WO2025076176A2

WO2025076176A2 - Cross-presenting dendritic cells

Info

Publication number: WO2025076176A2
Application number: PCT/US2024/049733
Authority: WO
Inventors: Yu Chun; Anna Karolina Palucka; Richard Barrett; Ananya Zutshi
Original assignee: Guardian Bio; Jackson Laboratory
Current assignee: Guardian Bio; Jackson Laboratory
Priority date: 2023-10-04
Filing date: 2024-10-03
Publication date: 2025-04-10
Anticipated expiration: 2026-04-04
Also published as: WO2025076176A3

Abstract

The disclosure relates to methods of producing dendritic cells and compositions comprising dendritic cells.

Description

CROSS-PRESENTING DENDRITIC CELLS

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/587,883, filed October 4, 2023, which is incorporated by reference herein in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (J022770144WO00-SEQ-HJD.xml; Size: 4,507 bytes; and Date of Creation: October 3, 2024) is herein incorporated by reference in its entirety.

BACKGROUND

The immune system maintains human health by balancing immunogenic and tolerogenic responses to ensure healthy aspects of the human body are not attacked. Various diseases, including cancer, chronic infectious, and autoimmune diseases overcome this delicate immune balance to develop, persist, and threaten the health. For example, cancer can overcome this balance by suppressing proper dendritic cells (DCs) function. Dendritic cells are specialized immune cells that detect and internalize pathogens, tumor cells, and other antigens. Once internalized, these antigens are processed into smaller peptides and presented on the cell surface using MHC (Major Histocompatibility Complex) class I molecules, a process referred to as cross-presentation. By presenting antigens on MHC class I molecules, cross-presenting DCs can activate CD8⁺ T cells. These T cells, once activated, become cytotoxic T lymphocytes that can target and destroy infected or malignant cells. By impeding differentiation, maturation, and antigen uptake of DCs, cancer prevents these cells from initiating and coordinating an immune response against a tumor.

Ex vivo generated DCs have been a long sought after therapeutic method for circumventing this dysfunction. Dendritic cells can be harvested from a patient (or derived ex vivo from precursor cells from a patient), loaded with tumor antigens in a laboratory, and then re-infused into the patient to induce a targeted immune response against a tumor, for example. Cross-presenting DC vaccines have shown potential in various cancer types. They aim to harness the body's immune response to recognize and destroy tumor cells. While most applications have been in oncology, there is also potential to use this technology in infectious diseases by loading DCs with pathogen-derived antigens. Many attempts at such DC therapies have failed, however, due to a lack of understanding about DC biology and the roles of different DC subtypes. Often, methods used to differentiate these cells from stem cells or other immune cells (such as monocytes) ex vivo have failed to elicit proper immune responses.

SUMMARY

The present disclosure provides, in some aspects, a platform for the ex vivo production of dendritic cells, referred to herein as “Guard DCs,” that have increased therapeutic potential, relative to native, conventional DCs observed in humans. One key recent advancement in the field of DC biology is the identification of the DC subtypes responsible for driving anti-tumor immunity - the conventional dendritic cells (eDCs) type 1 and 2 (cDCl and cDC2). Unfortunately, the difficulty of producing these subtypes of cells ex vivo has hampered their translation into the therapeutic setting. Academically, protocols have been developed using feeder layers of mouse cells that help facilitate the differentiation of hematopoietic stem cells (HSCs) to these eDC subtypes. But these feeder layers, among other components in the protocols, are not clinically compliant and replacing their influence with clinically compliant reagents has been elusive.

The Guard DCs produced by the methods of the disclosure include a mixed population of dendritic cell subtypes derived from CD34+ hematopoietic progenitor cells and are functionally distinct from native DCs. For example, the Guard DCs display classical biomarkers for cDCl and cDC2 subtypes, yet all Guard DC subtypes exhibit a cDCl-like functionality (e.g., including CD141⁺/IRF8⁺ cells). Additionally, the Guard DCs produced by the methods of the disclosure secrete supraphysiological levels of IL- 12 (for example, orders of magnitude higher than native blood DCs, e.g., 5-100 times higher). The Guard DCs provided herein also continue to take up antigen after maturation/activation, exhibit morphological differences such as increased size (e.g., ~85pm), relative to native DCs (e.g., 70pm), and the Guard DC subtypes exhibit an uncharacteristic overlap in function. For example, both subtypes can efficiently stimulate CD8+ T cells in an antigen-specific manner (a function conventionally attributed to only cDCl subtypes). Also, Guard DCs with a primarily cDC2/CDlc+ phenotype are capable of high IL-12 secretion and crosspresentation. Finally, many of the T cells derived from the Guard DCs are polyfunctional, meaning they can perform multiple functions at the same time, which is rare and important in cancer immunity. For example, specific CD8+ T cells produce multiple cytokines (e.g., IFNy and TNFa) and have the ability to release lytic granules (e.g., CD107a exocytosis) at the same time to kill tumor cells. Thus, the platform technology provided herein, in some aspects, is used to produce dendritic cell subtypes with improved functionality from HSCs (e.g., including several different sources of HSCs) using clinically compliant methods, thus enabling the efficient production of effective dendritic cell therapies for patients.

The Guard DC subtypes of the disclosure express classical DC markers (e.g., HLA- DR, CD141, CDlc, CDl lc, CD40, CD80, CD86 and CLEC9A) but lack monocyte markers (e.g., CD 14 and CD64) and secret high level of IL- 12 p70 in response to poly I:C stimulation, for example. Guard DCs also demonstrate the capacity for cross-presentation of tumor antigens and expansion of cytotoxicity CD8⁺ T cells.

Unexpectedly, the data provided herein demonstrates that inflammatory type II interferon (IFNy) cooperatively promotes human cDCl-like and inflammatory cDC2-like differentiation, even in the presence of opposing signals, such as anti-inflammatory IL-4. This was unexpected, in part, because IFNy, while previously used to active/mature dendritic cells (following expansion and differentiation), has not been shown to promote differentiation of dendritic cells. Yet, when combined with IL-4, in some embodiments, IFNy drives HPCs toward the dendritic cell fate, particularly a cDCl-like (e.g., CD141+, CLEC9A+, or IRF8⁺) fate.

Also surprising were the data showing that the addition of StemRegeninl to the expansion phase not only contributed to a higher yield of progenitor cells, but the reagent also contributed to a higher yield of progenitor cells that later differentiate into a Guard DC subtype exhibiting certain characteristics of cDCl cells (i.e., cDCl-like cells).

Thus, some aspects related to a method of producing DCs, the method comprising: (a) expanding a population of human CD34⁺ hematopoietic progenitor cells (HPCs) in a first culture medium; and (b) differentiating the human CD34⁺ HPCs in a second culture medium comprising IFNy or a functional analog thereof, thereby producing a population of differentiated cells comprising DCs, wherein the DCs comprise CD141⁺ or CLEC9A⁺ cells and CDlc⁺/CLEC9A" cells. In some embodiments, the second culture medium comprises IFNy. In some embodiments, the second culture medium further comprises interleukin-4 (IL- 4) or a functional analog thereof. In some embodiments, the second culture medium further comprises IL-4.

In some embodiments, step (a) comprises culturing a starting population of human CD34⁺ HPCs in a first culture medium to produce an expanded population of human CD34⁺ HPCs; and step (b) comprises culturing the expanded population of human CD34⁺ HPCs in the second culture medium to produce the population of DCs.

In some embodiments, the method further comprises activating the DCs. In some embodiments, the activating comprises exposing the DCs to activation molecules, optionally Toll-like receptor agonist molecules and/or cytokines.

In some embodiments, the first culture medium is serum-free medium. In some embodiments, the first culture medium comprises a cytokine selected from Fms-related tyrosine kinase 3 ligand (Flt3L), stem cell factor (SCF), interleukin-3 (IL-3), thrombopoietin (TPO), and StemRegininl (SRI). For example, the first culture medium may comprise Flt3L, SCF, IL-3, TPO, and SRI. In some embodiments, the first culture medium comprises about 30-300 ng/ml Flt3L, about 80-120 ng/ml SCF, about 16-24 ng/ml IL-3, about 40-60 ng/ml TPO, and about 345-515 ng/ml SRI. For example, the first culture medium may comprise about 100 ng/ml Flt3L, about 100 ng/ml SCF, about 20 ng/ml IL-3, about 50 ng/ml TPO, and about 430 ng/ml SRI. In some embodiments, the ratio of Flt3L:SCF:IL-3:TPO:SRl in the first culture medium is about 5:5:1:2.5:21.5.

In some embodiments, the second culture medium further comprises Flt3L, SCF, and granulocyte-macrophage colony- stimulating factor (GM-CSF). In some embodiments, the second culture medium comprises about 80-120 ng/ml Flt3L, about 16-24 ng/ml SCF, about 2-20 ng/ml GM-CSF, about 2-20 ng/ml IL-4, and about 0.8-8 ng/ml IFNy. For example, the second culture medium may comprise about 100 ng/ml Flt3L, about 20 ng/ml SCF, about 2.5 ng/ml GM-CSF, about 2.5 ng/ml IL-4, and about 1 ng/ml IFNy. In some embodiments, the ratio of Flt3L:SCF:GM-SCF:IL-4:IFNyin the second culture medium is about 100:20:2.5:2.5:1. In some embodiments, the second culture medium further comprises human serum, for example, at a concentration of about 2%. In some embodiments, the second culture medium further comprises serum, and the serum consists of human serum, for example, at a concentration of about 2%. In some embodiments, the second culture medium does not comprise non-human serum (e.g., fetal bovine serum). In some embodiments, the second culture medium does not comprise any serum at all.

In some embodiments, the human CD34⁺ HPCs are from umbilical cord blood, bone marrow, or peripheral blood (e.g., mobilized peripheral blood) of a subject. In some embodiments, the method further comprises obtaining the human CD34⁺ HPCs from umbilical cord blood, bone marrow, or peripheral blood of a subject. In some embodiments, the human CD34⁺ HPCs are adult human CD34⁺ HPCs, optionally wherein the adult CD34⁺ HPCs comprises CD34⁺CD38⁺ and CD34⁺CD38’ HPCs.

In some embodiments, the starting population of human CD34⁺ HPCs comprises about 100,000 to about 2 million human CD34⁺ HPCs. In some embodiments, the starting population of human CD34⁺ HPCs comprises about 100,000 to about 4 million human CD34⁺ HPCs.

In some embodiments, the expanded population of human CD34⁺ HPCs comprises about 10 million to about 100 million cells. In some embodiments, the expanded population of human CD34⁺ HPCs comprises about 10 million to about 200 million cells. In some embodiments, the expanded population of human CD34⁺ HPCs comprises about 25-fold to about 500-fold more CD34⁺ HPCs than the starting population of CD34⁺ HPCs. For example, the expanded population of human CD34⁺ HPCs may comprise about 50-fold to about 200- fold more CD34⁺ HPCs than the starting population of CD34⁺ HPCs.

In some embodiments, the culturing of step (a) is for about 5 to 12 days, optionally about 6 to 8 days. In some embodiments, the culturing of step (a) is for about 7 days. In some embodiments, the culturing of step (b) is for at least 5 days. For example, the culturing of step (b) may be for about 5 to 10 days, optionally about 6 to 8 days. In some embodiments, the culturing of step (b) is for about 7 days.

In some embodiments, the culturing of step (a) and/or step (b) is in an incubator with 5% CO₂ at 37 °C.

In some embodiments, at least 20%, at least 30%, or at least 40% of the population of differentiated cells are DCs. In some embodiments, about 30% to about 60% of the population of differentiated cells are DCs.

In some embodiments, the DCs secrete IL- 12 p70 in response to poly I:C stimulation.

In some embodiments, the DCs have the capacity to cross-present tumor antigen to CD8⁺ T cells.

In some embodiments, the population of differentiated cells comprises about 5%-30% CLEC9A⁺ DCs and/or about 70%-99% CDlc⁺ DCs. In some embodiments, the population of differentiated cells comprises about l%-30% CLEC9A⁺ DCs and/or about 70%-99% CDlc⁺ DCs.

In some embodiments, DCs of the differentiated population do not express detectable

CD 14 and/or CD64. Other aspects relate to a method of producing a dendritic cell vaccine, comprising pulsing the DCs of any one of the preceding paragraphs with antigenic material, optionally a tumor- specific peptide, tumor lysate, or a tumor-derived RNA, to produce loaded DCs; and exposing the loaded DCs to activating molecules, thereby producing activated dendritic cells.

In some embodiments, the activating molecules are selected from Toll-like receptor agonists, RIG-I-like receptor agonists, and cGAS-STING DNA sensing pathway agonists, optionally selected from Poly I:C, R848, LPS, 2'3'-Cyclic GMP-AMP (cGAMP), and defective interfering (DI) RNA.

Some aspects relate to a composition comprising a population of DCs produced by the process of any one of the preceding paragraphs.

Other aspects relate to a method of producing DCs, the method comprising: culturing a population of human CD34⁺ HPCs in a culture medium comprising IL-4 and IFNy to produce a differentiated population of human cells comprising DCs, wherein the DCs comprise CLEC9A⁺ cells and CD lc⁺/CLEC9A- cells.

Further aspects relate to a method of producing DCs, the method comprising: (a) culturing a population of adult human CD34⁺ HPCs in a first culture medium comprising Flt3L, SCF, IL-3, TPO, and SRI to produce an expanded population of human CD34⁺ HPCs; and (b) culturing the expanded population of adult human CD34⁺ HPCs in a second culture medium Flt3L, SCF, GM-CSF, IL-4, and IFNy to produce a differentiated population of human cells comprising DCs, wherein the DCs comprise CLEC9A⁺ cells and CDlc⁺/CLEC9A- cells.

Some aspects relate to a culture medium comprising: Flt3L, SCF, GM-CSF, IL-4, and IFNy. For example, the culture medium may comprise Flt3L (e.g., 80-120 ng/ml Flt3L), SCF (e.g., about 16-24 ng/ml SCF), GM-CSF (e.g., about 2-20 ng/ml GM-CSF), IL-4 (e.g., about 2-20 ng/ml IL-4), and IFNy (e.g., about 0.8-3 ng/ml IFNy). In some embodiments, culture medium further comprises human serum, for example, at a concentration of about 2%. In some embodiments, the culture medium does not comprise non-human serum.

Other aspects relate to a composition comprising: the culture medium of any one of the preceding claims; and human CD34⁺ HPCs and/or DCs. In some embodiments, the human CD34⁺ HPCs are from adult tissue.

