Engineered progenitor cells and methods of use thereof

Abstract

Disclosed herein are engineered progenitor cells and methods of using the same. Also disclosed herein are engineered cells differentiated from the engineered progenitor cells of the present disclosure. Also disclosed are methods of treating a disease in a subject by administering an engineered progenitor cell of the present disclosure or an engineered cell differentiated from the engineered progenitor cell.

Description

[0001] Cross-referenced applications

[0002] This application claims priority from European patent application No. EP22176819 filed on June 1, 2022, the entire contents of which are incorporated herein by reference.

[0003] Incorporation by reference into the sequence listing

[0004] This application contains a sequence listing that has been submitted through the patent office. The sequence listing, created on May 17, 2023, is 51207 bytes in size and is titled 212225.701601_PCT_SL.xml, and is incorporated herein by reference in its entirety.

[0005] field

[0006] The present disclosure generally relates to engineered dendritic progenitor cells and methods of use thereof.

[0007] background

[0008] Immunotherapy can be used to treat a variety of human diseases, such as infections, degenerative diseases, and cancer. In cancer, immunotherapy sometimes involves stimulating the patient's own immune system to attack cancer cells or other cellular components of a tumor.

[0009] Vaccinating patients with tumor-associated antigens (TAAs) to stimulate or enhance cancer-specific T lymphocytes is an attractive means of treating subjects through 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 through various mechanisms and present them to T lymphocytes, thereby initiating a potent effector response against tumors. DCs are also able to migrate between lymphoid and non-lymphoid tissues and regulate cytokine and chemokine gradients to control inflammation and lymphocyte homing. However, improving the therapeutic efficacy of DCs has been a challenge.

[0010] Immune cell engineering as described herein provides a method for improving the efficacy of DC vaccines. As described herein, the autologous cell-based platform capable of generating and amplifying cDC1 in vivo can effectively absorb and present tumor-associated antigens (TAA) and induce a strong and extensive T cell response against a variety of TAAs, making it an attractive therapy for a variety of cancers. Therefore, the present disclosure provides a method for engineering DC progenitor cells, which can efficiently generate cDC1 in vivo without the need for antigen loading in vitro, thus providing a method for a tumor-independent DC vaccine.

[0011] Summarize

[0012] The present invention discloses an in vitro cell composition comprising artificially synthesized partially differentiated dendritic cell progenitor cells, wherein the artificially synthesized partially differentiated dendritic cell progenitor cells have CD115 + 、CD11c - and Clec9A - In certain embodiments, the phenotype of the artificially synthesized partially differentiated dendritic cell progenitor cells further comprises a phenotype selected from the group consisting of CD11b - , MHCII - 、CD45R / B220 - and cKIT - One or more phenotypes of the present invention, as determined by flow cytometry.

[0013] The present invention also discloses cDC1 or cDC2 dendritic cells differentiated from the differentiation of the artificially synthesized partially differentiated dendritic cell progenitor cells described herein. In certain embodiments, the artificially synthesized differentiated cDC1 or cDC2 is an engineered dendritic cell expressing an interleukin or effector. In certain embodiments, the engineered dendritic cell expresses an interleukin, wherein the interleukin is IL12. In certain embodiments, the engineered dendritic cell expresses an effector, wherein 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.

[0014] The present invention also discloses an in vitro cell composition comprising artificially synthesized partially differentiated dendritic cell progenitor cells, wherein the artificially synthesized partially differentiated dendritic cell progenitor cells comprise a protein selected from the group consisting of CD115 + 、CD34 + 、CD3 - 、CD19 - 、CD335 - 、CD66b - 、CD10 - and CD14 - One or more phenotypes of the present invention, as determined by flow cytometry.

[0015] The present invention also discloses antigen presenting cells (APCs) differentiated from the artificially synthesized partially differentiated dendritic cell progenitor cells described herein. In certain embodiments, the APCs are engineered dendritic cells expressing interleukins or effectors. In certain embodiments, the engineered dendritic cells express interleukins, wherein the interleukins are IL12. In certain embodiments, the engineered dendritic cells express effectors, wherein the effectors are 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.

