Genetically engineered progenitor cells and methods of use

Abstract

Genetically engineered progenitor cells and methods of using the same are disclosed herein. Genetically engineered cells differentiated from the genetically engineered progenitor cells of the present disclosure are also disclosed herein. Also disclosed herein is a method of treating a condition in a subject by administering the genetically engineered progenitor cells of the present disclosure or the genetically engineered cells differentiated from the genetically engineered progenitor cells.

Description

Technical Field

[0001] Cross - referenced applications This application claims the benefit of priority to European Patent Application No. 22176819, filed on June 1, 2022, the entire content of each of which is incorporated herein by reference.

[0002] Incorporation by reference of the sequence listing This application includes a sequence listing filed via the Patent Center. The sequence listing named 212225.701601_PCT_SL.xml, created on May 17, 2023, and having a size of 51,207 bytes, is incorporated herein by reference in its entirety.

[0003] Field The present disclosure generally relates to engineered dendritic progenitor cells and methods of using the same.

Background Art

[0004] Immunotherapy can be utilized for the treatment of various human diseases such as infectious diseases, degenerative conditions, and cancer. In cancer, immunotherapy may involve stimulating the patient's own immune system to attack cancer cells or other cellular components of the tumor.

[0005] The generation or enhancement of cancer - specific T lymphocytes by vaccinating patients against tumor - associated antigens (TAAs) represents an attractive means of treating subjects by immunotherapy. One type of cancer vaccine involves the use of dendritic cells (DCs). DCs are a family of immune cells that have the ability to capture TAAs and present them to T lymphocytes through various mechanisms, priming a strong effector response against the tumor. DCs can also migrate between lymphoid and non - lymphoid tissues and control inflammation and lymphocyte homing by changing cytokine and chemokine gradients. However, it has been difficult to improve the efficacy of DCs for therapeutic use.

Summary of the Invention

[0006] As described herein, immune cell genetic manipulation provides a means for improving the efficacy of DC vaccines. A self-cell-based platform capable of generating and expanding cDC1 in vivo as described herein efficiently takes up and presents tumor-associated antigens (TAAs), induces a strong and broad T cell response against multiple TAAs, thereby making cDC1 an attractive therapeutic agent against a wide range of cancers. Accordingly, the present disclosure provides a methodology for efficiently generating cDC1 in vivo and for generating DC precursors that do not require ex vivo antigen addition, and thus provides a means for tumor agnostic DC vaccines.

[0007] [Overview] An in vitro cell composition comprising synthetically partially-differentiated dendritic cell progenitors, wherein the synthetically partially-differentiated dendritic cell progenitors have a phenotype of CD115 + , CD11c - , and Clec9A - as determined by flow cytometry is disclosed herein. In some embodiments, the phenotype of the synthetically partially-differentiated dendritic progenitor cells further comprises one or more phenotypes selected from CD11b - , MHCII - , CD45R / B220 - , and cKIT - when determined by flow cytometry.

[0008] Also disclosed herein are differentiated cDC1 or cDC2 dendritic cells differentiated from synthetically partially differentiated dendritic cell precursors described herein. In some embodiments, the synthetically differentiated cDC1 or cDC2 are genetically engineered dendritic cells that express an interleukin or effector. In some embodiments, the genetically engineered dendritic cell expresses an interleukin, and the interleukin is IL12. In some embodiments, the genetically engineered dendritic cell expresses an effector, and the effector is selected from the group consisting of extracellular vesicle-internalizing receptor (EVIR), FMS-like tyrosine kinase 3 ligand (FLT3L), IL-12, TNF-α, IL-1, IL-2, IL-6, CXCL8, interferon (IFN), GM-CSF, and G-CSF.

[0009] An in vitro cell composition comprising a synthetically partially differentiated dendritic cell precursor, wherein the synthetically partially differentiated dendritic cell precursor, when determined by flow cytometry, has one or more phenotypes selected from CD115 + , CD34 + , CD3 - , CD19 - , CD335 - , CD66b - , CD10 - , and CD14 - is also disclosed herein.

[0010] Also disclosed herein are antigen presenting cells (APCs) differentiated from synthetically partially differentiated dendritic cell precursors described herein. In some embodiments, the APCs are genetically engineered dendritic cells that express an interleukin or effector. In some embodiments, the genetically engineered dendritic cell expresses an interleukin, and the interleukin is IL12. In some embodiments, the genetically engineered dendritic cell expresses an effector, and the effector is selected from the group consisting of extracellular vesicle internalization receptor (EVIR), FMS-like tyrosine kinase 3 ligand (FLT3L), GM-CSF, IL-6, IL-12, IFNα2β, IFNγ, SCF, and TNF-α.

[0011] The following steps: (a) obtaining shortly-expanded hematopoietic stem / progenitor cells (HSPCs), and (b) CD115, if determined by flow cytometry + , CD11c - , and Clec9A - In an amount sufficient to differentiate HSPC cells into synthetically partially differentiated dendritic cell precursors having the phenotype of, contacting the shortly-expanded HSPCs with a synthetic medium containing or not containing FMS-like tyrosine kinase 3 ligand (FLT3L) and GM-CSF, and containing IL-1, IL-2, IL-4, IL-6, IL-12, CXCL8, G-CSF, TNF-α, IFNa, PGE2, or retronectin. Also disclosed herein is a method of making synthetically partially differentiated dendritic cell precursors, which includes the step of contacting the HSPCs in a medium containing FBS, L-glutamine, SCF, TPO, FLT3L, IL-3, IL-6, and IL-1b, thereby further comprising the step of making HSPCs that are shortly-expanded prior to the contacting step of (b).

[0012] The following steps: (a) shortly-expanded CD34 +Steps to obtain human hematopoietic stem progenitor cells (human HSPC); and (b) CD115 when determined by flow cytometry + , CD34 + , CD3 - , CD19 - , CD335 - , CD66b - , CD10 - , and CD14 - To differentiate HSPC cells into synthetically partially differentiated dendritic cell precursors having one or more phenotypes selected from, in an amount sufficient to differentiate, with or without IFNγ, IL-12, retronectin, TNF-α, or UM729, a synthetic medium containing FMS-like tyrosine kinase 3 ligand (FLT3L), IL-3, IL-6, TPO, and SCF, and contacting the synthetic medium with short-term expanded human HSPC. A method for producing synthetically partially differentiated dendritic cell precursors is also disclosed herein.

[0013] A pharmaceutical composition for use in the treatment of a condition, comprising the following components: (a) an in vitro cell composition as described herein, and (b) a pharmaceutically acceptable excipient, diluent, or carrier, is also disclosed herein. A method of treating a subject in need thereof, comprising the step of administering to the subject a pharmaceutical composition comprising the following components: (a) an in vitro cell composition as described herein, and (b) a pharmaceutically acceptable excipient, diluent, or carrier, is also disclosed herein. In some embodiments, the condition is cancer. In some embodiments, the pharmaceutical composition further comprises an interleukin or an effector. In some embodiments, the differentiated cDC1 or cDC2 dendritic cells are genetically engineered dendritic cells that express an interleukin or an effector. In some embodiments, the APC is a genetically engineered dendritic cell that expresses an interleukin or an effector. In some embodiments, the interleukin is IL-12. In some embodiments, the effector is selected from the group consisting of extracellular vesicle internalization receptor (EVIR), FMS-like tyrosine kinase 3 ligand (FLT3L), IL-12, TNF-α, IL-1, IL-2, IL-6, CXCL8, interferon (IFN), GM-CSF, and G-CSF. In some embodiments, the effector is not expressed on the cells of cancer.

[0014] The novel features of the exemplary embodiments are set forth with particularity in the appended claims. A better understanding of the features and advantages will be obtained from the following detailed description of the exemplary embodiments that utilize the principles of the disclosed systems and methods, as well as the reference to the appended drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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Mode for Carrying Out the Invention

[0016] Overview Synthetically differentiated dendritic cell precursors (or DCPs) are disclosed herein. The synthetically differentiated dendritic cell precursors as described herein can be partially differentiated from host precursor cells. Thus, the synthetically differentiated dendritic cell precursors as described herein are partially differentiated cells. In some embodiments, the synthetically differentiated dendritic cell precursors as described herein are not fully differentiated.

[0017] The synthetically differentiated dendritic cell precursors as described herein can naturally differentiate into dendritic cells such as cDC1, cDC2, or immature dendritic cells upon administration to a host. As disclosed herein, the synthetically differentiated dendritic cell precursors of the present disclosure differentiate into such dendritic cells to a greater extent when administered to a subject compared to the administration of otherwise equivalent dendritic cells such as monocyte-derived dendritic cells (moDC) or conventional type 1 DC (cDC1) cells. Furthermore, the synthetically differentiated dendritic cell precursors of the present disclosure are capable of differentiating into dendritic cells in the presence of a tumor and are thus capable of differentiating in the presence of inflammation and immunosuppressive cytokines associated with the presence of the tumor.

[0018] The synthetically differentiated dendritic cell precursors as described herein are host precursor cells, e.g., CD34 +It can be differentiated from human hematopoietic stem progenitor cells. In some embodiments, the synthetically differentiated dendritic cell precursors as described herein are partially differentiated cells. In some embodiments, the synthetically differentiated dendritic cell precursors as described herein are not fully differentiated. In some embodiments, the synthetically differentiated dendritic cell precursors as described herein can differentiate in vitro into antigen-presenting cells (APCs), cDC2, monocytes, immature dendritic cells, or combinations thereof. Further, in some embodiments, the synthetically differentiated dendritic cell precursors of the present disclosure can differentiate into APCs, cDC2, monocytes, immature dendritic cells, or combinations thereof in the presence of a tumor and are thus capable of differentiating in the presence of inflammation and immunosuppressive cytokines associated with the presence of the tumor.

[0019] The synthetically differentiated dendritic cell precursors can be genetically engineered to express interleukins and / or effectors to stimulate the production of tumor-specific T cells. In some examples, co-expression of an extracellular vesicle internalization receptor (EVIR) such as interleukin-12 or an effector such as FMS-like tyrosine kinase 3 ligand (FLT3L) results in differentiated dendritic cells (e.g., cDC1, cDC2, APC, monocytes, or immature dendritic cells) that generate tumor-specific T cells that reduce tumor growth, inhibit tumorigenesis, or both. Further, the presence of an effector and / or interleukin (whether co-expressed by the differentiated dendritic cells or added exogenously) results in the generation of tumor-specific T cells without the need to supply tumor antigens (e.g., whether exogenously or through expression by the dendritic cells).

