Generation of dendritic cells

Abstract

The presently claimed and described technology provides methods of generating dendritic cells using hematopoietic stem cells and a feeder layer including at least one murine cell line expressing human notch ligands. The presently claimed and described technology also provides for cellular immunotherapy using the generated dendritic cells.

Description

MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCGENERATION OF DENDRITIC CELLSRELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 746,775, filed January 17, 2025, the content of which is hereby incorporated by reference in its entirety into this disclosure.BACKGROUND

[0002] Dendritic cells (DCs) play a crucial role in initiating and regulating immune responses by processing antigens from pathogens and captured cancer cells and presenting them to T cells. This natural adjuvant property of DCs has made them an attractive platform for therapeutic vaccines against malignancy and infectious diseases and which has been evaluated in hundreds of clinical trials. The accessibility of abundant monocytes from peripheral blood and the straightforward protocols for their in vitro generation have established monocyte-derived DCs (Mo-DCs) as a dominant platform for cell adjuvants in clinical trials. Recent advancements in techniques for isolating sufficient quantities of naturally circulating conventional type II DCs (cDC2) and plasmacytoid DCs (pDCs) have facilitated their inclusion in clinical trials. Although these various sources of DCs have clearly shown safety and ability to induce T cell responses, long-term clinical efficacy is still elusive.

[0003] Paradoxically, the conventional type I DC (cDCl) have yet to be evaluated as a therapeutic vaccine platform in humans, despite substantial and compelling evidence from animal models demonstrating their crucial role for eliciting anti-tumor immune responses. cDCls are superior to other DC subtypes in their ability to cross present and cross prime cellular antigens and induce potent CD8+ cytotoxic T lymphocytes (CTL) mediated tumor control. The presence of cDCl transcripts in many human tumor types is associated with good prognosis and responsiveness to therapy such as checkpoint inhibitors. Furthermore, the potential of cDCl as cellular vaccine vehicles has been validated in animal models including humanized mice, and their superiority over MoDC vaccines has been established. Whilst these findings identify cDCl as the more attractive vaccine candidate, their extremely low frequency in blood and lack of robust clinically translatable in vitro propagation protocols has limited their therapeutic application to date.

[0004] Previous culture systems have generated human cDCl from CD34+ progenitorMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCcells derived from sources such as cord blood (CB), mobilized peripheral blood (mPB), and bone marrow (BM). However, these systems lack efficiency in terms of scalability, purity, and functional quality required for clinical-grade cDCl products.BRIEF SUMMARY

[0005] The inventors have established a method for the large scale generation of conventional type I dendritic cell subset (cDCl) cells in serum free culture conditions. The generation of the cDCl cells may be done in vivo, in vitro, or ex vivo.

[0006] One aspect of the disclosure is a serum free method for generating cells, the method comprising: differentiating hematopoietic stem cells into conventional type 1 dendritic cells, wherein the hematopoietic stem cells are differentiated with a feeder layer comprising at least one murine cell line expressing human notch ligands and at least one cytokine in a serum free media, wherein the at least one cytokine is selected from the group consisting of Fms-like tyrosine kinase receptor 3 ligand (Flt3L), Stem cell factor (SCF), Thrombopoietin (TPO), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin-4 (IL-4), and Interleukin- 15 (IL- 15).

[0007] One aspect of the disclosure is a serum free culture system for generating cells, the serum free culture system comprising: hematopoietic stem cells, a feeder layer, wherein the feeder layer comprises at least one murine cell line expressing human notch ligands and at least one cytokine, wherein the at least one cytokine is selected from the group consisting of Fms-like tyrosine kinase receptor 3 ligand (Flt3L), Stem cell factor (SCF), Thrombopoietin (TPO), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin-4 (IL-4), and Interleukin- 15 (IL- 15) and wherein the hematopoietic stem cells are differentiated into conventional type 1 dendritic cells in a serum free media.

[0008] In an aspect, the hematopoietic stem cells are differentiated with a combination of Flt3L, SCF, GM-CSF, IL-4, and / or IL-15.

[0009] In an aspect, the hematopoietic stem cells are differentiated in a media for at least about 1 day, alternatively at least about 2 days, alternatively at least about 3 days, alternatively at least about 4 days, alternatively at least about 5 days, alternatively at least about 6 days, alternatively at least about 7 days, alternatively at least about 8 days, alternatively at least about 9 days, alternatively at least about 10 days, alternatively at least about 11 days, alternatively at least about 12 days, alternatively at least about 13 days, alternatively at least about 14 days, alternatively at least about 15 days, alternatively at least about 16 days, alternatively at least about 17 days, alternatively at least about 18 days.MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PC

[0010] In an aspect, the hematopoietic stem cells have been expanded prior to differentiation.

[0011] In an aspect, the hematopoietic stem cells have been expanded with a mouse stromal cell feeder layer prior to differentiation.

[0012] In an aspect, the expanded hematopoietic stem cells are expanded in a media for at least about 1 day, alternatively at least about 2 days, alternatively at least about 3 days, alternatively at least about 4 days, alternatively at least about 5 days, alternatively at least about 6 days, alternatively at least about 7 days, alternatively at least about 8 days, alternatively at least about 9 days, alternatively at least about 10 days, alternatively at least about 11 days, alternatively at least about 12 days, alternatively at least about 13 days, alternatively at least about 14 days, alternatively at least about 15 days.

[0013] In an aspect, the expanded hematopoietic stem cells are cryopreserved for future use.

[0014] In an aspect, the at least one cytokine is a human recombinant cytokine.

[0015] In an aspect, the hematopoietic stem cells are human cells.

[0016] In an aspect, the hematopoietic stem cells are CD34+hematopoietic cells.

[0017] In an aspect, the CD34+hematopoietic cells are from cord blood, mobilized peripheral blood, non-mobilized peripheral blood, or are bone marrow derived.

[0018] In an aspect, the murine cell line are from mouse bone marrow stromal cells.

[0019] In an aspect, the mouse bone marrow stromal cells are selected from the group consisting of MS-5 and OP9.

[0020] In an aspect, the murine cell line expresses human DLL1.

[0021] In an aspect, the murine cell line is human DLL1 expressing OP9 cells.

[0022] In an aspect, the method is performed in vivo, in vitro, or ex vivo.

[0023] In an aspect, the method is performed ex vivo or in vitro, wherein the method further comprises a cell culture vessel, wherein the differentiation of the hematopoietic stem cells occurs within the cell culture vessel.

[0024] In an aspect, the cell culture vessel is selected from the group consisting of a cell culture flask, a cell culture plate, a cell culture dish, a cell culture tube, a cell culture insert, a chamber slide, and a chambered cover glass.

[0025] In an aspect, the hematopoietic stem cells are differentiated on the feeder layer.

[0026] One aspect of the disclosure is a method of treating a subject having cancer, the method comprising administering the conventional type 1 dendritic cells generated to the subject.MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PC

[0027] One aspect of the disclosure is a method of performing cellular immunotherapy in a subject having cancer, the method comprising administering the conventional type 1 dendritic cells generated to the subject.

[0028] In an aspect, the cancer is selected from the group consisting of genitourinary cancer, prostate cancer, myeloma, head and neck cancer, high-positive MSI cancer, and metastatic cancer.

[0029] One aspect of the disclosure is a cellular immunotherapy composition comprising the conventional type 1 dendritic cells generated.

[0030] In an aspect, the cellular immunotherapy composition is used to treat or prevent cancer.

[0031] In an aspect, the cancer is selected from the group consisting of genitourinary cancer, prostate cancer, myeloma, head and neck cancer, high-positive MSI cancer, and metastatic cancer.

[0032] In an aspect, the cellular immunotherapy composition is formulated as a vaccine.

[0033] In an aspect, the conventional type 1 dendritic cells are adjuvants.

[0034] In an aspect, the vaccine is administered intramuscularly, subcutaneously, intranodally, intratumorally, or intravenously.

[0035] These and other advantages, aspects, and novel features of the present disclosure, as well as details of an illustrated embodiment thereof, will be more fully understood from the following description and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Various aspects of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0037] FIGs.1 A - IF show the generation of human eDC 1. FIG.1 A is a representative dot plots showing the frequency of cDCls generated in feeder free or OP9 DLL1 feeders using Serum free or containing culture media and are identified as CD 141 and CLEC9A double positive cells. FIGs. IB and 1C display the frequency and number of cDCls normalized to the number of input cells used on day one of differentiation. Colored bars indicate culture conditions; Light green (far left bar) (FF + RPMI +10% FBS), dark green (second bar from left) (OP9 DLL1 + RPMI +10% FBS), light blue (third bar from left) (FF + SFEM II) and dark blue (far right bar) (OP9 DLL1 +SFEM II) respectively. (Mean± SDMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCshown; Mann Whitney test, n=4). FF = Feeder Free, SFEM = Serum Free Expansion Media.FIGs. ID depicts the frequency and FIG. IE depicts the yield of cDCls generated from expanded CD34+ cells from cord blood (CB, n=9), G-CSF mobilized peripheral blood (mPB, n=3), non-mobilized peripheral blood (PB, n=7), and bone marrow (BM, n=3) are shown. Medians shown on graphs for each cell source by horizontal lines. FIG. IF shows a robust and consistent generation of cDCl from cord blood HSCs. DCs were generated using eight independent donors in three independent culture systems. Each symbol represents a donor, bars indicate the three independent cultures (Mean ± SD shown; two tailed non-parametric paired t test, n=8 donors per culture).