[0016] The present invention also discloses a method for preparing the artificially synthesized partially differentiated dendritic cell progenitor cells, the method comprising: (a) obtaining transiently expanded hematopoietic stem / progenitor cells (HSPCs); and (b) contacting the transiently expanded HSPCs with a synthetic culture medium, wherein the synthetic culture medium comprises FMS-like tyrosine kinase 3 ligand (FLT3L) and GM-CSF, and includes or does not include IL-1, IL-2, IL-4, IL-6, IL-12, CXCL8, G-CSF, TNF-α, IFNa, PGE2 or recombinant human fibronectin fragment (retronectin), the content of which is sufficient to differentiate the HSPC cells into cells with CD115 + 、CD11c - and Clec9A - The phenotype of the artificially synthesized partially differentiated dendritic cell progenitor cells is determined by flow cytometry. In certain embodiments, the method further comprises contacting the HSPCs with a culture medium comprising FBS, L-glutamine, SCF, TPO, FLT3L, IL-3, IL-6, and IL-1b, thereby preparing a transiently expanded HSPC prior to the contacting in step (b).

[0017] The present invention also discloses a method for preparing the artificially synthesized partially differentiated dendritic cell progenitor cells, the method comprising: (a) obtaining a transiently expanded CD34 + (b) contacting the transiently expanded human HSPCs with a synthetic culture medium comprising FMS-like tyrosine kinase 3 ligand (FLT3L), IL-3, IL-6, TPO, and SCF, with or without IFNγ, IL-12, recombinant human fibronectin fragment, TNF-α, or UM729, in an amount sufficient to differentiate the HSPCs into cells having a phenotype selected from the group consisting of CD115 + 、CD34 + 、CD3 - 、CD19 - 、CD335 -、CD66b - 、CD10 - and CD14 - Artificially synthesized partially differentiated dendritic cell progenitor cells having one or more phenotypes as determined by flow cytometry.

[0018] The present invention also discloses a pharmaceutical composition for treating a disease, comprising: (a) an in vitro cell composition as described herein, and (b) a pharmaceutically acceptable excipient, diluent, or carrier. Also disclosed herein is a method for treating a disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising: (a) an in vitro cell composition as described herein, and (b) a pharmaceutically acceptable excipient, diluent, or carrier. In certain embodiments, the disease is cancer. In certain embodiments, the pharmaceutical composition further comprises an interleukin or effector. In certain embodiments, the differentiated cDC1 or cDC2 dendritic cell is an engineered dendritic cell expressing an interleukin or effector. In certain embodiments, the APC is an engineered dendritic cell expressing an interleukin or effector. In certain embodiments, the interleukin is IL-12. In certain embodiments, 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. In certain embodiments, the effector is not expressed on cancer cells.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The novel features of the exemplary embodiments are set forth with particularity in the appended claims. The following detailed description sets forth exemplary embodiments utilizing the principles of the disclosed systems and methods, and the features and advantages will be better understood by reference to the following detailed description and accompanying drawings which set forth illustrative embodiments:

[0021] Figures 1A-1C Partial differentiation of host hematopoietic stem / progenitor cells into dendritic cell progenitors (DCPs) as described herein is described. Figure 1A The protocol for preparing DCPs is outlined; Figure 1B Shows the purity of isolated DCPs; Figure 1C The phenotypes of the isolated DCPs are shown.

[0022] Figures 2A-2K The ability of various dendritic cells, including monocyte-derived dendritic cells (moDCs), conventional type I dendritic cells (cDC1s), and the DCPs of the present disclosure to generate mature cDC1s in tumor-free animals is described. Figure 2AA protocol for adoptive transfer of dendritic cells is outlined. Figures 2B-2D and Figure 2K The moDC of the present disclosure is shown Figure 2B and 2K )、cDC1( Figure 2C ) and DCPs( Figure 2D ) phenotype. Figure 2B and 2K Shown are FACS analyses of the same data using different inclusion criteria to determine the percentage of cells with the moDC phenotype. Figures 2E-2F It was shown that dendritic cells induced cDC1( Figure 2E ) and cDC2( Figure 2F ) differentiation. Figure 2G Demonstrates engraftment of donor-derived cells following infusion. Figures 2H-2J Shown is the infusion of the moDCs disclosed herein ( Figure 2H )、cDC1( Figure 2I ) or DCPs( Figure 2J ) after donor-derived cells differentiate into various dendritic cell types in the spleen.

