24 hours post-transfection. Expression levels as recited herein are given for a population of macrophages at a concentration of 4xl06 / ml (which equates to 2x106 cells per cm2). In the Examples, the macrophages were transfected, isolated by centrifugation, re-suspended in TexMACs buffer supplemented with IL-3 and IL-14, and incubated at 37°C under 5% CO2. Those skilled in the art would be aware of equivalent conditions suitable to determine secreted protein concentration.

[0310] In some embodiments, the macrophage is engineered to overexpress IL- 10, wherein the secreted IL-10 protein level is greater than about 300pg / ml. Suitably, the secreted IL-10 protein is greater than about: 300pg / ml or 400pg / ml or 500pg / ml or 600pg / ml or 700pg / ml or 800pg / ml or 900pg / ml or l,000pg / ml or 2,000pg / ml or 3,000pg / ml or 4,000pg / ml or 5,000pg / ml or 6,000pg / ml or 7,000pg / ml or 8,000pg / ml or 9,000pg / ml or 10,000pg / ml or l l,000pg / ml.

[0311] Suitably, these IL- 10 protein levels may be measured by culturing the macrophages as described above, wherein the concentration of macrophages in the medium is 4 x 106 cells / ml, which equates to2xl06cells / cm2, and measuring the concentration of the protein in the culture medium. In preferred embodiments, the macrophage secretes IL- 10 at a culture supernatant concentration of at least 10,000pg / ml, 15,000pg / ml or 20,000pg / ml, when cultured in vitro at a cell concentration of 4xl06 / ml. In a particularly preferred embodiment, the IL-10 is secreted at a culture supernatant concentration of 49,000 pg / ml or greater. In some embodiments, the engineered macrophage may secrete IL- 10 at levels 1000 fold greater than non-engineered, nonpolarised hMDMs. Non-engineered, non-polarised hMDMs are described in the art, for example in WO2019175595.

[0312] In some embodiments, the macrophage secretes IL- 10 at a culture supernatant concentration of at least 100,000pg / ml, at least 200,000pg / ml, at least 300,000pg / ml, at least 400,000pg / ml, at least 500,000 pg / ml or at least 600,000 pg / ml, wherein the IL- 10 protein levels may be measured by culturing the macrophages as described above, wherein the concentration of macrophages in the medium is 4 x 106cells / ml. In some embodiments, the macrophage secretes IL-10 at the same level as, or at a higher level than, a non-mobilised macrophage which has been engineered to express IL- 10 in the same way.

[0313] In some embodiments, the macrophage is engineered to express MMP9, wherein the secreted MMP9 protein level is greater than about 200ng / ml. Suitably, the secreted MMP9 protein is greater than about: 300ng / ml or 400ng / ml or 500ng / ml or 600ng / ml or 700ng / ml or 800ng / ml or 900ng / ml or l,000ng / ml. Suitably the secreted MMP9 protein level is between about 200ng / ml and 2000ng / ml. In preferred embodiments, the secreted MMP9 protein level is greater than 200ng / ml. In one embodiment, the secreted MMP9 protein level is greater than 1500ng / ml. Suitably the engineered macrophage (comprising IL- 10 and MMP9) has a secreted MMP9 protein level that is greater than the average level of secreted MMP9 protein in macrophages engineered with IL- 10 alone. In some embodiments, the engineered macrophage (comprising IL- 10 and MMP9) has a secreted MMP9 protein level that is at least equal to the average level of secreted MMP9 protein in non-polarised, non-transfected macrophages. Suitably, the overall MMP activity of the engineered macrophage for use according to the invention is also higher than the overall MMP activity of a macrophage engineered with IL- 10 alone. In other embodiments, the overall MMP activity of the engineered macrophage is greater than that of an untransfected macrophage. In a preferred embodiment, the MMP activity of the engineered macrophage is at least 1.5 times greater than that of an untransfected macrophage. Suitably, these IL- 10 and MMP9 protein levels may be measured -by culturing the macrophages as described above, wherein the concentration of macrophages in the medium is 4 x 106 cells / ml, which equates to2xl06cells / cm2, and measuring the concentration of the protein in the culture medium. In some embodiments, the engineered macrophage may secrete greater amounts of other matrix metalloproteases. In particular embodiments, the engineered macrophage may secrete Matrix Metalloproteinase-3 (MMP3) in 10-fold greater amounts than non-engineered, non-polarised cells. In particular embodiments, the engineered macrophage may secrete Matrix Metalloproteinase- 10 (MMP10) in 10-fold greater amounts than non-engineered, non-polarised cells.

[0314] In some embodiments, the macrophage secretes MMP9 at the same level as, or at a higher level than, a non-mobilised macrophage which has been engineered to express MMP9 in the same way.

[0315] In some embodiments, the macrophages that are engineered to express human IL- 10 and human MMP9 may be used in the treatment of ACLF.

[0316] In some embodiments, the macrophage has a pro-restorative phenotype and is anti-inflammatory and anti-fibrotic. Such a phenotype is defined further herein.

[0317] The macrophage engineered to express IL- 10 and MMP9 may be genetically engineered in any suitable way. For example, using viral or non-viral vectors, DNA or RNA constructs or gene editing using any suitable technology. Thus, in some embodiments, the macrophage may be engineered with one or more exogenous coding sequences. These exogenous coding sequences may encode IL- 10 and / or MMP9 and / or they may encode gene editing proteins such as CRISPR, nickases, and the like, that are capable of altering the cell’s genome to increase the expression of endogenous IL- 10 and / or endogenous MMP9. This could be achieved by editing the promoter or enhancer sequence, for example.

[0318] Known viral vectors for transfecting macrophages include lentivirus, adenoviruses and adeno- associated viruses (AAV).

[0319] As used herein, “coding” refers to the ability of sequences of nucleotides, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of macromolecules in biological processes such as a defined sequence of amino acids. Thus, a coding sequence may be any suitable nucleic acid sequence which provides the instructions to synthesise the relevant entity (e.g., IL-10 or MMP9). The coding sequences may be included in the same vector / construct or on different vectors / constructs.

[0320] As used herein, “exogenous” refers to any material, notably genetic material, introduced from or produced outside a particular cell. As used herein, in some embodiments, the exogenous coding sequence or engineered macrophage encode / express IL- 10 or MMP9. In some embodiments, the IL- 10 and / or MMP9 are human. It will be understood by those skilled in the art that variations to the sequence of these genes / coding sequences is also encompassed in this invention. Ideally, the genes / coding sequences are human. The genes / coding sequences may be codon optimised. The gene / coding sequences may be adjusted, and it will be appreciated if the reference sequence is RNA, then a different nucleotide may be present in a DNA vector.

[0321] In some embodiments, the macrophage is engineered using a nucleic acid vector. In some embodiments the macrophage is transfected via electroporation. Other suitable methods of transfection include nucleofection.

[0322] In some embodiments, the mRNA is delivered to the macrophage via nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles, which may be as described in, for example, U.S. Patent No. 8,058,069, U.S. Patent No. 8,492,359, U.S. Patent No. 8,822,668, U.S. Patent No. 9,364,435, U.S. Patent No. 9,504,651, and U.S. Patent No. 11,141,378.

[0323] The engineered macrophages may have use in therapy, e.g. for use in treating an inflammatory condition in a subject, and / or a fibrotic condition.

[0324] Preferably, administration of the engineered macrophage to a subject is not associated with an inflammatory response. In particular, administration of the engineered macrophage to a subject is preferably not associated with an increased concentration of inflammatory cytokines in the plasma, such as IL- lb and / or TNF-a.

[0325] Overexpression is understood as “excessive” or higher levels of expression of a gene, such as that caused by increasing the frequency of transcription of a gene. Thus, it may also be viewed as above wild type or normal levels of expression. Overexpression can be defined with reference to the amount of protein produced for a population of cells, or by reference to a fold increase from wild type or normal levels of expression. Expression levels may be described as the amount of protein secreted per volume of cell culture. However, increased transcription may not necessarily lead to increased amounts of secreted protein.

[0326] A cell which overexpresses IL- 10 and / or MMP9 comprises coding sequences expressing IL- 10 and / or MMP9 at a higher level than in non-engineered cells. As described above, overexpression may be achieved through the introduction of exogenous nucleic acid encoding IL- 10 and / or MMP9 such as mRNA, or genetic modification which stimulates expression of IL- 10 and / or MMP9 from endogenous coding sequences. An engineered macrophage which overexpresses IL- 10 and / or MMP9 may not necessarily secrete a greater amount of IL-10 and / or MMP9 than a non-engineered macrophage.

[0327] As used herein, over-expression relates to the artificial expression of a gene in increased quantity, relative to the expression level of the gene without the artificial modification, which may be referred herewith as wild-type or natural macrophage. As used in the Examples, expression levels were quantified at between 16 to 24 hours post-transfection. Expression levels as recited here are given for a population of macrophages at a concentration of 4xl06 / ml (which equates to 2x106 cells per cm2). In a preferred embodiment, over-expression is achieved via the introduction of an exogenous mRNA into the macrophage. In the Examples, the macrophages were transfected, isolated by centrifugation, re-suspended in TexMACs buffer supplemented with IL-3 and IL-14, and incubated at 37°C under 5% CO2. Those skilled in the art would be aware of equivalent conditions suitable to determine secreted protein concentration. The following levels were determined experimentally under these conditions:

[0328] In an embodiment, overexpression of IL-10 means the secreted IL-10 protein level is greater than about 300pg / ml. Suitably, the level of IL-10 expression is greater than about: 300pg / ml or 400pg / ml or 500pg / ml or 600pg / ml or 700pg / ml or 800pg / ml or 900pg / ml or l,000pg / ml or 2,000pg / ml or 3,000pg / ml or 4,000pg / ml or 5,000pg / ml or 6,000pg / ml or 7,000pg / ml or 8,000pg / ml or 9,000pg / ml or 10,000pg / ml or l l,000pg / ml. In preferred embodiments, the secreted IL-10 protein level is greater than 10,000 pg / ml.