[0020] Accordingly, synthetically differentiated dendritic cell precursors can be used as a therapeutic agent for targeting an organ-agnostic cancer therapeutic to a specific tumor antigen. As a result, administration of such synthetically differentiated dendritic cell precursors as part of a pharmaceutical composition to a subject having cancer can be used to treat cancer without using any knowledge of the antigens expressed on the cancer cells.

[0021] Definitions The terms used herein are for the purpose of describing particular instances only and are not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, as long as the terms “including,” “comprising,” “having,” “with,” or variations thereof are used in any detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0022] The term “about” or “substantially” means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, such as ±10%. Where a range and / or sub-range of values is provided, the range and / or sub-range is inclusive of the endpoints of the range and / or sub-range.

[0023] As used herein, the term “substantially” means a value that is close to 100% of a given value. For example, an expression system described herein that “substantially” does not express a transgene in the absence of an inducer can be shown to express less than 10% (e.g., less than 5%, less than 1%, less than 0.1%, or less than 0.01%) of the transgene as compared to the amount of transgene expressed in the presence of the inducer.

[0024] The terms "subject", "individual", or "patient" can be used interchangeably herein. "Subject" means a plant, an animal, or a microorganism such as, for example, bacteria, viruses, fungi, and protozoa. The subject can be a mammal. Mammals include, but are not limited to, non-human primates such as humans, chimpanzees, apes or other monkey species; livestock such as cows, horses, sheep, goats, pigs; pet animals such as rabbits, dogs (or Canidae), and cats (or Felidae); and any member of the Mammalian class including experimental animals such as rodents like rats, mice and guinea pigs.

[0025] The terms "host" and "donor" are used interchangeably herein to mean the organism from which the progenitor cells are isolated. The host can be a mammal as described herein. When isolating progenitor cells from a "host" and differentiating them into a therapeutic agent for administration to a "subject", the host and the subject do not need to be of the same class, genus, or species of animal.

[0026] The term "in vivo" means an event that occurs within the body of a subject.

[0027] The term "in vitro" means an event that occurs outside the body of a subject. An in vitro assay can include cell-based assays that can utilize viable or dead cells. An in vitro assay can also include cell-free assays that cannot utilize intact cells.

[0028] Dendritic cell precursor Synthetically partially differentiated dendritic cell precursors from host precursor cells are disclosed herein. As disclosed herein, host precursor cells include precursor cells derived from a host that are capable of partial or complete differentiation. In some examples, host precursor cells can be isolated from a host such as a mammal. In some embodiments, the precursor cells can be precursor cells isolated from bone marrow or blood, such as hematopoietic stem cells or progenitor cells. Examples of hematopoietic progenitor cells include hematopoietic stem cells, pluripotent progenitor cells, and myeloid and lymphoid progenitor cells. In some embodiments, the precursor cells can be dedifferentiated cells such as induced pluripotent stem cells or neural progenitor cells.

[0029] As disclosed herein, synthetically partially differentiated dendritic cell precursors can be prepared from host precursor cells by contacting the host precursor cells with a synthetic medium to induce partial differentiation. In some embodiments, the resulting synthetically partially differentiated dendritic cell precursors are capable of further differentiation into dendritic cells (i.e., the dendritic precursors are not fully differentiated).

[0030] Host precursor cells comprising human precursor cells are also disclosed herein. In some embodiments, the human precursor cells can be isolated from bone marrow or blood, such as cord blood CD34 + progenitor cells. In some embodiments, the human precursor cells are capable of undergoing partial differentiation into dendritic cell precursors. Thus, in some embodiments, dendritic cell precursors can be prepared by contacting human precursor cells (e.g., CD34 + progenitor cells) with a synthetic medium as described herein to induce partial differentiation. In some embodiments, dendritic cell precursors derived from human precursor cells are capable of undergoing in vitro differentiation into antigen-presenting cells (APCs), cDC2, monocytes, immature dendritic cells, or combinations thereof.

[0031] The synthetic medium for differentiation can contain an effective amount of effector sufficient to induce partial differentiation of the host progenitor cells. In some embodiments, the synthetic medium is at least about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 51 ng / mL, 52 ng / mL, 53 ng / mL, 54 ng / mL, 55 ng / mL, 56 ng / mL, 57 ng / mL, 58 ng / mL, 59 ng / mL, 60 ng / mL, 61 ng / mL, 62 ng / mL, 63 ng / mL, 64 ng / mL, 65 ng / mL, 66 ng / mL, 67 ng / mL, 68 ng / mL, 69 ng / mL, 70 ng / mL, 71 ng / mL, 72 ng / mL, 73 ng / mL, 74 ng / mL, 75 ng / mL, 76 ng / mL, 77 ng / mL, 78 ng / mL, 79 ng / mL, 80 ng / mL, 81 ng / mL, 82 ng / mL, 83 ng / mL, 84 ng / mL, 85 ng / mL, 86 ng / mL, 87 ng / mL, 88 ng / mL, 89 ng / mL, 90 ng / mL, 91 ng / mL, 92 ng / mL, 93 ng / mL, 94 ng / mL, 95 ng / mL, 96 ng / mL, 97 ng / mL, 98 ng / mL, 99 ng / mL, 100 ng / mL, 101 ng / mL, 102 ng / mL, 103 ng / mL, 104 ng / mL, 105 ng / mL, 106 ng / mL, 107 ng / mL, 108 ng / mL, 109 ng / mL, 110 ng / mL, 111 ng / mL, 112 ng / mL, 113 ng / mL, 114 ng / mL,115 ng / mL, 116 ng / mL, 117 ng / mL, 118 ng / mL, 119 ng / mL, 120 ng / mL, 121 ng / mL, 122 ng / mL, 123 ng / mL, 124 ng / mL, 125 ng / mL, 126 ng / mL, 127 ng / mL, 128 ng / mL, 129 ng / mL, 130 ng / mL, 131 ng / mL, 132 ng / mL, 133 ng / mL, 134 ng / mL, 135 ng / mL, 136 ng / mL, 137 ng / mL, 138 ng / mL, 139 ng / mL, 140 ng / mL, 141 ng / mL, 142 ng / mL, 143 ng / mL, 144 ng / mL, 145 ng / mL, 146 ng / mL, 147 ng / mL, 148 ng / mL, 149 ng / mL, 150 ng / mL, 151 ng / mL, 152 ng / mL, 153 ng / mL, 154 ng / mL, 155 ng / mL, 156 ng / mL, 157 ng / mL, 158 ng / mL, 159 ng / mL, 160 ng / mL, 161 ng / mL, 162 ng / mL, 163 ng / mL, 164 ng / mL, 165 ng / mL, 166 ng / mL, 167 ng / mL, 168 ng / mL, 169 ng / mL, 170 ng / mL, 171 ng / mL, 172 ng / mL, 173 ng / mL, 174 ng / mL, 175 ng / mL, 176 ng / mL, 177 ng / mL, 178 ng / mL, 179 ng / mL, 180 ng / mL, 181 ng / mL, 182 ng / mL, 183 ng / mL, 184 ng / mL, 185 ng / mL, 186 ng / mL, 187 ng / mL, 188 ng / mL, 189 ng / mL, 190 ng / mL, 191 ng / mL, 192 ng / mL, 193 ng / mL, 194 ng / mL, 195 ng / mL, 196 ng / mL, 197 ng / mL, 198 ng / mL, 199 ng / mL, 200 ng / mL, 205 ng / mL, 210 ng / mL, 215 ng / mL, 220 ng / mL, 225 ng / mL, 230 ng / mL, 235 ng / mL, 240 ng / mL, 245 ng / mL, 250 ng / mL, 255 ng / mL, 260 ng / mL, 265 ng / mL, 270 ng / mL, 275 ng / mL, 280 ng / mL, 285 ng / mL, 290 ng / mL, 295 ng / mL, 300 ng / mL, 305 ng / mL, 310 ng / mL, 315 ng / mL, 320 ng / mL, 325 ng / mL330 ng / mL, 335 ng / mL, 340 ng / mL, 345 ng / mL, 350 ng / mL, 355 ng / mL, 360 ng / mL, 365 ng / mL, 370 ng / mL, 375 ng / mL, 380 ng / mL, 385 ng / mL, 390 ng / mL, 395 ng / mL, 400 ng / mL, 405 ng / mL, 410 ng / mL, 415 ng / mL, 420 ng / mL, 425 ng / mL, 430 ng / mL, 435 ng / mL, 440 ng / mL, 445 ng / mL, 450 ng / mL, 455 ng / mL, 460 ng / mL, 465 ng / mL, 470 ng / mL, 475 ng / mL, 480 ng / mL, 485 ng / mL, 490 ng / mL, 495 ng / mL, 500 ng / mL, 505 ng / mL, 510 ng / mL, 515 ng / mL, 520 ng / mL, 525 ng / mL, 530 ng / mL, 535 ng / mL, 540 ng / mL, 545 ng / mL, 550 ng / mL, 555 ng / mL, 560 ng / mL, 565 ng / mL, 570 ng / mL, 575 ng / mL, 580 ng / mL, 585 ng / mL, 590 ng / mL, 595 ng / mL, 600 ng / mL, 605 ng / mL, 610 ng / mL, 615 ng / mL, 620 ng / mL, 625 ng / mL, 630 ng / mL, 635 ng / mL, 640 ng / mL, 645 ng / mL, 650 ng / mL, 655 ng / mL, 660 ng / mL, 665 ng / mL, 670 ng / mL, 675 ng / mL, 680 ng / mL, 685 ng / mL, 690 ng / mL, 695 ng / mL, 700 ng / mL, 705 ng / mL, 710 ng / mL, 715 ng / mL, 720 ng / mL, 725 ng / mL, 730 ng / mL, 735 ng / mL, 740 ng / mL, 745 ng / mL, 750 ng / mL, 755 ng / mL, 760 ng / mL, 765 ng / mL, 770 ng / mL, 775 ng / mL, 780 ng / mL, 785 ng / mL, 790 ng / mL, 795 ng / mL, 800 ng / mL, 805 ng / mL, 810 ng / mL, 815 ng / mL, 820 ng / mL, 825 ng / mL, 830 ng / mL, 835 ng / mL, 840 ng / mL, 845 ng / mL, 850 ng / mL, 855 ng / mL, 860 ng / mL, 865 ng / mL, 870 ng / mL, 875 ng / mL, 880 ng / mL,It contains an effector at 885 ng / mL, 890 ng / mL, 895 ng / mL, 900 ng / mL, 905 ng / mL, 910 ng / mL, 915 ng / mL, 920 ng / mL, 925 ng / mL, 930 ng / mL, 935 ng / mL, 940 ng / mL, 945 ng / mL, 950 ng / mL, 955 ng / mL, 960 ng / mL, 965 ng / mL, 970 ng / mL, 975 ng / mL, 980 ng / mL, 985 ng / mL, 990 ng / mL, 995 ng / mL, or 1000 ng / mL.