[0038] FIGs.2A-2E show large scale cDCl generation. FIG.2 A are bar graphs depict the frequency (left) and yield of cDCls (right) in a feeder free (FF - green), OP9 feeder (OP9-brown) and OP9 feeder expressing human DLL1 (DLLl-blue) (Mann Whitney test, n=5).FIGs 2B and 2C show expanded CD34+ cells were differentiated for 12 days on mouse stromal cell feeder layers. The feeders either lack or expressed different levels of DLL4 [OP9-DLL410 / med / hior highest levels of DLL4] or DLL1 [OP9-DLLllo / med / hior highest levels of DLL I]43. Bar graphs (a) show the frequency and number (b) of cDCls generated in different feeder layer cultures. cDCl numbers (b) are normalized to the input cells on day 0. FIG.2D shows inhibition of notch signaling using the y-secretase inhibitor DAPT blocks the large-scale generation of cDCls from expanded CD34+ cells. Open bars show control conditions and colored bars denote treatment with DAPT (all culture conditions contain OP9-DLL1 feeder cells). Symbols represent individual donors (Mann Whitney test, n=5). FIG.2E are bar graphs showing the frequency (top row) and yield (bottom row) of cDCls generated from different CD34+ cell sources as depicted using OP9 feeder layer expressing human DLL1. The bars represent the cytokine combinations used for DC differentiation: green (left bar) (Flt3L + SCF + GM-CSF: FSG), blue (middle bar) (FSG+ IL4: FSG4) and red (right bar) (FSG4+ IL15: FSG4+15) respectively. Each dot represents an independent donor (Wilcoxon matched-pairs signed rank test). Mean ±SD shown for all graphs.

[0039] FIGs.3 A - 3F show phenotype and transcriptome profiling of in vitro generated cDCls. FIG.3A are histogram overlays that show the expression of cDCl specific markers CLEC9A, CADM1 and XCR1 on CD141 positive cells generated in feeder free (FF), OP9 feeders (OP9) or OP9-DLL1 feeder cultures (DLL1). Dark histograms show FMO controls and medium grey histograms show specific staining in each culture condition. FIG. 3B is principal component analysis showing cDCl clustered according to which feeder layers theyMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCwere generated; cDC2 generated from the same OP9 feeder layer culture are shown for comparison. DLLl cDCl were clustered in the lower left quadrant; FF cDCl and OP9_cDCl were clustered in the upper left quadrant. FIG. 3C is a heatmap showing the variance stabilizing transformation (vst) normalized expression of cDCl -specific genes in each culture condition and as a comparison, the expression of the same genes in cDC2s generated in the OP9 feeder layer. FIG. 3D shows DEGs between cDCl generated using different methods; Venn diagrams show overlap of upregulated (red, left) and downregulated genes (blue, right) from pairwise comparisons of cDCls generated in three different culture conditions. DEGs were identified using DESeq2, and cut-off used for selecting DEGs are absolute log2-fc >1 and adjusted p-value < 0.05. FIG. 3E are heatmaps showing the variance stabilizing transformation (vst) normalized expression of genes associated with DC functions in each culture condition. FIG. 3F are Ingenuity Pathway Analysis (IP A) illustrating the top 10 most significantly activated (brown; left) or inactivated (blue; right) pathways on cDCls generated in DLL1 expressing feeder cultures in comparison with feeder free culture. The color intensity indicates the activation Z-score and length indicates the statistical significance. The red (dashed) line indicates the threshold value set for the analysis (p = 0.05). P-value is calculated by IPA using right-tailed Fisher's Exact Test and is adjusted using the Benjamini-Hochberg method for multiple hypothesis test correction.

[0040] FIGs.4A and 4B show expression of DC associated and lineage markers on in vitro differentiated cells from different cultures. FIG. 4A is a high-dimensional viSNE analysis examining the expression of key DC markers derived from CD34+ cells under various conditions. The cDCl cluster specifically expresses CD141 and Clec9A, while the cDC2 cells specifically express CD14, CD206, CD209, or FCERIA. Both fractions exhibit variable levels of CD1 lc and CleclOA expression. FIG.4B shows expression of different lineage and myeloid markers on the non cDCl (CD141-Clec9A-)generated in different culture conditions.

[0041] FIG. 5A depicts hierarchical clustering of sample Euclidian distance calculated using the vst normalized counts of the top 1000 most variable genes across samples. The analysis shows that cDCls generated in feeder free and OP9 feeders cluster together compared to the cDCls generated on the OP9 feeders expressing DLL1 and DI, D2 and D3 indicates three independent donors of CD34+ cells. FIG. 5B depicts in vitro generated cDCls exhibit a similar gene expression profile to in vivo cDCls: Transcriptome profile of in vitro generated cDCls from different culture conditions are aligned with the naturally circulating DCs, DCMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCprecursors or MoDCs using the Stemformatics Human DC Atlas platform. FIG. 5C is a heatmap generated using Stemformatics showing the proximity of in vitro generated cDCls from different culture conditions (rows), to previously published datasets of human cDCls isolated from blood (in vivo), or generated in vitro (in cultures) or in vivo in humanized mouse models engrafted with human CD34+ cells (columns). FIG. 5D is a volcano plot showing differentially expressed genes between DLLl cDCl and FF cDCl. Log2 fold change is plotted on the x-axis, and statistical significance (negative log 10 of the Benjamini-Hochberg adjusted p-value) is shown on y-axis. Adjusted p value cutoff is 0.05, and logiFC cutoff is 1. Red (right), significantly upregulated genes in DLL1; blue (left), significantly upregulated genes in FF.

[0042] FIGs. 6 A and 6B is an Ingenuity Pathway Analysis that illustrates the top 10 inactivated (blue; right) or activated (brown; left) pathways on in vitro generated cDCls. The color intensity indicates the activation Z-score and length indicates the statistical significance. The red (dashed) line indicates the threshold value set for the analysis (p = 0.05). P-value is calculated by IPA using right-tailed Fisher's Exact Test and is adjusted using the Benjamini-Hochberg method for multiple hypothesis test correction. FIG.6A is a comparison of cDCls generated using DLL1 feeder vs OP9 feeder. FIG.6B is a comparison of cDCl generated in OP9 feeder vs feeder-free condition.

[0043] FIGs. 7A-7D show functional characterization of in vitro generated cDCl.FIG. 7A are bar plots that illustrate the change in surface expression of cDCl specific molecules, costimulatory molecules, maturation markers and inhibitory molecules on cells unstimulated (green; left bars) or stimulated with TLR adjuvants (poly LC+R848) (red; right bars). The plots show the change in mean fluorescence intensity of each molecule assessed by flow cytometry. Each dot represents individual donors (paired t-test, n=3). FIG. 7B are bar plots show the cytokine secretion by unstimulated (green; left bars) or stimulated with TLR adjuvants (poly LC+R848) (red; right bars). Each dot represents independent donors (paired t test). FIG. 7C shows functional characterization of in vitro generated cDCl: Cytokine production by cDCls activated with TLR adjuvants and measured using intracellular cytokine staining. The light blue, left bars represent the cDCls generated in feeder free cultures and dark blue, right bars represent cDCls generated in DLL1 -expressing feeder layers (Wilcoxon matched-pairs signed rank test, n=7). FIG. 7D shows chemotaxis towards CCL-19: in vitro generated cDCls were stimulated with or without TLR adjuvants for 16 hours and used for testing the chemotaxis towards CCL-19. The bar plot shows the percentage of input cellsMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCmigrated towards CCL-19 normalized to the input control cells (paired t test, n=5).

[0044] FIGs. 8A-8C show antigen presentation and CD8+ T cell priming by human cDCl. FIG. 8A shows activation of NY-ESO-1 SLL-specific T cells measured by IFNy ELISPOT following stimulation with HLA-A2+, unpulsed cDCl (light green; left) or cDCl pulsed with cognate SLL peptide (brown; right bar) (Unpaired t test, n=3). FIG. 8B shows celltrace deep red-labelled dying K562 tumor cells were cocultured with in vitro generated cDCl and uptake of dead K562 tumor cells by cDCl was analyzed by flow cytometry by measuring the frequency of cDCls positive for celltrace deep red fluorescence (red; right bar) (n=3). FIG.8C shows cross-presentation of the NY-ESO-1 SLL epitope to a SLL specific T cell line by cDCl following uptake of dead K562 with or without the expression of full-length NY-ESO1 (paired t test, n=3)

[0045] FIGs. 9A - 9C show cytokine production and cross presentation capacity of cDCl generated using different culture methods. FIG. 9A shows cytokine production by cDCls generated with or without serum: The bar graphs depict the difference in the ability of cDCls generated with (green; left bar) or without (blue; right bar) serum in response to TLR (poly I:C+ R848) adjuvants (Wilcoxon matched-pairs signed rank test, n=4). FIGs. 9B-C show antigen presentation by cDCls generated with or without feeder layer: (FIG. 9B) Bar graph shows cellular antigen cross presentation by FF cDCl (green; left bar) and DLLl cDCl (blue; right bar) illustrated as the fold difference in the IFNy spots normalized to the spots in control conditions (K562 without NY-ESO1 expression; n=3). (FIG. 9C) Bar graph illustrates IFNy production by SLL specific T cells cocultured with SLL peptide-unpulsed or pulsed cDCl (fluorescent green; two left bars = FF cDCl, blue; two right bars =DLLl_cDCl) (paired t test, n=3).DETAILED DESCRIPTIONI. Introduction

[0046] Dendritic cells (DC) are a link between innate and adaptive immune system and play a crucial role in initiating, maintaining, and balancing immune homeostasis. They are professional antigen presenting cells that capture antigens from invading infectious agents or malignant cells and present to the naive or antigen experienced effector cells. DC are natural adjuvants making them an attractive option for cellular therapy. DC based-vaccines remain the sole approved cancer vaccine. Despite their established safety and efficacy in numerous trials against cancers and infections, long-term clinical benefits have been modest. Most trialsMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PChave employed DCs derived from blood monocytes (MoDC), but emerging evidence underscores the unique role of cDCl in triggering potent antitumor immune responses and their intratumoral infiltration with favorable prognoses in many cancers. However, the scarcity of cDCl in peripheral blood and challenges in generating them in vitro have hindered their widespread application as cellular vaccines.