In some embodiments, the dendritic cells express: (i) a higher level of CD80 and/or CCR7; and/or (ii) a lower level of PDL1 and IL10R; relative to monocyte-derived dendritic cells, optionally wherein the dendritic cells and monocyte-derived dendritic cells are derived from the same donor.

In some embodiments, the dendritic cells express: (i) a higher level of IL- 12 p70 (e.g., at least 50-fold, at least 60-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85- fold, or at least 90-fold higher); and/or (ii) a lower level of IL- 10 (e.g., at least 5-fold or at least 10-fold lower); relative to monocyte-derived dendritic cells, optionally wherein the dendritic cells and monocyte-derived dendritic cells are derived from the same donor.

In some embodiments, the dendritic cells are capable of expanding a higher number of IFNy- secreting CD8⁺ T cells (e.g., at least 10-fold, at least 15-fold, or at least 20-fold expansion) that are antigen specific to melanoma antigens such as MART-1 or gplOO, relative to monocyte-derived dendritic cells, optionally wherein the dendritic cells and monocyte- derived dendritic cells are derived from the same donor.

In some embodiments, the dendritic cells exhibit greater persistence of an immunogenic phenotype in a tumor microenvironment (e.g., express a higher level of surface expression of HLA-DR), relative to monocyte-derived dendritic cells, optionally wherein the dendritic cells and monocyte-derived dendritic cells are derived from the same donor.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A-1D. Characterization of CD34-derived DCs from mobilized blood cultured with Flt3L, GM-CSF and TNFa. A. CD34+ cells isolated from G-CSF mobilized blood were cultured in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 50 ng/ml GM-CSF and 10 ng/ml TNFa for 9-16 days at 37°C 5% CO2 incubator. FACS plots illustrate the gating strategy for DC subsets on day 13 of culture. B. The kinetics of total cell number expanded from one CD34+ cell. C. The percentage different DC subsets in the culture. D. The cell number of different DC subsets derived from one CD34+ cell.

Figs. 2A-2E. IL-6 inhibits DC differentiation. A. CD34+ cells isolated from cord blood were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3E, 100 ng/ml SCF and 20 ng/ml IE-3 for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3E, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, and 2.5 ng/ml IE-4 for another 7 days at 37°C 5% CO2 incubator. FACS plots illustrate the gating strategy for DC subsets. B. CD34-dervied DCs from CD34+ cells isolated from bone marrow were cultured as in A. C. IL-6 and IL-6R at different concentration (20, 10, 5 ng/mL each) were supplemented in the 7- day differentiation phase. D. Bar plots illustrate the percentage and total number of different DC subsets expanded from one cord blood CD34+ cells. E. Bar plots illustrate the percentage and total number of different DC subsets expanded from one bone marrow CD34+ cells.

Figs. 3A-3B. DLL-1 showed little effects on DC differentiation. A. CD34+ cells isolated from cord blood were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF and 20 ng/ml IL-3 for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, and 2.5 ng/ml IL-4 for another 7 days at 37°C 5% CO2 incubator. DLL-1 at different concentration (10, 5, 1 ng/mL) were supplemented in the 7-day differentiation phase. B. Bar plots illustrate the percentage and total number of different DC subsets expanded from one cord blood CD34+ cells.

Figs. 4A-4C. SRI promotes cDCl-like differentiation. A. CD34+ cells isolated from cord blood were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 20 ng/ml IL-6, and 50 ng/ml TPO for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF. 2.5 ng/ml GM- CSF, and 2.5 ng/ml IL-4 for another 7 days at 37°C 5% CO2 incubator. SRI (1 pM), VPA(1 mM), UM729 (50 nM), or DLL-1 (5 ng/mL) were supplemented in the 7-day differentiation phase. B. Bar plots illustrate the percentage and total number of different DC subsets expanded from one cord blood CD34+ cells. C. Bar plots illustrate the percentage and total number of different DC subsets expanded from one bone marrow CD34+ cells.

Figs. 5A-5D. TPO and SRI consistently improve the output of cDCl-like cells. A. CD34+ cells were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3 for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, and 2.5 ng/ml IL-4 for another 7 days at 37°C 5% CO2 incubator. Different combination of 20 ng/ml IL-6, 50 ng/ml TPO and SRI (1 pM) were supplemented in the 7-day differentiation phase. B. Bar plots illustrate the percentage and total number of different DC subsets expanded from one cord blood CD34+ cells. C-D. Bar plots illustrate the percentage and total number of different DC subsets expanded from two bone marrow CD34+ cells. Figs. 6A-6E. IFNy consistently promotes cDCl-like differentiation. A. CD34+ cells were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, and SRI (1 pM) for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, and 2.5 ng/ml IL-4 for another 7 days at 37°C 5% CO2 incubator. IFNP (10 ng/ml), IFNy (10 ng/ml), TNFa (10 ng/ml), or DLL-1 (10 ng/mL) were supplemented in the 7-day differentiation phase. B-D. Bar plots illustrate the percentage and total number of different DC subsets expanded from CD34+ cells derived from cord blood, bone marrow, and G-CSE mobilized blood. E. Bar plots illustrate the percentage and total number of CLEC9A+ DCs expanded from bone marrow CD34+ cells. n=4 with ratio paired t test.

Figs. 7A-7D. Polyvinyl alcohol inhibit total DC differentiation. A. CD34+ cells were first expanded in polyvinyl alcohol (PVA) culture medium or StemSpan™ medium supplemented with different combination of cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, and SRI (1 pM) for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, 2.5 ng/ml IL-4 and IFNy (2.5 ng/ml) for another 7 days at 37°C 5% CO2 incubator. B. Bar plots illustrate the total cell expansion from CD34+ cells derived from cord blood, bone marrow at day 14. C-D. Bar plots illustrate the percentage and total number of different DC subsets expanded from CD34+ cells derived from cord blood or bone marrow.

Figs. 8A-8B. CD34-derived DC express high level of costimulatory molecules. A.

CD34+ cells were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, and SRI (1 pM) for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM- CSF, 2.5 ng/ml IL-4 and IFNy (1 ng/ml) for another 7 days at 37°C 5% CO2 incubator. Total DCs including cDCl-like cells and cDC2-like cells were enriched by FACS-sorting from cord blood, bone marrow, and G-CSF mobilized blood from melanoma patients at day 14. B. Sorted DCs were then stimulated with DC activation cocktails including 10 pg/ml of poly I:C and/or 200ng/ml of CD40L and/or tumor supernatant. The percentage of DCs expressed costimulatory molecules were evaluated after overnight stimulation by FACS. Figs. 9A-9D. CD34-derived DC express high level of IL-12 p70. A-B. CD34+ cells were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, and SRI (1 M) for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, 2.5 ng/ml IL-4 with or without IFNy (1 ng/ml) for another 7 days at 37 °C 5% C CO2 incubator. Bar plots illustrate the percentage and total number of different DC subsets expanded from CD34+ cells derived from bone marrow and G-CSF mobilized blood from melanoma patients at day 14. C-D. Total DCs were enriched by FACS-sorting. Sorted DCs were then stimulated with DC activation cocktails including 10 pg/ml of poly I:C, 200 ng/ml of CD40L and/or tumor supernatant for 18 hours. IL-12p70 production were measured in the supernatant by ELISA.

Figs. 10A-10B. DCs capture tumor antigens, process, and present tumor antigens to antigen specific CD8+ T cells. A. CD34+ cells from melanoma patients were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, and SRI (1 pM) for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, 2.5 ng/ml IL-4 with or without IFNy (1 ng/ml) for another 7 days at 37°C 5% CO2 incubator. Total DCs or sorted cDCl-like cells and cDC2-like cells were enriched by FACS-sorting. Sorted DCs were then stimulated with 10 pg/ml poly I:C and loaded with MART-1 long peptides for overnight. Cross-presentation of MART- 1 by DCs were evaluated by the capacity to stimulate MART-1- specific CD8+ T cell lines at 1:10 ratio and IFNy were measured in the culture supernatant by ELISA. B. cDC2-like cells were stimulated with 10 pg/ml poly I:C, and/or lOOng/ml CD40L, and loaded with MART- 1 long peptides overnight in the presence of tumor supernatant. IFNy production in the culture supernatant of cDC2-like cells with MART-l-spefic CD8+ T cell lines.

Figs. 11A-11D. DCs capture tumor antigens, process, and present tumor antigens, and expand antigen-specific CD8+ T cells. A. CD34+ cells from melanoma patients were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, and SRI (1 pM) for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml, GM-CSF, 2.5 ng/ml IL-4 with or without IFNy (1 ng/ml) for another 7 days at 37°C 5% CO2 incubator. Total DCs were enriched by FACS-sorting. Sorted DCs were then stimulated with 10 pg/ml poly I:C in the presence or absence of tumor supernatant and loaded with MART-1 long peptides for overnight. Loaded DCs were then coculture with autologous CD8+ T cells for 12 days in the presence of IL-2. The percentage of MART-1 specific CD8+ T cells were measured by MART-l-loaded HLA-A2-tetramer by FACS. B. MFI of MART-l-tetramer+ cells. C. CD8+ T cells were restimulated with MART-1 9-mer peptides for 5 hours for intracellular cytokine production. The percentage of MART- 1 specific CD8+ T cells with the capacity to secret IFNy were measured by FACS. D. The percentage of muti-functional/polyfunctional MART-1 specific CD8+ T cells were measured by their capacity to express one or multiple effectors including IFNy, TNFa, or CD107a, a surrogate marker for lytic granule release.

Fig. 12. CD8+ T cells expanded by DCs kill tumor cells. A. CD34+ cells from melanoma patients were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, and SRI (1 pM) for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM- CSF, 2.5 ng/ml IL-4, with or without IFNy (1 ng/ml) for another 7 days at 37°C 5% CO2 incubator. Total DCs were enriched by FACS-sorting. Sorted DCs were then stimulated with 10 pg/ml poly I:C in the presence or absence of tumor supernatant and loaded with MART-1 long peptides for overnight. Loaded DCs were then coculture with autologous CD8+ T cells for 12 days in the presence of IL-2. CD8+ T cells were cocultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled melanoma cells (Me275 and Z80-mel) or K562 at different ratio for 4 hours. The specific killing of tumor cells was measured by Pl-staining by FACS.

Fig. 13. Workflow for functional validation of CD34-dervived DCs. CD34+ cells were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, and SRI (1 pM) for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, 2.5 ng/ml IL-4 and IFNy (1 ng/ml) for another 7 days at 37 °C 5% CO2 incubator. Total DCs as well as cDCl-like cells or cDC2-like cells were enriched by FACS-sorting. Sorted DCs were then activated and loaded with tumor antigens. The functionality of DCs were evaluated by the expression of costimulatory molecules, IL- 12 p70 secretion, the capacity to cross-present tumor antigen to specific CD8+ T cells line, and the capacity to expand cancer-specific cytotoxic CD8+ T cells with effector molecular expression.

Figs. 14A-14C. Guard-cDCl demonstrate superior ability to stimulate immunity and generate strong effector CD8+ T cells over moDCs. To generate Guard-cDCl, CD34+ cells were first expanded in StemSpan™ medium supplemented with expansion cytokine mix (100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, and 1 pM SRI ) for 7 days, followed by differentiation in Ex- Vivo 15 media supplemented with 2% human serum and differentiation cytokine mix (100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, 2.5 ng/ml IL-4, 1 ng/ml IFNy) for another 7 days. Total eDCs were enriched by FACS-sorting, followed by activation with 10 pg/mL poly I:C and 200 ng/mL CD40L, and loaded with melanoma peptides for 6 hours before cryopreservation in CRYOSTOR™ CS10 freezing media. To generate monocyte-derived DCs (moDCs), total monocytes were first enriched with DYNABEADS™ Untouched™ Human Monocyte Kit, differentiated in Ex- Vivo 15 media supplemented with 1000 lU/ml GM-CSF and 1000 lU/ml IL-4 for 5 days, followed by activation with maturation cocktail (1000 lU/mL IFNy, 20 ng/mL TNFa, and 200 ng/mL CD40L) for 16 hours and loaded with melanoma peptides for the last 4 hours before cryopreservation. Fig. 14A. Bar plots illustrate immune- stimulating IL-12p70 and immune- suppressive IL- 10 production in the supernatant after cryorecovery and exposed to tumor conditioned medium for 18 Hr. Two independent experiments from one donor. Fig. 14B. DCs were then cocultured with HLA-A2-matched naive CD8+ T cells at 1:100 ratio for 12 days in the presence of IL-2. CD8+ T cells were restimulated with MART-1 9-mer peptides for 5 hours for intracellular cytokine production. Bar plot illustrates the number of MART-1 specific CD8+ T cells with the capacity to secret IFNy by FACS. Fig. 14C. The percentage of poly-functional MART-1 specific CD8+ T cells were measured by their capacity to express one or multiple effector molecules including IFNy, TNFa, or CD107a. Bar plot illustrates the number of MART-1 specific CD8+ T cells with the capacity to secret different number of effectors by FACS.

Fig. 15. Guard-cDCls demonstrate higher CCR7 expression ex vivo compared to moDCs, indicating that Guard-cDCls migrate to lymph nodes, overcoming migration issues of moDCs in vivo. Bar plots illustrate lymph node-homing receptor CCR7 expression on Guard DCs or moDCs after cryorecovery and exposed to tumor conditioned medium for 18 Hr. Two independent experiments from one donor. DETAILED DESCRIPTION

Dendritic cells (DCs) are antigen-presenting cells (APCs) essential for initiating and driving T cell immunity. It is well established that DCs respond to viruses and vaccines through their innate sensors, allowing them to initiate adaptive antiviral or anti-tumor effector T and B cell responses. In humans, DCs include three major subsets with different phenotypes, tissue localizations, and functions. For example, blood and lung CD141+ classical DCs (cDCl) are the most potent in cross-presentation of antigens from dying cells; blood and lung CDlc+ classical DCs (cDC2) drive the differentiation of mucosal effector CD8+ T cells in response to influenza virus; and blood plasmacytoid DCs rapidly produce type I interferons in response to many viruses. Thus, cancer vaccines delivered by DCs offers a promising therapeutic candidate for patients with compromising immunity.