[0023] Figures 3A-3I Differentiation of the DCPs of the present disclosure following systemic administration to tumor-bearing animals is described. Figure 3A The procedure for drug administration is shown. Figure 3B The gating strategy used to identify intratumoral donor-derived cDC1 and cDC2 is shown. Figure 3C Flow cytometric analysis of tumor-derived cells and splenocytes is shown. Figures 3D-3E and 3H-3I showed that donor-derived DCPs were efficiently differentiated into cDCs. Figure 3D and 3H Pie charts showing the relative proportions of differentiated cDCs and other cell types are shown, each derived from the same data, with the data in each figure processed using different inclusion criteria. Figure 3E and 3I Pie charts showing the relative proportions of differentiated cDCs and other cell types are shown separately based on the same data, with the data in each figure processed using different inclusion criteria. Figures 3F-3G The tumor ( Figure 3F ) and spleen ( Figure 3G ) in the donor-derived cDCs.

[0024] Figures 4A-4F The identification of interleukins (ILs) that promote T cell activation by cDC1s-like cells is described. Figure 4A The experimental procedure is described. Figures 4B-4C The effects of ILs on the differentiation of DCPs into cDC1s were shown. Figures 4D-4FWe show that IL-12 enables cDC1-like cells to promote potent IFNγ production by OT-I and OT-II T cells.

[0025] Figures 5A-5H Differentiation of DCPs expressing IL-12 or IL-2 in tumor-free animals is shown. Figure 5A Shown are flow cytometric analyses of cDC1-like cells transduced with lentiviral vectors expressing IL-12 or IL-2 and GFP. Figure 5B and 5H The transduced cells were shown to secrete IL-12 and IL-2 by ELISA. Figure 5H yes Figure 5B Alternative representations of the same data shown, but with different curve fitting criteria. Figure 5C The administration protocol of transduced DCPs is shown. Figures 5D-5E Flow cytometric analysis of splenocytes is shown, demonstrating transduced (GFP+) donor-derived cells. Figures 5F-5G Transduced donor-derived DCPs were shown to efficiently differentiate into cDCs.

[0026] Figures 6A-6C The activation of antigen-specific T cells from DCPs expressing IL-12 and a tumor antigen (OVA) is described. Figure 6A It is a schematic diagram of the experiment. Figures 6B-6C Flow cytometry of splenocytes is shown, demonstrating robust expansion of OVA-reactive T cells.

[0027] Figures 7A-7G showed that DCPs expressing IL-12 and tumor antigens blocked tumorigenesis. Figure 7A It is the process of the experiment. Figures 7B-7C Flow cytometry of blood collected from mice 4 days after tumor invasion is shown, demonstrating activated CD4 + and CD8 + The proportion of T cells. Figure 7D The levels of OVA-reactive T cells in the same blood samples are shown. Figure 7E The changes in tumor volume over time after tumor invasion are described. Figures 7F-7G Robust expansion of OVA-reactive effector T cells in the spleen was demonstrated.

[0028] Figures 8A-8F showed that DCPs expressing IL-12 and tumor antigens inhibited tumor growth. Figure 8A It is the process of the experiment. Figure 8B The DCPs of the present disclosure were shown to inhibit tumor growth. Figures 8C-8F Shows blood ( Figure 8C ),spleen( Figure 8D ), tumor-draining lymph nodes (tdLN) ( Figure 8E ) and tumors ( Figure 8F ) in the presence of OVA-reactive T cells.