[0032] In some embodiments, the synthetic medium can contain a mixture of effectors. For example, the synthetic medium can contain at least 2, 3, 4, 5, 6, 7, 8, 9, or more than 10 effectors. In some examples, the effector can be a cytokine. Examples of cytokines include IL-1, TNF-α, TPO, SCF, IL-3, IL-6, IL-12, IL-4, CXCL8, FLT3L, GM-CSF, IFNa, PGE2, Retronectin, UM729, and G-CSF. In some embodiments, the synthetic medium contains a mixture of GM-CSF and FLT3L. In some embodiments, IFNa is IFNa2b. In some embodiments, the synthetic medium contains a mixture of GM-CSF, FLT3L, SCF, and IFNa2b. In some embodiments, the synthetic medium does not contain UM729.

[0033] Synthetically partially differentiated dendritic cell precursors differentiated from host precursor cells using a synthetic medium as described herein are different from naturally occurring dendritic precursors or mature dendritic cells. For example, synthetically partially differentiated dendritic cell precursors can differentiate into mature dendritic cells (e.g., cDC1 or cDC2) or immature dendritic cells under conditions where naturally occurring dendritic cells cannot differentiate. For example, synthetically partially differentiated dendritic cell precursors can differentiate into mature dendritic cells in the presence of inflammatory or immunosuppressive cytokines such as those secreted by tumors. For example, synthetically partially differentiated dendritic cell precursors derived from human precursor cells can differentiate into antigen-presenting cells (APCs), cDC2, monocytes, immature dendritic cells, or combinations thereof. Thus, in some embodiments, synthetically partially differentiated dendritic cell precursors can undergo differentiation into APCs, cDC2, monocytes, immature dendritic cells, or combinations thereof in the presence of inflammatory or immunosuppressive cytokines such as those secreted by tumors.

[0034] Synthetically partially differentiated dendritic cell precursors as disclosed herein can exhibit a specific phenotype that is different from naturally occurring dendritic cell precursors. For example, synthetically partially differentiated dendritic cell precursors can have a flow cytometry phenotype that is one or more of CD115 + , CD11b-neg, CD11c-neg, MHCII-neg, CD45R / B220-neg, cKIT-neg / low, and Clec9A-neg. Alternatively, in some embodiments, synthetically partially differentiated dendritic cell precursors can have a flow cytometry phenotype that is one or more of CD3 - , CD19 - , CD335 - , CD66b - , CD10 - , CD14 - , CD34 + , and CD115 + .

[0035] In some embodiments, the synthetically partially differentiated dendritic cell precursors can be genetically engineered dendritic cell precursors. For example, the synthetically partially differentiated dendritic cell precursors can be genetically engineered to co-express a transgene that, when expressed, works in concert with dendritic cells differentiated from the synthetically partially differentiated dendritic cell precursors to activate the subject's immune system. For example, genetically engineered dendritic cell precursors as described herein can be genetically engineered to co-express an interleukin, an effector, or both. Interleukins that can be co-expressed in the genetically engineered dendritic cell precursors include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40. In some embodiments, the interleukins described herein include interleukins derived from the same species as the host. For example, the interleukins described herein can include human interleukins when the dendritic cell precursors are derived from human cells. Effectors that can be co-expressed in the genetically engineered dendritic cell precursors include internalization receptors such as extracellular vesicle internalization receptor (EVIR); or cytokines such as IL-1, TNF-α, IL-6, IL-12, IL-2, CXCL8, FLT3L, GM-CSF, IFNa, PGE2, retronectin, and G-CSF. In some embodiments, the effector includes an effector derived from the same species as the dendritic cell precursor. For example, the effectors described herein can include human effectors when the dendritic cell precursors are derived from human cells.

[0036] Synthetically partially differentiated dendritic cell precursors can be included in an in vitro cell composition. In some examples, the in vitro cell composition can be used to prepare functional mature dendritic cells (e.g., antigen-presenting cells (APCs), monocytes, immature dendritic cells, cDC1 or cDC2) for use as a therapeutic agent in vitro. In some examples, the in vitro cell composition can be included in a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient, diluent, or carrier. In some embodiments, the pharmaceutical formulation can comprise an excipient. Excipients include those described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986). In some embodiments, excipients include buffers, preservatives, stabilizers, binders, compressants, lubricants, chelating agents, dispersion enhancers, disintegrants, flavoring agents, sweetening agents, coloring agents. Diluents include water; glycerol; methanol; ethanol; aqueous acids such as acetic acid, citric acid, maleic acid, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, etc.; alkali metal phosphates such as calcium phosphate; alkali metal sulfates such as calcium sulfate; alkali metal carbonates such as calcium carbonate; cellulose derivatives such as cellulose, microcrystalline cellulose, cellulose acetate, mannitol, fructose, dextrose, magnesium oxide, dextrin, glyceryl palmitostearate, kaolin, lactose, maltose, simethicone, sorbitol, starch, pregelatinized starch, talc, lactitol, xylitol; and / or their anhydrides, hydrates and / or pharmaceutically acceptable derivatives or combinations thereof.

[0037] A pharmaceutical composition containing an in vitro cell composition as described herein can be administered to a subject for treating the conditions described herein. In some embodiments, the pharmaceutical composition can further comprise an interleukin, an effector, or both. Interleukins that can be included in the pharmaceutical composition include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40. In some embodiments, the interleukin described herein includes an interleukin derived from the same species as the host. For example, the interleukin described herein can include human interleukin when the dendritic cell precursor is derived from human cells. Effectors that can be included in the pharmaceutical composition include internalization receptors such as extracellular vesicle internalization receptor (EVIR); or cytokines such as IL-1, TNF-α, IL-6, IL-12, IL-2, CXCL8, FLT3L, IFNa, and GM-CSF. In some embodiments, the effector includes an effector derived from the same species as the dendritic cell precursor. For example, the effector described herein can include a human effector when the dendritic cell precursor is derived from human cells.

[0038] Method for producing dendritic precursor cells Methods for generating synthetically partially differentiated dendritic cell precursors are also disclosed herein. When disclosed herein, synthetically partially differentiated dendritic cell precursors can be prepared by contacting host precursor cells with a synthetic culture medium as described herein. In some embodiments, host precursor cells can be expanded prior to the step of contacting with the synthetic culture medium. Expansion can include culturing host precursor cells isolated from a host-derived sample (e.g., bone marrow or blood) in an expansion culture medium. Such an expansion culture medium can include 10% FBS, 1% L-glutamine, 100 ng / mL SCF, 40 ng / mL TPO, 50 ng / mL FLT3L, 30 ng / mL IL-3, 30 ng / mL IL-6, and 30 ng / mL IL-1b. Similarly, expansion of human precursor cells (e.g., cord blood CD34 + precursor cells) isolated from a human-derived sample can include culturing the human precursor cells in an expansion culture medium that includes FLT3L, SCF, IL3, IL6, and TPO.

[0039] Partial differentiation of host precursor cells (e.g., expanded hematopoietic stem / precursor cells) into synthetically partially differentiated dendritic cell precursors can be accomplished by culturing the host precursor cells with a synthetic culture medium (e.g., a medium that includes cytokines or effectors) as described herein. In some examples, partial differentiation can occur over a period of at least 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, at least 120 hours, at least 144 hours, at least 168 hours, at least 192 hours, at least 216 hours, or at least 240 hours. After differentiation, the cells can be selected and isolated based on phenotype to generate purified synthetically partially differentiated dendritic cell precursors.

[0040] Therapeutic methods Also disclosed herein are methods of treating a condition in a subject in need thereof and pharmaceutical compositions for use in treating a condition. In some embodiments, the treatment method can include administering to the subject a synthetically partially differentiated dendritic cell precursor as described herein, an in vitro cell composition containing a synthetically partially differentiated dendritic cell precursor as described herein, or a pharmaceutical composition containing a synthetically partially differentiated dendritic cell precursor as described herein. In some embodiments, the treatment method can include administering to the subject a mature dendritic cell differentiated (differentiated in vitro) from a synthetically partially differentiated dendritic cell precursor as described herein, or an in vitro cell composition or pharmaceutical composition containing a mature dendritic cell differentiated from a synthetically partially differentiated dendritic cell precursor as described herein.

[0041] Administration to a subject can include administration by inhalation, into the ear, into the cheek, into the conjunctiva, dental, endocervical, endosinusial, intratracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intra-amniotic, intra-arterial, intra-articular, intra-biliary, intra-bronchial, intra-bursal, intracardiac, intra-cartilaginous, intradural, intramedullary, intracapsular, intra-corneal, intracoronal, intra-coronary, intracorpous cavernaosum, intradermal, intra-laminar, intraductal, intra-duodenal, intradural, intra-epidermal, intra-esophageal, intra-gastric, intra-gingival, intra-hippocampal, intra-ileal, intralesional, intraluminal, intra-lymphatic, intramedullary, intrathecal, intramuscular, intra-ocular, intra-ovarian, intra-pericardial, intraperitoneal, intra-pleural, intra-prostatic, intra-pulmonary, intrasinal, intraspinal, intra-synovial, intratendinous, intra-testicular, intrathoracic, intra-tubular, intra-tumoral, intra-tympanic, intra-uterine, intra-vascular, intra-venous, intravenous bolus, intravenous infusion, intravesical, intravitreal, iontophoresis, perfusion, laryngeal, intranasal, nasogastric, ophthalmic, oral, oro-pharyngeal, parenteral, percutaneous, peridural, perineural, periodontal, rectal, retrobulbar, sub-arachnoid, sub-conjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, tracheal, transtympanic, ureteral, urethral, vaginal, sub-orbital, parenchymal, intramedullary, intracerebroventricular, stereotactic, or any combination thereof.

[0042] In some embodiments, the treatment method can include the treatment of cancer in a subject. Examples of cancers include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone tumors, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, pediatric optic pathway / hypothalamic glioma, Hodgkin lymphoma, melanoma, islet cell carcinoma, Kaposi sarcoma, renal cell cancer, laryngeal cancer, leukemia, lymphoma, mesothelioma, neuroblastoma, non-Hodgkin lymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasma cell neoplasm, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer, and uterine cancer.

[0043] In some embodiments, the administration is sufficient to reduce the number and / or size of cancer cells. For example, if the cancer is a solid tumor cancer, the administration can result in a reduction in tumor size and / or inhibition of tumor development.

