Dendritic cell culture method

Abstract

The present disclosure relates to a method of obtaining an enriched type 1 conventional dendritic cell (DC1) population from hematopoietic progenitor cells (HPCs) using a differentiation cocktail that preferentially promotes the differentiation of HPCs into DC1 cells, the differentiated population of cells having a purity of at least 30% of DC1 cells. The population of enriched DC1 cells useful in the treatment of cancer, an inflammatory or autoimmune condition.

Description

[0001] Dendritic cell culture method

[0002] All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety.

[0003] Field of the Disclosure

[0004] The disclosure relates generally to compositions and methods for manufacturing dendritic cells. In particular, the invention relates to improved culture platforms for manufacturing dendritic cells.

[0005] Dendritic cells (DCs) are sentinels that scan the body for infected or malignant cells and initiate an appropriate immune response through their ability to take up and present many pathogen-derived and, in addition, tumour-associated antigens (TAAs) to T cells. For these reasons DCs have long been considered ideal candidates for therapeutic strategies aimed at eliciting an immune response to tumours. Indeed, one type of DCs, monocyte-derived DCs (MoDCs) have been extensively trialled as a cellular immunotherapy, but with limited success (only one FDA-approved vaccine to date). MoDCs were the first type of DC to be grown and characterised in the laboratory and were chosen for clinical development due to the existence of robust methods to generate sufficient numbers from blood monocytes for clinical use. MoDCs continue to be developed and show - at best - modest efficacy, with a recent report of a phase III trial of MoDCs for the treatment of glioblastoma (DCVax-L trial). Despite numerous clinical trials, only Provenge® has been FDA approved. It is now known that a different DC subtype called type 1 conventional DCs (DC1s) are likely the superior choice for a DC immunotherapy, not MoDCs. DC1s are also known as cDC1s, Clec9a+ DCs, Xcr1+ DCs, CD141+ DCs, CD103+ DCs, BDCA3+ DCs. Unfortunately, only a few DC1 therapies have successfully progressed to clinical trials to date due to three major hurdles: 1) their scarcity, and 2) a lack of suitable DC1 manufacturing methods, and 3) challenges with their delivery and activation after encountering tumours.

[0006] Several studies have shown that DC cell types can be generated in culture on a small scale. Naik et al., (J Immunol. 2005, 174(11):6592-7) showed that functional and phenotypic equivalents of mouse splenic CD8(+) and CD8(-) conventional dendritic cell (eDC) subsets (recently renamed DC1 and DC2, respectively) and plasmacytoid DCs (pDCs) can be generated in vitro when bone marrow cells are cultured with fms-like tyrosine kinase 3 ligand(FLT3L). Balan et al., (Cell Rep, 2018. 24(7): p. 1902-1915.) showed that signalling using a nonhuman (xeno) stromal cell line expressing the Notch ligand, Delta-Like Ligand 1 (DLL1) when differentiating cells from human CD34+ precursors lead to high yields of pDCs and both types of conventional DCs (DC1s and DC2s). Furthermore, the same group describes in US2021284963 a procedure for making pDCs from hematopoietic stem cells, using a Notch ligand. Hematopoietic precursors were expanded in a-MEM glutamax, FCS, and a cocktail of recombinant human cytokines: FLT3-L, SCF, IL-7, TPO. The cells were then differentiated into DC with the above cytokine cocktail and adherent OP9 or OP9-DLL1 (Notch) feeder cells.

[0007] Kirkling et al., Cell Rep, 2018. 23(12): p. 3658-3672. e6 reported that cultured hematopoietic progenitors with DC growth factor FLT3 ligand (FLT3L) yields very few DC1s (in humans) or only immature "DC1-like" cells (in the mouse). Co-culture with the combination of OP9 and DLL1 also greatly increased the yield of IRF8-dependent CD141+ DC1s from human bone marrow progenitors cultured with FLT3L. Reliance on stromal cells, especially a xenogeneic source, is limiting however, if one wants to produce clinical grade material for human use.

[0008] Working towards a clinical grade cell culture method, van Eck van der Sluijs, et al., (Cancer Immunol Immunother, 2021. 70(11): p. 3167-3181) developed a clinically applicable culture method using donor-derived G-CSF mobilized CD34+ hematopoietic progenitor cells (HPCs) to generate DC1s, DC2s and pDCs, amongst other cells. CD34+ HPCs isolated from G-CSF mobilized blood cells were cultured with Stem Regininl (SR1 , an agonist of the aryl hydrocarbon receptor), ascorbic acid (AA), human serum (HS), FLT3L, rhSCF and rhTPO. After harvest, the cells were washed and resuspended in medium supplemented with rhGM-CSF, rhIFN-a and rhFLT3L. At the end of the culture, modest numbers (5 million DC1s per million initial progenitor cells) were generated. The data showed mostly an enrichment in the DC2s (13-23%) after 14 days with fewer pDCs and DC1s (5%) along with a 100-fold expansion of total numbers.

[0009] Seet and Crooks of University California describe in EP16797322 an enrichment of CLEC9A+ dendritic cells, by culturing in stem cell factor (SCF), FLT3 ligand (FLT3L), thrombopoietin (TPO), IL-3 and GM-CSF; and stromal cells which expresses a notch ligand.

[0010] There is a need in the art for culture methods that can promote differentiation of precursor cells into dendritic cells, predominantly of the DC1 subtype in an efficient, cost-effective, scalable and timely manner.Summary of the Disclosure

[0011] The present disclosure is based on an improved method for culturing hematopoietic progenitor cells (HPCs) which are also referred to as hematopoietic stem cells (HSC) or hematopoietic stem and progenitor cells (HSPCs) to promote their differentiation into type I conventional dendritic cells (DC1s). The methods of the disclosure are particularly suitable for large scale production of DC1s. The ability to generate these cells in vitro offers tremendous opportunities to study their properties and fortheir use in immunotherapeutic methods including antigen pulsing and adaptive cell transfer.

[0012] In a first aspect, there is provided a method of obtaining a type 1 conventional dendritic cell (DC1) population, the method comprising contacting hematopoietic progenitor cells (HPCs) with a differentiation cocktail ex vivo or in vitro, wherein the differentiation cocktail comprises (1) FLT3L (fms-related tyrosine kinase 3 ligand), (2) IFN-a (interferon alpha), (3) GM-CSF (granulocyte colony stimulating factor), (4) BATF3 (basic leucine zipper ATF-like transcription factor 3, (5) a notch ligand and (6) an adhesion molecule.

[0013] Preferably, the HPCs are differentiated in a suitable cell culture medium that supports expansion and differentiation of HPC CD34+ cells into dendritic cells. Examples include Cellgenix® GMP DC medium, StemSpan™-AOF medium, TexMACS™ medium, CTS AIM V™ medium and X-VIVO™-15 which are serum free. In a particular example, the HPCs are differentiated in CellGenix® GMP DC Medium which is a type of CellGenix medium comprising salts, sugars, amino acids, vitamins and buffers, phenol red, p-mercaptoethanol and L-glutamine. In a further example, the cell culture medium comprises additionally one or more of HS (human serum), SR1 (StemRegenin 1) and L-AA (L-ascorbic acid).

[0014] In certain examples, the CD34+ cells may be contacted with one or more stem cell viability factors. Examples include stromal elements supportive of CD34+ HPC survival or medium conditioned by being previously inoculated onto stromal feeder layers which secrete factors and proteins conducive to the maintenance and survival of CD34+ HPCs. Examples of factors include one or more of stem cell factor (SCF), thrombopoietin (TPO), pleiotrophin, HoxB4, HoxA9, Wnt3a, Notch 1 , and Jagged-1.

[0015] In certain examples, the notch ligand is a delta-like family ligand (DLL). In another example, the notch ligand is DLL1 or DLL4. In a further example, the notch ligand is DLL4.

[0016] In certain examples, the adhesion molecule is an integrin, such as vitronectin, fibronectin, collagen or laminin. Cells of all types interact with the extracellular matrix (ECM) proteins via integrins, multifunctional transmembrane receptors, which are required for adhesion and proliferation in many cell types including HPCs.In certain examples, the adhesion molecule is selected from VCAM1 , fibronectin or retronectin. In another example, the adhesion molecule is VCAM1.

[0017] In certain examples DLL1 or DLL4 and VCAM1 are coated on beads.

[0018] In certain examples DLL1 or VCAM1 is coated on beads. In other examples DLL4 or VCAM1 is coated on beads. In further examples both DLL1 and VCAM1 are coated on beads. In further examples both DLL4 and VCAM1 are coated on beads.

[0019] In certain examples, the BATF3 is provided as mRNA. Preferably, the BATF3 mRNA is provided to the CD34+ HPCs on day 1 of the differentiation phase. Preferably this corresponds to day 7 or day 8 of the total culture time of CD34+ HPCs. The HPCs may be contacted with BATF3 mRNA according to any method known in the art that provides for delivery of the mRNA intracellularly. Examples of such transformation methods include contacting the HPCs with lipid nanoparticles (LNPs) encapsulating BATF3 mRNA (referred as BATF3 mRNA LNPs), nucleofection or electroporation of BATF3 mRNA. Preferably, the BATF3 mRNA is encapsulated within lipid nanoparticles. LNPs are known in the art, and typically contain an ionizable cationic lipid, a neutral lipid, a cholesterol, a pegylated lipid and water. In a particular example, the LNPs of the disclosure comprise the ionizable cationic lipid, SM-102, the phospholipid, 1 ,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and the PEGylated lipid, DMG-PEG 2000 at the molar ratio of 50:38.5:10:1.5.

[0020] In certain examples, the HPCs are co-cultured with the BATF3 mRNA LNPs for a period of time sufficient to allow the HPCs to be transfected with the mRNA. Whilst the LNPs remain in the culture for the entire differentiation phase the majority of the LNPs are typically taken up by the stem cells within hours. In some examples, the HPCs are contacted with the BATF3 mRNA LNPs prior to their transfer into wells i) that are coated with a notch ligand and an adhesion molecule or ii) that contain beads coated with a notch ligand and an adhesion molecule. In certain examples the HPCs are electroporated to introduce BATF3 mRNA and then allowed to rest for 5 to 12 hours before adding both a notch ligand and an adhesion molecule, both or either of which may be presented as coatings on beads or coated directly onto the culture plates.

[0021] In certain examples, the differentiation cocktail medium is replenished or renewed periodically. The differentiation cocktail may be replenished during the differentiation of the HPCs. The differentiation cocktail may be replenished once, but may be replenished two, three, four times or more as required, e.g. every 5, 6, 7, or 8 days. The HPCs may be contacted with the differentiation cocktail for as long as required for the optimal emergence of DC1 cells. Typically, the cells will be contacted with the differentiation cocktail for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 10 days or up to at least 20 days.In certain examples, the cell culture medium during the differentiation phase is also replenished or renewed periodically. The cell culture medium may be replenished once, but may be replenished two, three, four times or more as required, e.g. every 5, 6, 7, or 8 days.

[0022] In some examples the population of cells is assessed by flow cytometry using the marker Clec9A+. In certain examples, the DC1 cells are assessed by flow cytometry using markers Clec9a+ andXCR1+. In certain examples the DC1 cells are assessed by flow cytometry using markers Clec9A+ and XCR1 + and one or more of the markers CD370, CD141 (BDCA-3) and CADM1.