[0029] Figures 9A-9M Vaccination using DCPs engineered to express IL-12 and EVIR is described. Figures 9A-9M DCPs expressing IL-12 and EVIR were shown to inhibit tumor growth. Figure 9A It is the process of the experiment. Figure 9B Summary of the time course of tumor growth following infusion of DCPs expressing IL-12 and EVIR. Figures 9C-9F Flow cytometric analysis of the tumor microenvironment is shown. Figure 9G The scheme for mixed administration of melanoma cells is shown. Figure 9H Summary of tumor growth over time following infusion of DCP expressing IL-12 and EVIR. Figures 9I-9M Flow cytometry of spleen and tdLN is described, showing the presence of OVA- and non-OVA-reactive T cells.

[0030] Figures 10A-10J Vaccination using DCPs engineered to express IL-12 and FLT3L is described. Figure 10A Flow cytometry of transduced cDC1-like cells is shown. Figure 10B It shows that FLT3L is produced by the transduced cells as detected by ELISA. Figure 10J yes Figure 10B Alternative representations of the same data shown, but with different curve fitting criteria. Figure 10C This is the process of DCP infusion study. Figure 10D Tumor growth over time after DCP infusion is summarized. Figure 10E Shown are the serum levels of transgenic cytokines at different time points after the last DCP infusion. Figures 10F-10G Flow cytometric analysis of intratumoral T cells is shown, showing CD8 + and CD4 + Robust expansion of T cells. Figures 10H-10I Flow cytometric analysis of T cells in tdLN is shown.

[0031] Figures 11A-11L Shown is a comparison between DCPs, moDCs, and cDC1 of the present disclosure, all of which express IL-12 and FLT3L. Figure 11A It is the process of the experiment. Figure 11B Tumor growth over time after dendritic cell infusion is summarized. Figure 11C and 11D Flow cytometric analysis of tumors from treated mice is shown. Figure 11E Shown are flow cytometric analyses of tdLNs from treated mice. Figures 11F-11H Analysis of in vitro activated tumor-derived T cells is shown, demonstrating robust T cell activation. Figures 11I-11L showed a massive infiltration of T cells in the tumor microenvironment of mice that received DCPs engineered to express IL-12 and FLT3L.

[0032] Figures 12A-12H Vaccination against another type of cancer using the DCPs of the present disclosure is described. Figure 12A It is the process of the experiment. Figure 12B Tumor growth over time after DCP infusion is summarized. Figures 12C-12D Flow cytometry of tumors is shown, demonstrating robust activation of T cells. Figures 12E-12F Flow cytometry of tdLNs is shown, demonstrating robust infiltration of T cells. Figure 12G showed remodeling of the tumor microenvironment and CD8 in tumors of mice receiving DCPs engineered to express IL-12 and FLT3L. + Large-scale expansion of T cells. Figure 12H Shows the Figure 12G Shown are pie charts of the same data, but processed using different macrophage inclusion criteria.

[0033] Figures 13A-13H Depicts CD34 + Differentiation of human hematopoietic stem and progenitor cells into dendritic cell progenitors (DCPs) as described herein. Figure 13A The cell expansion protocol for the preparation of DCPs is outlined. Figure 13B Shown was identified as CD3 - 、CD19 - 、CD335 - 、CD66b - 、CD10 - 、CD14 - 、CD34 + and CD115 + Gating strategy for DCPs enrichment. Figure 13C The percentages of DCPs in the presence or absence of enrichment treatment are shown. Figure 13D A cell expansion protocol for preparing DCPs in the presence or absence of the stem cell expansion enhancer UM729 is outlined. Figure 13E The effect of UM729 addition on CD34 T cells obtained from two different donors is shown. + The impact of human hematopoietic stem and progenitor cells. Figure 13F The in vitro preparation protocol of DCPs is outlined. Figure 13G Shows the CD34 + and CD115+ Fluorescence activated cell sorting (FACS) of cells. Figure 13H Shown are the percentages of DCPs observed in APCs and other cells after 7 days of expansion and 7 days of differentiation, respectively.