[0044] The method of treating cancer does not necessarily require knowledge of tumor antigens. In some embodiments, the administration of a composition containing synthetically partially differentiated dendritic cells or mature dendritic cells differentiated therefrom does not require the administration or co-expression of antigens expressed on the cells of the cancer. Rather, the administration of a composition containing synthetically partially differentiated dendritic cells or mature dendritic cells differentiated therefrom provides a targeted cross-treatment of cancer. In some embodiments, the effector is not expressed on the cells of the cancer.

Example

[0045] For a better understanding of the present disclosure and its numerous advantages, the following examples are given by way of illustration and without limiting the scope of the present disclosure.

[0046] Example 1: Generation of dendritic cell precursors (DCPs) from mouse hematopoietic cells Dendritic precursor cells (DCPs) were prepared from mouse hematopoietic stem / progenitor cells using a two-step protocol (Figure 1A).

[0047] Step 1: Expansion and proliferation of hematopoietic stem / progenitor cells The expansion and proliferation step uses a medium that supports the maintenance and expansion of hematopoietic stem / progenitor cells (HSPCs). This medium contains RPMI 1640 medium (referred to as complete RPMI medium) supplemented with 10% FBS, 1% L-glutamine, 1% penicillin-streptomycin, with 100 ng / mL SCF, 40 ng / mL TPO, 50 ng / mL FLT3L, 30 ng / mL IL-3, 30 ng / mL IL-6, and 30 ng / mL IL-1β added.

[0048] Total mouse bone marrow (BM) cells were isolated from the long bones of C57BL / 6 mice and depleted of red blood cells (RBCs) by incubation in 5 - 10 mL of RBC lysis buffer (catalog number R7767-100ML) for 5 minutes. Subsequently, the BM cells were passed through a 70 μm cell strainer (catalog number 352350), washed in complete RPMI medium, and resuspended and plated (1 - 3×10 6 cells / mL) in the above HSPC medium. The cells were cultured in HSPC medium for 2 days. Subsequently, the floating cells were collected and replated at the same density in HSPC medium for an additional 1 day to further remove the remaining adherent cells.

[0049] Step 2: Differentiation of HSCPs into DCPs Floating HSPCs (end of day 3 of step 1) were harvested and washed once in complete RPMI medium. Subsequently, the harvested cells were cultured in a medium that supports cDC1 differentiation. This medium contains complete RPMI medium supplemented with 200 ng / mL FLT3L and 5 ng / mL GM-CSF. The cells were plated at a density of 1 - 3×10 6 cells / mL. After 3 days of differentiation in cDC1 medium, an equal volume of cDC1 medium was added to each well. After an additional 2 days (end of day 5 of step 2), the cell cultures contained 20 - 50% of cells designated as DCP. Subsequently, major lineage positive cells (CD5 + , CD45R / B220 + , CD11b + , CD19 + , Ly6C / G + , TER119 + ) were depleted through negative selection, enabling further enrichment of DCP. The final purity of DCP after negative selection at the end of the protocol (end of day 8 combining steps 1 and 2) was 60 - 90% (Figure 1B).

[0050] After enrichment, DCP exhibited the following phenotype by flow cytometry analysis: CD115 + , CD11b-neg, CD11c-neg, MHCII-neg, CD45R / B220-neg, cKIT-neg / low, and Clec9A-neg (Figure 1C). These DCPs are different from common dendritic cell precursors (CDP) as CDP expresses Clec9A, and different from pre-cDC1 as pre-cDC1 expresses Clec9A and CD11c.

[0051] Thus, this example demonstrates a simple protocol for the efficient generation and enrichment of DCP from mouse BM.

[0052] Example 2: Excellent in vivo differentiation of DCP into conventional type I and type II DCs (cDC1 and cDC2) Next, the ability of different types of DCs to form cDC1 in tumor-free mice was investigated (Figures 2A - 2K).

[0053] To enable tracking of donor-derived cells in congenic CD45.2 C57BL / 6 mice, BM cells were isolated from CD45.1 mice. Different types of DCs were generated by differentiating mouse BM cells in different differentiation media (Figure 2A). Three types of DCs were compared: (i) Monocyte-derived dendritic cells (moDCs) were generated using an established protocol that included incubating BM cells in complete RPMI medium supplemented with 100 ng / mL GM-CSF and 40 ng / mL IL4 (referred to as moDC medium). BM cells were cultured at a density of 2 - 3×10 6 cells / mL in moDC medium for 2 days. Subsequently, non-adherent and weakly adherent cells were collected and re-plated at a density of 2 - 3×10 6 / mL and cultured for an additional 6 days in moDC medium (with one addition of fresh moDC medium). Figures 2B and 2K show the phenotype of the moDC culture at the end of the differentiation protocol (end of day 8; note that moDC cultures may contain some macrophages) and prior to injection into recipient CD45.2 mice. Figures 2B and 2K illustrate two gating strategies; Figure 2K identifies MHCII-high moDCs, and Figure 2B shows MHCII + moDCs with MHCII levels ranging from low to high. (ii) Conventional type I DCs (cDC1s) are professional antigen-presenting cells, and their important role in anti-tumor immunity has been demonstrated in recent years. Thus, cDC1 cells can function as a novel DC type for vaccination applications. cDC1 cells were generated. Briefly, BM cells were directly plated at a density of 2×10 6 cells / mL in cDC1 medium. Cells were cultured for a total of 16 days by adding 1 mL of fresh cDC1 medium every 3 - 4 days. Figure 2C shows the phenotype of the cDC1 cells at the end of the differentiation protocol (end of day 16) and prior to injection into recipient CD45.2 mice. (iii) DCP was generated as shown in Example 1. Figure 2D shows the phenotype of DCP at the end of the protocol (end of day 8) and prior to injection into recipient CD45.2 mice.

[0054] Each type of DC prepared as described above was injected systemically (via the tail vein) into syngeneic CD45.2 mice without any prior conditioning of the mice. 2×10 6 Two doses of DC of cells (in 200 μL of PBS) were administered at 3-day intervals, and recipient mice (4 mice per condition) were sacrificed 4 days after the second DC injection, and the phenotype of donor-derived CD45.1 + cells was analyzed (Figure 2A). Control mice (3 mice) were administered PBS. The spleen was thoroughly disrupted on a 70 μm cell strainer, and RBCs were depleted using RBC lysis buffer; subsequently, the cells were washed in PBS containing 10% FBS and 2 mM EDTA prior to analysis. The data showed substantial chimerism among cDCs (both cDC1 and cDC2; more than 15% of the donor-derived cells were cDC1) in mice administered DCP, but negligible amounts of donor-derived cDCs were seen in mice injected with moDC or cDC1 cells (Figures 2E - 2F). In addition to substantial cDC chimerism after DCP injection, DCP-treated mice had a higher frequency of donor-derived cells (Figure 2G), indicating the superior longevity of the injected DCP compared to other types of DC.

[0055] CD45.1 + Analysis of splenocytes revealed that moDC-derived cells did not contain cDC or double-negative (DN; CD11b-neg, CD11c + , MHC-II + , CD8a-neg) immature DCs (Figure 2H). Also, only a minority of cDC1-derived cells were either cDC1, cDC2, or DN DCs (Figure 2I). Importantly, the majority of the induced DCP-derived cells were cDC1, cDC2, or DN DCs (Figure 2J).

[0056] Statistical analysis: One-way ANOVA using Tukey's multiple comparison test for (e - g). P-values are coded as follows: * : P < 0.05; ** : P < 0.01; *** : P < 0.001; and **** : P < 0.0001.

[0057] Therefore, this example demonstrates that DCP can efficiently generate cDC1, cDC2, and immature DCs in mice, while conventional moDCs or mature cDC1s cannot.

[0058] Example 3: DCP differentiates into intratumoral cDC1 and cDC2 after systemic injection Example 2 showed that, unlike moDCs and mature cDC1s, DCP can efficiently differentiate into cDC1 and cDC2 after systemic injection in tumor-free mice. The ability of DCP to differentiate in the presence of inflammation and upregulation of immunosuppressive cytokines in tumor-bearing mice. Subsequently, DCP was injected into tumor-bearing mice to study the migration and phenotype of donor-derived cells in both the spleen and tumor.

[0059] MC38 cancer cells (5×10 5 cells) were injected into the right flank of CD45.2 mice (8 mice per condition). Subsequently, tumor-bearing mice were injected with DCP generated and enriched according to the 2-step protocol shown in Example 1. Enriched DCP (2×10 6 cells in 200 μL of PBS) was injected systemically via the tail vein on both the 5th and 8th days after tumor injection. Control mice (8 mice) were administered PBS. Mice were sacrificed 4 days after the second DCP injection. The spleen and tumor of each mouse were analyzed for donor-derived (CD45.1 +)Analysis was performed regarding the presence of cells. Figure 3A shows the workflow of the experiment. At the end of the experiment, the spleen was thoroughly disrupted on a 70 μm cell strainer, and RBCs were depleted using RBS lysis buffer; subsequently, the cells were washed in PBS containing 10% FBS and 2 mM EDTA before analysis. Tumor-derived single cells were isolated using conventional enzymatic digestion.

[0060] Figure 3B shows the gating strategy regarding the identification of donor-derived cDC1 and cDC2 within the tumor. Flow cytometry analysis of tumor-derived cells and splenocytes revealed that donor CD45.1 + cells infiltrated the tumor and spleen after systemic injection (Figure 3C) and efficiently differentiated into cDCs (Figures 3D–3E and 3H–3I). In both the tumor and spleen, approximately half of all cDC1s were donor-derived (CD45.1 + ), indicating substantial chimerism of the majority of professional antigen-presenting cells after DCP injection in naïve mice (Figure 3F–3G). The chimerism of cDC2 was relatively low but was readily detectable in all mice.

[0061] Therefore, this example demonstrates that DCP efficiently generates cDC1 and cDC2 in the tumors and spleens of tumor-bearing mice without prior conditioning of the mice.

[0062] Example 4: Identification of Cytokines That Enable Differentiation of DCPs into Co-stimulatory cDC1 Cytokines were screened in an in vitro screening study regarding the ability to maintain the potential for differentiation of DCPs into cDC1 while enabling the T cell co-stimulatory ability of the cDC1 obtained during co-culture with T cells.

[0063] To study differentiation, DCPs were cultured in cDC1 medium supplemented with interleukin (IL) selected at different concentrations. To study T cell co-stimulation, the same cytokines were used during co-culture of antigen-loaded cDC1-like cells and antigen-specific T cells.