[0023] In a further example, the method further comprises expanding the HPCs in an expansion medium prior to differentiating the HPCs. In some examples, the expansion medium comprises a cell culture medium comprising at least one of HS, AA and SR1 and an expansion cocktail comprising FLT3L, SCF, and TPO. In one example, the HPCs are cultured in the same cell culture medium as that used for differentiation. More particularly, the cell culture medium comprises CellGenix® GMP DC medium as described herein. In one example, the expansion medium does not comprise DLL1 or DLL4 in combination with VCAM1.

[0024] In a particular example, the expansion medium comprises a cell culture medium (e.g. CellGenix® GMP DC Medium) comprising FLT3L, SCF, TPO, HS, SR1 and L-AA.

[0025] In certain examples, the cell culture medium for the expansion and differentiation phase comprises CellGenix® GMP DC Medium supplemented with one or more of human serum (HS), ascorbic acid (AA) and SR1. In one example, human serum is provided in the medium at concentration of about 2% (v / v).

[0026] In certain examples, the expansion medium is replenished or renewed periodically. The expansion medium may be replenished once, but may be replenished two, three, four times or more as required, e.g. every 5, 6, 7, or 8 days. The HPCs may be expanded for a time sufficient to increase the number of HPCs. Typically, the cells will be expanded for at least 5 days, at least 6 days, at least 7 days, at least 8 days or at least up to 10 days. In certain examples, the HPCs are cultured in differentiation medium for about 5 to 7 days, preferably about 7 days.

[0027] In a particular example, the method comprises culturing CD34+ HPCs in a cell culture expansion medium supplemented with SR1 , wherein the HPCs are expanded for a period of about 5 to 7 days, preferably about 7 days in an expansion cocktail comprising FLT3L, SCF, and TPO, followed by differentiating the HPCs in a differentiation cocktail comprising GM-CSF, IFN-a, FLT3L, BATF3 mRNA, VCAM1 and DLL1 or DLL4 for a period of about 5 to 7 days, preferably about 7 days. Other factors such as TPO, pleiotrophin, HoxB4, HoxA9, Wnt3a, Notchl and Jagged-1 may be used alone or in combination with the expansion medium.In some examples, the cell culture medium is also supplemented with HS and ascorbic acid (AA).

[0028] In a particular example, the HPCs are co-cultured with LNPs encapsulating BATF3 mRNA. The differentiation phase preferably comprises DLL1 or DLL4 in combination with VCAM1. In one example, the combination comprises DLL1 and VCAM1 , in another example, the combination comprises DLL4 and VCAM1. DLL1 and VCAM1 , or DLL4 and VCAM1 may be provided on a solid support e.g. a cell culture plate, or a particle or bead (e.g. magnetic bead).

[0029] In a further example, the total culture period is about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 20 days or about 27 days. In some examples, the total culture period is up to about 20 days.

[0030] As described herein, the methods of the disclosure result in the production of DC1 cells which are significantly greater in number and purity compared to the published methods such as described in the van Eck van der Sluijs (van Eck van der Sluijs J et al, (2021) Cancer Immunology, Immunotherapy 70:3167). In certain examples, the differentiated DC1 cells express one or more markers selected from CLEC9A+, CADM1 , XCR1+, BTLA, CD26, DNAM-1 / CD226.

[0031] In some examples, the methods of the invention are able to produce at least 4 x 106DC1 cells per 106expanded HPCs, or at least 5 x 106DC1 cells per 106expanded HPCs originally isolated from cord blood or G-CSF mobilized samples respectively. This corresponds to a least about 50 - 100 x 106DC1 per starting or sourced HPCs (before the expansion phase) respectively. In one example, the methods of the invention result in at least a two-fold expansion, at least a three-fold expansion, at least a four-fold expansion, at least a five-fold expansion, at least a six-fold expansion or greater in the number of DC1 cells compared to the number of starting or sourced HPCs prior to expansion and differentiation.

[0032] In one example, the methods of the invention produce an enriched population of DC1 cells. For example, an enriched population of DC1 cells comprises a population of expanded and differentiated HPCs comprising at least 10% DC1 cells or greater. In another example, the enriched population of DC1 cells comprises at least 20% DC1 cells, at least 30% DC1 cells, or at least 40% DC1 cells or at least 50% DC1 cells or at least 60% or at least 80% DC1 cells. In a further example, the enriched population of DC1 cells comprises about 30-60% DC1 cells. In a further example, the enriched population of DC1 cells comprises about 75-80% DC1 cells. In one example, the enriched population of DC1 cells can be further purified by methods such as flow cytometry wherein the DC1 cells are sorted based on expression of Clec9A+. In one example, the enriched population of DC1 cells can be further purified by methods such as flow cytometry wherein the DC1 cells are sorted based on expression of Clec9A+, and XCR1 + markers.In one example the enriched population of DC1 cells can be further purified by methods such as flow cytometry wherein the DC1 cells are sorted based on expression Clec9A+ and XCR1 + and one or more of the markers CD370, CD141 (BDCA-3) and CADM1.

[0033] The HPCs according to the methods may be obtained from any suitable source. Sources include but are not limited to embryonic yolk sac, the embryonic aorta-gonad-mesonephros region, fetal liver or spleen, adult spleen, adult bone marrow, fetal bone marrow, peripheral blood and umbilical cord blood. Therefore, the methods may further comprise a preliminary step of providing a sample comprising HPCs. The HPCs may be from any desired mammalian species, e.g. human, other primate, or domestic, laboratory or livestock animals, such as a rodent rat or guinea pig), lagomorph (e.g. rabbit), cat. In certain examples, the HPCs are from a mouse, dog, pig, cow, horse, sheep or goat. In certain examples, the HPCs are human. In certain examples, the HSCS are derived from a human donor. In some examples, the HPCs are derived from G-CSF mobilised blood, e.g. PBMCs.

[0034] The HPC containing sample may be contacted directly with the differentiation cocktail described herein. However, it may be desirable to enrich the HPC sample for HPCs and / or expand the HPCs in the sample before such differentiation. This may involve a positive selection for cells expressing markers associated with HPCs. Additionally, or alternatively, enrichment for HPCs may involve negative selection to remove cells which are not HPCs. HPCs typically do not display lineage specific markers. Thus, it may be desirable to deplete the sample of HPC containing cells expressing lineage specific markers (so-called "lineage positive cells"). Such enrichment or depletion may be performed in addition to, or as an alternative to, an expansion step as described above.

[0035] In some examples, expansion (i.e. proliferation) of the HPCs, or a sample of cells comprising HPCs is induced prior to contacting the cells with the differentiation cocktail. This allows for the number of HPCs to be increased prior to differentiation, thus increasing the final yield of DC1s. Thus, the methods may additionally comprise providing a sample of HPCs and expanding the HPCs prior to contacting the cells with the differentiation cocktail.

[0036] In some examples, the sample of cells is depleted of cells expressing lineage specific markers in order to obtain an enriched HPC population and subsequently to expand the number of HPCs in the sample. In some examples, the sample is enriched for cells expressing CD34+. Methods of enriching forCD34+cells are known in the art. In one example, the cells are selected for CD34+cells using a commercial kit e.g. EasySep Human CD34 Positive Selection Kit II as used herein or CD34+coated microbeads. In another example the CD34+cells are selected using fluorescence activated cell sorting (FACS) a flow cytometry technique

[0037] It is understood that the enriched DC1 cell population obtained by contacting HPCs with the differentiation cocktail may additionally include other cell types, including plasmacytoiddendritic cells (pDC) and DC2 cells. In some examples the % purity of each of these type is less than the % purity of DC1 cells. In some examples the DC1 population contains less than 10% DC2 cells. In some examples the DC1 population contains less than 10% HSC (progenitor) cells. In some examples the DC1 population contains less than 10% DC2 and less than 10% HSC (progenitor) cells.

[0038] In another aspect, the disclosure provides a population of cells enriched for DC1 cells obtained by, or obtainable by a method described herein.

[0039] In another aspect , there is provided a population of cells enriched for type 1 conventional dendritic cell (DC1) cells wherein the % purity of DC1 cells in the population of cells is greater than 30%.

[0040] In some examples the population of cells contains less than 10% progenitor cells.

[0041] In some examples the % purity is measured by flow cytometry. In some examples the population of cells is assessed by flow cytometry using markers Clec9A+.

[0042] In some examples the population of cells is assessed by flow cytometry using the markers Clec9A+ and XCR1+.

[0043] In some examples the population of cells is assessed by flow cytometry using the markers Clec9A+ and XCR1 + and additionally one of the markers CD370, CD141 (BDCA-3), and CADM1.

[0044] In some examples the population of cells contains less than 10% progenitor cells. In some examples the population of cells contains less than 10% DC2 cells. In some examples the population of cells contains less than 10% progenitor cells and less than 10% DC2 cells.

[0045] In some examples the population of cells was obtained from culturing hematopoietic progenitor cells (HPCs) in a first culture step comprising an expansion medium to obtain expanded HPCs and then in a second culture step comprising a differentiation cocktail ex vivo, the second step results in preferentially differentiating the expanded HPCs into DC1 cells . In some examples the differentiation cocktail comprises 1) FLT3L (fms-related tyrosine kinase 3 ligand), 2) IFN-a (interferon alpha), 3) GM-CSF (granulocyte colony stimulating factor), 4) BATF3 (basic leucine zipper ATF-like transcription factor s) a notch ligand and 6) an adhesion molecule.

[0046] In some examples the culturing of the hematopoietic progenitor cells (HPCs) in a first culture step and then in a second culture step, results in the generation of at least 2 million DC1 type cells per million expanded cells.The population of enriched DC1 cells may be utilised in various ways. For example, the cells may be administered directly to a subject or formulated for administration to a subject directly.

[0047] Thus, in a further aspect, the disclosure provides an enriched DC1 cell population obtained or obtainable by a method described herein for use, or when used in medicine. In another aspect, the disclosure provides an enriched DC1 cell population obtained or obtainable by a method described herein for use, or when used in the treatment of any suitable condition as described in further detail below. In another aspect, the disclosure provides an enriched DC1 cell population obtained or obtainable by a method described herein, in the manufacture of a medicament for the treatment of any suitable condition as describe in further detail below. In certain examples, it may be desirable to expose the DC1 cells to an antigen for presentation to T cells. This is described in the art as “antigen pulsing”. It may be desirable that the antigen is cross-presented to T cells via MHC class I molecules.

[0048] In another aspect, the disclosure provides a method comprising contacting in vitro or ex vivo the enriched DC1 cell population obtained, or obtainable by a method described herein with an antigen.

[0049] In another aspect, the disclosure provides a source of DC1s that can present antigen to T cells in vitro for activating T cells. In one example, this could be for a diagnostic purposes, for example as a means to determine which antigens are immunogenic to naive T cells.

[0050] In some examples, the enriched DC1 cell population may be contacted with an adjuvant, such as toll-like receptor agonist, Sting agonist or CD40 agonist, preferably in vitro. This may occur at approximately the same time as contacting the antigen, or at or after administration to the recipient subject. The type of antigen may be selected based on the desired immune response. The antigen pulsed DC1 population may then be administered to a subject or formulated for administration to a subject. The subject may be same individual from whom the HPCs were originally derived (e.g. autologous) or not (e.g allogeneic).

[0051] In another aspect, the disclosure provides an antigen-pulsed enriched DC1 cell population obtained, or obtainable by a method of the disclosure for use in the treatment of any suitable condition as described herein. The antigen pulsed enriched DC population may further be contacted with T cells in vitro or ex vivo in order to generate or stimulate T cells (e.g. cytotoxic T cells (CTLs), but also helper T cells or regulatory T cells (T regs) specific for the antigen.