[0047] To address these limitations, a novel serum-free culture system is disclosed that enables the scalable generation of billions of human cDCls from CD34+ progenitors cells derived from cord or peripheral blood. This robust protocol provides access to enriched, functional cDCl suitable for therapeutic use and fundamental biological research, overcoming previous barriers to clinical translation.II. Definitions

[0048] Before continuing to describe the present disclosure in further detail, it will be understood that the materials, methods, and examples are illustrative only and not intended to be limiting. Methods and materials are described herein for use in the present invention and other, suitable methods and materials known in the art can also be used. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0050] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0051] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0052] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation ofMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCone or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0053] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0054] By "consisting of' is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of' indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of' indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0055] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0056] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PC

[0057] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0058] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0059] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0060] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0061] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCBiological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0062] Cluster of differentiation may be abbreviated as CD

[0063] Cytotoxic T lymphocytes may be abbreviated as CTL or CTLs

[0064] Chimeric antigen receptor may be abbreviated as CAR

[0065] Conventional Type 1 dendritic cells may be abbreviated as "cDCl", "cDCls", or "conventional DC 1 " " .

[0066] Delta like Canonical Notch Ligand 1 may be abbreviated as "DLL1".

[0067] Dendritic cells may be abbreviated as "DC" or "DCs".

[0068] Ethylenediaminetetraacetic acid may be abbreviated as “EDTA”.

[0069] Ex Vivo: As used herein, the term “ex vivo” refers to events that occur outside of an organism (e.g., animal, plant, or microbe or cell or tissue thereof). Ex vivo events may take place in an environment minimally altered from a natural (e.g., in vivo) environment.

[0070] Fetal bovine serum may be abbreviate as “FBS” and fetal calf serum may be abbreviated as “FCS”.

[0071] Fms-like tyrosine kinase receptor 3 ligand may be abbreviated as "Flt3L".

[0072] Granulocyte-macrophage colony-stimulating factor may be abbreviated as "GM-CSF" or "GMCSF".

[0073] Granzyme B maybe abbreviated as “GrB”

[0074] Hematopoietic stem cells may be abbreviated as "HSC" or "HSCs".

[0075] Interferon Gamma may be abbreviated as “ZFNy”

[0076] Interleukin-3 may be abbreviated as "IL-3" or "IL3", friterleukin-4 may be abbreviated as "IL-4" or "IL4", Interleukin-5 maybe abbreviated as "IL-5", Interleukin-7 may be abbreviated as "IL-7", Interleukin- 10 may be abbreviated as "IL-10" or "IL10", friterleukin-12 may be abbreviated as "IL-12" or "IL12", Interleukin- 13 may be abbreviated as "IL-13" orMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PC"IL13", and Interleukin- 15 maybe abbreviated as "IL-15" or "IL15

[0077] In Vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0078] In Vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0079] Monocyte-derived dendritic cells may be abbreviated as "MoDC" or "MoDCs".

[0080] N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic Acid may be abbreviated as “HEPES”.

[0081] Phosphate-buffered saline may be abbreviated as “PBS”.

[0082] Plasmacytoid dendritic cells may be abbreviated as "pDC" or "pDCs".

[0083] Polyinosinic:polycytidylic acid may be abbreviated as “poly I:C or poly(I:C)”, poly I:C mixed with the stabilizers carboxymethylcellulose and polylysine may be abbreviated as “poly IC:LC”.

[0084] Resquimod may be abbreviated as R484

[0085] Stem cell factor may be abbreviated as "SCF".

[0086] Thrombopoietin may be abbreviated as "TPO".

[0087] Toll-like receptor activation may be abbreviated as "TLR activation".

[0088] Tumor necrosis factor a may be abbreviated as “TNFa”.

[0089] Type I Interferon may be abbreviated as “IFN-2ab”.

[0090] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.III. System for the Generation of Dendritic Cells

[0091] eDC 1 s play a non-redundant role in eliciting antitumor immune responses, as clearly demonstrated by studies using animal models that lack cDCls, or by enhancing cDCl frequency and function with Flt3L or poly I:C, respectively. In vivo studies in animal models have demonstrated the superiority of eDC 1 -based cellular vaccines and their ability to directlyMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCpresent antigens to effector cells. An intra-tumoral cDCl vaccine alone is sufficient for tumor rejection without the need for additional steps for tumor antigen loading. The success of neoadjuvant therapies and cell therapies, such as CAR-T and adoptive T cell therapy (ACT), heavily depends on endogenous cDCls. Several neoadjuvant therapies have demonstrated a positive correlation between durable response and the frequency of cDCls in patients, corroborating observations from animal models.

[0092] The extremely low frequency of circulating cDCls in PBMC poses significant challenges for isolating them in adequate quantities, as current isolation procedures yield only about 3-6 million cells, which is insufficient for therapeutic purposes4. Furthermore, circulating cDCls in many cancer patients was significantly impaired in both quality and quantity compared to those in healthy donors, creating additional challenges for their successful therapeutic application. DC-based immunotherapy requires multiple rounds of vaccination, and DC yield is a limiting factor that determines the number and frequency of vaccinations; administering higher doses more frequently may be critical for boosting the immune response to eradicate the tumor. In current DC vaccine regimens, which primarily use monocyte-derived DCs, patients receive three to eight injections depending on the DC yield, with each dose containing between one million and ten million cells. Here, we demonstrate that our novel serum-free culture system can generate over 400 million cDCls from one million mobilized peripheral blood HSCs, which is sufficient for more than 10 doses of vaccine, assuming 20 million cDCls per dose. This treatment regimen is currently unachievable with existing methods. Routine stem cell mobilization protocols successfully harvest enough HSCs; sufficient CD34+ (158 ± 133 x 106) cells can be mobilized from cancer patients for DC generation, thus highlighting the real-world application of our method. Our platform can also generate an average of 2.9 billion cDCls from one million CB CD34+ cells, which can serve as a source for an allogeneic cDCl covering common HLA haplotypes by leveraging international cord blood banking programs as a source of allogeneic HSCs. This approach will facilitate the development of ready-to-vaccinate products for cancer patients, thereby reducing vaccine access time, sparing them from costly and complex HSC harvesting procedures, and providing options for those unable to undergo HSC mobilization.

[0093] Notch signaling has previously been shown to play a key role in the development of mouse and human cDCls; here we demonstrate that leveraging the notch-signaling requirement in combination with an optimized cytokine cocktail and serum free media enabled large-scale in vitro generation of human cDCl closely resembling their in vivo counterparts.MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCThe in vitro derived DCs exhibited bona fide cDCl phenotype including expression of Clec9A, XCR1, CADM1 and functions such as the production of IL-12 and IFN-I and III in response to TLR stimulation. They readily take up and cross-present tumor cell antigens, prime naive CD8+ T cells and drive the antitumor immune response in preclinical models, validating their potential as cellular vaccines. When compared to cDCl generated in the absence of Notch signaling, DLLl cDCls expressed higher levels of CCR7, CXCL9, TNF, IFNL1, TNFSF, and TLR8 transcripts, correlating with increased cytokine production upon exposure to TLR adjuvants. This suggests that Notch signaling not only increases cDCl numbers but also enhances their function, as previously reported in mice.

[0094] Significantly, our serum-free culture system, devoid of xenogeneic serum, demonstrates the capability for large-scale production of bona fide functional cDCls, making it readily adaptable and scalable to a fully GMP-compliant system for clinical translation. This platform offers several opportunities for refinement of vaccine products including comparison of adjuvants for cDCl activation, evaluation of different sources of antigens, such as shared tumor-associated antigens or neoantigens for cancer immunotherapy and antigen loading e.g. through mRNA based approaches. The potential for genetic modifications to improve cDCls adjuvanticity is also now feasible. For example, DLLl cDCls could be loaded with mRNA encoding chemokines, cytokines, or siRNA to have inhibitory molecules ablated, thereby enhancing their effector function. Finally, it affords scientists the ability to investigate in-depth the biochemical and functional characterization of cDCls, helping to understand their biology, ontogeny, and the role of specific genes. In the future, it may even be possible to generate tolerogenic DCs, thereby expanding the utility of these understudied antigen presenting cells.

[0095] Optimization of human cDCl generation

[0096] We previously established a two-step culture system consisting of a 7-day HSC expansion phase, followed by a 12-day differentiation process, and successfully generated CD141 + Clec9A+ cDCls that are identical to their human blood counterparts in gene expression profile, phenotype, and function. Despite the presence of bona fide cDCls, the cultures were highly heterogeneous, consisting of a major fraction of undifferentiated HSCs, cDC2 / MoDC-like cells and a low proportion (<10%) of cDCl. The low yield, serum requirement and heterogeneity prompted us to explore alternative measures to develop a serum-free culture system, which can preferentially generate large numbers and higher frequency of cDCls to bypass additional enrichment procedures to establish a GMP systemMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCfor cDCl based cancer immunotherapy.

[0097] Notch signaling is an evolutionarily conserved juxtacrine signaling pathway essential for cell fate determination and plays a crucial role in HSC differentiation. In the context of DCs, the interaction of DLL 1 or DLL4 with Notch receptors on precursor cells initiates signaling cascades that drive their differentiation towards the cDCl lineage. Therefore, we investigated whether augmenting Notch signaling would improve cDCl yields. To achieve this, we generated a feeder line by expressing human DLL1 in murine bone marrow stromal cells (OP9), and confirmed that it enhanced the differentiation of human cDCls from HSCs when incorporated into culture systems (FIGs. 1A-C). The human DLL1 expressing feeder line increased the percent output of CD141+Clec9A+ cells (cDCls) to >40% of the culture progeny compared to <10% achieved by our previously reported feeder free culture system (FIG. IB). The feeder layer itself did not contribute to enhanced cDCl generation, as confirmed by cultures using OP9 feeder layers (FIG. 2A-B). However, the expression of variable levels of murine DLL1 or DLL4 on OP9 feeders correlated with the cDCl frequency in the culture (FIG. 2A-C). Additionally, treatment with a Notch signaling y-secretase inhibitor, DAPT, abrogated cDCl generation (FIG. 2D). The new human DLL1 expressing feeder cell line (hereafter referred to as DLL1, FIG. IB) was incorporated into all subsequent cultures.