[0064] DCP was generated as described in Example 1. The enriched DCP was cultured in cDC1 medium supplemented with various ILs (IL-2, IL-12, IL-15, IL-18, IL-21, IL-23, or IL-27) at the indicated concentrations in the range of 2 - 20 ng / mL. 1 mL of IL-supplemented cDC1 medium was added to DCP every 3 - 4 days for 15 days (Figure 4A). After 15 days, cells were analyzed for the presence of cDC1 (identified as + CD103 + ), and compared to DCP cultured in cDC1 medium without IL. Flow cytometry analysis demonstrated that IL-18 and IL-21 induced early activation of DCP (indicated by an increased proportion of CD86 + CD103-neg cells), and impaired the differentiation of DCP into cDC1 (indicated by a decreased proportion of CD103 + CD86-neg cells), while the other ILs (IL-2, IL-12, IL-15, IL-23 and IL-27) did not impair the differentiation of DCP into cDC1 (Figures 4B - 4C).

[0065] To study the effect of the above-mentioned ILs on T cell polarization, subsequently, OVA-added cDC1-like cells were co-cultured with OVA-specific CD8 + (OT-I) or CD4 + (OT-II) T cells in the presence of the selected ILs (3 independent replicates per condition). Briefly, 50,000 cDC1 were seeded into U-bottom 96-well plates with 0.5 mg OVA protein (vac-stova) or OVA peptide SIINFEKL for 4 hours. The cDC1 were washed and then co-cultured with OT-I cells for 3 days or with OT-II cells for 5 days. Intracellular staining with antibodies against interferon-γ (IFNγ) was used to measure T cell activation according to the manufacturer's protocol with the BD Golgi Stop kit (Catalog number 554715). The results showed that IL-12 could induce robust IFNγ production by both OT-I and OT-II T cells (Figures 4D - 4F).

[0066] Statistical analysis: One-way ANOVA using Tukey's multiple comparison test for (d - e). All samples are compared to the "untreated" sample. P-values are coded as follows: * : P < 0.05; ** : P < 0.01; *** : P < 0.001; and **** : P < 0.0001.

[0067] Therefore, this example demonstrates that IL-12 does not impair the differentiation of DCPs into cDC1 and enhances the co-stimulatory ability of cDC1 progeny in vitro.

[0068] Example 5: DCPs genetically engineered to express IL-12 and IL-2 differentiate into cDCs in mice As shown in Example 4, exogenous IL-12 and IL-2 did not prevent the differentiation of DCPs into cDC1s in vitro; furthermore, IL-12 enhanced the co-stimulatory ability of cDC1-like cells. Therefore, the transgenic expression of either IL-12 or IL-2 was investigated for its ability to support the differentiation of DCPs into cDC1s and cDC2s in vivo. For this purpose, lentiviral vectors (LVs) expressing either IL-12 or IL-2 together with the green fluorescent protein (GFP, SEQ ID NO: 1), a marker gene, named IL12-P2A-GFP (SEQ ID NO: 5) LV and IL2-P2A-GFP (SEQ ID NO: 3) LV, respectively, were generated. A monocistronic LV expressing only GFP, named GFP LV, was also generated. Flow cytometric analysis of mouse BM cells transduced with LV at a multiplicity of infection (MOI) of 350 in HSPC medium and subsequently differentiated in cDC1 medium showed robust expression of GFP 5 days after transduction, at which point the transduced cells mainly contained DCPs (Figure 5A; differences in GFP expression between LVs may be due to their different infectivities affected by the size of the transgene). Furthermore, cells transduced with IL12-P2A-GFP LV and IL2-P2A-GFP LV secreted IL-12 and IL-2, respectively, into the cell culture medium when evaluated by ELISA 2 weeks after transduction, at which point the transduced cells mainly contained cDC1s (Figure 5B and Figure 5H; "C" indicates concentration). Therefore, LV enables robust transduction of DC precursors and sustained expression of either IL-12 or IL-2 in cDC progeny.

[0069] Next, the in vivo differentiation potential of untransduced DCPs (UT) and DCPs transduced with IL12-P2A-GFP LV, and IL2-P2A-GFP LV or GFP LV was investigated. DCPs were generated from the BM of CD45.1 mice as described in Example 1. The enriched DCPs were adjusted to 1.5×10 6Transferred to cDC1 medium at a concentration of cells / mL and simultaneously transduced with LV at an MOI of 350. The transduced DCPs were harvested 12 hours after transduction and injected into recipient mice (5 mice per condition). Each mouse was administered 2×10 6 DCPs; the recipient mice were sacrificed 4 days after injection of the DCPs, and splenocytes (isolated by grinding the spleen as described in Example 2) were analyzed for the presence of donor-derived (CD45.1 + ) cells (Figure 5C).

[0070] Flow cytometric analysis of splenocytes revealed that the majority of CD45.1 + cells expressed GFP (Figures 5D - 5E), indicating efficient LV transduction, transgene expression, and lack of counterselection of transduced cells in vivo (differences in GFP expression between LVs may be due to their different infectivities affected by the size of the transgene). Importantly, transduction of DCPs with IL-12 or IL-2 did not prevent their differentiation into cDC1 and cDC2 (Figures 5F - 5G), which was consistent with the in vitro studies shown in Example 4. However, IL-2 slightly enhanced cDC1 generation while IL-12 slightly enhanced cDC2 generation.

[0071] Statistical analysis: (f) One-way ANOVA using Tukey's multiple comparison test. P-values are coded as follows: * : P < 0.05; ** : P < 0.01; *** : P < 0.001; and **** : P < 0.0001.

[0072] Thus, this example demonstrates that transgenic expression of IL-12 does not impair the differentiation of DCPs into cDC1 in vivo.

[0073] Example 6: DCPs genetically engineered to express IL-12 and tumor antigens promote the expansion of tumor antigen-specific T cells in mice As shown in Examples 4 and 5, IL-2 and IL-12 do not prevent the differentiation of DCPs into cDC1s in vitro and in vivo. The in vitro data of Example 4 also show that IL-12 can activate both CD4 + and CD8 + T cells. To investigate whether IL-12 and IL-2 can activate antigen-specific T cells in vivo, either cytokine was co-expressed with the truncated intracellular OVA (dOVA, SEQ ID NO: 7) sequence in DCPs by LV transduction. For transduction, enriched DCPs were transferred into cDC1 medium at a concentration of 1.5×10 6 cells / mL and simultaneously transduced with an LV encoding GFP, dOVA, IL2-P2A-dOVA (SEQ ID NO: 9) or IL12-P2A-dOVA (SEQ ID NO: 11) at an MOI of 350. Cells were harvested 12 hours after transduction, and 0.7×10 6 transduced DCPs were injected into tumor-free mice. Figure 6A shows a schematic diagram of the experiment.

[0074] Transduced DCPs were injected in tumor-free mice (5 mice per condition). Recipient mice were sacrificed 10 days after vaccination, and splenocytes (isolated by grinding the spleen as described in Example 2) were stained with OVA 257-264 -conjugated dextramers according to the manufacturer's protocol (Immunex) to identify OVA-reactive T cells. Flow cytometry data demonstrated that expression of IL-12 could dramatically increase OVA-reactive T cells, with approximately 12.8% of all T cells in the spleen being OVA-reactive after a single DCP vaccination (Figure 6B). It should be noted that DCPs expressing dOVA alone and DCPs expressing dOVA together with IL-2 induced a much weaker OVA-specific response. To study the phenotype of OVA-reactive T cells in vaccinated mice, splenocytes were also stained with anti-CD44 and anti-CD62L antibodies. Flow cytometry analysis showed that IL12 effector (CD44 +It was revealed that the expansion and proliferation of CD62L-neg) OVA-reactive T cells were strongly enhanced (Figure 6C).

[0075] Statistical analysis: (b - c) One-way ANOVA using Tukey's multiple comparison test. P-values are encoded as follows: * : P < 0.05; ** : P < 0.01; *** : P < 0.001; and **** : P < 0.0001.

[0076] Therefore, this example demonstrates that DCP co-expressing IL-12 and tumor antigen increases tumor antigen-specific T cells in vivo.

[0077] Example 7: DCP genetically engineered to express IL-12 together with tumor antigen inhibits tumorigenesis in mice To study the antitumor ability of DCP_IL2-P2A-dOVA cells and DCP_IL12-P2A-dOVA cells, a prophylactic vaccination study was conducted.

[0078] As described in Example 6 above, transduced DCP was generated. Subsequently, both transduced DCP and untransduced DCP were intravenously injected into tumor-free mice (0.7 × 10 6 cells per mouse; 6 mice per condition). Subsequently, one week after vaccination, B16-OVA cancer cells (5 × 10 5 cells) were subcutaneously inoculated into the mice (Figure 7A).

[0079] Four days after tumor challenge, blood samples were collected from the mice and circulating T cells were analyzed by flow cytometry. Co-expression of IL-12 and dOVA increased effector T cells (CD44 + and CD8 + T cells in both) (Figure 7B - Figure 7C). Staining of T cells using OVA + conjugated dextramers showed that all circulating CD8 257-264 + ​Approximately one-third of the T cells were shown to be OVA-reactive after vaccination with DCP_IL12-P2A-dOVA (Figure 7D). Conversely, IL-2 stimulated a relatively weak immune response against OVA.

[0080] Vaccination of mice with DCP_IL12-P2A-dOVA cells completely prevented tumor growth (Figure 7E). Conversely, DCP_IL2-P2A-dOVA cells did not completely prevent tumor growth. Twenty-six days after vaccination (19 days after tumor challenge), the spleens of the mice were isolated and purified. Flow cytometry analysis showed robust expansion of OVA-reactive effector T cells in mice vaccinated with DCP_IL12-P2A-dOVA cells (Figures 7F-7G), which was consistent with the results obtained in tumor-free mice (see Example 6 above). Conversely, IL-2 stimulated a relatively weak immune response against OVA.

[0081] Statistical analysis: (B-D, F-G) One-way ANOVA using Tukey's multiple comparison test. The # symbol in f-g indicates a comparison between the two groups of interest using an unpaired Student's t test. P values are coded as follows: * : P < 0.05; ** : P < 0.01; *** : P < 0.001; and **** : P < 0.0001.

[0082] Thus, this example demonstrates that DCPs co-expressing IL-12 and tumor antigen increase tumor antigen-specific T cells and protect mice from tumor challenge.

[0083] Example 8: DCP Genetically Engineered to Express IL-12 with Tumor Antigen Inhibits Tumor Growth Due to its superior efficacy, DCP cells expressing IL12 were prepared and tested as DCP vaccination in a therapeutic setting including vaccination of tumor-bearing mice.