[0052] In another aspect, the enriched DC1 cell population may be used to stimulate T cells engineered to express a receptor, for example chimeric antigen receptor (CAR) T cells, orspecific T cell receptor (TCR)-expressing T cells. The T cells in the population may be allowed to expand in culture in order to increase the number or proportion of T cells in the population which are specific for the antigen. The T cells may then be administered to a subject orformulated for such administration. Preferably, the T cells are autologous i.e. they are derived from the same subject, or from genetically identical subjects, but may also be used in an allogeneic setting i.e. from a different donor.

[0053] Preferably, the enriched DC1 cell population is used for the treatment of cancer, or an inflammatory or autoimmune condition, especially a condition characterized by ineffective and / or undesirable CTL activity. The condition to be treated may be selected from:

[0054] -solid tumours, such a colorectal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, skin cutaneous metastatic melanoma, thyroid cancer. - autoimmune diseases, including rheumatoid arthritis and other types of chronic or acute arthritis or arthropathies with an immune component, systemic lupus erythematosus (which is known to involve particularly high levels of cell death), scleroderma, Sjogren syndrome, autoimmune (particularly Type I) diabetes, thyroiditis, and other organ-specific immune diseases, including psoriasis;

[0055] - neurologic diseases, including multiple sclerosis, myasthenia gravis, and other neurologic immune-mediated diseases. Also included are gastrointestinal diseases, including Crohn's disease, colitis, celiac disease and hepatitis;

[0056] - cardiovascular diseases, which are now recognised to have a significant immune-mediated component, including atherosclerosis, cardiomyopathy, rheumatic fever, endocarditis, vasculitis, and other immune-mediated cardiovascular diseases;

[0057] - immune-mediated respiratory diseases, including emphysema, respiratory airways infections, and other immune-mediated respiratory diseases;

[0058] - allergic processes and hypersensitivity reactions (type I, II, III, and IV), including asthma, rhinitis, and other immune-mediated hypersensitivity reactions;

[0059] - transplant or graft rejection and graft versus host disease, as occurs during or subsequent to, for example, organ transplant, tissue graft, blood transfusion, bone marrow transplant;

[0060] - degenerative processes, such as neurodegenerative processes, which implicate immune competent cells such as microglia.

[0061] - immunopathological responses to infectious agents, including septic shock syndromes;

[0062] In some examples, the enriched DC1 cell population is administered with a check point inhibitor such as PD-1 inhibitor, CTLA-4 inhibitor or LAG-3 inhibitor.

[0063] In some examples the enriched DC1 cell population is administered with T cells, CAR-T, NK cells and / or adjuvants (e.g. STING agonists).

[0064]

[0065] Figure 1 shows the preferred embodiment of the method and results from the generation of cDC1 from CD34+progenitors using coated DLL1 + VCAM1 and BATF3 mRNA LNPs overexpression.

[0066] A. The detailed method of cDC1 generation from the van Eck van der Sluijs method and this invention (new improved method). B. Cord blood and C. G-CSF-mobilised CD34+progenitor cells were cultured according to one of the embodiments of the invention.

[0067] Figure 2 shows results from use of different commercially available GMP-approved culture media for DC1 production from cord blood HPCs, following the van Eck van der Sluijs prior art method.

[0068] (A). CellGenix® GMP DC Medium (Cellgenix), StemSpan-AOF, TexMACS, CTS AIM V or (B).

[0069] X-Vi vo-15.

[0070] Figure 3 shows results from use of Notch ligands DLL1 or DLL4 in the presence or not of VCAM1 adhesion molecule for producing DC1s from CD34+ cord blood HPCs, from 3 independent donors (D03, D15, D22), expanded for seven days as shown in Figure 1A and then differentiated on plates coated with: DLL1 alone, DLL4 alone or in combination with VCAM1. (A). The number of DC1 (106) obtained per 106expanded CD34+HPCs. (B). The % of DC1 cells at the end of the culture.

[0071] Figure 4 shows results from the use of different adhesion molecules, some that bind VLA-4 (including VCAM1 , fibronectin and retronectin) and others like laminin which does not bind to VLA-4 in the culture media for producing DC1s from CD34+ cord blood HPCs (experiments from three independent donors) using the van Eck van der Sluijs prior art method shown in Figure 1. (A). The number of DC1 (106) obtained per 106expanded CD34+cord blood HPCs and (B). The purity of DC1 (%) at the end of culture analysed by flow cytometry.

[0072] Figure 5 shows results from use of adhesion molecules laminin and poly-L-lysine in the van Eck van der Sluijs prior art method on differentiation of cord blood CD34+ HPC differentiation to DC1s. (A). The number of DC1 (106) obtained per 106expanded CD34+HPCs derived from cord blood and (B). The purity of DC1 (%) at the end of the culture analysed by flow cytometry.

[0073] Figure 6 shows results from DC1 differentiation from cord blood CD34+ HPCs in the presence of various transcription factors (BATF3, IRF8, PU.1 , DC-script) cultured according to the van Eck van der Sluijs prior art method shown in Figure 1 , in the presence of DLL1 alone or DLL1 +VCAM1. (A). The number of DC1 (106) obtained per 106expanded CD34+cord blood HPCs and (B). The purity of DC1 (%) at the end of culture analysed by flow cytometry.

[0074] Figure 7 shows results from DC1 differentiation from three donors of G-CSF mobilised CD34+ HPCs in the presence of DLL1 and VCAM1 or DLL4 and VCAM1 with or without BATF3 mRNA delivered in LNPs. (A). The number of DC1 (106) obtained per 106expanded CD34+HPCs and (B). The purity of DC1 (%) at the end of culture determined by flow cytometry.

[0075] Figure 8 shows results from use of BATF3 mRNA comparing electroporation or LNPs as the delivery method. (A). Cord blood CD34+HPCs or (B). G-CSF mobilised CD34+HPCs.

[0076] Figure 9 shows results from different time frame culture processes. The experiment was performed using cord blood derived CD34+ HPCs from 3 different donors (D20, D21 , D22). Figure 10 shows a comparison of the number of cDC1 generated per initial / starting HPC isolated from G-CSF mobilized donor samples using the best method of this invention compared to the van Eck van der Sluijs method.

[0077] Figure 11 shows a comparison of (A) the number of DC1 (106) per 106expanded HPCs and (B) the percentage (%) of DC1 (purity) when the DLL1 and VCAM1 ligands were attached to beads of different sizes and doses or with DLL1 and VCAM1 ligands plated onto the wells. CD34+ cord blood cells from healthy donors were isolated, expanded for 7 days, and differentiated using either: the van Eck van der Sluijs prior art method, or the method of the disclosure wherein DLL1 and VCAM1 are coated onto culture plates (D+V) or DLL1 and VCAM1 are attached / coated onto the beads (D-V). 1x dose consists of 10,000 beads with a size of 6.8pm cultured with 10,000 expanded HPCs. For other bead sizes, an equivalent total surface area to that of 10,000 beads of 6.8pm is used. 2x doses consists of 20,000 beads with a size of 6.8pm cultured with 10,000 expanded HPCs, with equivalent surface area adjustments for other bead sizes.

[0078] Figure 12 shows a comparison of (A) The number of cDC1 (106) per 106expanded HPCs and (B) the percentage (%) of cDC1 (purity) when the DLL1 and VCAM1 ligands were attached to either beads or coated onto the wells. G-CSF-mobilised CD34+progenitor cells from healthy donors were isolated, expanded for 7 days, and differentiated using either: van Eck van der Sluijs method, D+V (coated DLL1 and VCAM1 ligands), or D-V (DLL1 and VCAM1) beads in the absence or presence of BATF3-mRNA-LNPs.Figure 13 shows G-CSF mobilised CD34+ HPCs from healthy donors isolated and cultured using either the van der Sluijs prior art method or the method of disclosure (comprising BATF3, DLL1 and VCAM1) or a modified method of the disclosure with either ascorbic acid (L-AA), human serum (HS) orStemRegenin-1 (SR1) during the expansion and / or differentiation phases. (A), the number of DC1 (xl 06) per 106expanded CD34+ hematopoietic stem cells cultured, and (B). the purity (%) of DC1 are shown in bar graphs. -L-AA-L-AA: the CD34+ progenitors were expanded and differentiated in absence of L-AA. -L-AA+gs: CD34+ progenitors were expanded in absence of L-AA, but L-AA is present during the differentiation phase.

[0079] Figure 14 shows G-CSF-mobilised CD34+ progenitor cells from either healthy donors or cancer patients were cultured according to the methods of the disclosure (B+D+V refers to the inclusion of BATF3 + DLL1 + VCAM1) compared to the van Eck van der Sluijs prior art method (A). The number of DC1 (106) per 106expanded CD34+haematopoietic stem cells cultured and (B). The purity (%) of DC1 are shown in bar graphs. P1 to P3 are healthy donors, MM01 and MM02 are derived from Multiple myeloma patients, DLBCL is derived from Diffuse large B cell lymphoma patient, MCL is derived from Mantle cell lymphoma patient.

[0080] Figure 15 shows G-CSF-mobilised CD34+ progenitor cells from either healthy donors or cancer patients that were isolated and cultured according to the new improved method using Batf3 mRNA LNPs + DLL1 + VCAM1 compared to the van Eck van der Sluijs prior art method. The CD34+ cells were expanded for either 5 days and differentiated for 5 days (5+5) or 7 days (5+7). The rest of the expanded cells were further expanded for two more days (7 total expansion days) and differentiated for 5 days (7+5) or 7 days ((7+7), our current improved protocol). (A). The number of cDC1 (106) per 106expanded CD34+ haematopoietic stem cells cultured and the (B). purity (%) of cDC1 are shown in bar graphs. P1-P3 are healthy donors, MM01 and MM02 are derived from Multiple myeloma patients, DLBCL is derived from Diffuse large B cell lymphoma patient, MCL is derived from Mantle cell lymphoma patient.

[0081] Figure 16 shows G-CSF-mobilised CD34+ progenitor cells from healthy donors that were enriched for CD200 and cultured according to Example 1.

[0082] (A). The number of cDCI (106) per 106expanded CD34+haematopoietic stem cells with or without CD200 enrichment and the (B). purity (%) of cDC1 are shown in bar graphs. P1 to P3 are healthy donors.

[0083] Figure 17 shows results of DC1 number and purity from cord-blood derived expanded CD34+ HPC cells differentiated for up to 20 days using either the van Eck van der Sluijs prior art methodor using additionally DLL1 +VCAM1. (A). The number of DC1 (106) per 106expanded CD34+HPC cells and the (B). purity (%) of DC1 are shown in bar graphs.

[0084] Figure 18 shows results of fold change of cDC1 per expanded HSCs and % DC1s from a GMP cell culture system for differentiating expanded CD34+ cells.

[0085] Detailed Description

[0086] General Techniques and Selected Definitions

[0087] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0088] Each example described herein is to be applied mutatis mutandis to each and every other example of the disclosure unless specifically stated otherwise.

[0089] Those skilled in the art will appreciate that the disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0090] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, cell biology and immunology. Such procedures are described, for example,

[0091] Hematopoietic Stem Cell Protocols (K.D. Bunting and C-K. Qu, 2014, Springer protocols), particularly pp211-221 and In Vitro Human Haematopoietic Stem Cell Expansion and Differentiation (YK. Bozhilov, I. Hsu, EJ. Brown, AC. Wilkinson, Cells, 2023).

[0092] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

[0093] The term “consists of’ or “consisting of’ shall be understood to mean that a method, process or composition of matter has the recited steps and / or components and no additional steps or components.