Manufacturing DCs for vaccines, especially in a therapeutic context like cancer immunotherapy, presents several challenges. The process of obtaining, differentiating, modifying, expanding, and formulating DCs for clinical use requires meticulous care, and the complex biology of these cells adds layers of complexity. Some of the primary challenges include the source of progenitor cells from which the DCs are derived; achieving the right maturation stage of DCs (balancing the ability of the cells to capture antigen and induce a robust immune response rather than tolerance), reproducibility, and scaling up the production of DCs while maintaining consistency and quality.

The methods and compositions described herein, in some aspects, are used to expand a mixture of DC subtypes from a variety of CD34⁺ HPC sources ranging from cord blood to adult bone marrow and mobilized blood. The DCs produced by the methods of the disclosure express many classical DC markers (e.g., HLA-DR, CD141, CDlc, CDllc, CD40, CD80, CD86 and CLEC9A), lack monocyte markers (e.g., CD14 and CD64), and secrete a high level of IL- 12 p70 in response to poly I:C stimulation. These DCs also demonstrate the capacity to cross-present tumor antigens and expand cytotoxicity CD8+ T cells.

Thus, methods of producing DCs of the disclosure, in some aspects, comprise: (a) expanding a population of human CD34+ HPCs in a first culture medium; and (b) differentiating the human CD34+ HPCs in a second culture medium comprising interferon gamma (IFNy) or a functional analog thereof, thereby producing a population of differentiated cells comprising DCs, wherein the DCs comprise CD141+ or CLEC9A+ cells and CDlc+/CLEC9A- cells. In some embodiments, the methods comprise culturing a starting population of human CD34+ HPCs in a first culture medium (e.g., an expansion culture medium) to produce an expanded population of human CD34+ HPCs, and culturing the expanded population of human CD34+ HPCs in a second culture medium (e.g., a differentiation medium) to produce the population of DCs.

Some methods of the disclosure comprise culturing a population of human CD34⁺ HPCs in a culture medium comprising IL-4 (e.g., about 2-20 ng/ml IL-4) and IFNy (e.g., about 0.8-3 ng/ml IFNy) to produce a differentiated population of human cells comprising DCs, wherein the DCs comprise CD141⁺ or CLEC9A⁺ cells and CDlc⁺/CLEC9A" cells.

Other methods of the disclosure comprise culturing a population of adult human CD34+ HPCs in a first culture medium comprising Flt3L (e.g., about 30-300 ng/ml Flt3L), SCF (e.g., about 30-300 ng/ml SCF), IL-3 (e.g., about 16-24 ng/ml IL-3), TPO (e.g., about 40-60 ng/ml TPO), and SRI (e.g., about 345-515 ng/ml SRI) to produce an expanded population of human CD34+ HPCs; and culturing the expanded population of adult human CD34+ HPCs in a second culture medium Flt3L (e.g., 80-120 ng/ml Flt3L), SCF (e.g., about 16-24 ng/ml SCF), GM-CSF (e.g., about 2-20 ng/ml GM-CSF), IL-4 (e.g., about 2-20 ng/ml IL-4), and IFNy (e.g., about 0.8-3 ng/ml IFNy) to produce a differentiated population of human cells comprising DCs, wherein the DCs comprise CD141⁺ or CLEC9A+ cells and CDlc+/CLEC9A- cells.

Hematopoietic Progenitor Cell Expansion

A hematopoietic progenitor cell (HPC, also referred to as hematopoietic stem cell — HSC) is a type of stem cell found in the bone marrow and umbilical cord blood that has the capability to differentiate into any of the blood cell types, including white blood cells, red blood cells, and platelets. These cells play a pivotal role in the body's hematopoiesis process, which is the formation of blood cellular components. Hematopoietic progenitor cells can be broadly categorized into two main types based on their differentiation potential: hematopoietic stem cells (HSCs), which have the dual ability to either self-renew (create more HSCs) or differentiate into any blood cell lineage; and multipotent hematopoietic progenitor cells, which can give rise to more than one type of blood cell but are limited in their potential compared to the hematopoietic stem cells. The distinction between these two types is based on their differentiation and self-renewal capabilities. CD34+ HPCs refer to a subset of HPCs that express the CD34 protein on their surface. CD34 is a cell surface marker and is often used as an identifying and isolating factor for hematopoietic progenitor and stem cells from bone marrow, peripheral blood, and umbilical cord blood. The presence of the CD34 marker on the cell surface is used as a primary marker to identify and isolate HPCs. Flow cytometry is a commonly used technique to detect and quantify CD34+ cells in a sample. CD34+ HPCs, in some instances, are mobilized from the bone marrow into the peripheral blood using certain drugs (for example, G-CSF or plerixafor). These drug mobilize more HSCs from the bone marrow and into peripheral blood, increasing the number accessible in peripheral blood.

In some embodiments, a population of HPCs (e.g., CD34+ HPCs) comprises HSCs. In some embodiments, a population of HPCs (e.g., CD34+ HPCs) comprises multipotent HPCs. In some embodiments, a population of HPCs (e.g., CD34+ HPCs) comprises HSCs and multipotent HPCs. HPCs (e.g., CD34+ HPCs), in some embodiments, are adult HPCs; that is, the HPCs are obtained from adult sources, such as adult bone marrow, peripheral blood, or umbilical cord blood/tissue. In some embodiments, adult CD34+ HPCs comprises CD34+CD38+ and CD34+CD38- HPCs. CD38 is a cell surface glycoprotein with multiple functions, including roles in cell adhesion, signal transduction, and calcium signaling. CD38 serves as a marker to distinguish certain populations of hematopoietic progenitor cells. CD38+ hematopoietic progenitor cells represent a specific subset of HPCs. These cells are more mature compared to the primitive (earliest) progenitors which are typically CD38-. Thus, CD34+CD38- cells represents primitive hematopoietic stem/progenitor cells that have high proliferative potential, while CD34+CD38+ cells are more differentiated compared to CD34+CD38- cells and have more limited proliferative potential.

The HPCs used in the methods of the disclosure may be obtained from umbilical cord blood, bone marrow, or peripheral blood (including mobilized peripheral blood) of a human subject, for example. In some embodiments, HPCs are from human umbilical cord blood. In some embodiments, HPCs are from human bone marrow. In some embodiments, HPCs are from human peripheral blood (blood that circulates throughout the body's vascular system, as opposed to blood located in the bone marrow or other organs). Peripheral blood also includes, for example, plasma, red blood cells, white blood cells and platelets. Peripheral blood also includes peripheral blood mononuclear cells and peripheral blood stem cells.

A population of human CD34+ HPCs is considered a “starting population” if it has not been cultured (e.g., expanded). For example, a starting population represents the cells initially plated or inoculated in cell culture medium. The health, confluence, and growth rate of this population will typically influence the results of downstream steps. In some embodiments, a starting population is the total mix of cells prior to any gating or sorting.

By contrast, an expanded population of cells has been cultured and grown to achieve a higher number of cells compared to the starting population of cells. This expansion can occur in a controlled environment, such as a cell culture dish, flask, or bioreactor, and often under specific conditions conducive to cell growth and division. Cell expansion, in some embodiments, involves providing cells with the necessary nutrients, growth factors, cytokines, optimal temperature, and gas exchange conditions to promote their proliferation.

A starting population of human HPCs (e.g., CD34+ HPCs), in some embodiments, comprises about 100,000 to about 2 million human HPCs. In other embodiments, a starting population of human HPCs (e.g., CD34+ HPCs) comprises about 100,000 to about 4 million human HPCs. For example, the starting population may comprise about IxlO⁴ to 2xl0⁶, about IxlO⁴ to IxlO⁶, about IxlO⁴ to 2xl0⁵, about IxlO⁴ to IxlO⁵, about IxlO⁴ to 2xl0⁴, about 2xl0⁴ to 2xl0⁶, about 2xl0⁴ to IxlO⁶, about 2xl0⁴ to 2xl0⁵, about 2xl0⁴ to IxlO⁵, about 3xl0⁴ to 2xl0⁶, about 3xl0⁴ to IxlO⁶, about 3xl0⁴ to 2xl0⁵, about 3xl0⁴ to IxlO⁵, about 4xl0⁴ to 2xl0⁶, about 4xl0⁴ to IxlO⁶, about 4xl0⁴ to 2xl0⁵, about 4xl0⁴ to IxlO⁵, about 5xl0⁴ to 2xl0⁶, about 5xl0⁴ to IxlO⁶, about 5xl0⁴ to 2xl0⁵, about 5xl0⁴ to IxlO⁵, about 6xl0⁴ to 2xl0⁶, about 6xl0⁴ to IxlO⁶, about 6xl0⁴ to 2xl0⁵, about 6xl0⁴ to IxlO⁵, about 7xl0⁴ to 2xl0⁶, about 7xl0⁴ to IxlO⁶, about 7xl0⁴ to 2xl0⁵, about 7xl0⁴ to IxlO⁵, about 8xl0⁴ to 2xl0⁶, about 8xl0⁴ to IxlO⁶, about 8xl0⁴ to 2xl0⁵, about 8xl0⁴ to IxlO⁵, about 9xl0⁴ to 2xl0⁶, about 9xl0⁴ to IxlO⁶, about 9xl0⁴ to 2xl0⁵, or about 9xl0⁴ to IxlO⁵ human HPCs. In some embodiments, the starting population comprises about 100,000, about 500,000, about 1,000,000, about 1,500,000, about 2,000,000, about 2,500,000, about 3,000,000, about 3,500,000, or about 4,000,000 human HPCs.

An expanded population of cells, in some embodiments, comprises about 10 million to about 300 million cells. For example, the expanded population may comprise about IxlO⁷ to IxlO⁸, about 2xl0⁷ to IxlO⁸, about 3xl0⁷ to IxlO⁸, about 4xl0⁷ to IxlO⁸, about 5xl0⁷ to IxlO⁸, about 6xl0⁷ to IxlO⁸, about 7xl0⁷ to IxlO⁸, about 8xl0⁷ to IxlO⁸, or about 9xl0⁷ to IxlO⁸. In some embodiments, an expanded population of cells comprises 25-fold to about 50- fold, about 25-fold to about 100-fold, 25-fold to about 200-fold, or about 25-fold to about 500-fold more cells than the starting population of cells. In some embodiments, an expanded population of cells comprises about 50-fold to about 100-fold, 50-fold to about 200-fold, or about 50-fold to about 500-fold more cells than the starting population of cells. For example, an expanded population of cells may comprise about 25-fold, about 50-fold, about 75-fold, about 100-fold, about 200-fold, about 250-fold, about 300-fold, about 400-fold, or about 500- fold more cells than the starting population of cells.

Expansion Conditions and Culture Medium A first culture media, in some embodiments, is an expansion culture medium. An expansion culture medium is a culture medium that promotes the rapid growth and proliferation of cells, thereby increasing the number of cells. The expansion culture medium of the disclosure comprises a cytokine selected from Fms-related tyrosine kinase 3 ligand (Flt3L), stem cell factor (SCF), interleukin-3 (IL-3), thrombopoietin (TPO), and StemRegininl (SRI). In some embodiments, the expansion culture medium comprises Flt3L. In some embodiments, the expansion culture medium comprises SCF. In some embodiments, the expansion culture medium comprises IL-3. In some embodiments, the expansion culture medium comprises TPO. In some embodiments, the expansion culture medium comprises SRI. In some embodiments, the expansion culture medium comprises SRI and any one or more of Flt3L, SCF, IL-3, and TPO. In some embodiments, the expansion culture medium comprises SRI and any two or more of Flt3L, SCF, IL-3, and TPO. In some embodiments, the expansion culture medium comprises SRI and any three or more of Flt3L, SCF, IL-3, and TPO. In some embodiments, the expansion culture medium comprises Flt3L, SCF, IL-3, TPO and SRI.

Fms-related tyrosine kinase 3 ligand (Flt3L) is a growth factor important in the development and regulation of the immune system. Specifically, Flt3L binds and activates the Fms-related tyrosine kinase 3 (Flt3) receptor, which is found on the surface of various hematopoietic progenitor cells. This interaction plays a pivotal role in hematopoiesis (the process of blood cell formation) and in the generation of DCs. The concentration of Flt3L in an expansion culture medium, in some embodiments, is about 30-300 ng/ml. For example, the concentration of Flt3L in an expansion culture medium may be about 30-250 ng/ml, about 30-200 ng/ml, about 30-150 ng/ml, about 30-100 ng/ml, about 30-50 ng/ml, about 40-300 ng/ml, about 40-250 ng/ml, about 40-200 ng/ml, about 40-150 ng/ml, about 40-100 ng/ml, about 40-50 ng/ml, 50-250 ng/ml, about 50-200 ng/ml, about 50-150 ng/ml, about 50-100 ng/ml, or about 50-50 ng/ml. In some embodiments, the concentration of Flt3L in an expansion culture medium is about 100 ng/ml.

Stem cell factor (SCF), also known as KIT-ligand or c-kit ligand, is a cytokine that plays a fundamental role in hematopoiesis, the process by which blood cells are formed. SCF exerts its biological effects by binding to its receptor, called c-Kit or CD117, which is a receptor tyrosine kinase. The concentration of SCF in an expansion culture medium, in some embodiments, is about 80-120 ng/ml SCF. For example, the concentration of SCF in an expansion culture medium may be about 80-115 ng/ml, about 80-110 ng/ml, about 80-105 ng/ml, about 80-100 ng/ml, about 80-95 ng/ml, about 80-90 ng/ml, about 90-120 ng/ml, about 90-115 ng/ml, about 90-110 ng/ml, about 90-105 ng/ml, about 90-100 ng/ml, about 100-120 ng/ml, about 100-115 ng/ml, or about 100-110 ng/ml. In some embodiments, the concentration of SCF in an expansion culture medium is about 100 ng/ml. For example, the concentration of SCF in an expansion culture medium may be about 80 ng/ml, about 85 ng/ml, about 90 ng/ml, about 95 ng/ml, about 100 ng/ml, about 105 ng/ml, about 110 ng/ml, about 115 ng/ml, or about 120 ng/ml.

Interleukin-3 (IL-3) is a cytokine that plays an important role in the regulation of blood cell production, or hematopoiesis. IL-3 acts on hematopoietic progenitor cells to promote the survival, proliferation, and differentiation of various cell lineages, especially those of the myeloid lineage, including dendritic cells. The concentration of IL-3 in an expansion culture medium, in some embodiments, is about 15-25 ng/ml. For example, the concentration of IL-3 in an expansion culture medium may be about 15-20 ng/ml or about 20- 25 ng/ml. In some embodiments, the concentration of IL-3 in an expansion culture medium is about 20 ng/ml. For example, the concentration of IL-3 in an expansion culture medium may be about 15 ng/ml, about 20 ng/ml, or about 25 ng/ml.