[0098] GMP compliance protocols do not allow the use of supplements from xenogeneic origins for cellular vaccines; therefore, we evaluated the adaptability of our culture system to serum-free conditions using SFEM II medium. Notably, even without DLL1 feeders, cultures in serum-free media showed enhanced differentiation of cDCl compared to those with serum (FIG. 1 A-C). The combination of serum-free media with DLL1 yielded the highest quantities of cDCls (FIG. 1 B-C), averaging 2.8-fold more DCs compared to serum containing DLL1 cultures. This optimized protocol successfully generated substantial numbers of cDCls from HSCs obtained from various sources, including CB, non-mobilized peripheral blood (PB) mobilized adult peripheral blood (mPB), and bone marrow (BM)(FIG.1D-E, FIG.2E). The addition of interleukin (IL)- 15 enhanced the cDCl generation in cultures with CB and PB CD34+ cells. Historically, existing culture systems have yielded between 0.25 to 28.7 cDCls per CB-HSC at best. In contrast, our optimized system achieves a remarkable yield of 2900 cDCls per HSC from CB-derived HSCs. Furthermore, our culture system represents a significant advancement in generating large quantities of cDCls from adult blood, producing 436 cDCls per mobilized adult CD34+ HSC compared to 2-6 cDCls using current published methods, and 404 cDCls per adult bone marrow-derived CD34+ HSCMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCcompared to 0.4 to 4 cDCls using current published methods as shown in Table 1.

[0099] Table 1

[0100] Moreover, our system consistently generates cDCls at comparable frequencies and yields from different donors and from cryopreserved expanded HSCs across various time points, underscoring the remarkable reproducibility and robustness of the culture system (FIG.IF).

[0101] In vitro generated cDCls comparison

[0102] We next compared the phenotype of cDCls generated in feeder free (FF), OP9 feeder and DLL1 feeder culture conditions. All conditions generated bona fide cDCl, confirmed by expression of cDCl specific markers including the C-type lectin receptor Clec9a, cell adhesion molecule CADM1 and chemokine receptor XCR1 on CD 141+ cells (FIG. 3A). Unbiased high dimensional viSNE analysis performed on cells from different culture conditions identified a distinct cluster of Clec9A+and CD141+cDCl significantly enriched in DLL1 cultures (FIG.4A). In vitro generated cDCls expressed CDlc as previously reported, but lacked other cDC2 or inflammatory DC-associated markers such as CD206, CD14, FCERIA, and CD209 (FIG. 4A). Clusters of cells expressing these cDC2 associated markers were enriched in the FF cultures. A large cluster of cells enriched in the FF and serum containing cultures did not express markers of either DC subset, but expressed CD33 indicative of myeloid populations. Both cDCl and cDC2 subsets expressed variable levels of CD1 lc and CleclOA (FIG. 4A). The non cDCl cells in the cultures were mostly comprised of myeloid (CD33+ and CD172a+) cells, with no evidence of T, B, or NK cell generation (FIG. 4B). In summary, a combination of serum free media and DLL1 feeders favors the differentiation of expanded CD34+ cells towards cDCl and suppresses development of other cell subsets or DC subsets in the culture.

[0103] To further confirm the identity of the in vitro generated cDCls, we compared the transcriptome profile of FF cDCl, OP9_cDCl and DLLl cDCls. Bulk RNA sequencing was performed on cells sorted as cDCls (CD 141+ Clec9A+) from all culture conditions andMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCcDC2 (CD141- Clec9A- CDlc+ FCERIA+) from OP9 cultures (OP9_cDC2) identifying unique signatures associated with DCs in the different culture conditions. Principal component analysis showed highest variance (PCI- 73%) between the in vitro generated cDCls and cDC2. The PC3 variance clustered the DLLl cDCl apart from the FF cDCl and OP9_cDCl (FIG. 3B). Hierarchical clustering based on the top 1000 most variable genes (DEG) also demonstrated that the DLLl cDCl from three independent donors clustered together and separately from the FF cDCl and OP9_cDCl (FIG. 5 A). Despite the differences in culture conditions, all cDCls expressed hallmark cDCl associated genes including THBD, CLEC9A, CADM1, XCR1, TLR3, CLNK, WDFY4, C1ORF54, BATF3, ID2 and IRFZ (FIG.3C). Projection onto the Stemformatics human DC Atlas confirmed that the in vitro generated cDCls, regardless of culture conditions, aligned with human cDCl signatures collated from publicly available datasets (FIG. 5B). Moreover, DLLl cDCl closely aligned with in vivo human cDCls compared to OP9_cDCl and FF cDCl suggesting they represent a more physiologically relevant phenotype (FIG. 5C). Collectively these data demonstrate that DLLl cDCl closely resemble their human in vivo counterparts.

[0104] The differential expression analysis identified 1,346 upregulated genes in DLL 1 cDCls when compared to FF cDCls, and 992 upregulated genes in DLL 1 cDCls when compared to OP9_cDCls comparison, with 795 genes upregulated in both comparisons (FIG. 3D). In contrast, in DLLl cDCls, we identified 288 and 251 downregulated genes respectively, with 126 of these being commonly downregulated (FIG. 3D). Additionally, cDCls from feeder-free and OP9 feeder conditions exhibited a higher degree of gene expression similarity, with only 55 genes upregulated in OP9 and 150 genes upregulated in FF (FIG. 3D). Most of the differentially expressed genes (DEGs) between DLLl cDCls and FF cDCl were genes of unknown function and / or not previously associated with cDCl biology (FIG. 5D). Certain specific genes associated with DC functions were found significantly upregulated between DLLl cDCls and FF cDCls (FIG. 3E). For example, cDCls from DLL1 feeder cultures expressed highest levels of CXCL9, CCR7, CCL19 and CCR10 indicating a potential for migration and / or interaction with effector cells. They also exhibited elevated expression of interleukin receptors (IL11RA, IL1R2, IL10RA, and IL21RA), cytokines (IL-15, TNF) and TLRs (TLR-8 and 10), while cytosolic sensor STING1 was downregulated. Additionally, cDCls from the DLL1 feeder express higher levels of inhibitory molecules including LILRA4 (ILT7), BTLA, LAG3 and immunoregulatory molecules such as IL4I1, IDO1, and IDO2. They also expressed immunostimulatory TNF Receptor Superfamily Members associated with inflammatory response and notch signalingMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCassociated molecules (FIG. 3E).

[0105] To unravel the functional differences between DCs grown on different conditions, we performed QIAGEN Ingenuity Pathway Analysis (IP A) using DEGs. The SI 00 family signaling pathways, phagosome formation, Class A / l (rhodopsin receptor) and G-PCR coupled receptor signaling pathways were augmented in DLLl cDCl versus both cDCI FF and cDCl_OP9 (FIG. 3F, FIG. 6A-B). Overall, the pathway analysis suggest that DLLl cDCl were programmed towards an activated DC phenotype, with improved antigen capture, cargo transport capability, and chemokine production, indicative of potential functional superiority compared to cDCls generated in the feeder-free or OP9 cultures.

[0106] Functional characterization of in vitro generated cDCls

[0107] cDCl express TLR3 and TLR8 and respond to their respective agonists Poly I:C and R848 by upregulating costimulatory molecules and producing type I and III interferons and inflammatory cytokines. DLLl cDCls treated with a combination of Poly I:C and R848 for 16 hrs, down regulated the cDCl specific markers Clec9a, CADM1 and XCR1 and upregulated CC-motif chemokine receptor 7 (CCR7), lymphocyte activation marker CD69, along with costimulatory molecules CD40, CD80, CD86, CD83 and MHC II, indicating DC activation (FIG. 7A). PD-L1 and CD200 showed a trend towards upregulation, while BTLA, TIM3 and LAG3, were downregulated (FIG. 7A). Activated cDCls secreted IL-27, IL-6, TNF-a, IFN-P, IFN- X, CXCL-10, and IL- 12; all cytokines that play a crucial role in regulating DC and adaptive immune effector cell functions (FIG. 7B). DLLl cDCl produced higher levels of CXCL10, IL-12, IL-29 and TNFa compared to FF cDCl, indicating potentially enhanced functionality to recruit and prime T cells, as observed in their gene expression profile (FIG. 7C). Similarly, cDCls generated in serum free conditions were superior in their ability to produce CXCL10, IL-12, IL-29 and TNF compared to serum supplemented cultures. (FIG. 9A). Consistent with their upregulation of CCR7, activated cDCls migrated towards the cognate CCL-19 chemokine (FIG. 7D). Collectively these assays demonstrate that in vitro generated cDCls can sense and respond to danger signals, similar to their in vivo counterparts. Thus, we demonstrate that the optimized protocol using DLL1 expressing feeder cells enhances cDCl functionality as well as yields.

[0108] In vitro generated cDCls cross present soluble and cellular antigens to activate naive and memory CD8+ T cells and provide therapeutic efficacy

[0109] cDCl are adept at cross-presentation of cell derived antigens. We validated the antigen presentation ability of in vitro generated cDCls using an HLA-A*0201 restricted experimental system with NY-ESO-1 as a model antigen. DLLl cDCl pulsed with NY-MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCES01-SLL peptide activated SLL-specific memory T cell lines (FIG. 8A). DLLl cDCl readily ingested dead tumor cells, processed and cross-presented the SLL epitope following uptake of dead tumor cells expressing full-length NY-ESO-1 antigen (FIG. 8B-C). DLLl cDCls exhibited an enhanced ability to cross present cellular antigens compared to FF cDCl, even though both were equally efficient in presenting the SLL peptide (FIG. 9B-C).