[0094] The term “about”, as used herein when referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass variations of ±20% or ±10%, morepreferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0095] Conventional dendritic cells

[0096] Dendritic cells (DC) are key orchestrators of immune responses. A specific subset of DCs are conventional DCs (eDCs) which are especially adept at presenting exogenous and endogenous antigens to T cells and resulting T cell proliferation, survival and effector function. For the avoidance of doubt, reference to DC1 also means cDC1. eDCs can be divided into at least two distinct subsets cDC1 and cDC2, also referred to as DC1s and DC2s, respectively. DC1 depend on the transcription factors BATF3, IRF8, and ID2 for their development and selectively express the chemokine receptor XCR1 + and the C-type lectin receptor DNGR-1 / CLEC9A+. Expression of the integrin aE (CD103) is also commonly used as an additional marker to identify DC1 in mouse tumours, while BDCA3 is used for the same purpose in humans. DC1s excel at cross-presenting exogenous antigens (e.g. tumour antigens) to CD8+T cells and are key cells for the generation of cytotoxic effector T cell responses.

[0097] DC1s can be identified as CD141+Clec9A+ DCs. In addition, some DC1 markers show conserved expression in both mouse and human, such as Clec9A, CD26, CADM1 and BTLA. In the peripheral lymphoid and non-lymphoid organs such as the lung, gut and LN, DC1s also can also be identified as CD103+CD11b- DCs.

[0098] Practically the DC1s can be identified based on flow cytometry using specific surface protein expression markers as described herein and once sorted (collected) confirmed by their functional capacity to cross-prime T-cells against engulfed cell-associated antigen. In one example, DC1s can be sorted based on expression of Clec9A+, and XCR1+ markers. In another example in vitro generated DC1s can be sorted based on expression of one or more of CD11c+, Clec9A+, XCR1+, HLA-DR+ markers.

[0099]

[0100] cells

[0101] Hematopoietic progenitor cells (HPCs) are multipotent cells that are capable of giving rise to all blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). As multipotential cells they also have the capacity to selfrenew. Cell types referred to as hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), or hematopoietic multipotent progenitor cells (MPPs) also have this activity and are included within the term "HPC" as used herein. HPC and HSC are thus used interchangeably throughout.

[0102] HPCs are characterized by the ability to regenerate a multilineage hematopoietic system when introduced into an immunodeficient host. A multilineage hematopoietic system will contain all blood cells and their progenitors, introduced in such circumstances will be able to sustain multilineage haematopoiesis long term (for example, for the lifespan of the animal model) or transiently (for example, for 4 months or less).

[0103] The positive expression or negative expression (expression profile) of certain cell surface proteins may be used to identify HPCs, for example CD34+. HPCs are lineage negative (Lin ) i.e. they do not express lineage-specific markers such as CD3, CD14, CD16, CD19, CD20 and CD56. The expression profile of other cell surface proteins suitable for identifying HSCs will be known to one skilled in the art.

[0104] HPCs obtained from any source are suitable for use in the present methods. Sources of HPCs include any tissue which contains any amount of HPCs. These tissues may include but are not limited to embryonic yolk sac, the embryonic aorta-gonadmesonephros region, fetal liver or spleen, adult spleen, adult and fetal bone marrow, untreated peripheral blood and umbilical cord blood. HPCs can also be mobilised into the peripheral blood and spleen tissue from the bone marrow by cytokine treatment, e.g. with G-CSF (granulocyte-macrophage colony stimulating factor) or GM-CSF, and optionally cyclophosphamide. The HPCs may be from any desired mammalian species, e.g. human, other primate, or domestic, laboratory or such as a rodent (e.g. mouse, rat or guinea pig), lagomorph (e.g. rabbit), cat, dog, pig, cow, horse, livestock animals sheep or goat. In certain embodiments, the HPCs are human. In certain embodiments, the HPCS are derived from human donors.

[0105] It may be desirable to enrich a sample for HPCs. HPCs suitable for use in the methods of the disclosure may be purified from a sample by any suitable technique or device. Positive or negative selection may be employed to enrich for, or deplete of, respectively cells expressing certain surface proteins associated with particular cell lineages or stages of differentiation. In one example a sample of HPCs maybe enriched prior to differentiation, by positive selection for cells expressing CD200.Since HPCs are lineage negative, a sample of cells can be enriched for HPCs by negative selection for cells expressing one or more lineage specific markers (CD3, CD14, CD16, CD19, CD20 and CD56).

[0106] HPC expansion medium

[0107] Once a population of cells containing HPCs has been obtained, the HPCs in this population may be expanded. The expansion step may occur before or after the cell population is enriched for HPCs. The expansion step may comprise, for example, culturing in hematopoietic bioreactors, or co-culturing with stromal elements supportive of HPC expansion / survival, or culturing in media conditioned by being previously inoculated onto stromal feeder layers which secrete factors and proteins conducive to the maintenance, survival and expansion of HPCs. The expansion step may comprise contacting said cell population with one or more of SCF, FLT3L, IL-3 and IL-6, for example, contacting said cell population with one or more of SCF, FLT3L, IL-3 and IL-6. Additional reagents may include CD200 orTPO.

[0108] DC1 differentiation cocktail

[0109] The term “differentiation cocktail” is used herein to refer to the combination of cytokines and / or growth factors which are used to differentiate HPCs into DC1 cells. In certain examples, the differentiation cocktail comprises FLT3L (fms-related tyrosine kinase 3 ligand), IFN-a (interferon alpha), GM-CSF (granulocyte colony stimulating factor), BATF3 (basic leucine zipper ATF-like transcription factor), DLL1 or DLL4 and VCAM1.

[0110] The differentiation cocktail is contacted with HPCs in vitro or ex vivo, but typically under cell culture conditions. It will be appreciated that the cell culture medium may contain additional factors which have roles other than promoting differentiation of HPCs to DC1 cells. These components are not regarded as part of the differentiation cocktail.

[0111] The cell culture medium may be a conventional medium for growing cells, e.g. CellGenix® GMP DC Medium as described herein. The medium may comprise components such as antibiotics (e.g. penicillin, streptomycin), nutrients (e.g. glutamine), heat-inactivated fetal calf serum, human serum or similar, etc.

[0112] The cell culture medium may also contain components for maintenance of HPC properties such as viability and potency which may be used individually or in combination. Such factors include ascorbic acid (AA) and StemRegeninl (SR1). In some examples, AA is provided at a concentration of about 30-50pg / ml, preferably about 50pg / ml. In some examples, SR1 is provided at concentration of about 1pM.The HPCs may remain in contact with the differentiation cocktail for at least 6 days, e.g. at least 7, at least 8, at least 9 or at least 10 days. The contact may be maintained until sufficient differentiation has taken place. Typically contact will be maintained for about 7 days.

[0113] Preferably, GM-CSF is provided at a concentration of about 800 lU / ml. Preferably, FLT3L is provided at a concentration of about 100 ng / ml. Preferably, IFN-a is provided at a concentration of about 1000 lU / ml. Preferably, the DLL1 or DLL4 is provided at a concentration of about 8pg / ml. Preferably, the VCAM1 is provided at a concentration of about 2pg / ml. Preferably, the BATF3-mRNA is provided by encapsulation in LNPs at a concentration of about 1000 ng / mL of BATF3 mRNA-LNPs in 100 uL medium per 10,000 HSCs.

[0114] Additional reagents may be also included that boost the differentiation into primarily DC1s. For example Ashayeripanah, Mitra et al. (Cell Reports, Volume 45, Issue 1 , 116772) and Lui et al (Bioarchives https: / / doi.org / 1064898 / 2026.01.14.699162) show that inclusion of IL4 into a differentiation medium helps promote cDC1 generation / differentiation.

[0115] Provision of the proteins / ligands.

[0116] Any of the protein additives used in either the expansion medium, the differentiation cocktail or in the cell culture medium can be natural or recombinant proteins, mimics or muteins. Typical sources of these are Aero Biosystems (VCAM-1 , DLL4, Notch, FLT3L, IFN-a, TPO, GM-CSF, SCF, IL-3, IL-6, wnt3A), Peprotech (TPO, SCF, ApoE3), Thermofisher (IFN-a) or Creative Biolabs (DLL1). Proteintech supplies GMP grade recombinant proteins (FLT3L, IFN-a, IL-3, IL-6, SCF, TPO, GM-CSF, wnt3A).

[0117] Uses of the DC1 cells

[0118] DC1 cells may be utilized as a form of anti-cancer cell therapy. These cells can be administered to patients through various routes, including intravenous or intratumoral injection. Prior to administration, DC1 cells may be loaded ex vivo with patient-derived tumor cell lysates or tumor-associated antigens or one or a cocktail of tumour antigen derived peptides, in the presence or not of a Toll-like receptor (TLR) agonist or adjuvant to enhance their therapeutic potential. Alternatively, DC1 cells can be engineered such as with a chimeric antigen receptor (CAR) to improve tumour uptake and antigen presentation, or to express specific genes known to enhance their function (such as CCR7, Galectin-9, IL-12, TNF-a) or inhibit the expression of genes involved in immunosuppressive pathways (such as TGFp, interleukin 10 (IL-10), adenosine, VEGF, indoleamine 2,3 dioxygenase 1 (ID01), indoleamine 2,3-dioxygenase 2 (ID02), tryptophan 2-3-dioxygenase (TDO), lactate, hypoxia, arginase, and prostaglandin E2). In certain cases, DC1 cells may be injected directly into the patient without prior antigen loading.The DC1 therapy may be associated with other treatment such as a check-point inhibitor treatment T-cell checkpoint inhibitors (including but not limited to, anti-CTLA4 (such as Ipilimumab) anti-PD1 (such as Pembrolizumab, Nivolumab, Cemiplimab), anti-PDL1 (such as Atezolizumab, Avelumab, Durvalumab), anti-PDL2, anti-BTLA, anti-LAG3, anti-TIM3, anti-VISTA, anti-TIGIT, and anti-KIR), radiotherapy or chemotherapeutic agent for synergistic antitumour efficacy. The DC1 cells may be derived from the patient for a personalised treatment or used as an off-the-shelf strategy (allogeneic sources of progenitors). The DC1 cells may be first cryopreserved and stored until their utilisation.

[0119] The cell population comprising DC1 cells are also useful for inducing various types of immune responses, for example, those involving T cells or Treg cells. They may be particularly useful in immune responses caused by immunogenic cell death, by taking up cellular debris from dead or dying cells (or even absorbing the entire cell) and presenting processed fragments to T cells.

[0120] In vitro, or following administration to a subject, the enriched DC1 cell population can induce an immune response to a target antigen with which they have been contacted. The immune response may be the proliferation of T cells, which may be cytotoxic T lymphocytes (CTL) or helper T cells. The immune response can be the proliferation of both CD8+T cells and CD4+T cells and may involve the proliferation of both types of T cell in any given immune response. The cell population comprising DC1 cells may also stimulate and induce proliferation of Treg cells. Given that Treg cells may be capable of modulating the response of other cells of the immune system against an antigen in other ways, e.g. inhibiting or suppressing their activity, the effect on the immune system as a whole may be to modulate (e.g. suppress or inhibit) the response against that antigen. Thus, an immune response can also comprise modulating the response to an antigen (e.g. inhibiting or suppressing).