Thrombopoietin (TPO) is a glycoprotein hormone that primarily regulates the production of platelets (thrombocytes) by the bone marrow. It plays a central role in the process of thrombopoiesis, which is the formation of platelets from their precursor cells, megakaryocytes. The concentration of TPO in an expansion culture medium, in some embodiments, is about 40-60 ng/ml. For example, the concentration of TPO in an expansion culture medium may be about 40-55 ng/ml, about 40-50 ng/ml, about 40-45 ng/ml, about 50- 60 ng/ml, or about 50-55 ng/ml. In some embodiments, the concentration of TPO in an expansion culture medium is about 50 ng/ml. For example, the concentration of TPO in an expansion culture medium may be about 40 ng/ml, about 45 ng/ml, about 50 ng/ml, about 55 ng/ml, or about 60 ng/ml.

StemRegenin 1 (SRI) is a small molecule compound that has been identified as a potent and specific antagonist of the aryl hydrocarbon receptor (AhR). SRI enhances the expansion of HSCs in culture. The concentration of SRI in an expansion culture medium, in some embodiments, is about and about 325-525 ng/ml. For example, the concentration of SRI in an expansion culture medium may be about 325-500 ng/ml, about 325-475 ng/ml, about 325-450 ng/ml, about 325-425 ng/ml, about 325-400 ng/ml, about 325-350 ng/ml, about 350-525 ng/ml, about 350-500 ng/ml, about 350-475 ng/ml, about 350-450 ng/ml, about 350-425 ng/ml, about 350-400 ng/ml, about 400-525 ng/ml, about 400-500 ng/ml, about 400-475 ng/ml, or about 400-450 ng/ml. In some embodiments, the concentration of SRI in an expansion culture medium is about 430 ng/ml. For example, the concentration of SRI in an expansion culture medium may be about 325 ng/ml, about 335 ng/ml, about 345 ng/ml, about 355 ng/ml, about 365 ng/ml, about 375 ng/ml, about 385 ng/ml, about 395 ng/ml, about 405 ng/ml, about 415 ng/ml, about 425 ng/ml, about 435 ng/ml, about 445 ng/ml, about 455 ng/ml, about 465 ng/ml, about 475 ng/ml, about 485 ng/ml, about 495 ng/ml, about 505 ng/ml, about 515 ng/ml, or about 525 ng/ml.

In some embodiments, an expansion culture medium comprises about 30-300 ng/ml Flt3L, about 80-120 ng/ml SCF, about 16-24 ng/ml IL-3, about 40-60 ng/ml TPO, and about 345-515 ng/ml SRI. In some embodiments, an expansion culture medium comprises about 100 ng/ml Flt3L, about 100 ng/ml SCF, about 20 ng/ml IL-3, about 50 ng/ml TPO, and about 430 ng/ml SRI.

In some embodiments, the ratio of Flt3L:SCF:IL-3:TPO:SRl in an expansion culture medium is about 5:5:1:2.5:21.5.

An expansion culture medium, in some embodiments, further comprises basal medium, which provides basic nutrients required for cell survival, including, for example, salts, sugars, and vitamins. In some embodiments, an expansion culture medium further comprises growth factors (e.g., proteins and/or hormones) to stimulate cell growth and proliferation. In some embodiments, an expansion culture medium further comprises human serum, which supplies additional growth factors, hormones, and other nutrients. In other embodiments, however, an expansion culture medium is serum-free.

Methods of the disclosure, in some embodiments, comprise culturing a starting population of human HPCs (e.g., CD34+ HPCs) in a (first) expansion culture medium to produce an expanded population of human HPCs. Culturing in the expansion culture medium, in some embodiments, is for about 5 to 12 days. For example, the culturing may be for about 5 to 10 days. In some embodiments, the culturing is for about 6 to 8 days. In some embodiments, the culturing is for about 7 days. For example, the culturing may be for about 5, about 6, about 7, about 8, about 9 or about 10 days. In some embodiments, the culturing is for at least 5 days (e.g., at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days).

In some embodiments, cells are cultured for no more than 15 days in any given method of the disclosure. In some embodiments, cells are cultured for no more than 20 days in any given method of the disclosure.

In some embodiments, cells are cultured in expansion culture medium for no more than 10 days in any given method of the disclosure. In some embodiments, cells are cultured in differentiation culture medium for no more than 10 days in any given method of the disclosure.

Cells, as is typically in the field, may be cultured in an incubator with 5% CO2 at 37 °C; however, other culture conditions may be used.

Dendritic Cell Differentiation

Dendritic cells (DCs) are a type of immune cell that play an important role in the adaptive immune response. They are a subset of white blood cells and are primarily known for their antigen-presenting capabilities. Dendritic cells, with their tree-like extensions (called dendrites), act as a bridge between the innate and adaptive immune systems. They capture and process antigens, and then migrate to lymph nodes where they present these antigens to T cells, thereby initiating an adaptive immune response. Dendritic cells display processed antigen fragments on their surface using molecules called major histocompatibility complex (MHC), which allows T cells to recognize and interact with the antigen. Dendritic cells originate from bone marrow precursors and can be found in various states of maturity throughout the body. Immature DCs are primarily involved in capturing antigens, whereas mature DCs are mainly involved in presenting antigens to T cells. There are various subtypes of dendritic cells with different functions, such as plasmacytoid dendritic cells and myeloid or conventional dendritic cells. Each subtype has distinct roles in the immune response.

The methods of the present disclosure may be used to produce dendritic cells from a population of HPCs, such as CD34+ HPCs.

Differentiation Conditions and Culture Medium

A second culture media, in some embodiments, is a differentiation culture medium. A differentiation culture medium is a growth medium used in cell culture that induces cells to differentiate into a specific cell type or form. Herein, a differentiation culture medium induces HPCs to differentiate into dendritic cells, including a mixed population of dendritic cell subtypes. Differentiation is the process by which unspecialized cells (e.g., HPCs) become specialized cells with specific functions, such as dendritic cells.

The differentiation culture medium of the disclosure comprises interferon gamma (IFNy) or a functional analog thereof. Interferon gamma is a type II interferon and a key cytokine with diverse immune-modulatory functions. It is principally produced by natural killer (NK) cells and CD4+ T-helper 1 (Thl) and CD8+ cytotoxic T cells. Upon activation, these cells release IFNy, which then exerts various effects on a range of immune cells. The concentration of IFNy in a differentiation culture medium, in some embodiments, is about 0.5-5 ng/ml. For example, the concentration of IFNy in a differentiation culture medium may be about 0.5-4.5 ng/ml, about 0.5-4 ng/ml, about 0.5-3.5 ng/ml, about 0.5-3 ng/ml, about 0.5- 2.5 ng/ml, about 0.5-2 ng/ml, about 0.5-1.5 ng/ml, about 0.5-1 ng/ml, about 1-3 ng/ml, about 1-2.5 ng/ml, about 1-2 ng/ml, about 1-1.5 ng/ml, about 1.5-3 ng/ml, about 1.5-2.5 ng/ml, or about 1.5-2 ng/ml. In some embodiments, the concentration of IFNy in an expansion culture medium is about 1 ng/ml. For example, the concentration of IFNy in a differentiation culture medium may be about 0.5 ng/ml, 0.6 ng/ml, 0.7 ng/ml, 0.8 ng/ml, 0.9 ng/ml, 1.0 ng/ml, 1.1 ng/ml, 1.2 ng/ml, 1.3 ng/ml, 1.4 ng/ml, or 1.5 ng/ml.

A functional analog refers to a molecule or compound that can perform the same or similar function as a particular protein but may not necessarily have the same structure or origin. The functional analog can be another protein, a peptide, a small molecule, or a synthetic compound, for example. A functional analog, in some embodiments, is a molecule or compound that can activate or repress signaling in the same biochemical pathway as a particular protein. In some embodiments, a functional analog directly binds to the same receptor or ligand as the particular protein, for example, to activate or repress the receptor or ligand. In other embodiments, a functional analog indirectly activates or represses the same receptor or ligand as the particular protein, for example, by binding to a molecule that signals upstream of the receptor or ligand in the same biochemical pathway. In yet other embodiments, a functional analog, binds to a molecule downstream from the receptor or ligand to which the particular protein binds to directly activate or repress signaling of the downstream molecule. In some embodiments, a functional analog is a protein or peptide analog. A protein or peptide analog may be a modified form of the original protein that retains or improves the desired function. The modification can involve the addition, removal, or substitution of amino acid residues. In some embodiments, a functional analog is a small molecule analog. Many proteins function by binding to specific molecules, such substrates, cofactors, or other proteins. A small molecule can be designed to bind to the same site and either activate or inhibit the protein's function, thereby serve as a functional analog, for example. In some embodiments, a functional analog is a synthetic compound - an entirely new molecule or synthetic polymer that can perform the function of a natural protein. Other functional analogs are contemplated herein. Interleukin-4 (IL-4) is a multifunctional cytokine that plays important roles in regulating immune responses, specifically in the context of T helper cell differentiation, B cell class switching, and the promotion of alternative macrophage activation. IL-4 is mainly produced by activated T cells, basophils, and mast cells. In some embodiments, the differentiation culture medium further comprises IL-4 or a functional analog thereof. The concentration of IL-4 in a differentiation culture medium, in some embodiments, is about 1- 20 ng/ml. For example, the concentration of IL-4 in a differentiation culture medium may be about 1-15 ng/ml, about 1-10 ng/ml, about 1-5 ng/ml, about 2-20 ng/ml, about 2-15 ng/ml, about 2-10 ng/ml, about 2-5 ng/ml, about 5-20 ng/ml, about 5-15 ng/ml, or about 5-10 ng/ml. In some embodiments, the concentration of IL-4 in a differentiation culture medium is about 2.5 ng/ml. For example, the concentration of IL-4 in a differentiation culture medium may be about 1 ng/ml, about 1.5 ng/ml, about 2 ng/ml, about 2.5 ng/ml, about 3 ng/ml, about 3.5 ng/ml, about 4 ng/ml, about 4.5 ng/ml, or about 5 ng/ml.

In some embodiments, the differentiation culture medium further comprises Flt3L, SCF, granulocyte-macrophage colony-stimulating factor (GM-CSF), or any combination of two to three of Flt3L, SCF, and GM-CSF. In some embodiments, the differentiation culture medium further comprises Flt3L, SCF, and GM-CSF. Thus, in some embodiments, a differentiation culture medium comprises IFNy, IL-4, Flt3L, SCF, and GM-CSF.

The concentration of Flt3L in a differentiation culture medium, in some embodiments, is about 80-120 ng/ml. For example, the concentration of Flt3L in a differentiation culture medium may be about 80-110 ng/ml, about 80-100 ng/ml, about 80-90 ng/ml, about 90-120 ng/ml, about 90-110 ng/ml, about 90-100 ng/ml, about 100-120 ng/ml, or about 100-110 ng/ml. In some embodiments, the concentration of Flt3L in a differentiation culture medium is 100 ng/ml. For example, the concentration of Flt3L in a differentiation culture medium may be about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 110 ng/ml, or about 120 ng/ml.

The concentration of SCF in a differentiation culture medium, in some embodiments, is about 15-25 ng/ml. For example, the concentration of SCF in a differentiation culture medium may be about 15-20 ng/ml or 20-25 ng/ml. In some embodiments, the concentration of SCF in a differentiation culture medium is about 20 ng/ml. For example, the concentration of SCF in a differentiation culture medium may be about 15 ng/ml, about 20 ng/ml, or about 25 ng/ml. Granulocyte-macrophage colony- stimulating factor (GM-CSF) is a cytokine that plays a crucial role in the stimulation of the growth and differentiation of hematopoietic progenitor cells. GM-CSF is involved in the production of granulocytes (neutrophils, eosinophils, and basophils) and monocytes, which can further differentiate into macrophages. The concentration of GM-CSF in a differentiation culture medium, in some embodiments, is about 2-20 ng/ml.

In some embodiments, a differentiation culture medium comprises about 80-120 ng/ml Flt3L, about 16-24 ng/ml SCF, about 2-20 ng/ml GM-CSF, about 2-20 ng/ml IL-4, and about 0.8-8 ng/ml IFNy. In some embodiments, a differentiation culture medium comprises about 100 ng/ml Flt3L, about 20 ng/ml SCF, about 2.5 ng/ml GM-CSF, about 2.5 ng/ml IL-4, and about 1 ng/ml IFNy.

In some embodiments, the ratio of Flt3L:SCF:GM-SCF:IL-4:IFNyin a differentiation culture medium is about 100:20:2.5:2.5:1.

A differentiation culture medium, in some embodiments, comprises human serum. The human serum may be present at a concentration of, for example, about l%-5%, about 1- 4%, about l%-3%, about 2%-5%, about 2%-4%, or ab out 2%-3%. In some embodiments, differentiation culture medium comprises human serum at a concentration of about 1%. In some embodiments, differentiation culture medium comprises human serum at a concentration of about 2%. In some embodiments, differentiation culture medium comprises human serum at a concentration of about 3%. In some embodiments, the only serum in the differentiation culture medium is human serum. That is, in some embodiments the differentiation culture medium does not comprise non-human serum, such as fetal bovine serum. In some embodiments, any serum in the differentiation culture medium consists of or consists essentially of human serum.

In some embodiments, a differentiation culture medium further comprises basic nutrients required for cell survival and growth (e.g., amino acids, vitamins, minerals, and/or glucose). In some embodiments, a differentiation culture medium further comprises hormones, growth factors, cytokines, and/or other bioactive compounds that signal the cell to begin the differentiation process.

Methods of the disclosure, in some embodiments, comprise culturing an expanded population of human HPCs (e.g., CD34+ HPCs) in a (second) differentiation culture medium to produce a population of DCs. Culturing in the differentiation culture medium, in some embodiments, is for about 5 to 12 days. For example, the culturing may be for about 5 to 10 days. In some embodiments, the culturing is for about 6 to 8 days. In some embodiments, the culturing is for about 7 days. For example, the culturing may be for about 5, about 6, about 7, about 8, about 9 or about 10 days. In some embodiments, the culturing is for at least 5 days (e.g., at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days).