[0034] Figures 14A-14B Detection of increased IFNγ and TNFα-producing CMV-specific CD8 + There are two antigen presentation pathways for T cells. Figure 14A Shows load pp65 495-504 HLA-A2 presentation of peptides, which mimics direct presentation. Figure 14B pp65 processed endogenously from native pp65 protein 495-504 Cross-presentation of peptides.

[0035] Figures 15A-15L CD34 + Differentiation of human hematopoietic stem and progenitor cells into dendritic cell progenitors (DCPs) as described herein. Figure 15A Overview of lentiviral vector-transduced CD34 + Cell expansion protocol for human hematopoietic stem and progenitor cells. Figure 15B Shown are flow cytometry of transduced DCPs used to determine transgene expression. Figure 15C Overview of the CD34 cells transduced with a lentiviral vector encoding αGD2-EVIR + Protocol for preparation of DCPs from human hematopoietic stem and progenitor cells. Figure 15D The authors outlined the GD2 uptake by evaluating DCPs transduced with GD2-EVIR compared with CTRL-dLNGFR. + or GD2 - Scheme for evaluating the function of GD2-EVIR-DCP using the capacity of tumor EVs. Figure 15E The progeny of GD2-EVIR-transduced DCPs showed a significant difference in the expression of GD2 compared to the progeny of CTRL-transduced DCPs. + Tumor EV uptake measured by flow cytometry. Figure 15F The results of the present study were summarized from CD34 T cells transduced with lentiviral vectors encoding FLT3L or FLT3L / IL-12. + Protocol for the preparation of DCPs from human hematopoietic stem and progenitor cells. Figure 15G The FLT3L-GFP LV-transduced cells were identified as CD34 + and CD115 + Flow cytometry of cellular DCPs. Figure 15H FLT3L-transduced cells or FLT3L -ELISA analysis of FLT3L production in the culture supernatant of DCPs transduced with a mixture of IL-12 and IL-12 (2:1 ratio) on day 14. Figure 15I A novel method for the isolation of CD34 T cells transduced with lentiviral vectors encoding IL12 or FLT3L / IL12 is described. + Protocol for the preparation of DCPs from human hematopoietic stem and progenitor cells. Figure 15J The IL12-dLNGFR LV-transduced cells were identified as CD34 + and CD115 + Flow cytometry of cellular DCPs. Figure 15K Shown is an ELISA analysis of IL12 produced in the culture supernatant of DCP progeny cells transduced with IL12 or with a FLT3L / IL12 mixture (2:1 ratio) on day 14. Figure 15L Shown are antigen-independent IFNγ production by CMV-specific T cells co-cultured with DCP progeny cells transduced with IL12 or a mixture of FLT3L / IL12 (2:1 ratio).

[0036] Detailed description

[0037] Overview

[0038] Disclosed herein are synthetically synthesized differentiated dendritic cell progenitors (or DCPs). The synthetically synthesized differentiated dendritic cell progenitors described herein can be partially differentiated from host progenitor cells. Thus, the synthetically synthesized differentiated dendritic cell progenitors described herein are partially differentiated cells. In certain embodiments, the synthetically differentiated dendritic cell progenitors described herein are not fully differentiated.

[0039] The artificially synthesized differentiated dendritic cell progenitor cells as described herein can naturally differentiate into dendritic cells, such as cDC1, cDC2 or immature dendritic cells, after being administered to a host. As disclosed herein, compared to the administration of other similar dendritic cells, such as monocyte-derived dendritic cells (moDCs) or conventional type 1 DC (cDC1) cells, the artificially synthesized differentiated dendritic cell progenitor cells of the present disclosure are more effectively differentiated into these dendritic cells when administered to a subject. In addition, the artificially synthesized differentiated dendritic cell progenitor cells of the present disclosure are capable of differentiating into dendritic cells in the presence of a tumor, and therefore are capable of differentiating in the presence of inflammatory and immunosuppressive cytokines associated with the presence of a tumor.