[0121] It may be particularly desirable to raise a Treg response against an antigen to which a subject exhibits, or is at risk of developing, an undesirable immune response. For example, it may be a self antigen against which an immune response occurs in an autoimmune disease. Examples of autoimmune diseases in which specific antigens have been identified as potentially pathogenically significant include multiple sclerosis (myelin basic protein), insulin-dependent diabetes mellitus (glutamic acid decarboxylase), insulin resistant diabetes mellitus (insulin receptor), coeliac disease (gliadin), bullous pemphigoid (collagen type XVII), auto-immune haemolytic anaemia (Rh protein), auto-immune thrombocytopenia (Gpllb / llla), myasthenia gravis (acetylcholine receptor), Graves' disease (thyroid-stimulating hormone receptor), glomerulonephritis, such as Goodpasture's disease (alphas(IV)NCI collagen), and pernicious anaemia (intrinsic factor). Under certain conditions, it may also be possible to tolerise a subjectagainst a particular antigen. Thus the disclosure provides a method for inducing tolerance in a subject towards an antigen, comprising administering to the subject a composition comprising the antigen and DC1s produced by the method described herein, or antigen pulsed DC1 cells produced by the method described herein and wherein the antigen and / or DC1s is administered or pulsed in the absence of an adjuvant.

[0122] DC1 cells are believed to be particularly important in the generation of CTL responses, so the immune response to be stimulated is preferably a CTL response. The immune response may involve production and / or proliferation of CTLs, which are typically T cells expressing CD8 and are capable of cytotoxic activity against cells displaying their cognate antigen in the context of MHC class I molecules. This will be useful to treat patient with solid tumour, as these CTLs will kill the tumour cells.

[0123] It will be understood that the cells produced by the methods of the disclosure may also be used for the prophylaxis and / or treatment of any condition in which it is desirable to induce a CTL response, such as cancer, or infection by an intracellular parasite or pathogen, such as a viral infection.

[0124] Pharmaceutical compositions

[0125] The enriched DC1 cell population produced by the methods of the disclosure may be used in the manufacture of a therapeutic composition.

[0126] The therapeutic composition may comprise a population of DC1 cells which can be administered separately by intravenous, intraperitoneal, enteral, or tracheal administration methods or in combination with other suitable compounds. The therapeutic composition may be formulated with additional excipients, stabilisers, medium or other materials well known to those skilled in the art. The formulation should be non-toxic, have a pH between 3 and about 10 and should not interfere with the efficacy of the therapeutic composition. Electrolytes such as, but not limited to, sodium chloride and potassium chloride can also be included. Typically, the cells may be resuspended in a biocompatible cell culture medium before injection. A cell culture medium that supports the maintenance, growth, and / or health of DC1 cells is suitable but in particular, serum-free medium, and animal-free may be preferred.

[0127] A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0128] Other combinations may include agents that inhibit immunosuppressive pathways, including but not limited to, inhibitors of TGFp, interleukin 10 (IL-10), adenosine, VEGF, indoleamine 2,3 dioxygenase 1 (ID01), indoleamine 2,3-dioxygenase 2 (ID02), tryptophan 2-3-dioxygenase(TDO), lactate, hypoxia, arginase, and prostaglandin E2. In another example, the DCs or a population of DCs of the present disclosure may be used in combination with T-cell checkpoint inhibitors, including but not limited to, anti-CTLA4 (such as Ipilimumab) anti-PD1 (such as Pembrolizumab, Nivolumab, Cemiplimab), anti-PDL1 (such as Atezolizumab, Avelumab, Durvalumab), anti-PDL2, anti-BTLA, anti-LAG3, anti-TIM3, anti-VISTA, anti-TIGIT, and anti-KIR.

[0129] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.1

[0130] EXAMPLES

[0131] Materials

[0132] Enrichment of CD34+progenitor cells from mobilised blood or cord blood

[0133] Cord blood units were obtained from Murdoch Children Research’s Institute. CD34 purification was performed using EasySep™ Human CD34 Positive Selection Kit II and isolated as per manufacturer’s instructions.

[0134] Apheresis G-CSF mobilised HPCs samples were obtained from Peter MacCallum Cancer Centre (Melbourne Australia). Typically G-CSF is administered to subjects who subsequently undergo apheresis and the mobilised HPCs are thus collected. Methods of this procurement are described in Rees et al Bone Marrow Transplantation 2022, 57(6); 1-3; Panch et al Transfusion.

[0135] 2015;55(2):265-74.; Roberts and Metcalf, Blood 86, 1600-1605 (1995); Sheridan et al Lancet, 1992; 3398794: 640-644.

[0136] CD34 purification was performed using human CD34 Microbead Kit and isolated as per manufacturer’s instructions.

[0137] Transfection of HSCs with BATF3 mRNA by electroporation

[0138] 200,000 pre-expanded CD34+ cells from cord blood or G-CSF-mobilised units were electroporated with 3pg BATF3 mRNA (BASE Facility, University Queensland, Australia) or with control GFP using Lonza 4D-Electroporator Unit with P3 Primary Cell Electroporator X Kit S. The pulse code used for electroporation was DS-150. Cells were immediately transferred into the complete expansion medium: CellGenix® GMP DC Medium comprising 2% human serum (HS), 1pM StemRegenin-1 (SR1) and 50 pg / ml ascorbic acid (AA) in the presence of 100ng / mL FLT3L, SCF and TPO. The cells were then rested in incubator overnight.

[0139] The mRNA was tagged with green fluorescent protein (GFP) to confirm that the mRNA was able to enter the cells and be translated.

[0140] Lipid nanoparticle transduction of cells

[0141] Lipid nanoparticles (LNPs) consisting of SM-102, 1 ,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and DMG-PEG 2000 at the molar ratio of 50:38.5:10:1.5 were used. Transfection of the LNPs with BATF3 mRNA was performed at concentration of 100ng of BATF3 mRNA LNPs per 10,000 expanded HSCs in the presence of 1pg / ml Apolipoprotein E3 (ApoE3). The LNPs were added to the culture i) prior to the transfer of the cells on notch ligand and adhesion molecule coated wells; or ii) with notch ligand and adhesion molecule coated beads.Coating plates with DLL1 , DLL4 or VCAM1

[0142] 50pl of 1xPBS containing 8pg / ml DLL1 or DLL4 in the presence of 2pg / ml VCAM1 was used to coat 96-well non-treated plates overnight. The next day, wells are washed with 1x PBS to remove excess proteins.

[0143] Coating streptavidin-beads with DLL1 or VCAM1-Fc-Avitag

[0144] Streptavidin (SA)-coated polystyrene beads of various sizes (6.8pm, 21.8pm, 45pm, or 105pm) were purchased from Spherotech. DLL1-Avitag and VCAM1-Avitag were purchased from AcroBiosystems. The SA-beads with DLL1-Avitag and VCAM1-Avitag were mixed at the mass ratio of 4:1 and centrifuged for 30 minutes.

[0145] Flow cytometry characterisation of DC1 cells and assessment of purity of DC1s

[0146] A portion of cells was harvested from the well and stained with antibodies specific for Clec9A, CD141 , CADM1 and XCR1 for 30 minutes at room temperature. The cells were washed twice in flow cytometry buffer and stain with propidium iodide for dead cell identification. Cells were analysed on flow cytometer for expression of DC1 markers (DC1 are Clec9A+,CD141+; in some examples DC1 were also CADM1+XCR1+ / -). Purity represents the percentage (%) of DC1 in the overall culture.

[0147] Example 1. DC1 differentiation from human CD34+ cells

[0148] A good manufacturing protocol (GMP)- compliant method for dendritic cell (DC) culture has been published previously (van Eck van der Sluijs J et al, (2021) Cancer Immunology, Immunotherapy 70:3167, see Figure 1 A). This method is referred to herein as “the van Eck van der Sluijs prior art method”. This method generates all types of DCs, namely DC1s, DC2s and plasmacytoid DCs from G-CSF mobilised CD34+ hematopoietic progenitor cells (HPCs), however numbers of DC1s are low (representing about 5% or about 6 x 106DC1s from 1 x 106initial donor-derived CD34+HPCs) suggesting that the method is not specific for DC1-differentiation, being the superior subset for promoting T helper 1 and cytotoxic T lymphocytes (CTL) responses which are important for tumour immunity.

[0149] The van Eck van der Sluijs prior art method comprises two phases of DC1 generation, the first corresponding to a 7 day expansion phase and the second phase corresponding to a 7 day differentiation phase. In the first phase, CD34+ hematopoietic progenitor cells (HPCs) are cultured CellGenix® GMP DC Medium comprising 2% human serum (HS), 1pM StemRegenin-1 (SR1) and 50 pg / ml ascorbic acid (AA) for 7 days in the presence of FLT3L, SCF and TPO. In the second phase, on day 7 cells are harvested, washed and resuspended in cell culture mediumsupplemented with a cocktail of 800 lU / ml rhGM-CSF, 1000 IIJ / mL rhIFN-a and 100 ng / mL rh FLT3L and cultured until day 14.

[0150] The present inventors have developed an improved method for promoting specifically DC1 generation which relies on a combination of three culture reagents that work together (in a synergistic way) to differentiate CD34+ HPCs into a cell population that is enriched in DC1s. This is explained further detail below and shown in Figure 1A.

[0151] First, CD34+ hematopoietic progenitor cells (HPCs) from either cord blood or healthy adult granulocyte colony stimulating factor (G-CSF) mobilised blood donor samples were obtained by using the EasySep Human CD34 Positive Selection Kit II (STEMCELL Technologies) or alternatively human CD34 microbeads (Miltenyi Biotech) as per manufacturer’s instructions. The donor sources were obtained from hospital biobanks. The isolated CD34+ cells were then frozen in 0.3 mL of fetal bovine serum (FBS, Bovogen) + 10% DMSO (Sigma Aldrich) aliquoted into 1mL cryovial tubes and stored in liquid nitrogen.

[0152] Day 0 to Day 7 - Expansion phase

[0153] On day 0, the frozen CD34+cells were thawed in a water bath at 37 °C. The cells were then subjected to an “expansion phase” whereby the cells were cultured for 7 days in up to 800 pl of CellGenix® GMP DC Medium (Cellgenix) with the following expansion additives: 1pM StemRegenin-1 (SR1 , Stemcell Technologies), 50 pg / ml L-ascorbic acid (L-AA, Sigma Aldrich), 2% human serum (HS), 100 ng / ml human recombinant tyrosine kinase ligand (rhFlt3L, HumanKine / Proteintech), 100 ng / ml human recombinant stem cell factor (rhSCF, Peprotech), 100 ng / ml human recombinant thrombopoietin (rhTPO, Peprotech) in 24 or 48-well culture treated plates and then incubated at 37 °C and 5% CO2 concentration in an incubator.

[0154] Day 7 to Day 14 - Differentiation phase

[0155] On day 6 or 7, 96-well plates were coated with 50 pl PBS containing 8 pg / ml delta-like canonical ligand (DLL1 , made in house, WEHI) and 2 pg / ml vascular cell adhesion molecule 1 (VCAM1 , made in house, WEHI) either overnight at 4 °C or for at least one hour at 37 °C.

[0156] On day 7, the cells were counted and 10,000 cells cultured in up to 200 pl of Cellgenix DC medium (Cellgenix) containing: 1 pM SR-1 (STEMCELL Technologies), 50 pg / ml L-AA (Sigma Aldrich) and 2% human serum, 100 ng / mL human rhFlt3L (Humankine), 62.5 ng / mL human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF, Peprotech), 1000 lU / ml interferon-a (IFN- a, ThermoFisher) with 100 ng of BATF3 mRNA-lipid nanoparticles (LNPs), in the presence of 1 pg / ml Apolipoprotein E3 (ApoE3, Peprotech). The cell suspension was then seeded on a 96-well plate previously coated with 8 pg / ml DLL1 (WEHI, Australia) and 2 pg / ml VCAM1 (WEHI, Australia). The cells were then incubated at 37 °C and 5% CO2.After 7 days of differentiation (day 14 of the overall culture), approximately 50 pl of the cells were harvested, stained with human antibody specific for Clec9a to identify the DC1 population (and thus the %). Numbers calculated are an extrapolation, assuming 106expanded cells were differentiated. For every 1 x 106CD34+HPCs, the method generated 5 - 10 x 10® DC1 cells at percentage of between 40-65% of the expanded cell population in relation to cord blood starting cells (Figure 1B), and between 1 - 5 x 10® DC1 cells at a percentage of between 20-50% of the expanded cell population in relation to G-CSF mobilised CD34+HPCs (Figure 1C).