[0110] We have successfully established a robust culture system for the large-scale generation of functional cDCls from HSCs, providing a potential platform for developing cDCl -based cellular vaccines and serving as a research tool for exploring their biology.

[0111] IV. Methods of Use

[0112] The generated conventional type 1 dendritic cells may be used in a method of treating a subject having cancer or in a method of performing cellular immunotherapy in a subject having cancer.

[0113] The generated conventional type 1 dendritic cells may also be formulated into a cellular immunotherapy composition, which may be used to treat or prevent cancer.

[0114] Non-limiting examples of cancers the subject may have include genitourinary cancer, prostate cancer, myeloma, head and neck cancer, high-positive MSI cancer, and metastatic cancer.

[0115] In certain embodiments, cellular immunotherapy composition is formulated as a vaccine. In an alternative embodiment, the conventional type 1 dendritic cells are adjuvants. The vaccine may be administered in a variety of ways including, but not limited to, intramuscularly, subcutaneously, intranodally, intratumorally, or intravenously.

[0116] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.EXAMPLES

[0117] Isolation of mononuclear cells from cord blood or buffycoats.

[0118] Cord blood units were obtained from NYC blood bank, Vitalant cord (US) orMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCfrom the Queensland Cord Blood Bank at the Mater (Brisbane) according to the relevant institute approved IRB protocols. Mobilized peripheral blood and bone marrow CD34+ cells were purchased from All Cells.

[0119] Blood samples and buffy coats were diluted to 1 : 1 and 1 :2 v / v respectively with IX PBS. Thirty mL of diluted samples were gently loaded to 15ml of ficoll / hypaque™ plus (Cytiva, Uppsala, Sweden) on a 50 ml Falcon tube. The tubes were centrifuged at 2000 rpm for 25 minutes at low break and acceleration at 20°C. Mononuclear cells (MNC) were carefully collected with a pipette and cells from two tubes were pooled together into a new 50 ml falcon tube and brought up to 50 ml with IX PBS. The cells were washed by spinning down at 2000 RPM for 10 minutes, cell pellets were pooled and resuspended in 25ml of in house MACS buffer prepared using 1XPBS supplemented with 2% FBS with final concentration of ImM EDTA (Sigma Aldrich, USA) and lOmM HEPES (Gibco, MT, USA). Cell viability was determined using 0.2% trypan blue (Gibco, MT, USA.

[0120] CD34+ hematopoietic stem cell enrichment

[0121] CD34+ cells were isolated from MNCs using LS columns or AutoMACS with CD34 MicroBead Kit UltraPure, human, (Miltenyi Biotec). Enriched MNC were washed by centrifuging at 500g for 5 minute and pellets were resuspended in 300uL MACS buffer. The MNCs incubated by adding 150 pL CD34 micro beads for 30 minutes at 2-8°C and further washed by adding 5 ml of MACS buffer and spinning down at 300g for 10 minutes. LS columns were prepared by washing with 3 ml of MACS buffer and the cell pellets were resuspended in 500 pL of MACS buffer and added to the LS columns. Columns were washed 3 times with 3 ml of MACS buffer and positively enriched cells were collected by adding 5ml of MACS buffer and using the plunger to a new 15 ml tubes. Isolated cells were counted and used for expansion cultures or cryopreserved for future use.

[0122] Generation and maintenance of Notch ligand expressing OP9 feeder layers

[0123] Mouse bone marrow stromal cells MS-5, OP9 and OP9 expressing different notch ligands were maintained in a-MEM glutamax + 20% FBS containing media supplemented with Sodium pyruvate, non-essential amino acid (NEAA), HEPEs and 0-Mercaptoethanol (0-ME) (All from Gibco, MT, USA ). The cells were subcultured on every 3-4 days by harvesting the cells using TrypLE (Gibco, MT, USA) and transferred to new flasks by 1:10 v / v ratio. Stromal feeder layers were prepared by seeding 2 X 105cells / well in 24 well plates and maintained overnight in 500 pL of a-MEM glutamax + 20% FBS media. The feeder layers were irradiated for 60Gy using X ray irradiator (RS-2000 X-Ray Irradiator, RADMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCsource technology). The media was carefully removed and expanded HSCs were seeded with DC differentiation media to the feeders.

[0124] The details of the feeders used for the cultures were given in the sequence depicted in FIG. 2B and C, 1) OP9 feeder cells purchased from ATCC, 2) OP9 feeder cells obtained from CIML, Marseille), 3) MS-5 feeder layer purchased from DSMZ. We obtained the OP9 feeder layers described by Mohtashami et al., from the lab of Dr J. C. Zuniga-Pflucker (Sunnybrook Research Institute, University of Toronto, Canada). They express low (4), medium (5) and high levels of DLL4 (6) (OP9-DLL4lo / med / hi) or highest levels of DLL4 (7) (OP9 DLL4) and low (8), medium (9) and high levels of DLL 1 (10) (OP9-DLLllo / med / hi) or the highest levels of DLL 1 (11) (OP9 DLL1). 12) OP9 expressing mouse DLL1 were obtained from CIML, Marseille).

[0125] Human DLL1 expressing feeder layer were generated by transducing the OP9 feeder layers (ATCC, VA, USA) with lentivirus expressing human DLL1. The expression system was generated by subcloning human DLL1 (HG11635-U, Sino biologicals, Nanjing, China) with aaccccgggcccgcatataatttaaatTTGGGCAGTCGGTGCGC (forward) and caaattttgtaatccagaggttgattgTTACACCTCAGTTGCTATGAC (reverse) primers to EFla-GFP-E2A- lentiviral expression plasmid. DLL1 positive lines were established by sorting the DLL1 and eGFP double positive cells.

[0126] DC generation

[0127] CD34+ cells were expanded and differentiated as described in the previously published protocol. Briefly 2.5 XI 04cells were expanded in U bottom 96 well plate in Stemspan (SFEM II) alone or Stemspan (SFEM II) + 10 % FBS containing media supplemented with combination of recombinant human cytokines FMS-like tyrosine kinase 3 (Flt3L), Stem Cell Factor (SCF) (100 ng / mL each), Interleukin-3 (IL-3) (20ng / ml) and Thrombopoietin (TPO) (50 ng / ml) for 7 days. Expanded CD34+ cells were cryopreserved or differentiated immediately in the absence of presence of stromal cell feeder layers (U bottom 96 well plate or 24 well plate) in Stem span (SFEM II) or RPMI +10% FBS supplemented with Sodium pyruvate, NEAA, HEPEs and p-ME. DCs were differentiated with recombinant human cytokines for 12 days in the presence of human Flt3L (100 ng / mL), SCF (20ng / ml), IL-4 (2.5 ng / ml), GMCSF (10 ng / ml) and on day 6 cultures were replenished by replacing half of the spent media with fresh media consist of 2X cytokines.

[0128] The protocol was further optimized by expanding the CD34+ cells at a lower cell density (IxlO4cells / ml) in stem span (SFEM II) as described above, which improved the viability and fold expansion. The expanded CD34+ cells were next differentiated in theMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCpresence of human Flt3L (100 ng / mL), SCF (20ng / ml), IL-4 (2.5 ng / ml), GMCSF (10 ng / ml), and IL-15 (5 ng / ml). On day 6 half of the media was carefully removed and replenished with fresh media and 2X concentration of cytokines. Cells were harvested on day 12 and viable count was determined and used for phenotyping or other functional assays. Recombinant human cytokines were purchased from R & D system (MN, USA) and Peprotech Inc (NJ, USA).

[0129] For determination of Notch sensitivity, cells were treated with y-secretase inhibitor XXI, compound E (at concentration of 0.5 or 1 pM, EMD Millipore, MA USA) or an equivalent volume of DMSO as control on both day 0 and day 6 of the differentiation culture.

[0130] The eDC 1 s generated with optimized protocols under different conditions such as feeder free (FF cDCl), on OP9 feeders (OP9_cDCl) and on human DLL1 expressing OP9 feeder (DLLl cDCl) were named accordingly and the term is used throughout the manuscript to represent each condition.

[0131] Cell enrichment and Cell sorting

[0132] Cells were resuspended in MACS buffer at a density of 10 million / 100 pL and incubated with biotinylated anti-human CD141 (M80-biotin) or CDlc (L161-biotin) (Biolegend, CA, USA) or CADM1 (3E1- biotin, MBL International, JP) antibody to final concentration of 1 pg in 100 pL and incubated for 5-8 minutes in room temperature. Cells were washed by adding 3 ml of MACS buffer and spinning at 500 g for 5 minutes. Cell pellets were resuspended in 100 pL of MACS buffer and 50 pL of anti-biotin beads and incubated for 20 minutes at 2-8°C. Cells were washed by adding 3 mL of MACS buffer by spinning at 500g for 5 minutes and resuspend in 500 pL of MACS buffer. Cells were enriched using LS columns as described earlier and used for downstream processing including functional assays or cell sorting. Cells were prepared for sorting by incubating with a cocktail of antibodies and live dead dye in MACS buffer for 30 minutes. Cells were washed once by adding 1 mL of MACS buffer and resuspend in 500 pL of MACS buffer and sorted in FACS Aria III. Sorted cells are collected in 5 ml FACS tubes consist of 200 pL of FBS and cells are washed and used for RNA extraction or functional assay.