[0329] In an embodiment, “relative overexpression” of IL- 10 in a culture of IL-10-engineered macrophages means the secreted IL-10 protein level in the culture is increased by about 100- 300pg / ml, or greater than about 300pg / ml, relative to the average wild-type protein secretion of a culture of wild-type macrophages cultured under the same conditions. Suitably, the increase in the level of IL-10 expression is greater than about: 300pg / ml or 400pg / ml or 500pg / ml or 600pg / ml or 700pg / ml or 800pg / ml or 900pg / ml or l,000pg / ml or 2,000pg / ml or 3,000pg / ml or 4,000pg / ml or 5,000pg / ml or 6,000pg / ml or 7,000pg / ml or 8,000pg / ml or 9,000pg / ml or 10,000pg / ml or l l,000pg / ml. In preferred embodiments, the secreted IL-10 protein level is greater than 10,000 pg / ml. Suitably, these IL-10 protein levels may be measured by culturing the macrophages as described above, wherein the concentration of macrophages in the medium is 4 x 106 cells / ml, which equates to 2xl06cells / cm2, and measuring the concentration of the protein in the culture medium. Accordingly, the macrophage secretes IL- 10 at a culture supernatant concentration of at least 10,000pg / ml when cultured in vitro at a cell concentration of 4xl06 / ml. In some embodiments, the engineered macrophage may secrete IL-10 at levels 1000 fold greater than non-engineered, non-polarised hMDMs. Non-engineered, non-polarised hMDMs are described in the art, for example in WO2019175595.

[0330] In an embodiment, expression of MMP9 means the secreted MMP9 protein level is between about 200ng / ml and 2000ng / ml. Suitably, the secreted MMP9 protein is greater than about: 300ng / ml or 400ng / ml or 500ng / ml or 600ng / ml or 700ng / ml or 800ng / ml or 900ng / ml or l,000ng / ml. Suitably the secreted MMP9 protein level is between about 200ng / ml and 2000ng / ml. In preferred embodiments, the secreted MMP9 level is greater than 200ng / ml. Suitably the engineered macrophage (comprising IL- 10 and MMP9) has a secreted MMP9 protein level that is greater than the average level of secreted MMP9 protein in macrophages engineered with IL- 10 alone. Suitably, the overall MMP activity of the engineered macrophage of the invention is also higher than the overall MMP activity of a macrophage engineered with IL- 10 alone. In preferred embodiments, the overall MMP activity of the engineered macrophage is at least 1.5 times greater than an untransfected macrophage.

[0331] In an embodiment, “relative overexpression of MMP9” in a culture of MMP9-engineered macrophages means that the secreted MMP9 protein level in the culture is increased by between about 200ng / ml and 2000ng / ml, relative to the average wild-type protein expression of a culture of wild-type macrophages cultured under the same conditions. Suitably, the increase in the level of secreted MMP9 protein is greater than about: 300ng / ml or 400ng / ml or 500ng / ml or 600ng / ml or 700ng / ml or 800ng / ml or 900ng / ml or l,000ng / ml, relative to the average wild-type protein expression of a culture of wild-type macrophages cultured under the same conditions. Suitably the increase in the level of secreted MMP9 protein level in the culture is between about 200ng / ml and 2000ng / ml, relative to the average wild-type protein expression of a culture of wild-type macrophages cultured under the same conditions. Suitably, these MMP9 protein levels may be measured -by culturing the macrophages as described above, wherein the concentration of macrophages in the medium is 4 x 106 cells / ml, which equates to 2xl06cells / cm2, and measuring the concentration of the protein in the culture medium. Accordingly, the macrophage secretes MMP9 at a culture supernatant concentration of at least 200ng / ml when cultured in vitro at a cell concentration of 4xl06 / ml. In preferred embodiments, the secreted MMP9 level is greater than 200ng / ml. Suitably the increase in the level of secreted MMP9 in a culture of IL-10- MMP9 engineered macrophages (i.e., comprising IL-10 and MMP9) is greater than the average increase in a culture of secreted MMP9 protein in macrophages engineered with MMP9 alone, relative to the average wild-type protein expression of a culture of wild-type macrophages cultured under the same conditions. Suitably, the overall MMP activity of the engineered macrophage of the invention is also higher than the overall MMP activity of a macrophage engineered with IL-10 alone. In some embodiments, these increases are synergistic. In preferred embodiments, the overall MMP activity of the engineered macrophage is at least 1.5 times greater than an untransfected macrophage.

[0332] In an embodiment, “relative underexpression of MMP9” in a culture of IL-10-engineered macrophages means that the secreted MMP9 protein level in the culture is decreased by between about 50ng / ml and lOOng / ml, between about lOOng / ml and 200ng / ml, between about 200ng / ml and 2000ng / ml, relative to the average wild-type protein expression of a culture of wild-type macrophages cultured under the same conditions. Suitably, the decrease in the level of secreted MMP9 protein is greater than about: lOOng / ml or 300ng / ml or 400ng / ml or 500ng / ml or 600ng / ml or 700ng / ml or 800ng / ml or 900ng / ml or l,000ng / ml, relative to the average wild-type protein expression of a culture of wild-type macrophages cultured under the same conditions. Suitably the decrease in the level of secreted MMP9 protein level in the culture is between about 200ng / ml and 500ng / ml, relative to the average wild-type protein expression of a culture of wildtype macrophages cultured under the same conditions.

[0333] The engineered macrophages (comprising IL- 10 and MMP9) secrete MMP9 at a level greater than the level of MMP9 expressed by macrophages engineered with IL-10 alone (See: Figure 1 : IL-10-MMP9 Trx vs IL- 10 Trx). In one embodiment, the macrophages are engineered with at least one DNA vector encoding IL- 10 and / or MMP9. It will be understood that a DNA vector may require one or more accessory sequences, such as a promoter, terminator, poly(A) signal sequence and the like.

[0334] A "promoter" is a nucleotide sequence which initiates and regulates transcription of a polynucleotide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. It is intended that the term "promoter" or "control element" includes full- length promoter regions and functional (e.g., controls transcription or translation) segments of these regions. In one embodiment, the DNA vector may include one or more liver specific promoters or cirrhosis specific promoters. In some embodiments, the DNA vector may comprise a CX3CR1 promoter, an insulin-like growth factor 1 (IGF1), or a CD1 IB promoter.

[0335] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a nucleic acid sequence is capable of affecting the expression of that sequence when the proper enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the nucleic acid sequence and the promoter sequence can still be considered "operably linked" to the coding sequence. Thus, the term "operably linked" is intended to encompass any spacing or orientation of the promoter element and the DNA sequence of interest which allows for initiation of transcription of the DNA sequence of interest upon recognition of the promoter element by a transcription complex.

[0336] Suitably the subject may be in need of treatment. Suitably therefore the subject may have a disease, condition or disorder or be at risk of developing a disease, condition or disorder. Suitably the subject may display one or more symptoms of a disease, condition or disorder.

[0337] The macrophages produced by the methods of the present invention may be for use in therapy. The macrophages of the present invention may be for use in treating an inflammatory condition and / or a fibrotic condition in a subject. As defined above, treating here can mean preventing, reducing or removing inflammation / fibrosis / organ damage. For example, the engineered macrophages maybe administered to a subject at an acute inflammation stage with the aim of preventing a chronic inflammatory condition. The engineered macrophages may also be administered to a subject at a chronic inflammation stage with the aim of preventing / reducing chronic fibrosis. The engineered macrophages may also administered to a subject who is experiencing an acute on chronic inflammatory state, such as acute on chronic liver failure (ACLF).

[0338] Suitably an acute disease or injury may be classed as a disease or injury with an onset of less than 24 weeks from cause. Suitably a chronic disease may be classed as a disease or injury which has persisted for more than 6 months. Suitably an acute-on chronic disease may be classed as a disease or injury with an onset of less than 24 weeks from cause in a patient that already has a chronic disease that has persisted for more than 6 months. Suitably, engineered macrophages of the present invention maybe administered to a subject with an acute occurrence to prevent transition to or increase of chronic inflammation and fibrosis.

[0339] Fibrosis refers to the deposition of extracellular matrix and connective tissue following tissue damage, which may result in replacing parenchymal tissue and eventually lead to scarring if in excess. In some embodiments, the condition is fibrosis.

[0340] Suitably, the condition is chronic organ damage associated with chronic inflammation. Suitably, the condition relates to the kidney, liver, or lung. For example, the condition maybe inflammatory liver damage, inflammatory kidney damage or inflammatory lung damage.

[0341] Suitably, the invention relates to a cell therapy product for inflammatory organ damage based on monocyte-derived macrophages genetically modified with payloads that induce a pro-restorative phenotype.

[0342] Suitably, the engineered macrophage has a pro-restorative phenotype and is anti-inflammatory and anti-fibrotic. The M2-like phenotype is pro-restorative. Whereas the Ml-like phenotype is pro-inflammatory.

[0343] The engineered macrophage includes exogenous coding sequence(s) for IL- 10 and MMP9. This exogenous coding sequence may be provided in any suitable way. This may be in the form of a nucleic acid vector, howsoever delivered, or genetic modification. For example, using viral or non-viral vectors, DNA or RNA constructs, or by gene editing using any suitable technique.