[0157] Thus, the modified production method was able to drive differentiation of the CD34+ HPCs towards a DC1 phenotype, and thus an enriched cell population of DC1s was achieved.

[0158] Example 2. Alternative cell culture medium requirements for production of DC1

[0159] To confirm the best GMP-grade cell medium to produce DC1 cells, the inventors expanded and differentiated cord blood CD34+cells in one of the following cell culture media: CellGenix® GMP DC Medium (Cellgenix, used in the van Eck van der Sluijs prior art method), StemSpan-AOF (STEMCELL Technologies), TexMACS (Miltenyi Biotec), CTSTM AIM VTM SFM (Gibco) or X-VIVO-15 (Lonza Bioscience) with the same expansion and differentiation method as that used in the van Eck van der Sluijs prior art method (namely FLT3L, SCF, and TPO, followed by GM-CSF, IFN-a and FLT3L). As shown in Figure 2A and B, the CellGenix® GMP DC Medium was found to be the best culture medium for DC1 differentiation and resulted in high purity compared to other GMP-grade mediums tested.

[0160] Accordingly, CellGenix® GMP DC Medium was selected as the preferred cell culture medium for expansion and differentiation of CD34+ HPCS to DC1s.

[0161] Example 3. DLL1 or DLL4 in the presence or absence of VCAM1 can increase the number and purity of DC1s

[0162] Previous studies have shown that the OP9 stromal cell line expressing Delta-like canonical Notch ligand 1 (DLL1) or Delta-like canonical Notch ligand 4 (DLL4) are able to promote DC1 differentiation and purity (Kirkling M et al., 2018 Cell Rep Jun 19;23(12):3658; Balan S et al., 2018 Cell Rep Aug 14; 24(7):1902). The use of stromal cells adds to the cost and complexity and can create regulatory hurdles. Therefore, the use of DLL1 and DLL4 proteins were explored as an alternative to the use of stromal cells. The inventors cultured CD34+cells as per the van Eck van der Sluijs prior art method and the cells expanded on 96-well plates precoated with 8 pg / mL of DLL1 (WEHI) or 8 pg / mL DLL4 (R&D Systems) protein alone or with 8 pg / mL DLL1 and 2 pg / ml VCAM1 (WEHI) or 8 pg / mL DLL4 (R&D Systems) and 2 pg / mL VCAM1. Figure 3A shows that a modest increase of 1.2 to 2-fold in the number of DC1 cells was obtainedfrom expanded and differentiated CD34+cord blood HPCs in presence of DLL1 or DLL4 alone compared to cells cultured according to the van Eck van der Sluijs prior art method.

[0163] It has been previously demonstrated that DLL4 in the presence of the adhesion molecule VCAM1 is able to further promote or sustain T cell differentiation from human pluripotent stem cells (Shukla S et al., (2017) Nat Methods May 14(5):531). The inventors hypothesized that VCAM1 may promote DC1 differentiation. To this end, 10,000 CD34+ HPCs were cultured and expanded as perthe van Eck van der Sluijs prior art method (Sluijs., 2021), on plates coated with 8 pg / ml DLL1 or DLL4 protein with or without 2 pg / ml of VCAM1. The addition of VCAM1 to DLL1 or DLL4 proteins resulted in a 3-5 fold increase in DC1 numbers (Figure 3A) as well as purity as evidenced by the percentage of DC1 cells (Figure 3B) compared to cells cultured in either DLL1 or DLL4 alone or van Eck van der Sluijs prior art method. In addition, as shown in Figure 3A and B, both DLL1 and DLL4 in presence of VCAM1 allow the production of high numbers of DC1.

[0164] Example 4. Fibronectin, retronectin and VACM1 can increase the number and purity of DC1s.

[0165] Other adhesion molecules, such as 2 pg / ml fibronectin (Roche), retronectin (Takara), laminin (rLMS115, made in house), laminin (L2020, SigmaAldrich) and 100 pg / ml poly-L-lysine (SigmaAldrich) were further explored. Cord blood CD34+ progenitor cells were used and expanded as per the van Eck van der Sluijs prior art method but differentiated in the presence of: 2 pg / ml of VCAM1 , 2 pg / mL of laminin (rLMS115) or 100 pg / ml poly-L-lysine, in presence or absence of 8 pg / ml DLL1 protein and compared to the van Eck van der Sluijs protocol. The data in Figure 4 showed that use of both 2 pg / ml of fibronectin, retronectin and VCAM1 , increased the number and purity of DC1 compared to cells cultured in the absence of these. The use of laminin and poly-L-lysine were found to be less effective at promoting differentiation of CD34+ cells into DC1s (Figure 5).

[0166] Example 5. Investiqation of alternative transcription factors

[0167] The transcriptional factors BATF3, IRF8, PU.1 and DC-script have been identified as important for dendritic cell development (Edelson et al., Pios One, 2011 ; Hildner et al., Science, 2008; Kurotaki and Tamura, J. Interferon Cytokines Res, 2016; Sichien et al., Immunity, 2016; Carotta et al., Immunity, 2010; Chopin et al., Immunity, 2019). Co-expression of BATF3, IRF8 and PU.1 have also been shown to reprogram human embryonic fibroblast to DC1-like cells (Rosa et al., Science Immunology, 2018). Adapting the use of DC-specific transcriptional factor(s) in an ex vivo setting to artificially make cells differentiate preferentially to DC1 (and keep the numbersof pDC and DC2 cells low) is however not trivial, various factors need to be considered. This examples compared different transcription factors on DC1 enrichment.

[0168] Cord blood CD34+HPCs from 3 donors were thawed and cultured for 7 days as per the expansion phase of the van Eck van der Sluijs prior art method but including 15 pmol of GFP (green fluorescent protein) RNA (transfection control). mRNA from dendritic cell transcription factors BATF3, IRF8, PU.1 and DC-script were delivered to the cells by 4D-nucleofector (Lonza) with pulse code DS150. Transfected cells were left to recover in the HPC expansion medium overnight. Then, 10,000 mRNA transfected cells were cultured with recombinant DLL1 (8 pg / mL) or recombinant DLL1 (8 pg / mL) + VCAM1 (2 pg / mL) coated plates in differentiation medium (as described in Example 1) and incubated at 37°C, 5% CO2 for 7 days. The number and percentage of DC1 in the culture following expansion and differentiation was assessed by fluorescence activated cell sorting (FACS).

[0169] As demonstrated in Figure 6, under the van Eck van der Sluijs prior art method, only cord blood CD34+HPCs transfected with BATF3 showed an increased number of DC1s (2.5 to 4 x 106) per 1 x 106expanded CD34+HPC when compared to GFP-transfected and either IRF8, PU.1 and DC-script-transfected HSCs (0.5-1 x106DC1) per l x 106CD34+HPCs. This result was unexpected as one would expect all transcription factors to work equally as taught from the abovementioned references. This was not the case for these particular cell types.

[0170] Interestingly, when BATF3 mRNA transfected HPCs were cultured in the presence of DLL1 or DLL1 + VCAM1 , the number of DC1 cells further increased. Indeed, in the presence of DLL1 , about 10 - 15 x 10® DCIs were obtained from BATF3 mRNA transfected CD34+HPCs. In contrast, only about 2 - 8 x 10® DC1s from GFP or IRF8, PU.1 and DC-script-transfected HSCs were obtained (Figure 6A). Again, this result was unexpected as BATF3 and the notch factor DLL1 are both DC1 promoting factors, but it appears from these results that they work synergistically and are not merely alternatives.

[0171] Furthermore, it was noted that when the BATF3 mRNA transfected HSCs were cultured in DLL1 and VCAM1 , the number of DC1s increased to between 3 - 4 x 10® DC1s per 10® HPCs and the percentage (purity) of DC1s increased to about 40% of the expanded CD34+HPCs. However, only 20% of DC1 cells was observed in BATF3 mRNA transfected HSCs in the presence of DLL1 only medium (Figure 6B).

[0172] These data show that BATF3 overexpression in HSCs, achieved through mRNA electroporation, enhances DC1 number and purity compared using other tested transcriptional factors HSCs. The DC1 number and purity are further increased in the presence of both DLL1 and VCAM1 stimulation.6. of method of the disclosure when G-CSF-mobilized

[0173]

[0174] The effect of BATF3 mRNA delivery was tested in the presence of DLL1 or DLL4 alone or in combination with VCAM1 in culture using adult CD34+HPCs from G-CSF mobilized donors. After expansion according to the method described in Example 1 , the inventors differentiated 10,000 CD34+HPCs on 8 pg / ml DLL4 or 8 pg / ml DLL1 with or without 2 pg / ml VCAM1 , in presence or absence of 100 ng BATF3 mRNA delivered via lipid nanoparticles.

[0175] As demonstrated in Figure 7A, overexpression of BATF3 in CD34+HPCs significantly increased (by about 1.5-fold) the number of DC1 cells compared to corresponding cultures of DLL1 and VCAM that did not contain BATF3 CD34+cells. This effect was more prominent when BATF3 CD34+cells were cultured in the presence of DLL1 or DLL4 and VCAM1 compared to corresponding cultures of DLL1 or DLL4 and VCAM that did not contain BATF3 CD34+cells.

[0176] Figure 7B demonstrated that the percentage of DC1s and thus the purity increased to between 30 and 40% when BATF3 CD34+ cells were cultured with DLL1 or DLL4 plus VCAM1.

[0177] This data confirmed that an increase in the number and purity of DC1 cells could be obtained when CD34+ cord blood cells or G-CSF mobilized cells were transfected with BATF3 and cultured in the presence of either DLL1 or DLL4 in combination with VCAM1.

[0178] Comparison of mRNA delivery strategies

[0179] Two mRNA delivery strategies were investigated, namely electroporation and the use of lipid nanoparticles (LNPs). To examine which delivery strategy was optimal for BATF3 delivery, CD34+HPCs were either:

[0180] 1) left untreated;

[0181] 2) electroporated at a cell density of 200,000 cells with either 3 pg GFP (control) or 3 pg BATF3 mRNA using a Lonza Nucleofector (electroporation code DS-150); or

[0182] 3) cultured at a cell density of 100,000 cells with 1pg / mL of ApoE3 (Peprotech) and 200ng of either GFP (control) or 200 ng of BATF3 mRNA delivered by LNPs.

[0183] Following transfection, the cells were then incubated at 37 °C 5% CO2 overnight. Then 25,000 expanded CD34+cells from each treatment group were cultured on 1) uncoated plates or 2) plates coated with 100 pl PBS containing 12 pg / ml DLL1 and 3 pg / ml VCAM1 , for seven days to differentiate the cells as described in Example 1.