[0133] DC Phenotyping and activation:

[0134] Cells were stained for surface markers with a cocktail of anti-human antibodies and viable cells were detected using LIVE / DEAD™ Fixable Blue Dead Cell Stain Kit (ThermoFischer, USA). Cells were stained for 30 minutes in IX PBS and washed once by adding 200pL of PBS and resuspend in 200pL of PBS. Secondary staining was performedMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCusing Streptavidin tagged flurochromes, incubated for an additional 25 minutes, and washed as described above. The following antibodies are used for DC phenotyping; CD141 (M80 -BV 785, PE / Dazzle 594), CDlc (L161 - BV510), Clec9A (8F9 -PE), CleclOA (H037G3 -APC), CD172a / b (SIRPa / b) (SE5A5 - PerCP / Cy5.5), CD206 (15-2, APC / Cyanine7, AF700, FITC), CD 14 (HCD14 -FITC, Pacific Blue, AF700), CD56 ( HCD56 - FITC, 5.1H11- APC), CD3 (UCHT1 - FITC, Pacific Blue), CD19 (HIB19 - FITC), XCR1 (S15046E - BV421), CD16 (3G8- FITC), CDllc (Bul5- Pacific Blue, FITC), CD33 (P67.6- APC / Cyanine7), FCERIA (AER-37 (CRA-1) - PE / Dazzle 594), CD209 (9E9A8- PE / Cyanine7, PerCP / Cy5.5. All reagents were purchased from Biolegend CA, USA. CADM1 (3E1- biotin, MBL international, JP) and secondary staining was performed with PE / Dazzle™ 594 Streptavidin (Biolegend). DC activation was assessed with the following combination of antibodies CCR7 (G043H7- APC / Cyanine7), CD69 (FN50- APC), CD40 (5C3 - APC / Cyanine7), CD80 (W17149D- PerCP / Cyanine5.5), CD83 (HB15e - PE, APC), HLADR (L243, PE / Cyanine7, BV 785), HLA-A2 (BB7.2- PE / Cyanine7), BTLA (MIH26 - PE / Cyanine7), TIM3 (F38-2E2-PerCP / Cyanine5.5), LAG3 (11C3C65 - BV785, PDL1 (29E.2A3 - PE / Cyanine7), CD200 (OX- 108- APC), all are purchased from Biolegend , CD86 (FUN-1 , BD Horizon™ R718, BD Bioscience, CA, USA). Cells were acquired in a LSR Fortessa BD biosciences (BD Bioscience, USA) and analyzed by FlowJo (BD Bioscience, OR, USA) FCS Express (De Novo Software, CA, USA) and viSNE analysis was performed using Cytobank (Beckman Coulter, USA).

[0135] RNA Sequencing

[0136] RNA isolation was performed using Qiagen micro RLT kit as per manufacturer’s protocol (QIAGEN LLC, MD, USA). Sorted cells were resuspended in 350 pL of RLT buffer and vortexed well to resuspend the cell pellet. The cell lysate was transferred to the gDNA depletion column and centrifuged at >8000g for 20seconds. The flow through was collected and frozen in -80 for future use. The frozen lysates were thawed, and mRNA was isolated as per the manufacturer’s protocol. The quality and quantity were analyzed using bioanalyzer with Agilent RNA 6000 Nano kits and samples had a RIN value above 9.5 (Agilent CA, USA). Illumina Stranded mRNA library was prepared using 100 ng of RNA from each samples. The sequencing procedures were carried out using NovaSeq 6000 SP Reagent Kit vl.5 (100 cycles). A paired end (50bp) sequencing with 60 million read coverage was performed using NovaSqq 6000 and the data was analyzed as described below (Illumina, CA, USA).

[0137] RNA-Seq PreprocessingMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PC

[0138] A total of 11 RNA-Seq libraries from DLLl cDCl (n = 3), FF cDCl (n = 3), OP9_cDCl (n = 3) and OP9_cDC2 (n = 2) were processed using the same pipeline for compatibility. Libraries were first evaluated for their quality using FastQC (v0.11.8, RRID:SCR_014583). Trim Galore! (vO.6.6, RRID:SCR_011847) was used to trim the adapter sequences with a quality threshold of 20. Reads were aligned to the human genome reference (hg38) using STAR aligner (v2.7.5b, RRID:SCR_004463). Gene level read counts are obtained by using Salmon (vl.2.1, RRID: SCR 017036) for all libraries.

[0139] Principal component and Sample Similarity analysis

[0140] We performed between-sample normalization using the variance stabilizing transformation (vst) from the DESeq2 (vl.30.1, RRID:SCR_015687) R package. The 1000 most variable genes were used to perform principal component analysis (PCA) as well as calculating the Euclidean distances between each sample. PCA plot was generated using plotly R package (v4.9.2.1) on the first and the third component. Heatmaps displaying Euclidean distance were generated using heatmaply (vl.1.0)51. R (v.4.1.0) was used to perform all bioinformatics analysis.

[0141] Differential Expression and Ingenuity Pathway Analysis

[0142] Differential expression analysis was performed using DESeq2 (vl.30.1, RRID:SCR_015687). Genes with less than 5 reads in total across all samples were filtered as inactive genes. A gene was considered differentially expressed if the Benjamini-Hochberg adjusted p-value is less than 0.05 and the absolute log2 fold change is greater than 1. To quantify and overlap differentially expressed genes from multiple comparisons, Venn diagrams were plotted using VennDiagram (vl.6.20, RRID:SCR_002414). VST normalized counts of cDCl signature genes and cDC2 signature genes were visualized on a heatmap. Differentially expressed genes from cDCl DLL1 vs FF and cDCl DLL1 vs OP9 were visualized on volcano plot using plotly R pakage. For each comparison, differentially expressed genes and their corresponding log2 fold change and adjusted p-value was used as input for Ingenuity Pathway Way (IPA) Core Analysis (v01-23-01, RRID:SCR_008653). Results of canonical pathway analysis for each comparison were ranked by -log 10 (p-value), and the top 10 pathways were visualized using barplots generated by ggplot2 (v3.3.5, RRID:SCR_014601).

[0143] Stemformatic Analysis

[0144] The transcriptome profile of in vitro generated eDC 1 s was compared to publicly available data sets using the Human DC Atlas on Stemformatics.

[0145] Functional assayMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PC

[0146] DC stimulation for cytokine production: In vitro generated DCs were resuspended in fresh culture media with Flt3L (lOOng / mL) and GMCSF (lOng / ml) in U -bottom 96 wells in the presence or absence of 5 pg / mL Poly I:C and 10 pM R848 (invivoGen, CA, USA). Cultures were harvested after 16 hrs and analyzed for the expression of costimulatory or inhibitory molecules and cells are stained as described above.

[0147] Culture supernatants from cDCls cultured for 16 hours with or without TLR ligands were analyzed for cytokine secretion using a Legendplex kit (BioLegend).

[0148] Intracellular cytokine staining of cDCls was performed by activating the DCs with TLR adjuvants as described above. Brefeldin A (lOpg / ml, Sigma Aldrich, Missouri, USA) was added to the cultures 1 hr after stimulation and maintained for another 6-7 hrs. Cells were washed and stained for cell surface markers (CD141-BV785, CADM1, CDlc-BV510, and CD206-AF700) and intracellular cytokines (IL-29 (MAB 15981, R& D system, MN, USA, conjugated with APC conjugation Kit- lightening Link -ab201807, Abeam, MA, USA), IL- 12 (p40 / p70) (Cl 1.5- PE, BD Bioscience), CXCL10 (J034D6 - PE / Cyanine7), and TNFa (MAbl 1- APC / Cyanine7, Biolegend)). Cells were permeabilized with Cytofix / Cytoperm kit from BD biosciences CA, USA.

[0149] Chemotaxis: DCs were enriched using CD141 -biotin antibody and anti-biotin antibody conjugated magnetic beads as described above and activated overnight with TLR adjuvants. Chemotaxis of activated DCs was evaluated by adding 5 X104cells on the upper chamber of cell culture inserts (8p, BD Falcon, USA) and the lower chambers holding media alone or media with lOOng / mL of CCL-19 (Peprotech Inc, Cranbury, NJ, USA). After 3 hrs. the inserts are removed, 20 pL of 15 p polybead polystyrene microspheres added to each well to assess the cell numbers (Polyscience, PA, USA), and cells from bottom chamber were harvested and stained for CD141 , Clec9a and CCR7. The percentage migration was calculated by [number of cells having migrated in response to CCL- 19 - number of cells having migrated spontaneously] / [total number of input cells] x 100.

[0150] Antigen Processing and Presentation Assays:

[0151] In vitro priming and expansion: Human CD45-enriched splenocytes were cultured with In vitro differentiated HLA-A2+ cDCl pulsed + / - SLLMWITQC peptide for 8 days in media containing IL-2 (lOOU / mL) IL7 (lOng / mL - both from Biosource) and 20ng / mL IL- 15 (Peprotech). Cultured splenocytes were stained as described above.

[0152] Dead cell uptake and Antigen cross presentation

[0153] K562 lines over expressing full length GFP E2A- NY ESO1 or GFP-E2A- OVA control was used as the source of cellular antigens for the assay. The lines were generated byMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCtransducing with lenti viral expression system with full length NY-ESO1- subcloned (HG15611-G Sino biologicals, Nanjing, China) with aaccccgggcccgcatataatttaaatATGCAGGCCGAAGGCC (forward) and caaattttgtaatccagaggttgattgTTAGCGCCTCTGCCCTG (reverse) primers. K562 lines were stained with DeepRED cell trace dye (1:1000, v / v) by incubating 10 million cells / mL in IX PBS for 15 minutes at 37°C with a ratio of the dye. Cells were washed once by adding RPMI +10 FBS containing media, resuspended in 1 mL of plain RPMI and incubated 55°C for 30 minutes. Cells were then ciyopreserved in IMDM+30% DMSO+ 10% DMSO containing media in LN2 for future use. For use, frozen cells were revived and incubated overnight in SFEM II for the dead cell uptake assay. cDCl were differentiated from A2+ cord blood donors and co-cultured with heat killed K562 lines overnight or + / - SLL peptide for 2hr. DC were then co-cultured with a NY-ESO-1 SLL-specific T cell line overnight and TFNy detected using an ELISPOT assay. DCs were enriched with CADM1 antibody and biotinylated beads to compare the antigen cross presentation efficiency between the DCs generated using feeder free and DLL1 feeder cultures.