[0344] The macrophage engineered to express IL- 10 and MMP9 may be genetically engineered in any suitable way. For example, using viral or non-viral vectors, DNA or RNA constructs, or by gene editing using any suitable technique.

[0345] The macrophage may be virally engineered. In such methods of genetic engineering, the exogenous coding sequence / payload is introduced into the macrophage using a virus, such as a lentivirus, adenovirus or AAV. The virus may provide the exogenous coding sequence / payload as an “extrachromosomal” construct, or the gene may be integrated into the genome of the macrophage. Viral engineering of macrophages needs careful techniques, as documented in the art, to prevent the macrophages phagocytosing the viruses.

[0346] Suitably, the engineered macrophage is non-virally engineered, for example using nucleic acid vectors including the exogenous coding sequences. The nucleic acid may be any suitable nucleic acid, including DNA and RNA. Suitably the macrophage is transfected with a DNA vector. Suitably the DNA vector is a naked DNA vector, such that it is not associated with proteins and / or lipids. Suitably the DNA vector is not derived from a viral genome. Optionally, the DNA vector is a non-integrating vector such that it can function without integrating into the chromosomes of the macrophage. Alternatively, the nucleic acid vector is a messenger RNA (mRNA) molecule.

[0347] Suitably, the DNA vector comprises at least one sequence encoding IL-10 and / or MMP9, operably linked to a promoter.

[0348] As used herein, “encoding” refers to the ability of specific nucleotide sequences, such as a gene or an mRNA, to serve as templates for synthesis of macromolecules such as proteins in a cell. A gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell.

[0349] Suitably, the engineered macrophage is a genetically engineered macrophage comprising an nucleic acid construct overexpressing IL-10 and expressing MMP9.

[0350] Suitably, the mRNA molecule expresses IL- 10 and / or MMP9. Alternatively worded, the mRNA includes the coding sequence for IL- 10 and MMP9, which are exogenous to the macrophage. The mRNA may be chemically modified. Said chemical modification may be any suitable modification, most notably to improve the half-life of the mRNA in the cell. Suitable modifications are discussed extensively herein.

[0351] A nucleic acid vector can be introduced into cells using any suitable transfection method, such as but not limited to: cationic liposome-mediated transfection, lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems, such as “gene guns”. Suitably, the macrophage is transfected with nucleic acid via electroporation. Other suitable methods of transfection include nucleofection.

[0352] The macrophage may suitably be engineered by altering the genome of the macrophage by gene editing. Gene editing permits permanent insertion of the exogenous coding sequence.

[0353] Numerous techniques of gene editing are known, including those that require the use of nucleases. Various nucleases are known that can be exploited to modify the genome, from base editing techniques, prime editing techniques to gene editing. Many nucleases are known - such as Zinc fingers, TALENs and guided nucleases. These may be guided by RNA (“RNA” guided nucleases) - such as the enzymes involved in CRISPR, including but not limited to Cas9, Cast 2a, Cast 3, Mad7 and the like. The macrophage itself may be subject to gene editing, or a progenitor cell may be gene edited prior to conversion to a macrophage.

[0354] Suitably, the macrophage is transfected with one or more free nucleic acids or vectors.

[0355] Suitably, the macrophage is transfected with one or more exogenous coding sequences for IL- 10 and MMP9.

[0356] Suitably, the macrophage is provided with least one exogenous coding sequence for IL- 10 and at least one exogenous coding sequence for MMP9.

[0357] Suitably, the macrophage is engineered to overexpress IL-10 and express MMP9.

[0358] Suitably, the macrophage is autologous or allogenic to the subject.

[0359] The invention also relates to a population of engineered macrophages as described herein. Said population may be for use as a cell therapy.

[0360] Suitably, the use comprises administering an effective amount of the engineered macrophage to the subject.

[0361] The invention also relates to a composition comprising the engineered macrophages of the invention or a population thereof. Suitably, the composition is a pharmaceutical composition.

[0362] Suitably, the engineered macrophages may be formulated into a pharmaceutical composition. Suitably the composition is suitable for administration to a subject. Suitably the composition is a liquid. Suitably the composition is an infusible liquid.

[0363] Suitably the engineered macrophages are for administration to a subject by any route. Delivery to a subject may be by local or systemic administration. In some embodiments, administration is by, e.g. local injection, nebulizer, systemic injection. Suitably the engineered macrophages are for administration to a subject by infusion. Suitably the engineered macrophages are for administration to a subject parenterally, suitably intravenously. Suitably the engineered macrophages are for administration to a subject by injection or infusion. Suitably the engineered macrophages are for administration to a subject intravenously by infusion.

[0364] The invention also relates to a method of improving the migration of monocytes to inflammation comprising the use of an engineered macrophage according to the first aspect of the invention, a population of macrophages according to the second aspect of the invention or a composition according to the third aspect of the invention.

[0365] Suitably the engineered macrophages, the engineered macrophages for use in therapy, or the engineered macrophages used in a method of improving the migration of monocytes to inflammation, have a chemoattractant effect on monocytes. The engineered macrophage may have a chemoattractant effect on monocytes in vitro or in vivo. Recruitment of host monocytes at the site of inflammation / damaged organ is advantageous for treating inflammatory conditions. In some embodiments, the engineered macrophages have a chemoattractant effect specifically on monocytes, and not other immune cell types. In some embodiments, the engineered macrophages localise to sites of fibrosis, and / or recruit monocytes to sites of fibrosis, when administered to a subject with fibrosis, such as a subject with a chronic inflammatory condition with a fibrotic element. Presence of the engineered macrophages or monocytes at sites of fibrosis may be determined by flow cytometry, as described in the Examples herein. In some embodiments, the site of fibrosis may be the lung or the liver. Producing an engineered macrophage expressing IL-10 and MMP9

[0366] The invention also relates to a method of producing an engineered macrophage expressing IL-10 and MMP9 comprising the method of producing a macrophage of the invention, and an additional step of transiently transfecting a macrophage with a combined mRNA construct comprising at least one sequence encoding IL- 10 and at least one sequence encoding MMP9. In some embodiments, the macrophage is contacted with an anti-inflammatory treatment after transfection. In some embodiments, the macrophage is contacted with IL4 and IL13. In other embodiments, the macrophage is contacted with IL-4, IL-13 and M-CSF. Suitably the engineered macrophages overexpress IL- 10 and have restored MMP activity as a result of coexpression with MMP9 (MMP activity is otherwise depressed by IL-10). Suitably the engineered macrophages are pro-restorative. Suitably the engineered macrophages produced can be used for cell therapy.

[0367] Notably, macrophages engineered to overexpress IL- 10, or IL- 10 and MMP9 display a pro- regenerative phenotype without further anti-inflammatory treatment following transfection. Suitably, the engineered macrophages produced are manufactured to a GMP-compliant standard. Suitably therefore the engineered macrophages and populations thereof are GMP-compliant.

[0368] In other embodiments, the method of producing an engineered macrophage comprises introducing a sequence encoding IL- 10 and / or MMP9 into the genome of the macrophage.

[0369] In preferred embodiments, in a population of engineered macrophages produced by the method, at least 80% of the macrophages secrete IL-10.

[0370] Anti-inflammatory treatment

[0371] In one embodiment, the method of producing an engineered macrophage, engineered with the combination of IL-10 and MMP9, comprises transient transfection of a macrophage with exogenous coding sequence(s) for IL- 10 and MMP9. These coding sequences may be provided via transfection with a nucleic acid, for example one or more mRNA molecules. The macrophage is provided with least one exogenous coding sequence for IL- 10 and at least one exogenous coding sequence for MMP9.

[0372] In some embodiments, the macrophage is contacted with an anti-inflammatory treatment after transfection with an exogenous coding sequence(s). Suitably the anti-inflammatory treatment comprises anti-inflammatory cytokines. In some embodiments the macrophage is contacted with an anti-inflammatory treatment comprising IL4 and IL13.

[0373] In some embodiments, the anti-inflammatory treatment may be added during the method of transfecting the macrophages.

[0374] The transfected macrophages may be contacted with these anti-inflammatory cytokines IL4 and IL13) for a period of about 2 hours to about 48 hours, suitably 4 hours to 40 hours, suitably 12 to 24 hours, optionally around 16 hours.

[0375] The transfected macrophages may be contacted with these anti-inflammatory cytokines ( IL4 and IL13) at a concentration of between 2ng / mL and 200ng / mL, suitably between 5ng / mL and 150 ng / mL, suitably between 10 ng / mL to 100 ng / mL, suitably between 15 ng / mL to 75 ng / mL, suitably between 20 ng / mL to 50 ng / mL.

[0376] Suitably the anti-inflammatory treatment is used as a solution.

[0377] For the step of contacting the macrophage with IL4+IL13, suitably cells are plated as follows: 2x106 hMDMs / cm2 at 4xlO6 / mL. The invention will now be described with reference to the following figures.

[0378] Figures

[0379] Figure 1. CD14 expression (MFI) on day 0 monocytes in mobilised (MA) and non-mobilised (LA) material before processing. Data shows mean ± SEM of mean fluorescence intensity (MFI) levels of CD14 for MA (N=7) and LA (N=7) materials

[0380] Figure 2. CCR2 surface expression in mobilised and non-mobilised material before processing.

[0381] (A) Monocyte marker expression (Live+CD14+CCR2+) in mobilized leukapheresis (MA) and

[0382] (B) steady state leukapheresis (LA) material. (C) % of day 0 monocytes expressing CCR2 in mobilised leukapheresis (MA) and non-mobilised leukapheresis (LA) material. Day 0 monocytes are monocytes present in leukapheresis material immediately following leukapheresis, before processing and differentiation into macrophages. (D) MFI expression of CD14+CCR2+ (from live population) on day 0 monocytes in Mobilized leukapheresis (MA) and non-mobilised leukapheresis (LA) material. No significant difference between groups in C, ***P=0.0001 (T- test, unpaired).