[0184] The inventors found that electroporation of CD34+cells impacted cell viability. Indeed, the cultures that were electroporated with the control GFP-mRNA had reduced DC1 number and purity compared to the non-electroporated condition despite the presence of DLL1 and VCAM1protein (Figure 8). However, there was no major impact on DC1 differentiation when using LNPs delivery of GFP-mRNA (Figure 8). Nevertheless, delivery of BATF3-mRNA via electroporation or LNPs compared to GFP-mRNA or GFP mRNA-LNPs resulted in a higher production (in terms of number and purity) of DC1 in the presence of DLL1 and VCAM1 (Figure 8).

[0185] 8. DC1 expansion and differentiation protocol

[0186] To reduce the DC1 culturing timeframe compared to prior art methods, several different culturing times were explored using cord blood samples from three different donors. Different protocols were investigated as explained further below and summarised in Table 1.

[0187] 1) reducing the time of each phase by two days (Protocol 1);

[0188] 2) using a single phase system which consists of a differentiation phase only from Day 1 to Day 10 (Protocol 2);

[0189] 3) adding SCF and TPO which are cytokines important for HPC proliferation and survival (Protocol 3 and 4).

[0190] Protocol 1 (Expansion 5 days followed by differentiation 5 days)

[0191] The expansion phase was reduced to 5 days and 10,000 expanded cord blood derived CD34+HSCs were transferred into the differentiation phase in the presence of 8 pg / ml DLL1 , 2 pg / ml VCAM1 and BATF3 mRNA delivered by LNPs; in a well of 96-well plate for a further 5 days. This process reduced the typical 14-day culture period (as described in Example 1) to a 10-day method. This process resulted in the production of an average of 65 x 106DC1 per 1 x 106starting donor CD34+HSCs and a purity of 40-50% DC1 % (Figure 9A and 9B) after the 10 day culture. When compared to the method of Example 1 at the same timepoint, the method of Process 1 generated a significantly higher number and purity of DC1s. When compared to the prior art method of Example 1 at the same time point (day 10), the method of process 1 generated similar or slightly higher DC1 numbers (65 x 106compared to 25 - 80 x 106DC1 per 1 x 106starting / donor HSCs, respectively) and with at least a 2-fold increase in purity at the day-10 timepoint. However, when comparing process 1 and the prior art method at their respective end points (day 10 vs. day 14), the prior art method yielded a 6-10-fold increase in DC1 number with comparable DC1 purity compared to the method of Process 1. This experiment suggests the possibility of shortening the DC1 manufacturing process from 14 to 10 days depending on the required number and purity of DC1s.

[0192] Protocol 2 (Differentiation for 10 days)

[0193] The two phase method of the van Eck van der Sluijs prior art method was compared to a one phase method lacking an expansion phase. Cord blood derived CD34+HSCs were thawedand immediately subjected to differentiation as described in Example 1 without an expansion phase for an overall culture time of 10 days.

[0194] The number of DC1 decreased to around 2.5 x106per 1 x 106by this method (Figure 9A). This was also reflected in the low percentage of DC1s after 10 days. These results indicate that the expansion phase of HSC is important in the DC1 production process.

[0195] Protocol 3 (Addition of SCF and TPO)

[0196] To investigate whether the addition of stem cell factor (SCF) and thrombopoietin (TPO) could improve DC1 production, SCF and TPO were added during the differentiation phase of a two-step process (described in Protocol 1) to produce Protocol 3. Alternatively, SCF and TPO were added to the 1 step process (described in Protocol 2) to produce Protocol 4. As shown in Figure 9A and B, the addition of SCF and TPO resulted in a slight decrease in DC1 cell number and no difference in DC1 purity compared to protocol 1. The addition of SCF and TPO in protocol 4 led to a very slight increase of DC1 number but a decrease in purity (10-20% compared to 30%). In other words, their presence may increase the proliferation / survival of all cells in the culture but do not necessarily favour DC1 differentiation specifically. This experiment suggests that additional SCF and TPO in the differentiation phase slightly improved the production of DC1 but not its purity. The results also suggest that again the omission of the differentiation phase and the inclusion of SCF and TPO from the beginning of any culturing does not give satisfactory numbers of DC1 cells nor a pure enough population.

[0197] Table 1 Various CD34+ HPC culture protocols for DC1 generation

[0198] Step 1 (Expansion) Step 2 (Differentiation)

[0199] Culture Reagents / ligands Culture period Reagents / ligands Culture period condition (days) (days) Van Eck van Flt3L, SCF, TPO 7 Flt3L, GM-CSF, IFNa 7

[0200] der Sluijs

[0201] method

[0202] The new Flt3L, SCF, TPO 7 Flt3L, GM-CSF, IFNa, LNP- 7 improved BATF3 mRNA, DLL1 ,

[0203] protocol VCAM1

[0204] Protocol 1 Flt3L, SCF, TPO 5 Flt3L, GM-CSF, IFNa, LNP- 5

[0205] BATF3 mRNA, DLL1 ,

[0206] VCAM1

[0207] Protocol 2 0 Flt3L, GM-CSF, IFNa, LNP- 10

[0208] BATF3 mRNA, DLL1 ,

[0209] VCAM1

[0210] Protocol 3 Flt3L, SCF, TPO 5 Flt3L, GM-CSF, IFNa, LNP- 5

[0211] BATF3 mRNA, DLL1 ,

[0212] VCAM1 and SCF, TPO

[0213] Protocol 4 0 Flt3L, GM-CSF, IFNa, LNP- 10

[0214] BATF3 mRNA, DLL1 ,

[0215]

[0216] VCAM1 and SCF, TPOExample 8 Direct comparison with the van Eck van der Sluijs prior art method Figure 10 shows a head-to-head comparison between the van Eck van der Sluijs prior art method and the method of the present disclosure for generating DC1 cells from starting CD34+ HPCs. The comparison also includes the number claimed in the van Eck van der Sluijs publication, so it is not a side by side comparison of the 2 methods “as worked by the inventors”. These methods are outlined in Figure 1.

[0217] For both methods, 500,000 to 800,000 CD34+ HPCs from G-CSF mobilized sample were harvested and purified by using human CD34 Microbead Kit (Miltenyi). The cells were expanded for 7 days in CellGenix® GMP DC Medium supplemented with 50pg / ml L-AA, 1pM SR-1 , 2% human serum, 10Ong of Flt3L, 10Ong / ml SCF and 10Ong / ml TPO. For the van Eck van der Sluijs prior art method, on day 7, the cells were harvested, washed and resuspended in CellGenix® GMP DC Medium supplemented with 50pg / ml L-AA, 1pM SR-1 , 2% human serum, 100ng / ml Flt3L, 800IU / ml GM-CSF and 1000IU / mL IFN-a for 7 days to induce differentiation to DC1 cells. For the method of the present disclosure, on day 7, expanded HPC cells were transfected with BATF3 mRNA by lipid nanoparticles in CellGenix® GMP DC Medium supplemented with 50pg / ml L-AA, 1pM SR-1 , 2% human serum, 100ng / ml Flt3L, 800IU / ml GM-CSF and 1000IU / mL IFN-a in 8pg / ml DLL1 and 2pg / ml VCAM1 coated cell culture plates and differentiated for 7 days.

[0218] On day 14, the cells were harvested and examined by flow cytometry as described herein. It is clear from the results of Figure 10 that the method of DC1 generation of the disclosure produced approximately 32 x 106to 141 x 106per 106initial HPCs, which provides a significant increase in the number of DC1 cells obtained (a range of about 5-24-fold increase compared to 6.3 x106number of DC1 generated and published by van Eck van der Sluijs).

[0219] Example 9. DC1 cell culture with DLL1-VCAM1 beads

[0220] This example investigated the use of beads coated with DLL1 and VCAM, the DLL1 and VCAM being specifically conjugated to beads. Streptavidin (SA)-coated polystyrene beads of various size (6.8pm, 21.8pm, 45pm, or 105pm) were purchased from Spherotech. DLL1-Avitag and VCAM1-Avitag were mixed at a mass ratio of 4:1 and centrifuged for 30 minutes. Avitag is a 15 amino acid peptide tag that binds to streptavidin. This sequence is Gly-Leu-Asn-Asp-lle-Phe-Glu-Ala-GIn-Lys-lle-Glu-Trp-His-Glu. In comparison to this conjugation to beads, experiments were concurrently run in which cell culture dishes were prepared by coating the wells with 1X PBS containing 8pg / mL of DLL1 and 2pg / mL of VCAM1 overnight at 4 °C, followed by two washes with 1X PBS as per Example 1.In both scenarios, CD34+HPCs were harvested and isolated from cord blood donors. The cells were then expanded for 7 days and differentiated according to each of the following conditions: (i) the van Eck van der Sluijs prior art method (in absence of BATF3) (ii) addition of the DLL1 and VCAM1 as a result of being coated onto a cell culture dish or (iii) addition of DLL1 and VCAM1 as a result of being coated to beads and added to the culture for 7 days prior to analysis for DC1 cells. 1 x dose consists of 10,000 beads with a size of 6.8pm cultured with 10,000 expanded HPCs. For other bead sizes, an equivalent total surface area to that of 10,000 beads of6.8pm was used. For example, if the total surface area of 10,000 beads of6.8pm is 1 ,000pm2, then 1 ,000pm2worth of 21.8pm, 45pm or 105pm beads is added. 2x doses consists of 20,000 beads with a size of 6.8pm cultured with 10,000 expanded HPCs, with equivalent surface area adjustments for other bead sizes, as described above. Figure 11 shows (A) The number of DC1 per expanded HPCs and(B) the percentage (%) of DC1 (purity) were determined.

[0221] The above experiment was repeated with G-CSF -mobilised CD34+HPCs and this time BATF3 was included in some of the expansion media cultures and delivered via mRNA encapsulated by LNPs as described herein.

[0222] The graphs shown in Figures 11 and 12 demonstrate that DLL1 and VCAM can be delivered to cell culture as ligand-coated beads. This is a convenient method of delivering these ligands.

[0223] Example 10. Ascorbic acid and human serum may not be required to produce DC1 cells.

[0224] G-CSF mobilised CD34+ HPCs from healthy donors were isolated and cultured according to the van Eck van der Sluijs prior art method (Figure 13) and compared to the method of the disclosure including the following cell culture medium conditions (i) including all additives, (ii) an absence of L-ascorbic acid (-LAA-LAA) during both the expansion and differentiation phases, (iii) an absence of StemReginin 1 (-SR1-SR1) during both the expansion and differentiation phases, (iv) an absence of human serum (-HS-HS) during both the expansion and differentiation phases, (v) an expansion phase with the absence of L-ascorbic acid but all additives present during differentiation (-LAA+gs), (vi) an expansion phase in the absence of StemReginin 1 but all additives present during differentiation (-SR+gs) wherein gs means that all additives are present during the differentiation phase (as per Example 1).

[0225] As shown in Figure 13, the inventors identified the most preferred additives / reagents for DC1 production by removing one reagent at a time from the differentiation and expansion phases. They found that the removal of i) L-ascorbic acid and 2) human serum during the expansion phase did not significantly impair the number or purity of DC1 cells produced.Example 11. Production of DC1 cells from cancer subjects and healthv donors bv the method of the invention.

[0226] G-CSF mobilised CD34+ HPCs from healthy donors or cancer subjects were isolated and cultured according to the method of the disclosure or according to the van Eck van der Sluijs prior art method. As shown in Figure 14, G-CSF mobilised HPCs isolated from healthy donors (P1-P3) or patients with multiple myeloma (MM) or lymphoma (DLBCL, MCL) were compared for DC1 production using the van Eck van der Sluijs prior art method and the method of the disclosure (DLL1 , VCAM1 , BATF3). The inventors confirmed that their protocol, regardless of source of the progenitor cells, resulted in both a higher DC1 cell production and increased cell purity compared to the Van Eck van der Sluijs prior art method.