[0084] B16-OVA cancer cells (5×10 5 cells) were subcutaneously inoculated into mice, and one week after tumor challenge, transduced or untransduced DCP (0.7×10 6 cells; 6 mice per condition) were intravenously injected (Figure 8A).

[0085] DCP_IL12-P2A-dOVA cells significantly inhibited tumor growth (Figure 8B). Furthermore, as shown by staining immune cells with OVA 257-264 -binding dextramers, DCP_IL12-P2A-dOVA cells increased OVA-reactive T cells in the blood, spleen, tumor-draining lymph nodes (tdLNs), and tumors of vaccinated mice (Figures 8C - 8F). Notably, in the tumors of mice vaccinated with DCP_IL12-P2A-dOVA cells, on average, approximately half of all CD8 + T cells were OVA-reactive, which is consistent with the strength of the observed anti-tumor response.

[0086] Statistical analysis: (b) Two-way ANOVA using Sidak's multiple comparison test. (c - f) Unpaired Student's t-test. P-values are coded as follows: * : P < 0.05; ** : P < 0.01; *** : P < 0.001; and **** : P < 0.0001.

[0087] Thus, this example demonstrates that DCP co-expressing IL-12 and tumor antigen increases tumor antigen-specific T cells and inhibits tumor growth.

[0088] Example 9: DCP genetically engineered to express IL-12 together with EVIR enables tumor antigen cross-presentation vaccination and inhibits tumor growth The studies shown in Examples 7-8 above used DCPs transduced with IL-12 along with defined surrogate tumor antigen (dOVA) to vaccinate mice bearing OVA-expressing tumors. Next, in a tumor antigen cross-sectional manner, i.e., without compelling the expression of any tumor antigen by DCP, the ability of DCPs transduced with IL-12 to elicit anti-tumor immunity was investigated.

[0089] The extracellular vesicle (EV) internalization receptor (EVIR) was used with an extracellular scFv domain targeting GD2, a disialogangloside expressed on the plasma membrane of both mouse and human melanomas and on their secreted EVs. Unlike conventional DC vaccination against defined tumor antigens, which involves in vitro antigen addition to DCs and elicits a T cell response only to the targeted (added) antigen, vaccination with DCs expressing EVIR can elicit a T cell response that may target any EV-associated tumor antigen. Notably, vaccination with DCs expressing EVIR does not require prior knowledge of tumor antigens, except for molecules (e.g., GD2) used to capture tumor EVs in vivo.

[0090] A bicistronic IL12-P2A-EVIR (SEQ ID NO: 17) LV for expressing both IL-12 and EVIR in DCP was generated. An IL12-P2A-dLNGFR (SEQ ID NO: 15) LV expressing IL-12 together with a control non-signaling receptor (truncated low-affinity nerve growth factor receptor, dLNGFR, SEQ ID NO: 13) lacking the extracellular scFv domain of EVIR was also generated. Subsequently, vaccination studies were conducted in tumor-bearing mice (Figure 9A). The same procedures as shown in Examples 5-8 above were used for the generation and transduction of DCP (DCP_IL12-P2A-EVIR or DCP_IL12-P2A-dLNGFR). Mice (8 mice per condition) were subcutaneously inoculated with B16-OVA-GD2 cancer cells (5×10 5 cells), and transduced DCP (1×10 6Cells) or PBS (as a mock treatment) were intravenously injected. To induce effector function of T cells at the time point shown in Fig. 9A, the mice were also treated with an anti-PD1 antibody.

[0091] Vaccination with DCP_IL12-P2A-EVIR cells resulted in significantly better tumor control than vaccination with DCP_IL12-P2A-dLNGFR cells lacking functional EVIR (Fig. 9B); it should be noted that this result was achieved without using OVA vaccination. Subsequently, the mice were sacrificed and the tumors were analyzed by flow cytometry. Flow cytometry analysis revealed that vaccination with DCP_IL12-P2A-EVIR cells led to an increase in tumor infiltration by activated CD4 + and CD8 + T cells compared to vaccination with DCP_IL12-P2A-dLNGFR lacking functional EVIR (Figs. 9C - 9F).

[0092] A mixture of B16 (1×10 5 cells) melanoma cells and B16-OVA-GD2 (3×10 5 cells) melanoma cells was subcutaneously inoculated into mice, and they were vaccinated with DCP_IL12-P2A-EVIR (8 mice) or DCP_IL12-P2A-dOVA cells (1×10 6 cells on days 3 and 6 after tumor challenge; 9 mice) or mock-treated (PBS; 5 mice). To induce effector function of T cells, all mice were also treated with an anti-PD1 antibody (Fig. 9G).

[0093] Vaccination with DCP_IL12-P2A-EVIR cells resulted in stronger tumor control than vaccination with DCP_IL12-P2A-dOVA cells (Figure 9H), indicating that EVIR can elicit anti-tumor immunity against B16 melanoma cells lacking OVA or GD2. Thus, vaccination with DCP_IL12-P2A-EVIR cells resulted in an increase in effector T cells in the tdLN (Figure 9I). Furthermore, vaccination with DCP_IL12-P2A-EVIR cells induced OVA-specific T cells in the tdLN and spleen (see comparison with PBS-treated mice), but their frequency was lower than expected compared to that in mice vaccinated with DCP_IL12-P2A-dOVA cells (Figures 9J–9K). Notably, analysis of the tdLN and spleen of mice vaccinated with DCP_IL12-P2A-EVIR cells demonstrated the expansion of non-OVA-reactive effector T cells compared to mice vaccinated with DCP_IL12-P2A-dOVA cells (Figures 9L–9M), which can explain the more significant tumor control against B16 tumors containing OVA-negative clones.

[0094] Statistical analysis: (b, h) Two-way ANOVA using Tukey's multiple comparison test. (c–f, i–m) One-way ANOVA using Tukey's multiple comparison test. The # symbol in j–k indicates comparison between the two groups of interest by unpaired Student's t test. P values are coded as follows: * : P < 0.05; ** : P < 0.01; *** : P < 0.001; and **** : P < 0.0001.

[0095] Thus, this example shows that DCP co-expressing IL-12 and EVIR inhibits tumor growth in a tumor-antigen agnostic fashion and is superior to vaccination against defined tumor antigens.

[0096] Example 10: DCP genetically engineered to express IL-12 and FLT3L enables highly effective tumor antigen cross-priming vaccination Vaccination studies were performed in mice by generating DCPs expressing either IL-12 or FLT3L by LV transduction and mixing two populations of DCPs. To track the transduced DCPs and their progeny, IL-12 was combined with dLNGFR while FLT3L was combined with GFP (both dLNGFR and GFP are neutral marker proteins).

[0097] To express FLT3L in DCPs, an FLT3L-P2A-GFP (SEQ ID NO: 19) LV was constructed and verified as done for the LV encoding IL-12 and IL-2 (see Examples 5a - b above). Briefly, flow cytometric analysis of mouse BM cells transduced with the LV at a multiplicity of infection (MOI) of 350 in HSPC medium and subsequently differentiated without exogenous FLT3L showed robust expression of GFP 5 days after transduction, when the transduced cells mainly contained DCPs (Figure 10A). Furthermore, cells transduced with the FLT3L-P2A-GFP LV secreted FLT3L into the cell culture medium when evaluated by ELISA 2 weeks after transduction, when the transduced cells mainly contained cDC1s (Figures 10B and 10J). Thus, the LV enables robust transduction of DC precursors and sustained expression of FLT3L in cDC progeny.

[0098] Subsequently, vaccination studies were performed in tumor-bearing mice (Figure 10C). Mice (10 mice per condition) were subcutaneously inoculated with B16-OVA melanoma cells (5×10 5 cells), and 1×10 6 DCP_IL12-P2A-dLNGFR cells and 2×10 6A mixture of DCP_FLT3L-P2A-GFP cells was intravenously injected twice (on the 3rd and 5th days after tumor challenge). To study DCPs expressing only one type of cytokine (i.e., either IL-12 or FLT3L), the cells were mixed with an appropriate number of control DCPs expressing either GFP or dLNGFR. Control mice were administered DCPs expressing only GFP and dLNGFR.

[0099] As shown in Figure 10D, the combination of DCP_FLT3L-P2A-GFP cells and DCP_IL12-P2A-LNGFR cells achieved better tumor control than DCP_IL12-P2A-dLNGFR (+ control DCP_GFP) cells. It should be noted that DCP_FLT3L-P2A-GFP (+ control DCP_dLNGFR) cells were not effective, indicating the synergistic activity of the combination of DCP_FLT3L-P2A-GFP cells and DCP_IL12-P2A-dLNGFR cells. To measure the systemic levels of transgenic cytokines in vaccinated mice, serum samples were collected from the tail vein on the 1st and 8th days after the last DCP injection. ELISA for serum IL-12 (BD 555256, BD Biosciences) and FLT3L (EMFLT3L, Invitrogen) showed an early and transient increase in transgenic cytokines in mice administered DCP_FLT3L-P2A-GFP and DCP_IL12-P2A-dLNGFR, followed by a rapid decrease to near baseline levels on the 8th day after vaccination (Figure 10E). Interestingly, flow cytometry analysis of intratumoral T cells revealed that more CD8 + and CD4 + T cells infiltrated after combined vaccination (Figures 10F - 10G). Furthermore, the combination of DCP_FLT3L-P2A-GFP and DCP_IL12-P2A-LNGFR induced the expansion and proliferation of effector CD8 + T cells and CD4 + T cells in the tdLN compared to all other vaccinated groups (Figures 10H - 10I).

[0100] Statistical analysis: (d) Two-way ANOVA using Tukey's multiple comparison test. (e - h) One-way ANOVA using Tukey's multiple comparison test. The # symbol in d indicates comparison between the two target groups by two-way ANOVA using Sidak's multiple comparison test. The # symbol in e - f indicates comparison between the two target groups by unpaired Student's t-test. P-values are coded as follows: * : P < 0.05; ** : P < 0.01; *** : P < 0.001; and **** : P < 0.0001.

[0101] Therefore, this example demonstrates that DCPs expressing IL-12 and FL3TL inhibit tumor growth in a tumor antigen-cross-reactive manner.

[0102] Example 11: DCPs genetically engineered to express IL-12 and FLT3L are superior to moDCs and cDC1 Next, the ability of transgenic FLT3L and IL12, which expand and proliferate T cells and confer the ability to control tumor growth, on other populations of DCs, namely moDCs or mature cDC1 (see Example 2 above), was investigated.