[0383] Figure 3. (A) Expression of macrophage marker CD14 on day 6 macrophages from mobilised leukapheresis (MA) and non-mobilised leukapheresis (LA) material. Day 6 macrophages are macrophages produced by differentiation of monocytes (which may be derived from mobilised or non-mobilised leukapheresis material) in TexMACS™ GMP Medium + 100 ng / mL of GMP Recombinant Human Macrophage-colony stimulating factor (M-CSF) in cell culture bags at a target density of 2 xl06 / cm2for 6 days. (B) Expression of macrophage marker CD206 on day 6 macrophages produced from mobilised leukapheresis (MA) and non-mobilised leukapheresis (LA) material. (C) Expression of macrophage marker 25F9 on day 6 macrophages produced from mobilized leukapheresis (MA) and non-mobilised leukapheresis (LA) material. (D) Foldchange in CD206 expression from day 0 monocytes to day 6 macrophages. No significant difference between mobilised and non-mobilised groups in A,B and D *P=0.045 in C (T-test, unpaired).

[0384] Figure 4. Phagocytic activity, expressed as percentage of macrophages which are CD14+pHrodo+ after differentiation from monocytes (i.e. percentage of cells phagocytosing), in mobilised leukapheresis (MA, N=8) and non-mobilised leukapheresis (LA, N=5) material. No significant difference between mobilised and non- mobilised groups was found (T-test, unpaired).

[0385] Figure 5. Viability of macrophages differentiated using IL-34 and / or M-CSF. Viability of macrophage populations differentiated from monocytes using various concentrations of IL-34 and / or M-CSF was tested. Viability was tested using flow cytometry with a DRAQ7 dye - the percentage of viable cells is the percentage of cells negative for DRAQ7 staining. Monocytes differentiated using M-CSF were in a medium where M-CSF was at a concentration of lOOng / mL. Monocytes differentiated using both IL-34 and M-CSF were present in a medium in which M-CSF was present at a concentration of lOOng / mL and IL-34 was present at a concentration of 200ng / mL. Otherwise, the concentrations of IL-34 used in the differentiation medium were as indicated in Figure 5. Monocytes were matured in the differentiation medium comprising M-CSF and / or IL-34 for 5 days. Otherwise, the macrophages were produced as described in Example 3. Each triangle / circle represents macrophages differentiated from monocytes of a different subject. Circles represent macrophages derived from non-mobilised monocytes, and triangles represent macrophages derived from mobilised monocytes. Figure 6. Macrophage surface marker expression on macrophages differentiated using IL-34. The surface phenotype of macrophages differentiated using IL-34 and / or M-CSF, as described in Example 5, was tested using flow cytometry as described in the Examples. Specifically, the fold change in the expression of the mature macrophage markers CD206 and 25F9 was tested. Success criteria for maturation into macrophage was at least a 5 -fold increase in CD206 and 25F9 surface expression compared to monocytes. Triangles represent macrophages derived from mobilised monocytes and circles macrophages derived from non-mobilised monocytes.

[0386] Figure 7. Phagocytic capability of macrophages differentiated using IL-34. The phagocytic capability of macrophages differentiated using IL-34 and / or M-CSF was tested using the pHrodo phagocytosis assay as described in the Examples. The percentage of total cells which are positive for both CD 14 and pHrodo was measured. Circles represent macrophages derived from nonmobilised monocytes.

[0387] Figure 8. Pro-regenerative polarisation capability of macrophages differentiated using IL-34. Mobilised-monocytes differentiated for 5 days with 200 ng / ml of IL-34 or 100 ng / ml M-CSF were polarised for 18 hours in TexMACS medium with or without IL-10. Expression of polarised macrophage markers were assessed by flow cytometry on day 6. Macrophages were gated using FSC-H / SSC-H followed by singlets selection based on FSC-A / FSC-H. Live cells were selected by gating DRAQ7- population in a DRAQ7 / FSC-H dot plot followed by gating the presented populations. Percentages of A) CD14+, B) CD14+CD86+and C) CD14+HLA-DR+expression in non-polarised (M-CSF+TM, circle symbol and IL-34+TM, square symbol) and polarised (M-CSF+IL-10, up-pointing triangle symbol and IL-34+IL-10, down-pointing triangle symbol) macrophages, indicative of the percentage of viable cells expressing the markers in each sample. MFI (Mean Fluorescence Intensity) ratio for D) CD 14, E) CD86 and F) HLA-DR, indicative of the level of marker expression in each sample. The data are shown here as ratios of MFI from polarised conditions vs the corresponding MFI from non-polarised conditions.

[0388] Figure 9. Macrophages matured from mobilised or non-mobilised monocytes are able to secrete proteins encoded by mRNA transfected into the macrophages .(A) Demonstrates secretion of human IL10 from Day 6 human macrophages generated from either mobilised apheresis (MA) donors (n=13) or steady state apheresis (LA) donors (n=2), secretion is assessed 16- 18h post transfection and normalised to lxlOA6 viable cells. (B) Demonstrates secretion of human MMP9 from Day 6 human macrophages generated from either mobilised apheresis donors (n=13) or steady state apheresis donors (n=2), secretion is assessed 16-18h post transfection and normalised to lxlOA6 viable cells.

[0389] Figure 10. Pro-regenerative polarisation capability of M-CSF matured macrophages from mobilised and non-mobilised monocytes. From the non-mobilised monocytes, three independent macrophage differentiations from the same donor were performed. From the mobilised monocytes, one macrophage differentiation was performed. Mobilised and non-mobilised monocytes differentiated for 5 days with 100 ng / ml M-CSF were polarised for 18 hours in TexMACS medium with (M-CSF+IL-10) or without (M-CSF+TM) 50ng / ml IL-10. Expression of polarisationmarkers, CD86 and HLA-DR, was assessed by flow cytometry on day 6. Macrophages were gated using FSC-H / SSC-H followed by singlets selection based on FSC- A / FSC-H. Live cells were selected by gating DRAQ7- population in a DRAQ7 / FSC-H dot plot. From the live population, the mean fluorescence intensity (MFI) of CD86 and HLA-DR was extracted. MFI ratio for (A) CD86 and (B) HLA-DR in non-polarised and IL- 10 polarised M- CSF matured macrophages from non-mobilised monocytes. MFI ratio for (C) CD86 and (D) HLA-DR for IL- 10 polarised M-CSF matured macrophages from non-mobilised and mobilised monocytes. The MFI data are shown here as ratios of MFI from polarised conditions vs the corresponding MFI from non-polarised conditions.

[0390] Examples

[0391] Materials and methods

[0392] Cell culture

[0393] Monocytes were isolated from a mobilised, or non-mobilised, leukapheresis product from healthy volunteers sourced from BioIVT. A “mobilised leukapheresis product” refers to a product obtained by leukapheresis of a subject who has been treated with a subject who has been treated with Filgrastim at a dose of lOpg / ml / kg for 4 consecutive days, and wherein the leukapheresis occurs less than 24 hours after the final dose of Filgrastim. Briefly, a sample was taken prior to CD14 isolation to ascertain cell counts and monocyte profiling. The volume of sample was adjusted to meet the required criteria for selection. CD14 cell isolation and separation of monocytes was carried out for non-mobilised material (LA) and mobilised material (MA) using the CliniMACS Prodigy with CliniMACS CD14 microbeads (medical device class III), wherein the CliniMACS Prodigy tubing set and program were altered for the mobilised material to allow for larger scale isolation of CD14 monocytes.

[0394] Upon completion of the isolation procedure, CD14+monocytes were counted from the positive target fraction bag, washed and resuspended in TexMACS (Miltenyi), plus lOOng / ml recombinant human M-CSF at a concentration of 4xlO6 / mL or 2xlO6 / mL. Cells were then seeded at a density of 2xl06 / cm2in culture bags.

[0395] Phagocytosis assay - as used in Example 4

[0396] The cells were prepared for flow cytometric analysis by resuspending them at a concentration of 2xl06 / ml in PBS + 0.5mM EDTA (Life Technologies). 50 pL of the cell suspension was dispensed into low adherence, round bottomed 96 well plates. 50 pL of resuspended pHrodo Beads (prepared as per manufacturer’s instructions) were added to the test wells. Cells were incubated for 2 hours at 37°C 5% CO2. At the end of the Ihr incubation, the plate was spun at 300xg, 4°C, 5 min, the supernatants were eliminated and the pellets were resuspended with 100 pL of 1 : 100 FcR block PEA solution / well. Following incubation for 15 min at 4°C in the dark, antibodies were added (see Table 1) to appropriate test wells and incubated for 20 min at 4 C. Cells were washed with PBS + 0.5mM EDTA and spun at 300g for 5 min. The supernatants were flipped off and cells resuspended in PBS + 0.5mM EDTA + 1 : 1000 DRAQ7. Cells were incubated for 5 min at 4 C. Cells were washed as before, then resuspended in lOOpl of PBS + 0.5mM EDTA + 0.1% human serum. 50pl of cells was acquired on the Novocyte3000 or Novocyte Quanteon (Agilent). Flow cytometry analysis was conducted on NovoExpress software and the following gating strategy was utilised to identify actively phagocytosing macrophages: “Cell gate” to exclude debris, “singlet gate” to exclude cell doublets, “live gate” to exclude dead cells, “CD14+ gate” to identify macrophages and “phRodo+ve gate” to measure the percentage of phagocytosing macrophages.