[0154] Various embodiments also include:

[0155] Example Clause A: A serum free method for generating cells, the method may include: differentiating hematopoietic stem cells into conventional type 1 dendritic cells, where the hematopoietic stem cells are differentiated with a feeder layer may include at least one murine cell line expressing human notch ligands and at least one cytokine in a serum free media, where the at least one cytokine is selected from the group having of Fms-like tyrosine kinase receptor 3 ligand (Flt3L), Stem cell factor (SCF), Thrombopoietin (TPO), Granulocytemacrophage colony-stimulating factor (GM-CSF), Interleukin-4 (IL-4), and Interleukin- 15 (IL-15).

[0156] Example Clause B : The method of Example Clause A, where the hematopoietic stem cells are differentiated with a combination of Flt3L, SCF, GM-CSF, IL-4, and / or IL-15.

[0157] Example Clause C: The method of Example Clause A or Example Clause B or of Example Clause A or Example Clause B, where the hematopoietic stem cells are differentiated in a media for at least about 1 day, alternatively at least about 2 days, alternatively at least about 3 days, alternatively at least about 4 days, alternatively at least about 5 days, alternatively at least about 6 days, alternatively at least about 7 days, alternatively at least about 8 days, alternatively at least about 9 days, alternatively at leastMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCabout 10 days, alternatively at least about 11 days, alternatively at least about 12 days, alternatively at least about 13 days, alternatively at least about 14 days, alternatively at least about 15 days, alternatively at least about 16 days, alternatively at least about 17 days, alternatively at least about 18 days.

[0158] Example Clause D: The method of any one of the preceding claims, where the hematopoietic stem cells have been expanded prior to differentiation.

[0159] Example Clause E: The method of any one of the preceding claims, where the hematopoietic stem cells have been expanded with a mouse stromal cell feeder layer prior to differentiation.

[0160] Example Clause F: The method of any one of Example Clauses A-E or of any one of Example Clauses A-E, where the expanded hematopoietic stem cells are expanded in a media for at least about 1 day, alternatively at least about 2 days, alternatively at least about 3 days, alternatively at least about 4 days, alternatively at least about 5 days, alternatively at least about 6 days, alternatively at least about 7 days, alternatively at least about 8 days, alternatively at least about 9 days, alternatively at least about 10 days, alternatively at least about 11 days, alternatively at least about 12 days, alternatively at least about 13 days, alternatively at least about 14 days, alternatively at least about 15 days.

[0161] Example Clause G: The method of any one of any one of Example Clauses A-F to 6, where the expanded hematopoietic stem cells are cryopreserved for future use.

[0162] Example Clause H: The method of any one of the preceding claims, where the at least one cytokine is a human recombinant cytokine.

[0163] Example Clause I: The method of any one of the preceding claims, where the hematopoietic stem cells are human cells.

[0164] Example Clause J: The method of any one of the preceding claims, where the hematopoietic stem cells are CD34+ hematopoietic cells.

[0165] Example Clause K: The method of any one of Example Clauses A- J, where the CD34+ hematopoietic cells are from cord blood, mobilized peripheral blood, non-mobilized peripheral blood, or are bone marrow derived.

[0166] Example Clause L: The method of any one of the preceding claims, where the murine cell line are from mouse bone marrow stromal cells.

[0167] Example Clause M: The method of any one of Example Clauses A-L, where theMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCmouse bone marrow stromal cells are selected from the group having of MS-5 and OP9.

[0168] Example Clause N: The method of any one of the preceding claims, where the murine cell line expresses human DLL1.

[0169] Example Clause O: The method of any one of any one of Example Clauses A-N to 14, where the murine cell line is human DLL1 expressing OP9 cells.

[0170] Example Clause P: The method of any one of the preceding claims, where the method is performed in vivo, in vitro, or ex vivo.

[0171] Example Clause Q: The method of any one of Example Clauses A-P, where the method is performed ex vivo or in vitro, where the method further may include a cell culture vessel, where the differentiation of the hematopoietic stem cells occurs within the cell culture vessel.

[0172] Example Clause R: The method of any one of Example Clauses A-Q, where the cell culture vessel is selected from the group having of a cell culture flask, a cell culture plate, a cell culture dish, a cell culture tube, a cell culture insert, a chamber slide, and a chambered cover glass.

[0173] Example Clause S: A method of treating a subject having cancer, the method may include administering the conventional type 1 dendritic cells generated using the method of any one of any one of Example Clauses A-R to 18 or the serum free culture system of any one of any one of Example Clauses A-R to 35 to the subject.

[0174] Example Clause T: The method of any one of Example Clauses A-S or of any one of Example Clauses A-S, where the cancer is selected from the group having of genitourinary cancer, prostate cancer, myeloma, head and neck cancer, high-positive MSI cancer, and metastatic cancer.

[0175] Example Clause U: A method of performing cellular immunotherapy in a subject having cancer, the method may include administering the conventional type 1 dendritic cells generated using the method of any one of any one of Example Clauses A-T to 18 or the serum free culture system of any one of any one of Example Clauses A-T to 35 to the subject.

[0176] Example Clause V: A cellular immunotherapy composition may include the conventional type 1 dendritic cells generated using the method of any one of any one of Example Clauses A-U to 18 or the serum free culture system of any one of any one of Example Clauses A-U to 35.MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PC

[0177] Example Clause W: The cellular immunotherapy composition of any one of Example Clauses A-V, where the cellular immunotherapy composition is used to treat or prevent cancer.

[0178] Example Clause X: The cellular immunotherapy composition of any one of Example Clauses A-W, where the cancer is selected from the group having of genitourinary cancer, prostate cancer, myeloma, head and neck cancer, high-positive MSI cancer, and metastatic cancer.

[0179] Example Clause Y : The cellular immunotherapy composition of any one of any one of Example Clauses A-X to 41, where the cellular immunotherapy composition is formulated as a vaccine.

[0180] Example Clause Z: The cellular immunotherapy of any one of Example Clauses A-Y, where the conventional type 1 dendritic cells are adjuvants.

[0181] Example Clause AA: The cellular immunotherapy of any one of Example Clauses A-Z or of any one of Example Clauses A-Z, where the vaccine is administered intramuscularly, subcutaneously, intranodally, intratumorally, or intravenously.

[0182] Example Clause AB : A serum free culture system for generating cells, the serum free culture system may include: hematopoietic stem cells, a feeder layer, where the feeder layer may include at least one murine cell line expressing human notch ligands and at least one cytokine, where the at least one cytokine is selected from the group having of Fms-like tyrosine kinase receptor 3 ligand (Flt3L), Stem cell factor (SCF), Thrombopoietin (TPO), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin-4 (IL-4), and Interleukin- 15 (IL- 15) and where the hematopoietic stem cells are differentiated into conventional type 1 dendritic cells in a serum free media.

[0183] Example Clause AC: The serum free culture system of Example Clause AB, where the hematopoietic stem cells are differentiated on the feeder layer.

[0184] Example Clause AD: The serum free culture system of Example Clause AB or Example Clause AC or of Example Clause AB or Example Clause AC, where the hematopoietic stem cells are differentiated with a combination of Flt3L, SCF, GM-CSF, IL-4, and / or IL-5.

[0185] Example Clause AE: The serum free culture system of any one of any one of Example Clauses AB-AD to 21, where the hematopoietic stem cells are differentiated in aMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCmedia for at least about 1 day, alternatively at least about 2 days, alternatively at least about 3 days, alternatively at least about 4 days, alternatively at least about 5 days, alternatively at least about 6 days, alternatively at least about 7 days, alternatively at least about 8 days, alternatively at least about 9 days, alternatively at least about 10 days, alternatively at least about 11 days, alternatively at least about 12 days, alternatively at least about 13 days, alternatively at least about 14 days, alternatively at least about 15 days, alternatively at least about 16 days, alternatively at least about 17 days, alternatively at least about 18 days.

[0186] Example Clause AF: The serum free culture system of any one of any one of Example Clauses AB-AE to 22, where the hematopoietic stem cells have been expanded prior to differentiation.

[0187] Example Clause AG: The serum free culture system of any one of Example Clauses AB-AF, where the expanded hematopoietic stem cells are expanded in a media for at least about 1 day, alternatively at least about 2 days, alternatively at least about 3 days, alternatively at least about 4 days, alternatively at least about 5 days, alternatively at least about 6 days, alternatively at least about 7 days, alternatively at least about 8 days, alternatively at least about 9 days, alternatively at least about 10 days, alternatively at least about 11 days, alternatively at least about 12 days, alternatively at least about 13 days, alternatively at least about 14 days, or alternatively at least about 15 days.

[0188] Example Clause AH: The serum free culture system of any one of any one of Example Clauses AB -AG to 24, where the expanded hematopoietic stem cells can be cryopreserved for future use.

[0189] Example Clause Al: The serum free culture system of any one of any one of Example Clauses AB-AH to 25, where the at least one cytokine is a human recombinant cytokine.

[0190] Example Clause AJ: The serum free culture system of any one of any one of Example Clauses AB-AI to 26, where the hematopoietic stem cells are human cells.

[0191] Example Clause AK: The serum free culture system of any one of Example Clauses AB-AJ, where the hematopoietic stem cells are CD34+ hematopoietic cells.

[0192] Example Clause AL: The serum free culture system of any one of Example Clauses AB-AK, where the CD34+ hematopoietic cells are from cord blood, mobilized peripheral blood, non-mobilized peripheral blood, or are bone marrow derived.MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PC

[0193] Example Clause AM: The serum free culture system of any one of any one of Example Clauses AB-AL to 29, where the murine cell line are from mouse bone marrow stromal cells.