[0103] DCPs, moDCs, and cDC1 were generated from the BM of CD45.1 mice as described in Example 2 and transduced using LV encoding FLT3L, IL-12, and / or marker genes as described in Example 10 above. Briefly, DCPs and moDCs were transduced at the end of day 8 of the differentiation protocol, and cDC1 was transduced at the end of day 16 of the differentiation protocol.

[0104] moDCs were placed at 1.5 × 10 in moDC medium 6Seeded at a concentration of cells / mL and transduced with FLT3L-P2A-GFP LV or IL12-P2A-dLNGFR LV at an MOI of 100 for 12 - 14 hours. cDC1 and DCP were seeded at a concentration of 1.5×10 6 cells / mL and transduced with FLT3L-P2A-GFP LV or IL12-P2A-dLNGFR LV at an MOI of 350 for 12 - 14 hours.

[0105] Subsequently, vaccination studies were conducted in tumor-bearing mice (Figure 11A). Mice (7 - 8 mice per condition) were subcutaneously inoculated with B16-OVA melanoma cells (5×10 5 ), and a mixture of 1×10 6 cells transduced with IL12-P2A-dLNGFR LV and 2×10 6 cells transduced with FLT3L-P2A-GFP LV was intravenously injected twice (on days 3 and 5 after tumor challenge). Control mice were administered PBS only.

[0106] The expression of IL12-P2A-dLNGFR and FLT3L-P2A-GFP inhibited tumor growth (compared to mock treatment), regardless of the DC type used. However, vaccination using DCP achieved better tumor control than vaccination using either cDC1 or moDC (Figure 11B). moDC did not inhibit tumor growth as well as cDC1 or DCP.

[0107] Flow cytometry analysis of tumors and lymph nodes from treated mice revealed that tumors treated with DCP-IL12-P2A-dLNGFR cells + DCP-FLT3L-P2A-GFP cells were strongly infiltrated by T cells, including the majority of those with an activated phenotype (Figures 11C - 11D); the strength of such an effect was much greater for DCP than for cDC1 and moDC. tdLNs showed similar results (Figure 11E). Single cell suspensions from tumors stimulated ex vivo with puromycin and ionomycin showed significantly stronger production of IFNγ, GZMB, and TNFA in samples from DCP-vaccinated mice compared to other groups (Figures 11F - 11H). Overall, tumors from mice vaccinated with DCP-IL12-P2A-dLNGFR cells + DCP-FLT3L-P2A-GFP cells showed a widely reprogrammed tumor microenvironment characterized by a residual epithelial (cancer cell) component and a markedly abundant T cell infiltrate (Figures 11I - 11L).

[0108] Statistical analysis: (b) Two-way ANOVA using Tukey's multiple comparison test. (c - h) One-way ANOVA using Tukey's multiple comparison test. The # symbol in b indicates a comparison between the two groups of interest by two-way ANOVA using Sidak's multiple comparison test. P values are coded as follows: * : P < 0.05; ** : P < 0.01; *** : P < 0.001; and **** : P < 0.0001.

[0109] Thus, this example demonstrates that DCPs expressing IL-12 and FL3TL are superior to moDCs and mature cDC1 and widely reprogram the tumor microenvironment into a form dominated by activated T cells.

[0110] Example 12: DCPs genetically engineered to express IL-12 and FLT3L are effective in various cancer types The main advantage of the DCP vaccination platform described in this specification is that this strategy does not rely on known tumor antigens. Therefore, DCPs have the potential to function as universal DC vaccines. To test the efficacy of DCP vaccination in various tumor models, MC38 cancer cells of colorectal cancer origin were used.

[0111] MC38 cancer cells (5×10 5 cells) were subcutaneously inoculated into mice (10 per condition), and a mixture of 1×10 6 DCP-IL12-P2A-dLNGFR cells and 2×10 6 DCP-FLT3L-P2A-GFP cells was intravenously injected twice (on days 3 and 5 after tumor challenge). Control mice were administered an equal number of DCPs transduced with a control LV (expressing only dLNGFR or GFP) (Figure 12A).

[0112] DCP-IL12-P2A-dLNGFR + DCP-FLT3L-P2A-GFP vaccination may represent a very effective tumor cross-sectional therapeutic agent or achieved robust MC38 tumor control (Figure 12B). Consistent with the results in the B16-OVA melanoma model, genetically engineered DCPs significantly enhanced tumor infiltration by CD8 + T cells and, to a lesser extent, CD4 + T cells (Figures 12C - 12D). Furthermore, the proportion of effector CD4 + and CD8 + T cells was significantly increased in the tDLN (Figures 12E - 12F). Overall, MC38 tumors in mice vaccinated with DCP-IL12-P2A-dLNGFR cells + DCP-FLT3L-P2A-GFP cells showed a widely reprogrammed immune microenvironment characterized by a reduction in myeloid cells including immunosuppressive macrophages and significantly enhanced CD8 + T cells (Figures 12G - 12H).

[0113] Statistical analysis: (b) Two-way ANOVA using Tukey's multiple comparison test. (c - f) Unpaired Student's t-test. P-values are coded as follows: * : P < 0.05; ** : P < 0.01; *** : P < 0.001; and **** : P < 0.0001.

[0114] Thus, this example demonstrates that DCPs expressing IL-12 and FL3TL broadly reprogram the tumor microenvironment into a form that is monopolized by activated T cells in both melanoma and colorectal cancer models.

[0115] Example 13: CD34 + Generation of human DCPs from progenitor cells Based at least in part on the preclinical efficacy of cytokine-armed mouse DCPs, a corresponding protocol for the generation of human DCPs was developed. Umbilical cord blood CD34 + progenitor cells were cultured in StemSpan SFEMII medium (Stem Cell Technologies; 09605) supplemented with StemSpan CD34 + Expansion Supplement (Stem Cell Technologies; 02691) containing FLT3L, SCF, IL3, IL6, and TPO in a U-bottom 96-well plate at an initial concentration of 5 × 10 4 cells / mL (Figure 13A). CD3 - , CD19 - , CD335 - , CD66b - , CD10 - , CD14 - , CD34 + and CD115 +The maximum enrichment of human DCP defined as such (Figure 13B) was obtained on day 7 (Figure 13C). Surprisingly, the addition of the stem cell expansion enhancer UM729 (Stem Cell Technologies; 72332) to the medium was detrimental to the yield of human DCP (Figures 13D - 13E). Since there is no mouse model that can sustain the differentiation of human DCP into bona fide cDC1 and cDC2, the ability of human DCP to differentiate into antigen-presenting cells (APCs) (including cDC1, cDC2, and moDC) was tested in vitro. For this purpose, DCP on day 7 was sorted by fluorescence-activated cell sorting (FACS) as CD34 + and CD115 + cells (Figures 13F - 13G) and further differentiated for 7 more days in StemSpam SFEMII medium (Stem Cell Technologies; 09605) supplemented with 50 units / mL penicillin (Gibco), 50 μg / mL streptomycin (Gibco), 20 ng / mL GM-CSF (Peproteh; 300-03), 100 ng / mL FLT3L (Peprotech; 300-19), 20 ng / mL SCF (Peprotech; 300-07), and 10 ng / mL (1000 IU / mL) IFNα2b (Invivogen; rcyc-hifna2b). Human DCP mainly differentiates into APCs (cDC1: CD66b - , CD3 - , CD19 - , CD14 - , CD141 + , and CLEC9A + ; cDC2: CD66b - , CD3 - , CD19 - , CD14 - , CD141 - , CLEC9A - , and CD1c + ; monocytes: CD66b - , CD3 - , CD19 - , and CD14 + ; and immature DC: CD66b - , CD3 - , CD19- , CD14 - , CD141 + , and CLEC9A - ) occurred, but the mock-sorted cells differentiated into a broader range of cell types (granulocytes: CD66b + ; T cells: CD66b - and CD3 + ; B cells: CD66b - , CD3 - and CD19 + ; and other cells: CD66b - , CD3 - , CD19 - , CD14 - , CD141 - , CLEC9A - , and CD1c - included) (Figure 13H), which demonstrated that CD34 + and CD115 + human DCPs are indeed dendritic cell precursors.

[0116] Statistical analysis: (h) Two-way ANOVA using the Sidak multiple comparison test. P-values are encoded as follows: **** : P < 0.0001.

[0117] Therefore, this example demonstrates that human DCPs, which have the ability to differentiate into bona fide antigen-presenting cells including cDC1 and cDC2, can be generated from CD34 + human hematopoietic stem progenitor cells.

[0118] Example 14: DCP-derived DCs are superior to moDCs in antigen presentation ability To further demonstrate that human DCPs are a source of professional APCs, the antigen-presenting capacity of their progeny (DCP progeny) obtained as shown in Example 13 (FIG. 13F) was evaluated and compared with conventional moDCs. To generate human moDCs, blood from healthy human donors was obtained from the Blood Transfusion Center (Lausanne, Switzerland) and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on Lymphoprep (Stem Cell Technologies; 07801). CD14 APCs were then isolated using magnetic beads (Miltenyi; 130-050-201) according to the manufacturer's instructions. + Isolate monocytes and incubate at 10 6 The cells were cultured at a concentration of 100 cells / mL in RPMI 1640 (Gibco; 21875-034) supplemented with 10% FBS (Gibco; 10270106), 100 U / Ml penicillin, 100 μg / mL streptomycin (Gibco; 15140-122), 2 mM glutamine (Gibco; 25030-024), 50 ng / mL GM-CSF (Peprotech; 300-03), and 50 ng / mL IL4 (Peprotech; 200-04).

[0119] Cytomegalovirus (CMV) protein pp65 (or its HLA-A2-restricted peptide, pp65 495-504 ) and HLA-A2-restricted CMV-specific T cells were used to assess antigen presentation. Two antigen presentation pathways were examined: (i) pp65, which mimics direct presentation; 495-504 Presentation of peptide-loaded HLA-A2 and (ii) endogenously processed pp65 from the native pp65 protein 495-504 Cross-presentation of peptides. To assay direct presentation and cross-presentation, respectively, pp65 495-504T cells were co-cultured in the presence of DCP progeny or moDCs that had been pre-exposed to (1 μg / mL for 1 hour at 37°C) or pp65 protein (ab43041, Abcam, 10 μg / mL for 2 hours at 37°C). Pulse-stimulated DCP progeny and moDCs were washed with 10% FBS (Gibco; 10270106), 100 U / Ml penicillin, 100 μg / mL streptomycin (15140-122, Gibco) in RPMI 1640 (Gibco; 21875-034), and then A2 / CMV / pp65 495-504 specific CD8 + T cells at a 1:1 ratio. The co-culture was maintained overnight at 37°C and for 4 hours in the presence of brefeldin A (1:1000; BD Biosciences, GolgiPlug, 51-2301KZ), and then stained for flow cytometry analysis. In each case, increased IFNγ and TNFα-producing CMV-specific CD8 + T cells, as shown by the presence of DCP progeny, were superior to moDCs (Figures 14A - 14B). It should be noted that moDCs lacked cross-presentation ability. Thus, these data indicate that human DCPs have the ability to generate progeny with superior antigen-presenting ability compared to those of conventional moDCs.