[0397] Phagocytosis assay - as used in Example 6

[0398] The cells were prepared for flow cytometric analysis by resuspending them at a concentration of lxl06 / ml in PBS + 0.5mM EDTA (Life Technologies) + 0.5% HSA / Alburex. 100 pL of the cell suspension was dispensed into low adherence, round bottomed 96 well. 100 pL of resuspended pHrodo Beads (prepared as per manufacturer’s instructions) were added to the test wells. Cells were incubated for 1 hour at 37°C 5% CO2. At the end of the Ihr incubation, the plate was spun at 300g, 4°C, 5 min, the supernatants were eliminated and the pellets were resuspended with lOOpL of 1 : 100 FcR block PEA solution / well. Following incubation for 15 min at 4°C in the dark, antibodies were added (see Table 1) to appropriate test wells and incubated for 15 min at 4 C in the dark. Cells were washed with PBS + 0.5mM EDTA and spun at 300g for 5 min. The supernatants were removed and cells resuspended in PBS + 0.5mM EDTA + 1 : 1000 DRAQ7. Cells were incubated for 5 min at 4 C in the dark. Cells were washed as before, then resuspended in lOOpl of PBS + 0.5mM EDTA. 50pl of cells was acquired on the Novocyte3000 or Novocyte Quanteon (Agilent). Flow cytometry analysis was conducted on NovoExpress software and the following gating strategy was utilised to identify actively phagocytosing macrophages: “Cell gate” to exclude debris, “singlet gate” to exclude cell doublets, “live gate” to exclude dead cells, “CD 14+ gate” to identify macrophages and “pHrodo+ve gate” to measure the percentage of phagocytosing macrophages.

[0399] Flow cytometry labelling - as used in Examples 2 and 3

[0400] Macrophages were resuspended at a concentration of lxl06 / ml in PBS + 0.5mM EDTA (Life Technologies) + FcR Block 1 : 100 (Miltenyi). 100 pl of cells were aliquoted into low adherence, round bottomed 96 well plates. Cells were incubated for 5 minutes, then appropriate antibodies were added (see Table 1) to appropriate test wells and left for 20 min at 4 C. Cells were washed with PBS + 0.5mM EDTA and spun at 300g for 5 min. The supernatants were flipped off and the cells resuspended in PBS + 0.5mM EDTA + 1 : 1000 DRAQ7. Cells were incubated for 5 min at 4 C. Cells were washed as before, then resuspended in lOOpl of PBS + 0.5mM EDTA + 0.1% human serum. 50pl of cells were acquired on the Novocyte3000 or Novocyte Quanteon (Agilent).

[0401] Flow cytometry labelling - as used in Examples 5 and 6

[0402] Macrophages were resuspended at a concentration of lxl06 / ml in PBS + 0.5mM EDTA (Life Technologies) + FcR Block 1 : 100 (Miltenyi). lOOpl of cells were aliquoted into low adherence, round bottomed 96 well plates. Cells were incubated for 5 minutes, then antibodies were added (see Table 1) to appropriate test wells and left for 20 min at 4 C. Cells were washed with PBS + 0.5mM EDTA and spun at 300g for 5 min. The supernatants were removed and the cells resuspended in PBS + 0.5mM EDTA + 1 : 1000 DRAQ7. Cells were incubated for 5 min at 4 C. Cells were washed as before, then resuspended in lOOpl of PBS + 0.5mM EDTA + 0.5% human serum / Albur ex. 50pl of cells were acquired on the Novocyte3000 or Novocyte Quanteon (Agilent).

[0403]

[0404] Table 1 - Antibodies used for flow cytometry

[0405] Example 1: Description of mobilised vs non-mobilised material

[0406] Non-mobilised leukapheresis is collected from patient circulation utilising Spectra Optia Apheresis System (Terumo BCT), running a standard mononuclear cell (MNC), A software programme. In contrast, mobilised leukapheresis occurs in patients who have been pre-treated with recombinant G-CSF (filgrastim). Briefly, filgrastim was administered subcutaneously to patients at a concentration of lOpg / kg / day over 4 consecutive days prior to the leukapheresis. Mobilised patients undergo leukapheresis up to 24h after the last filgrastim administration. Mobilised leukapheresis is collected from patient circulation utilising Spectra Optia Apheresis System (Terumo BCT), running a standard MNC, A software programme. Following leukapheresis (either mobilised or non-mobilised), monocytes are isolated by selecting CD14+ cells, essentially as described above.

[0407] Example 2: Characterisation of mobilised vs non-mobilised material Monocytes

[0408] To observe the maturation status of monocytes from mobilised Leukapheresis material (MA) and non-mobilise leukapheresis material (LA) flow cytometry was utilised to assess the surface marker expression of the typical monocyte markers CD45, CD14, and CCR2. No significant differences were found in the surface expression of CD14 on day 0 CD45+CD14+ monocytes when comparing mobilised leukapheresis material (MA) and non-mobilised leukapheresis material (LA) (Figure 1). “Day 0 monocytes” as used herein refers to monocytes immediately after isolating from the leukapheresis product, before processing and before the step of differentiating into macrophages in medium comprising M-CSF has begun.

[0409] Furthermore, expression of CCR2 was assessed on day 0 to identify differences in maturation stage between MA and LA. Mobilised material (MA) presented a more heterogeneous population of monocytes, (Figure 2A) while non-mobilised leukapheresis (LA) presented a homogeneous population of CD14+CCR2+ monocytes (Figure 2B). The percentage of CD14+CCR2+ cells out of the isolated CD14+ cells (on Day 0, before maturation to macrophages) was similar in monocytes isolated from mobilised apheresis (MA) and non-mobilised apheresis (LA), with a slight reduction in cells from mobilised apheresis (Figure 2 C). The CD14+CCR2L0Wmonocyte population was significantly increased in MA material (CD14+CCR2LOWin LA = 1.19 ± 0.37% vs MA = 9.12 ± 0.87%) (Figure 2D). The expression of CCR2 is upregulated in mature monocytes that emigrate from the bone marrow. The presence of a CCR2i0Wpopulation suggests that mobilised material consists of a monocytic pool containing CCR2+ mature, and CCR2i0Wimmature monocytes.

[0410] Example 3: Characterisation of macrophages manufactured from mobilised vs non-mobilised material

[0411] To mature macrophages from monocytes, monocytes were isolated (by selection for CD14 expression) from leukapheresis of non-mobilised (LA) or mobilised material (MA) as described above and then incubated in TexMACS™ GMP Medium + 100 ng / mL of GMP Recombinant Human Macrophage-colony stimulating factor (M-CSF) in cell culture bags at a target density of 2 xl06 / cm2for 6 days.

[0412] To characterise the resulting macrophages, surface marker expression was measured using flow cytometry at the end of the maturation (referred to as Day 6 in Fig. 3). More generally, day 6 macrophages refers to macrophages obtained by incubation of day 0 monocytes in medium comprising M-CSF for 6 days.

[0413] Successful macrophage maturation from monocytes is determined by upregulation in the functional macrophage mannose receptor (CD206) and maturation marker (25F9). Macrophages from LA and MA material express similar levels of CD14 (Fig.3A) and CD206 (Fig.3B). Macrophages from MA and LA express the maturation marker 25F9, however, MA expression is lower, suggesting the macrophages may be more immature (Fig.3C). Importantly, successful macrophage maturation was confirmed with over five-fold increase in CD206, over monocytes (Fig 3D) (3).

[0414] Example 4: Function of macrophages manufactured from mobilised vs non-mobilised material

[0415] Macrophages are professional phagocytes specialised in the removal of dying / dead cells and cellular debris. These phagocytic properties can be captured in vitro by flow cytometry quantification of the fluorescence levels of macrophages co-incubated with pH-sensitive pHrodoTM Red E. coli bioparticles. As reported in Figure 2, MA-derived monocytes comprise a heterogeneous population of CCR2+ and CCR2i0Wmonocytes. This heterogeneity was not detected in LA-derived monocytes which exhibited the classical CCR2+ expression. CCR21ow monocytes have been reported to have impaired phagocytic ability compared their CCR2+ counterparts (9). This heterogeneity is likely to negatively impact the phagocytic properties of MA-derived macrophages. Nonetheless, our data surprisingly shows no distinction between the phagocytic capacity of macrophages derived from mobilised (MA) and non-mobilised (LA) material, with both macrophage groups exhibiting high phagocytic levels (87.37% ± 3.87 and 88.82% ± 5.14 for MA-M(p and LA-M(p, respectively) (Fig. 4). These results are further supported by the similar expression of CD14 and CD206 in MA- and LA-derived Mcp (Fig.3A and B), which are hallmarks of a phagocytosis competent cell.

[0416] Conclusions

[0417] In conclusion, monocytes isolated from MA (G-CSF mobilised) material show a heterogeneous population, indicative of immature monocytes. LA (non-mobilised) material lacks CCR2 negative monocytes, and comprise a lower proportion of CCR2i0Wmonocytes, indicating a fully mature monocyte population. Despite these differences, both materials generate functional macrophages, as indicated by >5 fold-upregulation of the macrophage functional marker CD206. Notably, MA material have a lower expression of the macrophage maturation marker 25F9 than LA material, suggesting that the macrophages may be more immature than LA macrophages.

[0418] Despite the immaturity of the monocytes and macrophages from the MA material, they are equally capable to be manufactured to fully functional macrophages, comparable to that of the mature LA material, as illustrated by high and similar phagocytic capacity.

[0419] Example 5. Monocytes differentiated into macrophages using IL-34 produce macrophage populations with high proportions of viable macrophages.