The methods provided herein are used, in some embodiments, to produce a population of differentiated cells comprising CD141+ cells, CLEC9A+ cells and CDlc+/CLEC9A- cells. CD 141, also known as thrombomodulin, is a glycoprotein that is frequently used as a marker for the cDCl subtype of dendritic cells. CLEC9A (C-type lectin domain family 9 member A) is a type of receptor mainly found on subsets of dendritic cells, for example, plasmacytoid DCs and some myeloid DCs. CLEC9A+ dendritic cells are specialized in capturing and processing dead or dying cells, making them crucial in cross-presentation of antigens. Crosspresentation refers to the process by which dendritic cells capture antigens from tumor cells or virus-infected cells and then present these antigens to CD8+ T cells, which can lead to the activation of a specific immune response against these threats. CDlc, also known as BDCA-1 (Blood Dendritic Cell Antigen 1), is a member of the CD1 family of transmembrane glycoproteins similar to the MHC class I molecules. CDlc is primarily expressed on a subset of dendritic cells, often referred to as CDlc+ dendritic cells (or BDCA-1+ dendritic cells). These cells are found in blood and are a major population of dendritic cells in human peripheral blood. CDlc+ dendritic cells play a pivotal role in the immune system. They are proficient at presenting antigens to T cells, thereby activating adaptive immune responses. Unlike classical MHC molecules, which present peptide antigens, CDlc can present lipid antigens to T cells.

The population of differentiated cells produced by the methods of the disclosure comprise DCs, including a mixture of DC subtypes. In some embodiments, at least 20% of the cells of the population of differentiated cells are DCs. In some embodiments, at least 30% of the cells of the population of differentiated cells are DCs. In some embodiments, at least 40% of the cells of the population of differentiated cells are DCs. In some embodiments, at least 50% of the cells of the population of differentiated cells are DCs. In some embodiments, at least 60% of the cells of the population of differentiated cells are DCs. In some embodiments, about 30% to about 60% of the cells of the population of differentiated cells are DCs. In some embodiments, about 30% to about 80% of the cells of the population of differentiated cells are DCs. For example, about 30% to about 50%, about 30% to about 40%, about 40% to about 60%, or about 40% to about 60% of the cells of the population of differentiated cells are DCs.

In some embodiments, a population of differentiated cells comprises about 0.5%-30% CLEC9A+ DCs. For example, a population of differentiated cells may comprise about 0.5%- 20%, about l%-20%, about 5%-20%, about 5%-10%, about 10%-30%, about 10%-20%, or about 20%-30% CLEC9A+ DCs. In some embodiments, a population of differentiated cells comprises about 5%, about 10%, about 15%, about 20%, about 25%, or about 30% CLEC9A+ DCs. In some embodiments, a population of differentiated cells comprises no more than 50% CLEC9A+ DCs.

In some embodiments, a population of differentiated cells comprises about 70%-99% CDlc+ DCs. For example, a population of differentiated cells may comprise about 70%-98%, about 70%-95%, about 70%-90%, about 70%-85%, or about 70%-80% CDlc+ DCs. In some embodiments, a population of differentiated cells comprises about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% CDlc+ DCs. In some embodiments, a population of differentiated cells comprises no fewer than 50% CDlc+ DCs.

In some embodiments, DCs (e.g., Guard DCs) of the differentiated population do not express detectable CD 14 and/or CD64. CD 14 and CD64 are primarily and preferentially expressed on the cell surface of monocytes and macrophages. In some embodiments, DCs (e.g., Guard DCs) of the differentiated population do not express significant CD 14 and/or CD64. CD 14 and CD64 are primarily and preferentially expressed on the cell surface of monocytes and macrophages.

The differentiated cells produced by the methods of the disclosure exhibit several functionalities characteristic of DCs. In some embodiments, the DCs (e.g., Guard DCs) secrete IL- 12 p70 in response to poly EC stimulation. Interleukin- 12 (IL- 12) is a pro- inflammatory cytokine that plays a pivotal role in linking innate and adaptive immunity. IL- 12 is primarily produced by antigen-presenting cells (APCs) such as dendritic cells, macrophages, and B cells in response to microbial infections. The term "IL- 12 p70" specifically refers to the bioactive heterodimeric form of IL- 12, consisting of two subunits: p35 (IL- 12a) and p40 (IL-12P). These subunits are non-covalently associated, and together they form the active IL- 12 p70 cytokine. Because of its ability to stimulate cytotoxic lymphocytes and drive Thl responses, IL- 12 has been studied for its potential use in cancer immunotherapy. By promoting an anti-tumor immune response, IL- 12 can aid in the clearance of tumor cells.

Polyinosinic:polycytidylic acid (poly I:C) is a synthetic analogue of double- stranded RNA (dsRNA). In the context of biology and immunology, dsRNA is often associated with viral infections, as many viruses produce dsRNA at some point during their replication cycle. Consequently, cells have evolved to recognize dsRNA as a danger signal.

In some embodiments, the DCs have the capacity to cross-present tumor antigen to CD8+ T cells. Cross-presentation is a unique and vital immunological process, especially carried out by DCs, that allows the presentation of extracellular antigens on MHC class I molecules. This is distinct from the conventional pathway where antigens from within the cell (for instance, those derived from intracellular pathogens or tumor cells) are presented on MHC class I molecules. Cross-presentation is important for the initiation of CD8+ T cell responses against pathogens that do not directly infect antigen-presenting cells, as well as for anti-tumor immunity. Not all dendritic cells are equally efficient at cross-presentation. In humans, the BDCA3+ or CD141+ subsets of DCs are especially adept at this process.

Dendritic Cell Activation

Dendritic cells are professional antigen-presenting cells (APCs) that play a pivotal role in the initiation and regulation of immune responses. DCs capture and process antigens and present them to T cells to trigger an immune response. The process of dendritic cell activation involves maturation of the cells. In an immature state, DCs are highly efficient at capturing antigens but are poor at activating T cells. Upon encountering antigens, DCs undergo a maturation process. During maturation, DCs increase the expression of MHC molecules, co- stimulatory molecules (such as CD80 and CD86), and chemokine receptors (such as CCR7) that direct them to lymph nodes.

They also produce pro-inflammatory cytokines and chemokines that help in the recruitment and activation of other immune cells. Once matured, DCs can migrate to the nearest lymph node. In the lymph node, DCs present the processed antigen fragments on their surface via MHC molecules to naive T cells. This presentation can lead to the activation of T cells, initiating an adaptive immune response. A unique feature of some dendritic cells is the ability to cross-present exogenous antigens on MHC class I molecules, which generally present endogenous antigens. This is important for the activation of CD8+ cytotoxic T lymphocytes that can target and kill infected cells. Factors inducing dendritic cell maturation and activation include, for example, pathogen-associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRRs) like Toll-like receptors (TLRs), damage-associated molecular patterns (DAMPs), inflammatory cytokines, and CD40 ligand (CD40L) expressed on T cells. In some embodiments, the methods of the disclosure comprise activating DCs, for example, following expansion and differentiation. Activation, in some embodiments, comprises exposing the DCs to activation molecules, such as Toll-like receptor agonist molecules and/or cytokines. “Exposing” may involve, for example, culturing the DCs in the presence of the activation molecules, for example, for about 30 minutes to about 24 hours.

Non-limiting examples of activating molecules include Toll-like receptor agonists, RIG-I-like receptor agonists, and cGAS-STING DNA sensing pathway agonists. More specific non-limiting examples include Poly I:C, R848, LPS, 2'3'-Cyclic GMP-AMP (cGAMP), and defective interfering (DI) RNA.

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that play a key role in the innate immune system. They recognize distinct pathogen-associated molecular patterns (PAMPs) that are commonly found on pathogens but are distinct from host molecules. TLRs are expressed on various immune cells, including dendritic cells, macrophages, and some non-immune cells. Agonists for TLRs are molecules that bind to and activate these receptors, resulting in downstream signaling that typically leads to the production of pro-inflammatory cytokines and type I interferons. This activation enhances the innate immune response against the invading pathogen and plays a role in shaping the adaptive immune response.

RIG-I-like receptors (RLRs) are a family of cytoplasmic pattern recognition receptors that recognize viral RNA and play a significant role in the antiviral immune response. The main members of the RLR family include RIG-I (Retinoic acid-inducible gene I), MDA5 (Melanoma differentiation-associated protein 5), and LGP2 (Laboratory of genetics and physiology 2). Upon recognizing viral RNA, RLRs initiate signaling cascades that lead to the production of type I interferons and pro-inflammatory cytokines, thereby orchestrating an inflammatory response.

The cGAS-STING (cyclic GMP-AMP synthase - stimulator of interferon genes) pathway is a crucial component of the innate immune system responsible for sensing cytosolic DNA. This could be DNA from pathogens (like bacteria and viruses) or mislocalized self-DNA. When cGAS detects this DNA, it synthesizes a cyclic dinucleotide called cGAMP. This molecule then binds to and activates the endoplasmic reticulum protein STING. Activated STING triggers downstream signaling pathways, leading primarily to the production of type I interferons and other pro-inflammatory cytokines. Non-limiting examples of cGAS-STING pathway agonists include cGAMP, DMXAA (5,6- dimethylxanthenone-4-acetic acid), ADU-S100 (also known as MIW815), and other small molecule STING agonists.

Dendritic Cell Vaccine Production

Dendritic cell vaccines are a form of immunotherapy wherein dendritic cells are harvested, loaded with specific antigens (often tumor antigens), and then introduced into a patient to stimulate an immune response against targeted cells (like tumor cells). A general process for producing dendritic cell vaccines includes, for example: obtaining HPCs from bone marrow, peripheral blood, or umbilical cord tissue (e.g., from the patient to be treated with the vaccine); expansion and differentiation of the HPCs into DCs in accordance with the methods of the disclosure; loading of the DCs with target antigens (e.g., peptides, tumor lysates, tumor RNA, viral vectors, or mRNA transfection); and activation (also referred to in the field as maturation) of the antigen-loaded dendritic cells, for example, by exposing the DCs to one or more molecules that simulate an infection or inflammation, such as TLR ligands and/or cytokines (e.g., TNF-a, IL-ip, etc.). Following production of the DC vaccine, it is introduced into the patient, for example, by intradermal or subcutaneous injection, though other routes (e.g., intra nodal) are contemplated.

Additional Embodiments

Additional embodiments of the disclosure are encompassed by the numbered paragraphs below.

1. A method of producing dendritic cells, the method comprising:

(a) expanding a population of human CD34⁺ hematopoietic progenitor cells in a first culture medium; and

(b) differentiating the human CD34⁺ hematopoietic progenitor cells in a second culture medium comprising interferon gamma (IFNy) or a functional analog thereof, thereby producing a population of differentiated cells comprising dendritic cells, wherein the dendritic cells comprise CLEC9A⁺ cells and CD lc⁺/CLEC9A" cells.

2. The method of paragraph 1, wherein the second culture medium comprises IFNy.

3. The method of paragraph 1 or 2, wherein the second culture medium further comprises interleukin-4 (IL-4) or a functional analog thereof.

4. The method of paragraph 3, wherein the second culture medium further comprises IL-

4.

5. The method of any one of paragraphs 1-4, wherein (a) comprises culturing a starting population of human CD34⁺ hematopoietic progenitor cells in a first culture medium to produce an expanded population of human CD34⁺ hematopoietic progenitor cells; and (b) comprises culturing the expanded population of human CD34⁺ hematopoietic progenitor cells in the second culture medium to produce the population of differentiated dendritic cells.

6. The method of any one of paragraphs 1-5 further comprising activating the dendritic cells.

7. The method of paragraph 6, wherein the activating comprises exposing the dendritic cells to activation molecules, optionally Toll-like receptor agonist molecules and/or cytokines.

8. The method of any one of the preceding paragraphs, wherein the first culture medium is serum-free medium.

9. The method of any one of the preceding paragraphs, wherein the first culture medium comprises a cytokine selected from Fms-related tyrosine kinase 3 ligand (Flt3L), stem cell factor (SCF), interleukin-3 (IL-3), thrombopoietin (TPO), and StemRegininl (SRI).

10. The method of paragraph 9, wherein the first culture medium comprises Flt3L, SCF, IL-3, TPO, and SRI.

11. The method of paragraph 10, wherein the first culture medium comprises about SO- SOO ng/ml Flt3L, about 80-120 ng/ml SCF, about 16-24 ng/ml IL-3, about 40-60 ng/ml TPO, and about 345-515 ng/ml SRI.

12. The method of paragraph 11, wherein the first culture medium comprises about 100 ng/ml Flt3L, about 100 ng/ml SCF, about 20 ng/ml IL-3, about 50 ng/ml TPO, and about 430 ng/ml SRI.

13. The method of paragraph 10, wherein the ratio of Flt3L:SCF:IL-3:TPO:SRl in the first culture medium is about 5:5:1:2.5:21.5. 14. The method of any one of the preceding paragraphs, wherein the second culture medium further comprises Flt3L, SCF, and granulocyte-macrophage colony- stimulating factor (GM-CSF).

15. The method of paragraph 14, wherein the second culture medium comprises about 80- 120 ng/ml Flt3L, about 16-24 ng/ml SCF, about 2-20 ng/ml GM-CSF, about 2-20 ng/ml IL-4, and about 0.8-8 ng/ml IFNy.

16. The method of paragraph 15, wherein the second culture medium comprises about 100 ng/ml Flt3L, about 20 ng/ml SCF, about 2.5 ng/ml GM-CSF, about 2.5 ng/ml IL-4, and about 1 ng/ml IFNy.

17. The method of paragraph 16, wherein the ratio of Flt3L:SCF:GM-SCF:IL-4:IFNy in the second culture medium is about 100:20:2.5:2.5:1.

18. The method of any one of the preceding paragraphs, wherein the second culture medium further comprises human serum, optionally at a concentration of about 2%.

19. The method of any one of the preceding paragraphs, wherein the second culture medium further comprises serum, and the serum consists of human serum, optionally at a concentration of about 2%.

20. The method of any one of the preceding paragraphs, wherein the second culture medium does not comprise non-human serum.

21. The method of any one of the preceding paragraphs, wherein the human CD34⁺ hematopoietic progenitor cells are from umbilical cord blood, bone marrow, or peripheral blood of a subject.

22. The method of any one of the preceding paragraphs further comprising obtaining the human CD34⁺ hematopoietic progenitor cells from umbilical cord blood, bone marrow, or peripheral blood of a subject.