[0194] Example Clause AN: The serum free culture system of any one of any one of Example Clauses AB-AM to 30, where the mouse bone marrow stromal cells are selected from the group having of MS-5 and OP9.

[0195] Example Clause AO: The serum free culture system of any one of any one of Example Clauses AB-AN to 31, where the murine cell line expresses human DLL1.

[0196] Example Clause AP: The serum free culture system of any one of Example Clauses AB-AO, where the murine cell line is human DLL1 expressing OP9 cells.

[0197] Example Clause AQ: The serum free culture system of any one of any one of Example Clauses AB-AP to 33, where the system further may include a cell culture vessel, where the differentiation of the hematopoietic stem cells occurs within the cell culture vessel.

[0198] Example Clause AR: The serum free culture system of any one of Example Clauses AB-AQ, where the cell culture vessel is selected from the group having of a cell culture flask, a cell culture plate, a cell culture dish, a cell culture tube, a cell culture insert, a chamber slide, and a chambered cover glass.

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[0200] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCCLAIMS1. A serum free method for generating cells, the method comprising: differentiating hematopoietic stem cells into conventional type 1 dendritic cells, wherein the hematopoietic stem cells are differentiated with a feeder layer comprising at least one murine cell line expressing human notch ligands and at least one cytokine in a serum free media,wherein the at least one cytokine is selected from the group consisting of Fms-like tyrosine kinase receptor 3 ligand (Flt3L), Stem cell factor (SCF), Thrombopoietin (TPO), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin-4 (IL-4), and Interleukin- 15 (IL-15).

2. The method of claim 1, wherein the hematopoietic stem cells are differentiated with a combination of Flt3L, SCF, GM-CSF, IL-4, and / or IL-15.

3. The method of claim 1 or claim 2, wherein the hematopoietic stem cells are differentiated in a media for at least about 1 day, alternatively at least about 2 days, alternatively at least about 3 days, alternatively at least about 4 days, alternatively at least about 5 days, alternatively at least about 6 days, alternatively at least about 7 days, alternatively at least about 8 days, alternatively at least about 9 days, alternatively at least about 10 days, alternatively at least about 11 days, alternatively at least about 12 days, alternatively at least about 13 days, alternatively at least about 14 days, alternatively at least about 15 days, alternatively at least about 16 days, alternatively at least about 17 days, alternatively at least about 18 days.

4. The method of any one of the preceding claims, wherein the hematopoietic stem cells have been expanded prior to differentiation.

5. The method of any one of the preceding claims, wherein the hematopoietic stem cellsMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PChave been expanded with a mouse stromal cell feeder layer prior to differentiation.

6. The method of claim 4 or claim 5, wherein the expanded hematopoietic stem cells are expanded in a media for at least about 1 day, alternatively at least about 2 days, alternatively at least about 3 days, alternatively at least about 4 days, alternatively at least about 5 days, alternatively at least about 6 days, alternatively at least about 7 days, alternatively at least about 8 days, alternatively at least about 9 days, alternatively at least about 10 days, alternatively at least about 11 days, alternatively at least about 12 days, alternatively at least about 13 days, alternatively at least about 14 days, alternatively at least about 15 days.

7. The method of any one of claims 4 to 6, wherein the expanded hematopoietic stem cells are cryopreserved for future use.

8. The method of any one of the preceding claims, wherein the at least one cytokine is a human recombinant cytokine.

9. The method of any one of the preceding claims, where the hematopoietic stem cells are human cells.

10. The method of any one of the preceding claims, wherein the hematopoietic stem cells are CD34+hematopoietic cells.

11. The method of claim 10, wherein the CD34+hematopoietic cells are from cord blood, mobilized peripheral blood, non-mobilized peripheral blood, or are bone marrow derived.

12. The method of any one of the preceding claims, wherein the murine cell line are from mouse bone marrow stromal cells.MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PC13. The method of claim 12, wherein the mouse bone marrow stromal cells are selected from the group consisting of MS-5 and OP9.

14. The method of any one of the preceding claims, wherein the murine cell line expresses human DLL1.

15. The method of any one of claims 12 to 14, wherein the murine cell line is human DLL1 expressing OP9 cells.

16. The method of any one of the preceding claims, wherein the method is performed in vivo, in vitro, or ex vivo.

17. The method of claim 16, wherein the method is performed ex vivo or in vitro, wherein the method further comprises a cell culture vessel, wherein the differentiation of the hematopoietic stem cells occurs within the cell culture vessel.

18. The method of claim 17, wherein the cell culture vessel is selected from the group consisting of a cell culture flask, a cell culture plate, a cell culture dish, a cell culture tube, a cell culture insert, a chamber slide, and a chambered cover glass.

19. A serum free culture system for generating cells, the serum free culture system comprising:hematopoietic stem cells,a feeder layer, wherein the feeder layer comprises at least one murine cell line expressing human notch ligands and at least one cytokine,wherein the at least one cytokine is selected from the group consisting of Fms-like tyrosine kinase receptor 3 ligand (Flt3L), Stem cell factor (SCF), Thrombopoietin (TPO), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin-4 (IL-4), andMSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCInterleukin- 15 (IL- 15) andwherein the hematopoietic stem cells are differentiated into conventional type 1 dendritic cells in a serum free media.

20. The serum free culture system of claim 19, wherein the hematopoietic stem cells are differentiated on the feeder layer.

21. The serum free culture system of claim 19 or claim 20, wherein the hematopoietic stem cells are differentiated with a combination of Flt3L, SCF, GM-CSF, IL-4, and / or IL-5.

22. The serum free culture system of any one of claims 19 to 21, wherein the hematopoietic stem cells are differentiated in a media for at least about 1 day, alternatively at least about 2 days, alternatively at least about 3 days, alternatively at least about 4 days, alternatively at least about 5 days, alternatively at least about 6 days, alternatively at least about 7 days, alternatively at least about 8 days, alternatively at least about 9 days, alternatively at least about 10 days, alternatively at least about 11 days, alternatively at least about 12 days, alternatively at least about 13 days, alternatively at least about 14 days, alternatively at least about 15 days, alternatively at least about 16 days, alternatively at least about 17 days, alternatively at least about 18 days.

23. The serum free culture system of any one of claims 19 to 22, wherein the hematopoietic stem cells have been expanded prior to differentiation.

24. The serum free culture system of claim 23, wherein the expanded hematopoietic stem cells are expanded in a media for at least about 1 day, alternatively at least about 2 days, alternatively at least about 3 days, alternatively at least about 4 days, alternatively at least about 5 days, alternatively at least about 6 days, alternatively at least about 7 days, alternatively at least about 8 days, alternatively at least about 9 days, alternatively at least about 10 days, alternatively at least about 11 days, alternatively at least about 12 days,MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PCalternatively at least about 13 days, alternatively at least about 14 days, or alternatively at least about 15 days.

25. The serum free culture system of any one of claims 19 to 24, wherein the expanded hematopoietic stem cells can be cryopreserved for future use.

26. The serum free culture system of any one of claims 19 to 25, wherein the at least one cytokine is a human recombinant cytokine.

27. The serum free culture system of any one of claims 19 to 26, where the hematopoietic stem cells are human cells.

28. The serum free culture system of claim 27, wherein the hematopoietic stem cells are CD34+hematopoietic cells.

29. The serum free culture system of claim 28, wherein the CD34+hematopoietic cells are from cord blood, mobilized peripheral blood, non-mobilized peripheral blood, or are bone marrow derived.

30. The serum free culture system of any one of claims 19 to 29, wherein the murine cell line are from mouse bone marrow stromal cells.

31. The serum free culture system of any one of claims 19 to 30, wherein the mouse bone marrow stromal cells are selected from the group consisting of MS-5 and OP9.

32. The serum free culture system of any one of claims 19 to 31 , wherein the murine cell line expresses human DLL1.MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PC33. The serum free culture system of claim 32, wherein the murine cell line is human DLL1 expressing 0P9 cells.

34. The serum free culture system of any one of claims 19 to 33, wherein the system further comprises a cell culture vessel, wherein the differentiation of the hematopoietic stem cells occurs within the cell culture vessel.

35. The serum free culture system of claim 34, wherein the cell culture vessel is selected from the group consisting of a cell culture flask, a cell culture plate, a cell culture dish, a cell culture tube, a cell culture insert, a chamber slide, and a chambered cover glass.

36. A method of treating a subject having cancer, the method comprising administering the conventional type 1 dendritic cells generated using the method of any one of claims 1 to 18 or the serum free culture system of any one of claims 19 to 35 to the subject.

37. A method of performing cellular immunotherapy in a subject having cancer, the method comprising administering the conventional type 1 dendritic cells generated using the method of any one of claims 1 to 18 or the serum free culture system of any one of claims 19 to 35 to the subject.

38. The method of claim 36 or claim 37, wherein the cancer is selected from the group consisting of genitourinary cancer, prostate cancer, myeloma, head and neck cancer, highpositive MSI cancer, and metastatic cancer.

39. A cellular immunotherapy composition comprising the conventional type 1 dendritic cells generated using the method of any one of claims 1 to 18 or the serum free culture system of any one of claims 19 to 35.MSIP ref no: 240704-WOLathrop ref no: 773717:MTST-003PC40. The cellular immunotherapy composition of claim 39, wherein the cellular immunotherapy composition is used to treat or prevent cancer.

41. The cellular immunotherapy composition of claim 40, wherein the cancer is selected from the group consisting of genitourinary cancer, prostate cancer, myeloma, head and neck cancer, high-positive MSI cancer, and metastatic cancer.

42. The cellular immunotherapy composition of any one of claims 39 to 41, wherein the cellular immunotherapy composition is formulated as a vaccine.

43. The cellular immunotherapy of claim 42, wherein the conventional type 1 dendritic cells are adjuvants.

44. The cellular immunotherapy of claim 42 or claim 43, wherein the vaccine is administered intramuscularly, subcutaneously, intranodally, intratumorally, or intravenously.

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