[0420] To determine whether IL-34 can also be used to generate viable macrophages, the differentiation step described in Example 3 was adapted to replace the M-CSF with various concentrations of IL-34. In addition, maturation of macrophages from mobilised (MA) and non-mobilised, buffy coat (BC) material was assessed following a 5 day maturation period. The viability of macrophages differentiated using M-CSF alone, and a combination of M-CSF and IL-34, was also tested using flow cytometry. Specifically, macrophages were counted in BD Trucount™ Tubes along with a DRAQ7 stain. The Trucount tubes facilitate an absolute count based off comparing cellular events against a known number of beads (specific to each LOT) while the DRAQ7 stains the nuclei of dead and permeabilized cells, allowing for clear separation between viable macrophages and dead / dying cells or cellular debris. An IL-34 concentration of at least 200 ng / mL (or at least 50ng / mL per lx 106CD14+ cells) resulted in the highest macrophage viability, and viability was comparable when differentiating non-mobilised and mobilised monocytes (circles and triangles in the figure, respectively). The viability of macrophages differentiated using at least 50ng / mL per lx 106CD14+ cells was comparable to those differentiated using M-CSF. The viability measurements can be seen in Fig. 5.

[0421] Example 6. Differentiation of monocytes using IL-34 produces mature functional macrophages.

[0422] The surface phenotype and phagocytic capability of macrophages differentiated using IL-34 (and / or M-CSF) was examined to assess macrophage maturation and functionality. Macrophages were deemed successfully matured when an at least 5-fold increase in CD206 and 25F9 expression was measured in the macrophages differentiated from mobilised (MA) or nonmobilised (BC) monocytes relative to the monocytes of origin. Macrophage populations produced from non-mobilised (BC) monocytes were classed as functional when at least 70% of the population were CD14+pHrodo+, indicating phagocytosing macrophages (Fig. 7). Similar phagocytosis assay is performed on macrophages differentiated from mobilised (MA) monocytes. As can be seen in Fig. 6, differentiating monocytes to macrophages after 5 days incubation with lOOng / mL of MCSF, 200ng / mL IL-34, or both, resulted in macrophages expressing surface markers CD206 and 25F9 above the maturation threshold (over 5-fold than the monocytes), regardless of whether monocytes were mobilized (black triangles) or nonmobilized (circles). The data in Figures 6 and 7 also demonstrate that mature, functional macrophages can be generated using a variety of concentrations of IL-34.

[0423] Example 7. Measurement of macrophage phenotype following differentiation of macrophages using IL-34 or M-CSF and polarisation to a pro-regenerative phenotype

[0424] To further determine and compare the functionality of macrophages matured by incubating mobilised (MA) or non-mobilised (LA / BC) monocytes in media comprising IL-34 or M-CSF, the phagocytosis and polarisation capability of the macrophages were examined. Macrophages generated using 200ng / mL IL-34 or lOOng / mL M-CSF matured from MA or LA / BC monocytes were then further polarised to a pro-regenerative phenotype by incubation in the presence of 50ng / mL of IL- 10. Following incubation, the surface markers CD86 and HLA-DR of the macrophages were measured using flow-cytometry to estimate their polarisation state, where a reduction in CD86 and / or HLA-DR is indicative of a pro-regenerative phenotype. The polarisation capacity of IL-34 differentiated macrophage matured from mobilised monocytes is compared to that of M-CSF matured macrophages from mobilised monocytes.

[0425] No differences in the percentage of CD14+and CD14+HLA-DR+macrophages were detected between IL-10 polarised (M-CSF+IL-10) and the corresponding non-polarised control (M- CSF+TM), indicating that polarised and non-polarised samples contained a comparable percentage of viable macrophages. No differences were detected between 11-34 (up-pointing triangle and down-pointing triangle symbols) and M-CSF (circle and square symbols)) macrophages (Fig.8A and C). The percentage of CD14+CD86+macrophages decreased after IL- 10 polarisation in a comparable manner between IL-34 and M-CSF macrophages (Fig.8B), indicating that both IL-34 and M-CSF-differentiated macrophages successfully polarised macrophages generated from mobilised monocytes towards a pro-regenerative ‘M2 -like’ phenotype. The increase in CD14 expression levels after IL-10 polarisation was comparable between IL-34 and M-CSF macrophages (Fig.8D). The reduction in CD86 and HLA-DR after IL-10 polarisation followed the same trend in IL-34 and M-CSF macrophages (Fig. 8E and F). Overall, this data indicates that IL-34 and M-CSF differentiated macrophages generated from mobilised monocytes have comparable pro-regenerative polarisation capacity.

[0426] The pro-regenerative polarisation capability of M-CSF matured macrophages from mobilised monocytes was also assessed and compared to that from non-mobilised monocytes. To do so, three independent macrophage differentiations from the same donor of non-mobilised monocytes were performed using M-CSF, as described above. The macrophages were then either maintained non polarised (M-CSF+TM) or polarised with IL-10 as above (M-CSF+IL-10). As can be seen in Fig. 10 A and B, the MFI (Mean Fluorescence Intensity) of CD86 and HLA-DR, respectively, was measured in the polarised and non-polarised macrophages differentiated from non-mobilised monocytes. The measurement indicated a decrease in CD86 and HLA-DR expression in the macrophages that have been polarised with IL-10, expressed as a ratio of the expression of the corresponding non-polarised control.

[0427] The ratios of the expression of CD86 or HLA-DR in the polarised macrophages vs their corresponding non-polarised controls was compared in the macrophages differentiated from nonmobilised monocytes and macrophages differentiated from mobilised monocytes (One macrophage differentiation was performed from the mobilised monocytes). As can be seen in Fig. 10 C and D, the decrease in CD86 and HLA-DR expression was comparable between polarised M-CSF matured macrophages from non-mobilised and mobilised monocytes. Overall, this data indicates that M-CSF matured macrophages from non-mobilised and mobilised monocytes have at least comparable pro-regenerative polarisation capability.

[0428] Example 8. Comparison of secretion levels from mRNA-transfected macrophages that have been differentiated from mobilised (MA) or non-mobilised (LA) monocytes

[0429] To further characterise the functionality of macrophages matured from mobilised (MA) or nonmobilised (LA) monocytes, their ability to secrete proteins encoded by transfected mRNA was compared. To mature macrophages from monocytes, monocytes were isolated (by selection for CD14 expression) from leukapheresis of non-mobilised / steady-state (LA) or mobilised material (MA) as described above and then incubated in TexMACS™ GMP Medium + 100 ng / mL of GMP Recombinant Human Macrophage-colony stimulating factor (M-CSF) in cell culture bags at a target density of 2 xl06 / cm2for 5 days.

[0430] Following incubation, the macrophages were transfected by electroporation using a bi-cistronic mRNA encoding both IL- 10 and MMP9 linked by a P2A self-cleaving peptide at an mRNA concentration of 8 ug / 10A6 cells, essentially as described in WO 2024 / 074376. At 16- 18h post - transfection, the levels of IL-10 and MMP9 were measured in the media as follows. Media from the overnight (16- 18h) culture post transfection was collected and run through BioTechne’s Ella system to assess both human IL10 and human MMP9 levels in pg / ml simultaneously from the same sample. Data was then normalised by first presenting the data as the secretion per lxlOA6 cells by dividing the result by the cell concentration plated (3.5-4xlOA6 / ml) and finally multiplying that result by the viability percentage to get the secretion per lxlOA6 viable cells.

[0431] As can be seen in Figure 9, transfected macrophages differentiated from mobilised monocytes (MA) secreted both IL- 10 and MMP9 to a comparable or higher levels than transfected macrophages differentiated from non-mobilised (LA) monocytes..

[0432] References

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[0441] 10. Sloas, Christopher et al. “Engineered CAR-Macrophages as Adoptive Immunotherapies for Solid Tumors”. Front Immunol, vol. 12 (2021): 783305.

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[0444] 13. Stanley, E Richard, and Violeta Chitu. “CSF-1 receptor signaling in myeloid cells.” Cold Spring Harbor perspectives in biology vol. 6,6 a021857. (2014) doi : 10.1101 / cshperspect. a021857

[0445] 14. Philip, Joseph et al. “Adverse events associated with apheresis procedures: Incidence and relative frequency.” Asian journal of transfusion science vol. 7,1 (2013): 37-41. doi : 10.4103 / 0973-6247.106730

[0446] 15. Winters JL. Complications of donor apheresis. J Clin Apher. 2006 Jul;21(2): 132-41. doi: 10.1002 / jca.20039. PMID: 15880355.

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[0448] 17. Berger TG, Strasser E, Smith R, Carste C, Schuler- Thurner B, Kaempgen E, Schuler G. Efficient elutriation of monocytes within a closed system (Elutra) for clinical-scale generation of dendritic cells. J Immunol Methods. 2005 Mar;298(l-2):61-72. doi: 10.1016 / j.jim.2005.01.005. Erratum in: J Immunol Methods. 2005 Aug;303(l-2): 152. PMID: 15847797.

[0449] 18. Strasser EF, Eckstein R. Optimization of leukocyte collection and monocyte isolation for dendritic cell culture. Transfus Med Rev. 2010 Apr;24(2): 130-9. doi:

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[0458] 27. Engelmann C, Berg T. G-CSF treatment in decompensated liver disease: a double-edged sword? Hepatol Int. 2022 Oct;16(5):979-982. doi: 10.1007 / sl2072-022-10379-8. Epub 2022 Sep 2. PMID: 36053413; PMCID: PMC9525346.28. Kim WR, Mannalithara A, Heimbach JK, Kamath PS, Asrani SK, Biggins SW, Wood NL, Gentry SE, Kwong AJ. MELD 3.0: The Model for End-Stage Liver Disease Updated for the Modem Era. Gastroenterology. 2021 Dec;161(6): 1887-1895.e4. doi: 10.1053 / j.gastro.202L 08.050. Epub 2021 Sep 3. PMID: 34481845; PMCID: PMC8608337.