23. The method of any one of the preceding paragraphs, wherein the human CD34⁺ hematopoietic progenitor cells are adult human CD34⁺ hematopoietic progenitor cells, optionally wherein the adult CD34⁺ hematopoietic progenitor cells comprise CD34⁺CD38⁺ and CD34⁺CD38‘ hematopoietic progenitor cells.

24. The method of any one of the preceding paragraphs, wherein the starting population of human CD34⁺ hematopoietic progenitor cells comprises about 100,000 to about 2 million human CD34⁺ hematopoietic progenitor cells. 25. The method of any one of paragraphs 5-24, wherein the expanded population of human CD34⁺ hematopoietic progenitor cells comprises about 10 million to about 100 million cells.

26. The method of any one of paragraphs 5-24, wherein the expanded population of human CD34⁺ hematopoietic progenitor cells comprises about 25-fold to about 500-fold more CD34⁺ hematopoietic progenitor cells than the starting population of CD34⁺ hematopoietic progenitor cells.

27. The method of paragraph 26, wherein the expanded population of human CD34⁺ hematopoietic progenitor cells comprises about 50-fold to about 200-fold more CD34⁺ hematopoietic progenitor cells than the starting population of CD34⁺ hematopoietic progenitor cells.

28. The method of any one of the preceding paragraphs, wherein the culturing of (a) is for about 5 to 12 days, optionally about 6 to 8 days, preferably about 7 days.

29. The method of any one of the preceding paragraphs, wherein the culturing of (b) is for at least 5 days.

30. The method of paragraph 26, wherein the culturing of (b) is for about 5 to 10 days, optionally about 6 to 8 days, preferably about 7 days.

31. The method of any one of the preceding paragraphs, wherein the culturing of (a) and/or (b) is in an incubator with 5% CO2 at 37 °C.

32. The method of any one of the preceding paragraphs, wherein at least 20%, at least 30%, or at least 40% of the population of differentiated cells are dendritic cells.

33. The method of paragraph 32, wherein about 30% to about 60% of the population of differentiated cells are dendritic cells.

34. The method of any one of the preceding paragraphs, wherein the dendritic cells secrete IL- 12 p70 in response to poly I:C stimulation.

35. The method of any one of the preceding paragraphs, wherein the dendritic cells have the capacity to cross-present tumor antigen to CD8⁺ T cells.

36. The method of any one of the preceding paragraphs, wherein the population of differentiated cells comprises about 0.5%-30% CLEC9A⁺ dendritic cells and/or about 70%- 99% CDlc⁺ dendritic cells.

37. The method of any one of the preceding paragraphs, wherein dendritic cells of the differentiated population do not express detectable CD 14 and/or CD64.

38. A method of producing a dendritic cell vaccine, comprising: pulsing the dendritic cells of any one of the preceding paragraphs with antigenic material, optionally a tumor- specific peptide, tumor lysate, or a tumor-derived RNA, to produce loaded dendritic cells; and exposing the loaded dendritic cells to activating molecules, thereby producing activated dendritic cells.

39. The method of paragraph 38, wherein the activating molecules are selected from Tolllike receptor agonists, RIG-I-like receptor agonists, and cGAS-STING DNA sensing pathway agonists, optionally selected from Poly I:C, R848, LPS, 2'3'-Cyclic GMP-AMP (cGAMP), and defective interfering (DI) RNA.

40. A composition comprising a population of dendritic cells produced by the process of any one of the preceding paragraphs.

41. A method of producing dendritic cells, the method comprising: culturing a population of human CD34⁺ hematopoietic progenitor cells in a culture medium comprising IL-4 and IFNy to produce a differentiated population of human cells comprising dendritic cells, wherein the dendritic cells comprise CLEC9A⁺ cells and CD lc⁺/CLEC9A" cells.

42. A method of producing dendritic cells, the method comprising:

(a) culturing a population of adult human CD34⁺ hematopoietic progenitor cells in a first culture medium comprising Flt3L, SCF, IL-3, TPO, and SRI to produce an expanded population of human CD34⁺ hematopoietic progenitor cells; and

(b) culturing the expanded population of adult human CD34⁺ hematopoietic progenitor cells in a second culture medium Flt3L, SCF, GM-CSF, IL-4, and IFNy to produce a differentiated population of human cells comprising dendritic cells, wherein the dendritic cells comprise CLEC9A⁺ cells and CDlc⁺/CLEC9A" cells.

43. A culture medium comprising: Flt3L, SCF, GM-CSF, IL-4, and IFNy.

44. The culture medium of paragraph 43, further comprising human serum, optionally at a concentration of about 2%.

45. The culture medium of paragraph 43 or 44, wherein the culture medium does not comprise non-human serum.

46. A composition comprising: the culture medium of any one of paragraphs 43-45; and human CD34⁺ hematopoietic progenitor cells and/or dendritic cells.

47. The composition of paragraph 46, wherein the human CD34⁺ hematopoietic progenitor cells are from adult tissue. 48. The method of any one of the preceding paragraphs, wherein the dendritic cells express: (i) a higher level of CD80 and/or CCR7; and/or (ii) a lower level of PDL1 and IL10R; relative to monocyte-derived dendritic cells, optionally wherein the dendritic cells and monocyte-derived dendritic cells are derived from the same donor.

49. The method of any one of the preceding paragraphs, wherein the dendritic cells express: (i) a higher level of IL- 12 p70; and/or (ii) a lower level of IL- 10; relative to monocyte-derived dendritic cells, optionally wherein the dendritic cells and monocyte- derived dendritic cells are derived from the same donor.

50. The method of any one of the preceding paragraphs, wherein the dendritic cells are capable of expanding a higher number of IFNy- secreting MART-1 specific CD8+ T cells, relative to monocyte-derived dendritic cells, optionally wherein the dendritic cells and monocyte-derived dendritic cells are derived from the same donor.

51. The method of any one of the preceding paragraphs, wherein the dendritic cells exhibit greater persistence of an immunogenic phenotype in a tumor microenvironment, relative to monocyte-derived dendritic cells, optionally wherein the dendritic cells and monocyte-derived dendritic cells are derived from the same donor.

EXAMPLES

Expansion DCs from CD34+ HPCs with FS3TR-FSGM4g protocol.

Different formulations were tested to achieve the highest expansion on both cDCl and cDC2 from a variety of CD34⁺ HPC sources ranging from cord blood to adult bone marrow and mobilized blood.

Flt3L, GM-CSF, and TNFa yield cDC2-like cells but few cDCl-like cells.

Figures 1A-1D depict CD34-derived DCs from a culture supplemented with Flt3L, GM-CSF and TNFa. This formulation was tested for the production of different eDC subsets. CD34⁺ cells isolated from G-CSF mobilized blood were cultured in Ex- Vivo 15 media supplemented with 2% human serum and a cytokine mixture including 100 ng/ml Flt3L, 50 ng/ml GM-CSF and 10 ng/ml TNFa for 9-16 days at 37°C 5% CO2 incubator. Cell cultures were feed with fresh medium with cytokines every 2-5 days.

We observed the differentiation of CD14⁺ and CDla⁺ DCs. While CDla⁺ DCs also expressed CDlc, a marker for cDC2, we observed very few CD141⁺ CLEC9A⁺ cDCl. The yield of CDla⁺ DCs or CDlc⁺ cDC2 peaked at day 13. IL-6 inhibits eDC differentiation.

IL-6 is known to support many aspects of hematopoiesis and immunity, including HPC survival, and IL-6 has been used in many in vitro expansion protocol. Thus, we tested it first in two-step protocol, where CD34+ DCs were first to expand in StemSpan™ medium supplemented with FS3 cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF and 20 ng/ml IL-3 for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and FSGM4 cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, and 2.5 ng/ml IL-4 for another 7 days at 37 °C 5% CO2 incubator. FS3- FSGM4 culture resulted in both CDlc+ DCs and CLEC9A+ DCs from cord blood CD34+ cells and 10-fold less from bone marrow CD34+ cells. When we supplemented IL-6 alone (not shown) or IL-6 and IL-6R in the differentiation phase, we observed a dramatic decrease in both CDlc+ DCs and CLEC9A+ DCs. This decrease in eDC output was consistent between different source of CD34+ HPCs. Figures 2A-2E depict the inhibition of IL-6 on eDC differentiation.

DLL-1 showed little effects on eDC differentiation.

Notch signaling mediated via DLL-1 was shown to promote cDCl differentiation. Thus, we tested it in the two-step FS3-FSGM4 protocol in the differentiation phase and observed a little difference in both CDlc+ DCs and CLEC9A+ DCs from cord blood CD34+ cells. Figures 3A-3B depict the lack of benefit from DLLlon eDC differentiation.

SRI in HPC expansion phase promotes downstream cDCl differentiation in differentiation phase.

Different HPC-differentiation blockers were introduced to expand cord blood cells in allogenic hematopoietic stem cell transplantation. These included small molecule UM729; StemRegenin 1, an aryl hydrocarbon receptor antagonist and valproic acid (VPA), an epigenetic modifier. CD34+ cells isolated from cord blood were first expanded in StemSpan™ medium supplemented with FS36T cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 20 ng/ml IL-6, and 50 ng/ml TPO for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and FSGM4 cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, and 2.5 ng/ml IL-4 for another 6-7 days at 37°C 5% CO2 incubator. SRI (1 pM), VPA (1 mM), UM729 (50 nM), or DLL-1 (5 ng/mL) were supplemented in the 7-day differentiation phase. SRI was shown to increase cDCl output while inhibit the differentiation of CD14+ cells. Figures 4A- 4C depict the increase in cDCl output when SRI were supplemented in the expansion phase.

TPO and SRI in HPC expansion phase consistently improve the downstream output of cDCl-like cells from differentiation phase.

We wanted to test which combination would lead to the maximum output in both cDCl and cDC2. To this end, CD34+ cells were first expanded in StemSpan™ medium supplemented with FS3 cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3 for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and FSGM4 cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, and 2.5 ng/ml IL-4 for another 7 days at 37°C 5% CO2 incubator. Different combination of 20 ng/ml IL-6, and 50 ng/ml TPO and SRI (1 pM) were supplemented in the 7-day differentiation phase. We found the cDC2 were relatively consistent between different combinations, the addition of TPO and SRI (FS3TR) consistently gave rise to higher output of cDCl from both cord blood and bone marrow. Figures 5A-5D depict the increase in cDCl output when TPO and SRI were supplemented in the expansion phase. StemRegininl (SRI) promoted CD34+ cell proliferation without differentiation in the presence of SCF, Fit- 3 ligand, TPO and IL- 6.

IFNy improve the output of cDCl-like cells during differentiation.

The combination of IFNP, IFNy, and TNFa was shown to promote cDCl differentiation from induced pluripotent stem cells. We wanted to test whether the same combination would lead to the maximum output in both cDCl and cDC2. To this end, CD34+ cells were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, SRI (1 pM) and for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, and 2.5 ng/ml IL-4 for another 6-7 days at 37°C 5% CO2 incubator. IFNP (10 ng/ml), IFNy (10 ng/ml), TNFa (10 ng/ml), DLL-1 (10 ng/mL) were supplemented in the 7-day differentiation phase. We found the cDC2 were relatively consistent between different combinations, the addition of IFNy (FSGM4g) consistently gave rise to higher output of cDCl from cord blood, bone marrow and G-CSF mobilized CD34+ cells. Figures 6A-6E depict the increase in cDCl output when IFNy were supplemented in the differentiation phase.

In summary, we have created a protocol to maximize the output of CD34-derived DCs, CLEC9A+ DCs, from adult CD34+ cells. This is a critical step toward DCs vaccine tailored toward cancer patients. Furthermore, optimization could be adjust based on the culture including the culture plates, cytokine concentration and the length of culture due to the donor variability.

PVA inhibits total eDC differentiation.

PVA (poly vinyl alcohol) were shown to be superior to albumin in allowing long-term ex vivo expansion of HSC (Wilkinson, 2019). We thus tested the development of DCs from CD34+ cells using the expansion medium containing PVA to our improved culture condition using StemSpan™ medium. CD34+ cells were first expanded in PVA culture medium or StemSpan™ medium supplemented with different combination of cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, SRI (1 pM) and for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM-CSF, 2.5 ng/ml IL-4 and IFNy (2.5 ng/ml) for another 7 days at 37°C 5% CO2 incubator. In comparison to our improved culture condition, the cells cultured in PVA regardless of cytokine combinations yield substantial lower number of total cells as well as different DC subsets. Thus, medium containing PVA negatively impact the output of total DCs. Figures 7A-7D depict the inhibition of total eDC output when PVA were used in the expansion phase.

CD34-derived DCs express high level of costimulatory molecules.

CD34+ cells were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, SRI (1 pM) and for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM- CSF, 2.5 ng/ml IL-4 and IFNy (1 ng/ml) for another 7 days at 37°C 5% CO2 incubator. Total DCs as well as cDCl or cDC2 were enriched by FACS-sorting from cord blood, bone marrow, and G-CSF mobilized blood from melanoma patients at day 14. Sorted DCs were then stimulated with DC activation cocktails including lOpg/ml of poly EC and/or 200ng/ml of CD40L. The percentage of DCs expressed costimulatory molecules were evaluated after overnight stimulation by FACS. All DCs express HLA-DR and CD86, and substantial increase expression of CD40, CD70 and CD86 were observed with poly I:C stimulation in the presence of tumor supernatant. Figures 8A-8B depict CD34-derived DC express high level of costimulatory molecules.

IL-12p70 production of DC from CD34+ HPCs.

CD34+ cells were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, SRI (1 M) and for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM- CSF, 2.5 ng/ml IL-4 with or without IFNy (1 ng/ml) for another 7 days at 37°C 5% CO2 incubator. Bar plots illustrate the percentage and total number of different DC subsets expanded from CD34+ cells derived from bone marrow G-CSF mobilized blood from melanoma patients at day 14. Total DCs were enriched by FACS-sorting. Sorted DCs were then stimulated with DC activation cocktails including I Opg/ml of poly I:C, 200 ng/ml of CD40L and/or tumor supernatant. IL-12p70 production were measured in the supernatant by ELISA. Substantial amount of IL- 12 p70 were produced by CD34-derived DCs after poly I:C stimulation in the presence of tumor supernatant. Eigures 9A-9C depict CD34-derived DC express high level of IL- 12 p70.

DCs capture tumor antigens, process and present tumor antigens to antigen specific CD8+ T cells.