[0227] 12. A 7 -day expansion followed by a 7 -day differentiation is the optimal timeframe for of DC1 cell number and

[0228] G-CSF mobilised CD34+HPCs from either cancer patients or healthy donors were isolated and cultured for 5 days or 7 days and then differentiated for 7 days according to the method of the disclosure or according to the van Eck van der Sluijs prior art method (referred to as van Eck van der Sluijs protocol) (Figure 15). The method of the disclosure was as detailed in Example 9. The inventors identified that a protocol using 7 days of expansion and 7 days of differentiation (B+D+V, 7+7) offered the highest number and purity of DC1 compared to any other combination of expansion and differentiation timeframe in either van Eck van der Sluijs or B+D+V protocol. Indeed, B+D+V (7+7) showed approximately 5-8-fold higher number of DC1 and 10-fold higher purity of DC1 compared to the van Eck van der Sluijs prior art method (Van der Sluijs method 5+7 and 7+7); and thus, independently of the source of progenitor (patient or healthy donor-derived). In addition, the inventors identified that the B+D+V (7+7) condition (method of the disclosure) produced approximately 1 ,5x-fold more DC1 s per 106expanded HSC than 5 days of expansion and 7 days of differentiation timeframes (B+D+V (5+7)). The inventors noticed that a B+D+V (5+7) generates a higher number and purity of DC1 than the prior art method (a range of 0.5 to 4 x 106DC1 per expanded HPCs; with the variability mostly related to the health status of the HPC donor. Depending on the desired number of DC1s, the method of disclosure (7 days expansion + 7 days differentiation) may be thus reduced to 5 days expansion and 7 days differentiation.

[0229]

[0230] 13. An enhancement of the cells prior to the differentiation phase and the effect on the overall

[0231]

[0232] and DC1 cell numbers.G-CSF mobilised CD34+ HPCs from healthy donors were isolated. Prior to DC1 differentiation, HPCs were enriched for CD200 marker and subjected to either the van Eck van der Sluijs prior art method or one the preferred method of the disclosure (DLL1 , VCAM1 , BATF3, refers as B+D+V). As shown in Figure 16, CD200 enriched HPCs increased DC1 numbers (#cDC1 on the Y axis) and slightly enhance purity in both the van Eck van der Sluijs prior art method and the method of the disclosure. The inventors confirmed that CD200 enrichment prior to differentiation phase may be beneficial for DC1 production.

[0233] Example 14. Extending the differentiation phase.

[0234] If HPCs are obtained from healthy cord blood donors, cryopreserved and placed in a biobank so that they can be used in allogenic cell treatment, then there is less restriction on timeframes to produce a clinical product as there would be in an autologous cell treatment. The inventors thus demonstrated the expanded CD34+ HPCs cells from cord blood donors could be differentiated up to 20 days after expansion before exhaustion occurred. The experiment followed expansion of CD34+ HPCs cells for 7 days according to the van Eck van der Sluijs prior art method and followed by differentiation according to the van Eck van der Sluijs prior art method in the presence of DLL1+VCAM1 for up to 20 days. The results are shown in Figure 17. It would be expected that the inclusion of BATF3 would give rise to superior results to these obtained for the van Eck van der Sluijs method, in terms of numbers (#) and % purity. Therefore it would be expected that one of the embodiments of the method of the current invention could include differentiating the cells for up to 20 days.

[0235] Example 15. Preparation of a DC1 enriched culture usinq bioreactor flasks.

[0236] For a clinically useful product, several manufacturing challenges need to be overcome. One of these challenges is to make a batch that contains a large population of cells. The use of a G-Rex® system which is a closed cell culture vessel with a flat, gas permeable silicone membrane at its base, has been touted as being useful for T cells, natural killer cells and hematopoietic cells and to be practical and a cost effective way to scale up and scale out.

[0237] Using a GRex cell culture system, the following experiment was carried out to demonstrate its applicability to making dendritic cells, in particular DC1 cells.

[0238] The experiment involved firstly expanding CD34+ HPCs obtained from G-CSF mobilized blood, in an expansion media containing Cellgenix Expansion media and Stemregeninl , human serum and L-ascorbic acid, and FLT3L, SCF, TPO , in a polystyrene cell culture system (labelled as Academic) for 7 days and then differentiated in the presence of DLL4, VCAM1 and BATF3 mRNA in a GMP compliant GRex vessel (labelled as GMP) for a further 7 days. Figure 18 show% purity of the final cell population after the 14 days culture compared to results from differentiationin the polystyrene, Academic system. The results show an improvement on the number of DC1s (fold change) as well as an improvement in purity (% DC1s).

[0239] Several batches were made with GRex and the final cell populations were characterised by flow cytometry Clec9A+ markers and the results of the batches are summarised in the below Table 2. In this cell culture format consistently high purity batches (>30% DC1 cells) were produced.

[0240] Table 2: Impurity profile.

[0241] Markers Cell type % cells

[0242] CD34 Progenitors -6-7

[0243] Clec9A+CADM1 +CD141 +CDCc+ / - DC1 -55-65

[0244] CD1c+ DC2 -3-6.5 CD123+CD45Ra+ pDC -6-7

[0245] CD14+ CD19 CD56 T cells, B cells NK cells <0.1

[0246] cKIT Mast cells -5-10

[0247]

[0248] Negative for all above CD71 + Other cells -10-15

[0249] Example 16. Biological, functional activity.

[0250] Expanded CD34+cells from G-CSF mobilized donors were differentiated either in polystyrene PS) or the GRex culture system for 7 days. Total culture cells were then stimulated for 24 hours with 4pg / ml polylC and 1pg / ml R848. The stimulants activate DCs (including DC1) so that cytokines are produced. Stimulants may be required in any therapeutic DC1 cell population to boost activity,

[0251] The supernatants were then harvested and analysed using flow cytometry. The graph of Figure 19 shows the concentration (pg / ml) of each cytokine production. IFNL1 isn’t present when cells are unstimulated. However the presence of IFNL, IL12p70, CXCL10 upon stimulation indicates that the differentiated cells are functional.

Claims

CLAIMS:

1. A method of obtaining an enriched type 1 conventional dendritic cell (DC1) population, the method comprising contacting hematopoietic progenitor cells (HPCs) with a differentiation cocktail ex vivo or in vitro, wherein the differentiation cocktail comprises 1) FLT3L (fms-related tyrosine kinase 3 ligand), 2) IFN-a (interferon alpha), 3) GM-CSF (granulocyte colony stimulating factor), 4) BATF3 (basic leucine zipper ATF-like transcription factor 5) a notch ligand and 6) an adhesion molecule.

2. The method according to claim 1 , wherein the HPCs are contacted with a cell culture medium that supports expansion and differentiation of HPCs into dendritic cells, the cell culture medium comprising one or more of HS (human serum), SR1 (StemRegenin 1) and L-AA (L-ascorbic acid).

3. The method according to claim 1 or 2, wherein the HPCs are CD34+cells.

4. The method according to any one of claims 1 to 3, wherein the notch ligand is a deltalike family ligand (DLL) or mimic thereof.

5. The method according to claim 4, wherein the notch ligand is DLL-1 or DLL4.

6. The method according to any one of claims 1 to 5, wherein the adhesion molecule is selected from VCAM1 , fibronectin or retronectin.

7. The method according to claim 6, wherein the adhesion molecule is VCAM1.

8. The method according to any one of claims 1 to 7, wherein the BATF3 is provided as mRNA.

9. The method according to claim 8, wherein the BATF3 mRNA is encapsulated in lipid nanoparticles (LNPs).

10. The method according to any one of claims 1 to 9, wherein the differentiation cocktail comprises FLT3L, IFN-a, GM-CSF, BATF3, DLL1 or DLL4 and VCAM1.

11. The method according to any one of claims 1 to 10, wherein the HPCs are contacted with the differentiation cocktail for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 10 days, or at least 20 days.

12. The method according to any one of claims 1 to 11 , wherein the method comprises expanding the HPCs in an expansion medium prior to differentiating the HPCs.

13. The method according to claim 12, wherein the expansion medium comprises FLT3L, SCF, and TPO.

14. The method according to claim 12 or 13, wherein the HPCs are expanded in the expansion medium for at least 5 days, at least 6 days, at least 7 days, or at least 8 days.

15. The method according to any one of claims 1 to 14 comprising culturing CD34+ HPCs in an expansion medium supplemented with FLT3L, SCF, and TPO, wherein the CD34+ HPCs are expanded for a period of about 5 to 7 days, preferably about 7 days, followed by differentiating the CD34+ HPCs in a differentiation cocktail comprising GM-CSF, IFN-a, FLT3L, BATF3 mRNA, VCAM1 and DLL-1 or DLL4, preferably DLL-1 ;for a period of about 5 to 7 days, preferably about 7 days.

16. The method according to claim 15, wherein the expansion medium and the differentiation cocktail additionally includes a cell culture medium that is supplemented with one or more of HS (human serum), SR1 (StemRegenin 1) and L-AA (L-ascorbic acid).

17. The method according to claim15, wherein the BATF3 mRNA is provided to the differentiation cocktail as LNPs encapsulating the BATF3 mRNA.

18. The method according to claim15, wherein the DLL1 or DLL4 and VACM1 are coated on beads.

19. The method according to any one of claims 1 to 18, wherein the culturing of the HPCs includes expansion in an expansion medium followed by differentiation in a differentiation cocktail and wherein culturing occurs over a total culture period of about 10 days to about 20 days.

20. The method according to any one of claims 1 to 19, wherein the HPCs are obtained from embryonic yolk sac, the embryonic aorta-gonad-mesonephros region, fetal liver or spleen, adultspleen, adult bone marrow, fetal bone marrow, peripheral blood and umbilical cord blood, or G-CSF mobilised blood.

21. The method of claim 20, wherein the HPCs are enriched for CD34+ cells and expanded prior to contacting the cells with the differentiation cocktail.

22. A population of cells enriched in type 1 conventional dendritic cell (DC1) cells wherein the % purity of DC1 cells in the population of cells is greater than 30%.

23. The population of cells of claim 22 wherein the population of cells contains less than 10% progenitor cells.

24. A population of cells of claim 21 or 22 wherein the population of cells contains less than 10% DC2 cells25 A population of cells of any one of claims 22 to 24 wherein the population was obtained from culturing hematopoietic progenitor cells (HPCs) in a first culture step comprising an expansion medium to obtain expanded HPCs and then in a second culture step comprising a differentiation cocktail ex vivo, the second culture step preferential promoting the differentiation into DC1 cells,26 A population of cells of claim 25 wherein the differentiation cocktail comprises 1) FLT3L (fms-related tyrosine kinase 3 ligand), 2) IFN-a (interferon alpha), 3) GM-CSF (granulocyte colony stimulating factor), 4) BATF3 (basic leucine zipper ATF-like transcription factor 5) a notch ligand and 6) an adhesion molecule.27 A population of cells of claim 25 wherein the second culture step results in the generation of at least 2 million DC1 s per million expanded cells.

28. A population of cells according to any one of claims 22 to 27, or obtained or obtainable by the method according to any one of claims 1 to 21 for use, or when used, in the treatment of cancer, an inflammatory or autoimmune condition.

29. A population of cells according to any one of claims 22 to 27 or obtained or obtainable by the method according to any one of claims 1 to 21 for use, or when used, in the manufacture of a medicament for the treatment of cancer, an inflammatory or autoimmune condition.

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