[0459] 29. Peng Y, Khaled T, Liu J, et al. Data Request from the MELD Enhancement Subcommittee of the Liver and Intestinal Organ Transplantation Committee. Data Request ID: HR2011 02. June 7, 2011

[0460] 30. Chihara, T., et al. IL-34 and M-CSF share the receptor Fms but are not identical in biological activity and signal activation. Cell Death Differ 17, 1917-1927 (2010).

[0461] 31. Boulakirba, S et al. IL-34 and CSF-1 display an equivalent macrophage differentiation ability but a different polarization potential. Sci Rep 8, 256 (2018). Embodiments A method of producing macrophages, wherein the method comprises incubating monocytes obtained from a subject in a medium comprising M-CSF to produce macrophages, wherein the monocytes used were previously isolated from leukocytes obtained from the subject through leukapheresis, and wherein prior to the leukapheresis, the subject was treated with G-CSF. A method of producing macrophages, comprising:

[0462] (a) treating a subject with G-CSF;

[0463] (b) obtaining leukocytes from the subject by leukapheresis following the treatment with G-CSF;

[0464] (c) isolating monocytes from the leukocytes obtained by the leukapheresis; and

[0465] (d) incubating the monocytes in a medium comprising M-CSF to produce macrophages. A method of treating an inflammatory condition in a subject, which comprises administering to the subject the macrophage produced by the method of embodiment 1 or 2. A method of treating an inflammatory condition in a subject, comprising:

[0466] (a) treating a subject with G-CSF;

[0467] (b) obtaining leukocytes from the subject by leukapheresis following the treatment with G-CSF;

[0468] (c) isolating monocytes from the leukocytes obtained by the leukapheresis;

[0469] (d) incubating the monocytes in a medium comprising M-CSF to produce macrophages; and

[0470] (e) administering the macrophages to the subject. A macrophage for use in a method of treating an inflammatory condition in a subject, wherein the method comprises administering the macrophages obtained by the method of embodiments 1 or 2 to the subject. A macrophage for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was administered with G-CSF, subsequently leukocytes were obtained from the subject by leukapheresis, and monocytes were isolated from the leukocytes, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising M-CSF to produce said macrophage. A macrophage and G-CSF for use in a method of treating an inflammatory condition in a subject, wherein the method comprises (a) treating the subject with G-CSF;

[0471] (b) obtaining leukocytes from the subject by leukapheresis following treatment with G- CSF;

[0472] (c) isolating monocytes from the leukocytes obtained by the leukapheresis;

[0473] (d) incubating the monocytes in a medium comprising M-CSF to produce macrophages; and

[0474] (e) administering the macrophages to the subject.

[0475] 8. A macrophage and G-CSF for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was administered with G-CSF, subsequently leukocytes were obtained from the subject by leukapheresis, and monocytes were isolated from the leukocytes, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising M-CSF to produce said macrophage.

[0476] 9. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the macrophages are isolated by the selection of CD14+ cells.

[0477] 10. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the incubation in medium comprising M-CSF is for 5-7 days.

[0478] 11. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the concentration of the M-CSF in the medium is 10-150 ng / ml.

[0479] 12. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the concentration of the M-CSF in the medium is lOOng / ml.

[0480] 13. The method, the macrophage for use or the macrophage and G-CSF for use according to any of the preceding embodiments, wherein the G-CSF is recombinant G-CSF.

[0481] 14. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 13, wherein the recombinant G-CSF is selected from the group consisting of: Lenograstim, Filgrastim, Pegfilgrastim and Lipegfilgrastim.

[0482] 15. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 13 or 14, wherein the recombinant G-CSF is Filgrastim.

[0483] 16. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 15, wherein the Filgrastim is selected from the group consisting of: Zarzio, Granix, Nivestim, Releuko, Ratiograstim, Accofil, Tevagrastim and Neupogen.

[0484] 17. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 15 or 16, wherein the Filgrastim is Neupogen. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein one or more doses of the G-CSF are administered to the subject. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 18, wherein more than one dose is administered to the subject, and wherein the one or more doses are administered to the subject on consecutive days. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 18 or 19, wherein 4 or 5 doses of G-CSF are administered to the subject. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the G-CSF is administered at a dose of lOpg / kg / day. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the G-CSF is administered subcutaneously or intravenously, preferably subcutaneously. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the leukapheresis occurs less than 24 hours after the final dose of G-CSF is administered to the subject. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the macrophage is non-polarised. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the macrophage is capable of phagocytosis, optionally wherein the macrophage has at least an equivalent phagocytic capacity to a macrophage which has been produced from a monocyte obtained by leukapheresis from a subject who had not been treated with G-CSF prior to leukapheresis. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the percentage of the CD14+CCR2iow population out of the isolated monocyte population is significantly higher than the percentage of the CD14+CCR2i0Wpopulation in monocytes obtained by leukapheresis from a subject who had not been treated with G-CSF prior to leukapheresis. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the expression of surface marker CD14 and CD206 on the macrophages is equivalent to that on a macrophage which has been produced from a monocyte obtained by leukapheresis from a subject who had not been treated with G-CSF prior to leukapheresis. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the macrophage is capable of being be polarised to a pro-restorative, anti-inflammatory and / or anti- fibrotic phenotype. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 28, wherein the macrophage is capable of being polarised to a pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype by incubation in the presence of IL-4 and / or IL- 13 and / or IL- 10. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 28 or 29, wherein the method comprises a further step of polarising the macrophage to a pro-restorative, anti-inflammatory and / or anti- fibrotic phenotype. The method of treating an inflammatory condition, the macrophage for use or the macrophage and G-CSF for use according to embodiment 30, wherein the step of polarising the macrophage occurs before administration of the macrophage to the subject. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of embodiments 28-31, wherein the macrophages polarised to a pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype have an increased expression of CD 163 and CD206, and a reduced expression of HLA DR and CD86 compared to cells not polarised to a pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the method comprises a further step of engineering the macrophage. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 33, wherein the macrophage is engineered to express one or more cytokines, optionally wherein the macrophage is engineered to express a cytokine which induces polarisation to a pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 34, wherein the cytokine is IL-10. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiments 33-35, wherein the macrophage is engineered to overexpress IL-10 and / or MMP9. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiments 3-8, wherein the inflammatory condition is a chronic inflammatory condition with a fibrotic element, optionally wherein the condition is organ damage associated with chronic inflammation. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiments 3-8 or 37, wherein the inflammatory condition is a liver injury, optionally chronic liver injury. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiments 3-8, 37 or 38, wherein the condition is liver cirrhosis. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 39, wherein the liver cirrhosis resulted from at least one disease or condition selected from the group consisting of: non-alcoholic fatty liver disease (NAFL) (e.g., non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)), alcoholic liver disease (e.g., alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH)), mechanical trauma to the liver, biliary obstruction, autoimmune hepatitis, iron overload, Hepatitis B infection (HBV) and Hepatitis C infection (HCV). The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 39, wherein the liver cirrhosis resulted from steatotic liver disease (SLD), optionally wherein the steatotic liver disease is metabolic dysfunction-associated steatotic liver disease, metabolic-associated steatohepatitis, Met-ALD or Cryptogenic SLD. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 39, wherein the aetiology of the liver cirrhosis is:

[0485] (a) pure metabolic dysfunction-associated steatotic liver disease (MASLD),

[0486] (b) metabolic and alcohol related / associated liver disease (Met-ALD),

[0487] (c) HCV sustained virologic response (HCV SVR) with ongoing cirrhosis, or

[0488] (d) alcohol -related liver disease (ALD or Met-ALD) provided that the patient is confirmed to not be drinking alcohol above Met-ALD limits and has a value of <25 mg / 1 in a PEth alcohol test. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiments 39-42, wherein the liver cirrhosis is selected from compensated cirrhosis and decompensated cirrhosis. The method, the macrophage for use or the macrophage and G-CSF for use according to embodiments 39-43, wherein the subject exhibits or has recovered from or has become clinically stable following one or more clinical signs of hepatic decompensation selected from the list consisting of ascites, hepatic encephalopathy, variceal haemorrhage (variceal bleed), hepatorenal syndrom e / acute kidney injury (HRS / AKI) and / or Spontaneous Bacterial Peritonitis (SBP). The method, the macrophage for use or the macrophage and G-CSF for use according to embodiment 3-8, or 37-44, wherein the subject has acute-on-chronic liver failure. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the macrophages are autologous to the subject from whom the leukocytes are obtained. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding embodiments, wherein the G-CSF is administered to the same subject from whom the leukocytes are obtained by leukapheresis. The method of treating an inflammatory condition, the macrophage for use or the macrophage and G-CSF for use according to any one of embodiments 3-47, wherein the macrophages are autologous to the subject to whom the macrophages are administered.

Claims

CLAIMS1. A method of producing macrophages, wherein the method comprises incubating monocytes obtained from a subject in a medium comprising a Colony Stimulating Factor 1 Receptor (CSF-1R) agonist to produce macrophages, wherein the monocytes used were previously isolated from leukocytes obtained from the subject through leukapheresis, and wherein prior to the leukapheresis, the subject was treated with G-CSF.