CD34+ cells from melanoma patients were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Elt3L, 100 ng/ml SCE, 20 ng/ml IL-3, 50 ng/ml TPO, SRI (1 M) and for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Elt3L, 20 ng/ml SCE, 2.5 ng/ml, GM-CSF, 2.5 ng/ml IL-4 with or without IFNy (1 ng/ml) for another 7 days at 37°C 5% CO2 incubator. Sorted total DCs, or cDCl and cDC2 were enriched by FACS-sorting. Sorted DCs were then stimulated with 10 pg/ml poly I:C and loaded with MART-1 long peptides for overnight. Cross-presentation of MART- 1 by DCs were evaluated by the capacity to stimulate MART-l-spefic CD8+ T cell lines at 1:10 ratio for IFNy production. High level of IFNy production was found in the culture supernatant of cDC2 cocultured with MART-l-spefic CD8+ T cell lines in the presence of tumor supernatant. Figures 10A-10B depict DCs capture, process, and present tumor antigens to antigen specific CD8+ T cells.

DCs capture tumor antigens, process, and present tumor antigens, and expand antigen specific CD8+ T cells.

CD34+ cells from melanoma patients were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, SRI (1 M) and for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml, GM-CSF, 2.5 ng/ml IL-4 with or without IFNy (1 ng/ml) for another 7 days at 37°C 5% CO2 incubator. Total DCs were enriched by FACS-sorting. Sorted DCs were then stimulated with 10 pg/ml poly I:C and loaded with MART-1 long peptides for overnight. Loaded DCs were then coculture with autologous CD8+ T cells for 12 days in the presence of IL-2. The percentage of MART-1 specific CD8+ T cells were measured by MART- 1 -loaded HLA-A2-tetramer and restimulation with MART-1 9-mer peptides for intracellular cytokine production by LACS. Multifunctional MART-1 specific CD8+ T cells were expanded by antigen-loaded DCs the capacity to express multiple effectors including IFNy, TNEa, and CD107a, a surrogate marker for lytic granule release. Eigures 11A-11D depict the capacity of DCs to capture tumor antigens, process and present tumor antigens, and expand antigen specific CD8+ T cells.

CD8+ T cells expanded by DCs kill tumor cells.

CD34+ cells from melanoma patients were first expanded in StemSpan™ medium supplemented with cytokine mix including 100 ng/ml Elt3L, 100 ng/ml SCE, 20 ng/ml IL-3, 50 ng/ml TPO, SRI (1 pM) and for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Elt3L, 20 ng/ml SCE, 2.5 ng/ml, GM-CSF, 2.5 ng/ml IL-4 with or without IFNy (1 ng/ml) for another 7 days at 37°C 5% CO2 incubator. Total DCs were enriched by FACS-sorting. Sorted DCs were then stimulated with 10 pg/ml poly I:C and loaded with MART-1 long peptides for overnight. Loaded DCs were then coculture with autologous CD8+ T cells for 12 days in the presence of IL-2. CD8+ T cells were cocultured with CFSE-labeled melanoma cells (Me275 and Z80- mel) or K562 at different ratio for 4 hours to evaluate their capacity to kill tumor cells. As dose-dependent killing were found in CD8+ T cells expanded with DCs. Figure 12 depicts CD8+ T cells expanded by DCs kill tumor cells.

In conclusion, we can generate higher number of cDCls and cDC2s from HPCs from three different HPCs sources (cord blood, adult bone marrow, and G-CSF mobilized peripheral blood HPCs) and cancer patients (melanoma patients) using clinically compliant protocols. DCs meet expected phenotypic criteria. DCs make high amount of IL- 12 p70, express co- stimulatory molecules (CD40, CD80, CD86, and CD70). DCs capture tumor antigens, process, present tumor epitopes, and prime naive CD8+ T cells. CD8+ T cells primed by DCs can kill tumor cells.

Guard-cDCl demonstrate superior ability to stimulate immunity and generate strong effector CD8+ T cells over moDCs.

Monocyte-derived DCs (moDCs) has been used in multiple clinical trial for treatment of metastatic disease with some immunological response. To test the function of Guard DCs, we generated both Guard DCs to moDCs from the same donor and compared their capacity to stimulate immunity in vitro after recovery from cryopreservation. To account for the influence of suppressive tumor microenvironment, both Guard DCs or moDCs were exposed to tumor condition medium. Guard DCs expressed a higher level of CD80 and CCR7, but a lower level of PDL1 and IL10R. HLA-DR, CD40, CD86, CD70 were comparable between these two types of DCs. Furthermore, Guard DCs expressed a high level of IL- 12 p70 (90- fold more) and a lower level of IL-10 (13-fold lower) (Fig. 14A). To probe their capacity to prime CD8+ T cells, DCs were then cocultured with HLA-A2-matched naive CD8+ T cells at 1:100 ratio for 12 days, and MART-1 specific CD8+ T cells were measured by their capacity to express one or multiple effector molecules including IFNy, TNFa, or CD107a. A higher number of IFNy- secreting MART-1 specific CD8+ T cells was expanded by Guard DCs (22- fold) than moDCs (9-fold) (Fig. 14B). The higher number of specific CD8+ T cells was associated with an increase of different types of poly-functional CD8+ T cells (Fig. 14C). In summary, these in vitro data consistently demonstrated superior quality of Guard-cDC than moDCs in both their phenotypes as well as capacity to stimulate CD8+ T cell immunity.

Guard-cDCls overcome persistence issues that plagued moDCs in vivo, even in the TME. To test the persistence of Guard DCs in vivo, immunodeficient NSG-SGM3 mice were first implanted with 5xl0⁶ patient melanoma cells subcutaneously (s.c.). When tumors reach 100 mm³ at 3-weeks after implant, 2xl0⁵ Guard DCs or moDCs were cryo-recovered and injected into the tumor. Tumor and draining lymph nodes were snap-frozen in Tissue-Tek O.C.T. Compound (OCT) at 24 and 72 hours. Immunofluorescence staining was performed on tumors for human HLA-DR, CD 11c, and DAPI. While both Guard DCs or moDCs were found in patient-derived xenograft (PDX) tumor at 24 and 72 hours after injection, Guard DCs displayed healthier morphology with a higher level of surface expression of HLA-DR. Overall, Guard-cDCls showed greater persistence of an immunogenic phenotype in the tumor microenvironment (fluorescent microscopy images of data not shown).

Guard-cDCls migrate to lymph nodes, overcoming migration issues of moDCs in vivo.

CCR7 play a central role in mobilizing DCs to the draining lymph nodes and initiating an immune response. In contrast to moDCs, Guard DCs expressed higher levels of CCR7 ex vivo (Fig. 15). To test the capacity of Guard DCs to migrate into the draining lymph node in vivo, draining lymph nodes of immunodeficient NSG-SGM3 mice were snap-frozen in OCT at 24-Hr and 72-hour after receiving Guard DCs or moDCs intra-tumorally. Immunofluorescence staining was performed on the draining lymph node for human HLA- DR, CD 11c, and DAPI. In contrast to moDCs, the draining lymph node of Guard DCs were swelled and infiltrated with high number of DCs demonstrating that Guard-cDCls overcome the migration limitation that previously limited moDC clinical efficacy. Overall, Guard- cDCls had greater persistence of immunogenic phenotype in the lymph nodes (fluorescent microscopy images of data not shown). In summary, both the in vitro and in vitro data consistently demonstrate superior capacity of Guard-cDCs over moDCs to migrate to the draining lymph nodes.

Materials and Methods

Functional validation of CD34-derived DCs

Figure 13 depicts the Workflow for functional validation of CD34-dervived DCs. CD34+ cells were first expanded in STEMSPAN™ medium supplemented with cytokine mix including 100 ng/ml Flt3L, 100 ng/ml SCF, 20 ng/ml IL-3, 50 ng/ml TPO, SRI (1 pM) and for 7 days, followed by the differentiation in Ex- Vivo 15 media supplemented with 2% human serum and cytokine mix including 100 ng/ml Flt3L, 20 ng/ml SCF, 2.5 ng/ml GM- CSF, 2.5 ng/ml IL-4 and IFNy(l ng/ml) for another 7 days at 37°C 5% CO2 incubator. Total DCs as well as cDCl or cDC2 subsets were enriched by FACS-sorting. Sorted DCs were then activated and loaded with tumor antigens. The functionality of DCs were evaluated by the expression of costimulatory molecules, IL- 12 p70 secretion, the capacity to cross-present tumor antigen to specific CD8+ T cells line, and the capacity to expand cancer-specific cytotoxic CD8+ T cells with effector molecular expression.

Cells. Purified frozen cord blood (CB) CD34+ HPCs were purchased from Lonza®. Bone marrow CD34⁺ HPCs were enriched by CD34+ EASYSEP™ kits (STEMCELL technology) from Lonza®. G-CSF mobilized CD34+ HPCs and T cells were isolated from leukapheresis with prior approval by the institutional review board at the Baylor Institute Research (Dallas, TX).

Reagents. Recombinant human Flt3L (308-FK-100), SCF (255-SC-050), IL-3 (203-IL-010), TPO (288-TP-025), GM-CSF (7954-GM-010), IL-2 (10453-IL-050), IL-4 (204-IL-010), IL-6 (206-IL-010), IL-7 (11089-IL-010), IL-10 (11178-IL-010), and IFN-g (10067-IF-025) was from R&D systems. All peptides used here were HLA-A*0201 -restricted and synthesized by Bio-Synthesis (Lewisville, TX) with purity higher than 95% including: melan A/MART-1 27- 35 (ELAGIGILTV (SEQ ID NO: 1)), gplOO (KTWGQYWQV (SEQ ID NO: 2)), HIV gag 77-85 (SLYNTVATL (SEQ ID NO: 3)) and melan A/MART-1 16-40 (GHGHSYTTAEELAGIGILTVILGVL (SEQ ID NO: 4)). Tetramer were purchased from MBL. CFSE cell proliferation kit (C34554) and LIVE/DEAD fixable aqua dead cell stain kit (L34957) were from Life Technologies. Poly EC (vac-pic) were from INVIVOGEN®. Antibodies used in the study were purchased from BD, BIOLEGEND®, MILTENYI BIOTEC®, or THERMOFISHER®.

CD34-dervied DC culture. For experiments with CD34+ cells, the Expansion Medium was StemSpan™ (stem cells) supplemented with 1% penicillin- streptomycin and cytokine mix; and the Differentiation Medium is Ex- Vivo 15 (Lonza) supplemented with 2% human AB serum, 2mM L-Glutamine, 10 pM p-mercaptoethanol and cytokine mix. Cytokine mixes were listed in individual experiments. Isolation of DC subsets. DCs were stained with antibodies against HLA-DR-APC-AF780, CLEC9A-PE, CDl lc- V450, CDlc-PerCP-Cy5.5, CD141-APC and with an antibody cocktail against CD 14, CD 15 and CD66b in channel FITC. DCs were sorted as FITC- HLA- DR+ CD11C+, cDCl were sorted as FITC- HEA-DR+ CDl lc+ CEEC9A+ CDlc+/- CD141+ and cDC2 were sorted as FITC- HEA-DR+ CD1 lc+ CEEC9A- CDlc+ with FACS Symphony S6 (BD) using Diva software (BD).

Flow cytometry. For phenotypic analysis, cells were stained on ice with specific antibody cocktails in the presence of 10% human AB serum for 30 mins. After washing twice with PBS buffer containing 2% FBS and 2mM EDTA, cells were analyzed using FACS Symphony A5 (BD) and Flow Jo software (Tree Star, Ashland, OR).

T cell isolation. T cells were isolated from peripheral blood mononuclear cells (PBMCs) in CD34" fraction using Easysep CD8+ T cell isolation kit (STEMCEEE Technology, 17953) following the manufactures protocol. Isolated CD8+ T cells had purity >95%. In experiments with naive CD8⁺ T cells, CD8⁺ T cells were further stained with CCR7-FITC, CD56-PE, HEA-DR-PE and CD45RA-PerC-Cy5.5 antibody. CD45RA⁺CCR7⁺CD8⁺ naive T cells were sorted by FACS Fusion or FACS Symphony S6 (BD) with purity >99%.

MART-1 antigen presentation to CD8+ T cells. Mart- 1- specific CD8+ T cell lines were generated from HEA-A*0201+ melanoma patients. Briefly, PBMCs were resuspended in RPMI medium with 10% human AB serum and pulsed with 1 pM of MART-1 (EEAGIGIETV (SEQ ID NO: 1)) peptides. IE-2 (100 U/mE) was added on day 2 and day 7. At day 9, cells were stained with MART-l-HEA-A*0201 tetramer for 30 min, washed twice with PBS and tetramer positive cells were sorted with FACS ARIA™ (BD) using Diva software (BD). Specific CD8+ T cells were expanded in the presence of irradiated feeder cells (allogeneic peripheral blood mononuclear cells (PBMCs) and lymphoblastoid cell lines (ECEs)) at a responder to stimulator ratio of 1:500 and 1:100 together with anti-CD3 (OKT3, BD) and 50U/ml of IE-2. Next, 50,000 Mart- 1 -specific CD8+ T cells were co-cultured with 5,000 DCs in a 96-well plate in 200pL of complete RPMI media with 10% human AB serum and 200 ng/mL CD40L (R&D Systems). After 18hr of co-culture, cells were centrifuged, and supernatant harvested for assessment of IFN-y by ELISA (BIOLEGEND®). Antigen presentation experiments with autologous CD8+ T cells. HLA-A*0201+ or autologous CD8+ T cells were isolated from cryopreserved PBMCs using human CD8+ T cell enrichment kits (StemCell Technologies) following the manufacturer’s protocol. Isolated CD8+ T cells had a purity >95%. CD8+ T cells were labeled with 1 pM CFSE (Invitrogen) for 10 mins at room temperature and cocultured with sorted DCs at 1:100 DC:T ratio for 12 days in completed RPMI with 10% AB serum together with 10 ng/ml of IL-7 and 200 ng/ml of CD40L. At day 2 and day 7, 10 ng/ml of IL-2 were added in the culture. Lor tetramer analysis, samples were stained at room temperature for 30 min with APC-conjugated HLA- A*0201 tetramer and 10 min with CD3-PE (SK7, BD), CD8-PB (SKI, BD), CD4-PE-Cy7 (SK3, BD). After washing twice with PBS buffer containing 2% PBS and 2mM EDTA, the samples were acquired on a Symphony A5 (BD), and analyzed with PlowJo software (Tree Star, Ashland, OR).