[0120] Statistical analysis: (a) Unpaired t-test. P-values are coded as follows: *** : P < 0.001.

[0121] Thus, this example demonstrates that antigen-presenting cells derived from enriched human DCPs have superior antigen-presenting ability compared to those of conventional human monocyte-derived DCs.

[0122] Example 15: Efficient generation of transduced human DCPs On day 1, human DCP transduction was performed using concentrated lentiviral vectors (LVs) (Figure 15A). Prior to transduction, cells were transferred to RetroNectin (Takara; T100A)-coated wells and dmPGE2 (Stem Cell Technologies; 72372) was added to a final concentration of 10 μM. After 2 hours, cells were transduced with control dLNGFR-encoding LV at 300 MOI. This protocol generated over 80% transgene-expressing DCPs as evaluated by flow cytometry (Figure 15B). Subsequently, DCPs were transduced with functional anti-GD2 EVIR. Compared to control dLNGFR, GD2 + or GD2 -The functionality of GD2-EVIR-DCP was analyzed by evaluating the ability of GD2-EVIR-transduced DCPs to take up tumor EVs. Tumor EVs (tEVs) were isolated from a human melanoma cell line expanded in RPMI 1640 (Gibco; 21875-034) containing 10% FBS (Gibco; 10270106), 1.1 μM arginine (Sigma Aldrich), 0.48 μM asparagine (Sigma Aldrich), 11.25 μM glutamine (Gibco; 25030-024), 10 mM Hepes (Gibco; 15630-056), 100 U / Ml penicillin, and 100 μg / mL streptomycin (Gibco; 15140-122). For EV isolation, the cell medium at 20% confluence was replaced with the above medium modified to contain 5% EV-depleted FBS (obtained by ultracentrifugation of standard FBS at 134,000 g for 16 h at 4 °C followed by filtration through a 0.1 μm vacuum filtration bottle). After 4 days of culture, the medium was harvested for EV isolation by sequential ultracentrifugation: the medium was centrifuged at 500×g for 5 min, 200×g for 10 min, and 10,000×g for 30 min at 4 °C to remove dead cells and debris. Subsequently, the medium was ultracentrifuged at 134,000×g for 70 min at 4 °C using a Hitachi CP80NX ultracentrifuge. The pellet was washed with 35 mL of PBS and ultracentrifuged again at 134,000×g for 70 min at 4 °C and finally resuspended in PBS. GD2 + or GD2 -EVs were labeled with the membrane dye PKH26 (1:200, MIDI26-1KT, Sigma, PE) for 10 minutes at RT and washed twice with 0.1% BSA in PBS and once with PBS on Vivaspin 500 (300,000 MWCO PES, VS0152 Sartorius), and then added to control (dLNGFR) transduced or GD2-EVIR transduced DCP progeny at a concentration of 5 μg / mL (Figures 15C - 15D). The mean fluorescence intensity of PKH26 in transduced DCPs measured the next day by flow cytometry was GD2 by GD2-EVIR transduced DCP progeny compared to control transduced DCP progeny + showed enhanced uptake of tumor EVs, indicating that EVIR expression in human DCPs specifically enhances the uptake and internalization of GD2-positive tEVs (Figure 15E).

[0123] Subsequently, DCPs were transduced with LVs encoding FLT3L and IL-12. DCPs were transduced on day 1 with LVs encoding FLT3L-GFP, IL12-dLNGFR or dLNGFR, and on day 7, sorted for further 7-day culture in FLT3L-depleted StemSpam SFEMII medium (Stem Cell Technologies; 09605) supplemented with 50 units / mL penicillin (Gibco), 50 μg / mL streptomycin (Gibco), 20 ng / mL GM-CSF (Peproteh; 300-03), 20 ng / mL SCF (Peprotech; 300-07), and 10 ng / mL (1000 IU / mL) IFNa2b (Invivogen; rcyc-hifna2b) (Figure 15F). CD34 + and CD115 + DCPs identified as day 7 cells expressing CD34 and CD115 robustly expressed GFP from the FLT3L-GFP LV (Figure 15G). Also, ELISA analysis (EHFL3LG, Thermo Fisher) of the 14-day culture supernatant revealed efficient FLT3L production by FLT3L-transduced cells or a mixture of FLT3L- and IL12-transduced DCPs (2:1 ratio) (Figure 15H).

[0124] The ability of IL12 to enhance the T cell-stimulating ability of human DCP was evaluated (Figure 15I). Flow cytometry-based dLNGFR analysis revealed efficient transduction by IL12-dLNGFR LV on day 7 (Figure 15J). Furthermore, IL12 was detected by ELISA (431701, Biolegend) in the culture supernatant of IL12-transduced DCP progeny after 7-day differentiation in StemSpam SFEMII medium (Stem Cell Technologies; 09605) supplemented with 50 units / mL penicillin (Gibco), 50 μg / mL streptomycin (Gibco), 20 ng / mL GM-CSF (Peproteh; 300-03), 100 ng / mL FLT3L (Peprotech; 300-19), 20 ng / mL SCF (Peprotech; 300-07), and 10 ng / mL (1000 IU / mL) IFNα2b (Invivogen; rcyc-hifna2b) (Figure 15K). Notably, a mixture of IL12-transduced DCP progeny or FLT3L / IL12-transduced (2:1 ratio) DCP progeny induced antigen-independent IFNγ production by CMV-specific T cells co-cultured with DCP progeny (Figure 15L).

[0125] In summary, the present disclosure demonstrates that, similar to the mouse DCP of the previous examples, human DCP can be armed with effectors (such as EVIR, FLT3L, or IL12) to generate DCP progeny, and that these human DCP progeny can recapitulate the anti-tumor immune function of mouse DCP.

[0126] Sequence Listing TIFF2025518217000002.tif239170TIFF2025518217000003.tif254170TIFF2025518217000004.tif251170TIFF2025518217000005.tif253170TIFF2025518217000006.tif246170TIFF2025518217000007.tif250170TIFF2025518217000008.tif253170TIFF2025518217000009.tif252170TIFF2025518217000010.tif248170TIFF2025518217000011.tif246170TIFF2025518217000012.tif245170TIFF2025518217000013.tif249170TIFF2025518217000014.tif236170TIFF2025518217000015.tif233170TIFF2025518217000016.tif249170TIFF2025518217000017.tif245170TIFF2025518217000018.tif16170

[0127] Exemplary embodiments have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. Those skilled in the art will envision numerous variations, modifications, and substitutions. It should be understood that numerous alternatives to the embodiments described herein can be utilized. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A synthetically partially differentiated dendritic cell precursor, wherein the synthetically partially differentiated dendritic cell precursor expresses an effector, the effector being a combination of interleukin 12 (IL12) and either FMS-like tyrosine kinase 3 ligand (FLT3L) or extracellular vesicle internalization receptor (EVIR), or both.

2. An in vitro cell composition comprising the synthetically partially differentiated dendritic cell precursor described in Claim 1, wherein the synthetically partially differentiated dendritic cell precursor is determined by flow cytometry, and CD115 + CD11c - , and Clec9A - The above-mentioned in vitro cell composition having the phenotype.

3. When the phenotype of synthetically partially differentiated dendritic progenitor cells is determined by flow cytometry, CD11b - MHCII - CD45R / B220 - , and cKIT - The in vitro cell composition according to claim 2, further comprising one or more phenotypes selected from the following.

4. cDC1 or cDC2 dendritic cells differentiated from synthetically partially differentiated dendritic cell precursors as described in claim 1.

5. A differentiated cDC1 or cDC2 dendritic cell according to claim 4, wherein the genetically modified dendritic cell expresses interleukin-12 (IL12) as an effector.

6. The differentiated cDC1 or cDC2 dendritic cell according to claim 4, wherein the genetically modified dendritic cell expresses an effector, the effector being selected from the group consisting of extracellular vesicle internalization receptor (EVIR) and FMS-like tyrosine kinase 3 ligand (FLT3L).

7. Antigen-presenting cells (APCs) differentiated from synthetically partially differentiated dendritic cell precursors as described in claim 1.

8. The APC according to claim 7, wherein the genetically modified dendritic cell expresses interleukin-12 (IL12), an extracellular vesicle internalization receptor (EVIR), and another effector selected from the group consisting of FMS-like tyrosine kinase 3 ligand (FLT3L).

9. The following steps: (a) CD34 that has been rapidly amplified + Steps to obtain human hematopoietic stem progenitor cells (human HSPCs); and (b) When determined by flow cytometry, CD115 + , CD34 + , CD3 - , CD19 - , CD335 - , CD66b - , CD10 - , and CD14 - In an amount sufficient to differentiate HSPC cells into synthetically partially differentiated dendritic cell precursors having one or more phenotypes selected from, with or without IFNγ, IL-12, retronectin, TNF-α, or UM729, a synthetic medium containing FMS-like tyrosine kinase 3 ligand (FLT3L), IL-3, IL-6, TPO, and SCF, and contacting the synthetic medium with short-term expanded human HSPC A method for producing synthetically partially differentiated dendritic cell precursors, including [a specific component].

10. A pharmaceutical composition for use in the treatment of a condition, comprising the following components: (a) the in vitro cell composition according to claim 2; differentiated cDC1 or cDC2 dendritic cells according to any one of claims 4 to 6, or APC according to any one of claims 7 to 8; and (b) a pharmaceutically acceptable excipient, diluent, or carrier.

11. The pharmaceutical composition according to claim 10, wherein the condition is cancer.

12. The pharmaceutical composition according to claim 10, further comprising an interleukin or an effector.

13. The pharmaceutical composition according to claim 12, wherein the effector is IL-12.

14. The pharmaceutical composition according to claim 12, wherein the effector is selected from the group consisting of extracellular vesicle internalization receptor (EVIR), FMS-like tyrosine kinase 3 ligand (FLT3L), TNF-α, IL-1, IL-2, IL-6, CXCL8, interferon (IFN), GM-CSF, and G-CSF.

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