2. A method of producing macrophages, comprising:(a) treating a subject with G-CSF;(b) obtaining leukocytes from the subject by leukapheresis following the treatment with G-CSF;(c) isolating monocytes from the leukocytes obtained by the leukapheresis; and(d) incubating the monocytes in a medium comprising a CSF-1R agonist to produce macrophages.

3. A method of treating an inflammatory condition in a subject, which comprises administering to the subject the macrophage produced by the method of claim 1 or 2.

4. A method of treating an inflammatory condition in a subject, comprising:(a) treating a subject with G-CSF;(b) obtaining leukocytes from the subject by leukapheresis following the treatment with G-CSF;(c) isolating monocytes from the leukocytes obtained by the leukapheresis;(d) incubating the monocytes in a medium comprising a CSF-1R agonist to produce macrophages; and(e) administering the macrophages to the subject.

5. A macrophage for use in a method of treating an inflammatory condition in a subject, wherein the method comprises administering the macrophages obtained by the method of claims 1 or 2 to the subject.

6. A macrophage for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was administered with G-CSF, subsequently leukocytes were obtained from the subject by leukapheresis, and monocytes were isolated from the leukocytes, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising a CSF-1R agonist to produce said macrophage.

7. A macrophage and G-CSF for use in a method of treating an inflammatory condition in a subject, wherein the method comprises(a) treating the subject with G-CSF;(b) obtaining leukocytes from the subject by leukapheresis following treatment with G- CSF;(c) isolating monocytes from the leukocytes obtained by the leukapheresis;(d) incubating the monocytes in a medium comprising a CSF-1R agonist to produce macrophages; and(e) administering the macrophages to the subject.

8. A macrophage and G-CSF for use in a method of treating an inflammatory condition in a subject, wherein prior to treating the inflammatory condition the subject was administered with G-CSF, subsequently leukocytes were obtained from the subject by leukapheresis, and monocytes were isolated from the leukocytes, and wherein the macrophage was obtained by incubating said monocytes in a medium comprising a CSF-1R agonist to produce said macrophage.

9. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the macrophages are isolated by the selection of CD14+ cells.

10. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the incubation in medium comprising the CSF-1R agonist is for 5-7 days.

11. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the concentration of the CSF-1R agonist in the medium is 10-500 ng / ml.

12. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the concentration of the CSF-1R agonist in the medium is 100-200ng / ml.

13. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of claims 1-12, wherein the CSF-1R agonist is selected from the group consisting of: an agonist antibody, a small molecule agonist, a protein agonist, a peptide agonist and a combination thereof; optionally wherein the protein agonist is a growth factor or a cytokine.

14. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 13, wherein the CSF-1R agonist is selected from M-CSF, IL-34 and a combination thereof.

15. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 14, wherein the CSF-1R agonist is M-CSF.

16. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 14, wherein the CSF-1R agonist is IL-34.

17. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 16, wherein the concentration of IL-34 is 50-300ng / ml.

18. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 16 or 17, wherein the concentration of IL-34 is 200ng / mL, or at least 200ng / ml.

19. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 15, wherein the concentration of M-CSF is lOOng / ml, or at least lOOng / ml.

20. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the medium comprising the CSF-1R agonist comprises IL-34 and M-CSF.

21. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 20, wherein the medium comprises IL-34 at a concentration of 200 / ml, and comprises M-CSF at a concentration of lOOng / ml.

22. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the monocytes are present in the medium at a concentration of 2 x 106cells / mL or 4 x 106cells / mL.

23. The method, the macrophage for use or the macrophage and G-CSF for use according to any of the preceding claims, wherein the G-CSF is recombinant G- CSF.

24. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 23, wherein the recombinant G-CSF is selected from the group consisting of: Lenograstim, Filgrastim, Pegfilgrastim and Lipegfilgrastim.

25. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 23 or 24, wherein the recombinant G-CSF is Filgrastim.

26. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 25, wherein the Filgrastim is selected from the group consisting of: Zarzio, Granix, Nivestim, Releuko, Ratiograstim, Accofil, Tevagrastim and Neupogen.

27. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 25 or 26, wherein the Filgrastim is Neupogen.

28. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein one or more doses of the G- CSF are administered to the subject.

29. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 28, wherein more than one dose is administered to the subject, and wherein the one or more doses are administered to the subject on consecutive days.

30. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 28 or 29, wherein 4 or 5 doses of G-CSF are administered to the subject.

31. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the G-CSF is administered at a dose of lOpg / kg / day.

32. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the G-CSF is administered subcutaneously or intravenously, preferably subcutaneously.

33. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the leukapheresis occurs up to 24 hours after the final dose of G-CSF is administered to the subject.

34. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the macrophage is nonpolarised.

35. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the macrophage is capable of phagocytosis, optionally wherein the macrophage has at least an equivalent phagocytic capacity to a macrophage which has been produced from a monocyte obtained by leukapheresis from a subject who had not been treated with G-CSF prior to leukapheresis.

36. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the percentage of the CD14+CCR2iow population out of the isolated monocyte population is significantly higher than the percentage of the CD14+CCR2i0Wpopulation in monocytes obtained by leukapheresis from a subject who had not been treated with G-CSF prior to leukapheresis.

37. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the expression of surface marker CD14 and CD206 on the macrophages is equivalent to that on a macrophage which has been produced from a monocyte obtained by leukapheresis from a subject who had not been treated with G-CSF prior to leukapheresis.

38. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the macrophage is capable of being be polarised to a pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype.

39. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 38, wherein the macrophage is capable of being polarised to a pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype by incubation in the presence of IL-4 and / or IL- 13 and / or IL- 10.

40. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 38 or 39, wherein the method comprises a further step of polarising the macrophage to a pro-restorative, anti-inflammatory and / or anti- fibrotic phenotype.

41. The method of treating an inflammatory condition, the macrophage for use or the macrophage and G-CSF for use according to claim 40, wherein the step of polarising the macrophage occurs before administration of the macrophage to the subject.

42. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of claims 39-41, wherein the macrophages polarised to a prorestorative, anti-inflammatory and / or anti-fibrotic phenotype have an increased expression of CD 163 and CD206, and a reduced expression of HLA DR and CD86 compared to cells not polarised to a pro-restorative, anti-inflammatory and / or anti- fibrotic phenotype.

43. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the method comprises a further step of engineering the macrophage.

44. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 43, wherein the macrophage is engineered to express one or more cytokines, optionally wherein the macrophage is engineered to express a cytokine which induces polarisation to a pro-restorative, anti-inflammatory and / or anti-fibrotic phenotype.

45. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 44, wherein the cytokine is IL- 10.

46. The method, the macrophage for use or the macrophage and G-CSF for use according to claims 43-45, wherein the macrophage is engineered to overexpress IL- 10 and / or MMP9.

47. The method, the macrophage for use or the macrophage and G-CSF for use according to claims 3-8, wherein the inflammatory condition is a chronic inflammatory condition with a fibrotic element, optionally wherein the condition is organ damage associated with chronic inflammation.

48. The method, the macrophage for use or the macrophage and G-CSF for use according to claims 3-8 or 47, wherein the inflammatory condition is a liver injury, optionally chronic liver injury.

49. The method, the macrophage for use or the macrophage and G-CSF for use according to claims 3-8, 47 or 48, wherein the condition is liver cirrhosis.

50. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 49, wherein the liver cirrhosis resulted from at least one disease or condition selected from the group consisting of: non-alcoholic fatty liver disease (NAFL) (e.g., non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)), alcoholic liver disease (e.g., alcoholic fatty liver disease(AFLD) or alcoholic steatohepatitis (ASH)), mechanical trauma to the liver, biliary obstruction, autoimmune hepatitis, iron overload, Hepatitis B infection (HBV) and Hepatitis C infection (HCV).

51. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 49, wherein the liver cirrhosis resulted from steatotic liver disease (SLD), optionally wherein the steatotic liver disease is metabolic dysfunction-associated steatotic liver disease, metabolic-associated steatohepatitis, Met-ALD or Cryptogenic SLD.

52. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 49, wherein the aetiology of the liver cirrhosis is:(a) pure metabolic dysfunction-associated steatotic liver disease (MASLD),(b) metabolic and alcohol related / associated liver disease (Met-ALD),(c) HCV sustained virologic response (HCV SVR) with ongoing cirrhosis, or(d) alcohol -related liver disease (ALD or Met-ALD) provided that the patient is confirmed to not be drinking alcohol above Met-ALD limits and has a value of <25 mg / 1 in a PEth alcohol test.

53. The method, the macrophage for use or the macrophage and G-CSF for use according to claims 49-52, wherein the liver cirrhosis is selected from compensated cirrhosis and decompensated cirrhosis.

54. The method, the macrophage for use or the macrophage and G-CSF for use according to claims 49-53, wherein the subject exhibits or has recovered from or has become clinically stable following one or more clinical signs of hepatic decompensation selected from the list consisting of ascites, hepatic encephalopathy, variceal haemorrhage (variceal bleed), hepatorenal syndrom e / acute kidney injury (HRS / AKI) and / or Spontaneous Bacterial Peritonitis (SBP).

55. The method, the macrophage for use or the macrophage and G-CSF for use according to claim 3-8, or 47-54, wherein the subject has acute-on-chronic liver failure.

56. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the macrophages are autologous to the subject from whom the leukocytes are obtained.

57. The method, the macrophage for use or the macrophage and G-CSF for use according to any one of the preceding claims, wherein the G-CSF is administered to the same subject from whom the leukocytes are obtained by leukapheresis.

58. The method of treating an inflammatory condition, the macrophage for use or the macrophage and G-CSF for use according to any one of claims 3-57, wherein the macrophages are autologous to the subject to whom the macrophages are administered.

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