Intracellular cytokine staining. Intracellular cytokine and degranulation of CD8⁺ T cells was examined. Briefly, total cells were stimulated with 2.5 pM of specific peptide in the presence of anti-CD28 and anti-CD49d antibodies (BD, 347690). Monesin and Alexa flour 488-conjugated anti-CD107a were included during the culture period. Pollowing T cell stimulation, cells were stained with LIVE/DEAD Fixable Aqua dead cell stain kit (Thermofisher, L34957), followed by surface labeled with surface antibodies for CD3 and CD8, and then intracellularly labeled with antibodies specific for IFN-y and TNF-oc using BD Pharmingen Cytofix/Cytoperm and perm/wash reagents (55471). Samples were analyzed on FACS Symphony A5 (BD).

Cytotoxicity assay. Cytotoxic T lymphocyte (CTL) activity was measured using flow cytometry-based viability assay. Briefly, CD8+ T cells expanded by DCs were used as the effector cells. Effector cells were cultured along with 10,000 CFSE-labeled target cells at several different effector: target (E: T) ratios in a 96-well U-bottom microtiter plate in the presence of 25 g/mL of PI. Target cells including melanoma cells (Me275 and Z80-mel) and K562 were labeled with IpM CFSE for 10 mins. The plate was centrifuged at 1200 rpm for 30 sec before incubating at 37°C for 4 hours. At the end of incubation, the plate was analyzed by FACS symphony A5. The spontaneous killing was determined from targeting cells incubating with medium alone and the maximum killing was obtained by substituting the effector cells with 1% Triton X-100. Cytotoxicity was determined by the percentage of PI+ in CFSE+ target cells. The specific lysis was calculated as: [(experimental PI)-(spontaneous PI)]/[(maximum PI)-(spontaneous PI)] xlOO percentage.

ELISA. ELSA were performed following manufacture protocol. For human IFN-g, culture supernatant was tested with human IFN-g EEISA MAX Deluxe Set from BIOLEGEND® (430104). For human IL-12p70, culture supernatant was tested with human IL- 12 p70 ELISA MAX Deluxe Set from BIOLEGEND® (431704).

Monocyte-derived DC culture. Total monocytes were first enriched from PBMCs with DYNABEADS™ Untouched™ Human Monocyte Kit, differentiated in Ex- Vivo 15 media supplemented with 1000 lU/ml GM-CSF and 1000 lU/ml IL-4 for 5 days, followed by activation with maturation cocktail (1000 lU/mL IFNy, 20 ng/mL TNFa, and 200 ng/mL CD40L) for 16 hours and loaded with melanoma peptides for the last 4 hours before cryopreservation in CytoStor CS10.

Tumor model. NSG-SGM3 (NOD. Cg-Prkdc^scid ZZ2r₍g^{fmW /}Tg(CMV - IL3,CSF2,KITLG)lEav/MloySzJ; RRIDJMSR JAX:013062) obtained from The Jackson Laboratory (Bar Harbor, ME). All protocols were reviewed and approved by the Institutional Animal Care and Use Committee at The Jackson Laboratory (14005-1) and University of Connecticut Health Center (AP-200785-1125; Farmington, CT). De-identified human specimens were approved by The Jackson Laboratory Institutional Review Board. Patient tumor cells were injected s.c. into flank of the mice. Tumor size was monitored every 7 days with a caliper. Tumor volume (ellipsoid) was calculated as follows: (short diameter)² x long diameter/2. When tumors reach 100 mm³ at 3-weeks after implant, 200,000 Guard DCs or moDCs were thawed and injected into the tumor in 30 pL of PBS. At 24 and 72 hours after injection, tumor and draining lymph nodes were snap-frozen in OCT.

Immunofluorescence Staining (data described but not shown). Tissues were embedded in OCT (Sakura Finetek U.S.A.) and snap frozen in liquid nitrogen. Frozen sections were cut at 6 pm, air dried on Superfrost plus slides and fixed with cold acetone for five min. Tissue sections were first block with Background Buster, followed by treatment of Fc Receptor Block (Innovex Bioscience). The sections were then stained with mouse monoclonal antibodies to human HLA-DR- Alexa 555 (TAL1B5, Novus Biologicals), CDl lc-Alexa 647 (3.9, BD) for one hour at room temperature. Respective isotype antibodies were used as the control. Finally, sections were counterstained with 1 pg/ml of DAPI, mounted with Fluoromount (Thermo Fisher Scientific), acquired on a Leica Thunder microscope with Leica LAS X software, and analyzed using Imaris software (Bitplane).

Statistical analyses. Statistical analysis was performed in Prism (GraphPad, San Diego, CA). Legend is: ***P < 0.001, **P < 0.01, *P < 0.05, ns = not significant. Comparisons between any 2 groups were analyzed using the Mann- Whitney test or two-tailed t-test. Comparisons between any 3 or more groups were analyzed by analysis of variance (ANOVA).

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

What is claimed is: CLAIMS

1. A method of producing dendritic cells, the method comprising:

(b) differentiating the human CD34⁺ hematopoietic progenitor cells in a second culture medium comprising interferon gamma (IFNy) or a functional analog thereof, thereby producing a population of differentiated cells comprising dendritic cells, wherein the dendritic cells comprise CD141⁺ or CLEC9A⁺ cells and CD lc⁺/CLEC9A" cells.

2. The method of claim 1, wherein the second culture medium comprises IFNy.

3. The method of claim 1 or 2, wherein the second culture medium further comprises interleukin-4 (IL-4) or a functional analog thereof.

4. The method of claim 3, wherein the second culture medium further comprises IL-4.

5. The method of any one of claims 1-4, wherein (a) comprises culturing a starting population of human CD34⁺ hematopoietic progenitor cells in a first culture medium to produce an expanded population of human CD34⁺ hematopoietic progenitor cells; and (b) comprises culturing the expanded population of human CD34⁺ hematopoietic progenitor cells in the second culture medium to produce the population of differentiated dendritic cells.

6. The method of any one of claims 1-5 further comprising activating the dendritic cells.

7. The method of claim 6, wherein the activating comprises exposing the dendritic cells to activation molecules, optionally Toll-like receptor agonist molecules and/or cytokines.

8. The method of any one of the preceding claims, wherein the first culture medium is serum-free medium.

9. The method of any one of the preceding claims, wherein the first culture medium comprises a cytokine selected from Fms-related tyrosine kinase 3 ligand (Flt3L), stem cell factor (SCF), interleukin-3 (IL-3), thrombopoietin (TPO), and StemRegininl (SRI).

10. The method of claim 9, wherein the first culture medium comprises Flt3L, SCF, IL-3, TPO, and SRI.

11. The method of claim 10, wherein the first culture medium comprises about 30-300 ng/ml Flt3L, about 80-120 ng/ml SCF, about 16-24 ng/ml IL-3, about 40-60 ng/ml TPO, and about 345-515 ng/ml SRI.

12. The method of claim 11, wherein the first culture medium comprises about 100 ng/ml Flt3L, about 100 ng/ml SCF, about 20 ng/ml IL-3, about 50 ng/ml TPO, and about 430 ng/ml

SRI.

13. The method of claim 10, wherein the ratio of Flt3L:SCF:IL-3:TPO:SRl in the first culture medium is about 5:5:1:2.5:21.5.

14. The method of any one of the preceding claims, wherein the second culture medium further comprises Flt3L, SCF, and granulocyte-macrophage colony-stimulating factor (GM- CSF).

15. The method of claim 14, wherein the second culture medium comprises about 80-120 ng/ml Flt3L, about 16-24 ng/ml SCF, about 2-20 ng/ml GM-CSF, about 2-20 ng/ml IL-4, and about 0.8-8 ng/ml IFNy.

16. The method of claim 15, wherein the second culture medium comprises about 100 ng/ml Flt3L, about 20 ng/ml SCF, about 2.5 ng/ml GM-CSF, about 2.5 ng/ml IL-4, and about 1 ng/ml IFNy.

17. The method of claim 16, wherein the ratio of Flt3L:SCF:GM-SCF:IL-4:IFNy in the second culture medium is about 100:20:2.5:2.5:1.

18. The method of any one of the preceding claims, wherein the second culture medium further comprises human serum, optionally at a concentration of about 2%.

19. The method of any one of the preceding claims, wherein the second culture medium further comprises serum, and the serum consists of human serum, optionally at a concentration of about 2%.

20. The method of any one of the preceding claims, wherein the second culture medium does not comprise non-human serum.

21. The method of any one of the preceding claims, wherein the human CD34⁺ hematopoietic progenitor cells are from umbilical cord blood, bone marrow, or peripheral blood of a subject.

22. The method of any one of the preceding claims further comprising obtaining the human CD34⁺ hematopoietic progenitor cells from umbilical cord blood, bone marrow, or peripheral blood of a subject.

23. The method of any one of the preceding claims, wherein the human CD34⁺ hematopoietic progenitor cells are adult human CD34⁺ hematopoietic progenitor cells, optionally wherein the adult CD34⁺ hematopoietic progenitor cells comprise CD34⁺CD38⁺ and CD34⁺CD38‘ hematopoietic progenitor cells.

24. The method of any one of the preceding claims, wherein the starting population of human CD34⁺ hematopoietic progenitor cells comprises about 100,000 to about 4 million human CD34⁺ hematopoietic progenitor cells.

25. The method of any one of claims 5-24, wherein the expanded population of human CD34⁺ hematopoietic progenitor cells comprises about 10 million to about 200 million cells.

26. The method of any one of claims 5-24, wherein the expanded population of human CD34⁺ hematopoietic progenitor cells comprises about 25-fold to about 500-fold more CD34⁺ hematopoietic progenitor cells than the starting population of CD34⁺ hematopoietic progenitor cells.

27. The method of claim 26, wherein the expanded population of human CD34⁺ hematopoietic progenitor cells comprises about 50-fold to about 200-fold more CD34⁺ hematopoietic progenitor cells than the starting population of CD34⁺ hematopoietic progenitor cells.

28. The method of any one of the preceding claims, wherein the culturing of (a) is for about 5 to 12 days, optionally about 6 to 8 days, preferably about 7 days.

29. The method of any one of the preceding claims, wherein the culturing of (b) is for at least 5 days.

30. The method of claim 26, wherein the culturing of (b) is for about 5 to 10 days, optionally about 6 to 8 days, preferably about 7 days.

31. The method of any one of the preceding claims, wherein the culturing of (a) and/or (b) is in an incubator with 5% CO2 at 37 °C.

32. The method of any one of the preceding claims, wherein at least 20%, at least 30%, at least 40%, or at least 50% of the population of differentiated cells are dendritic cells.

33. The method of claim 32, wherein about 30% to about 60% of the population of differentiated cells are dendritic cells.

34. The method of any one of the preceding claims, wherein the dendritic cells secrete IL- 12 p70 in response to poly I:C stimulation.

35. The method of any one of the preceding claims, wherein the dendritic cells have the capacity to cross-present tumor antigen to CD8⁺ T cells.

36. The method of any one of the preceding claims, wherein the population of differentiated cells comprises about 20%-50% CD141⁺ dendritic cells, 0.5%-30% CLEC9A⁺ dendritic cells and/or about 70%-99% CDlc⁺ dendritic cells.

37. The method of any one of the preceding claims, wherein dendritic cells of the differentiated population do not express detectable CD 14 and/or CD64.

38. A method of producing a dendritic cell vaccine, comprising: pulsing the dendritic cells of any one of the preceding claims with antigenic material, optionally a tumor- specific peptide, tumor lysate, or a tumor-derived RNA, to produce loaded dendritic cells; and exposing the loaded dendritic cells to activating molecules, thereby producing activated dendritic cells.

39. The method of claim 38, wherein the activating molecules are selected from Toll-like receptor agonists, RIG-I-like receptor agonists, and cGAS-STING DNA sensing pathway agonists, optionally selected from Poly I:C, R848, LPS, 2'3'-Cyclic GMP-AMP (cGAMP), and defective interfering (DI) RNA.

40. A composition comprising a population of dendritic cells produced by the process of any one of the preceding claims.

41. A method of producing dendritic cells, the method comprising: culturing a population of human CD34⁺ hematopoietic progenitor cells in a culture medium comprising IL-4 and IFNy to produce a differentiated population of human cells comprising dendritic cells, wherein the dendritic cells comprise CD141⁺ cells, CLEC9A⁺ cells and CD lc⁺/CLEC9A" cells.

42. A method of producing dendritic cells, the method comprising:

(b) culturing the expanded population of adult human CD34⁺ hematopoietic progenitor cells in a second culture medium Flt3L, SCF, GM-CSF, IL-4, and IFNy to produce a differentiated population of human cells comprising dendritic cells, wherein the dendritic cells comprise CD141⁺ cells, CLEC9A⁺ cells, and CDlc⁺/CLEC9A" cells.

43. A culture medium comprising: Flt3L, SCF, GM-CSF, IL-4, and IFNy.

44. The culture medium of claim 43, further comprising human serum, optionally at a concentration of about 2%.

45. The culture medium of claim 43 or 44, wherein the culture medium does not comprise non-human serum.

46. A composition comprising: the culture medium of any one of claims 43-45; and human CD34⁺ hematopoietic progenitor cells and/or dendritic cells.

47. The composition of claim 46, wherein the human CD34⁺ hematopoietic progenitor cells are from adult tissue.

48. The method of any one of the preceding claims, wherein the dendritic cells express: (i) a higher level of CD80 and/or CCR7; and/or (ii) a lower level of PDL1 and IL10R; relative to monocyte-derived dendritic cells, optionally wherein the dendritic cells and monocyte-derived dendritic cells are derived from the same donor.

49. The method of any one of the preceding claims, wherein the dendritic cells express: (i) a higher level of IL- 12 p70; and/or (ii) a lower level of IL- 10; relative to monocyte- derived dendritic cells, optionally wherein the dendritic cells and monocyte-derived dendritic cells are derived from the same donor.

50. The method of any one of the preceding claims, wherein the dendritic cells are capable of expanding a higher number of IFNy- secreting MART-1 specific CD8+ T cells, relative to monocyte-derived dendritic cells, optionally wherein the dendritic cells and monocyte-derived dendritic cells are derived from the same donor.

51. The method of any one of the preceding claims, wherein the dendritic cells exhibit greater persistence of an immunogenic phenotype in a tumor microenvironment, relative to monocyte-derived dendritic cells, optionally wherein the dendritic cells and monocyte- derived dendritic cells are derived from the same donor.