[0040] Artificially differentiated dendritic cell progenitors as described herein can be differentiated from host progenitor cells, such as CD34 +Human hematopoietic stem and progenitor cells. In certain embodiments, the artificially synthesized differentiated dendritic cell progenitor cells as described herein are partially differentiated cells. In certain embodiments, the artificially synthesized differentiated dendritic cell progenitor cells as described herein are not fully differentiated. In certain embodiments, the artificially synthesized differentiated dendritic cell progenitor cells as described herein can differentiate into antigen presenting cells (APCs), cDC2s, monocytes, immature dendritic cells or a combination thereof in vitro. In addition, in certain embodiments, the artificially synthesized differentiated dendritic cell progenitor cells of the present disclosure can differentiate into APCs, cDC2s, monocytes, immature dendritic cells or a combination thereof when a tumor exists, and therefore can differentiate when inflammatory and immunosuppressive cytokines related to the presence of a tumor exist.

[0041] In order to stimulate the production of tumor-specific T cells, artificially synthesized differentiated dendritic cell progenitors can be engineered to express interleukins and / or effectors. In some cases, the co-expression of effectors (such as extracellular vesicle internalization receptors (EVIR) or FMS-like tyrosine kinase 3 ligands (FLT3L)) with interleukins (such as IL-12) will produce differentiated dendritic cells (such as cDC1, cDC2, APCs, monocytes or immature dendritic cells), which will produce tumor-specific T cells, thereby reducing tumor growth and / or inhibiting tumorigenesis. In addition, the presence of effectors and / or interleukins (whether co-expressed by differentiated dendritic cells or exogenously added) leads to the production of tumor-specific T cells, and the production of tumor-specific T cells does not require the provision of tumor antigens (e.g., whether exogenous or expressed by dendritic cells).

[0042] Therefore, artificially synthesized differentiated dendritic cell progenitors can be used as therapeutic agents to treat cancers for which the specific tumor antigens are unknown. Consequently, administering these artificially synthesized differentiated dendritic cell progenitors as part of a pharmaceutical composition to a subject with cancer, without knowing the antigens expressed on the cancer cells, can be used to treat cancers for which the antigens expressed on the cancer cells are unknown.

[0043] definition

[0044] The terminology used herein is for descriptive purposes only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "a" include the plural forms as well, unless the context clearly indicates otherwise. In addition, to the extent that the terms "include," "comprising," "having," "having," "including," or variations thereof are used in the detailed description and / or claims, these terms are intended to be encompassed in a manner similar to "comprising."

[0045] The term "about" or "approximately" means within an acceptable error range for a particular value recognized by one of ordinary skill in the art, e.g., plus or minus 10%. Where ranges and / or subranges of values ​​are provided, the ranges and / or subranges include the endpoints of the ranges and / or subranges.

[0046] As used herein, the term "substantially" refers to 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 mean that less than 10% (e.g., less than 5%, less than 1%, less than 0.1%, or less than 0.01%) of the transgene is expressed relative to the amount of transgene expressed in the presence of the inducer.

[0047] The terms "subject," "individual," or "patient" are used interchangeably herein. The "subject" refers to a plant, animal, or microorganism, including bacteria, viruses, fungi, and protozoa. The subject can be a mammal. The mammal can be any member of the class mammalia, including, but not limited to, humans, non-human primates (such as chimpanzees, apes, or other monkey species), farm animals (such as cattle, horses, sheep, goats, pigs), domestic animals (such as rabbits, dogs (or canines), and cats (or felines)), and laboratory animals (including rodents, such as rats, mice, and guinea pigs).

[0048] The terms "host" and "donor" are used interchangeably herein to refer to the organism from which the progenitor cells are isolated. The host can be a mammal as described herein. When the progenitor cells are isolated from a "host" and differentiated into a therapeutic agent for administration to a "subject," the host and the subject need not be of the same class, genus, or species.

[0049] The term "in vivo" refers to events that occur within the body of a subject.

[0050] The term "in vitro" refers to events that occur outside the body of a subject. In vitro assays can include cell-based assays, which can use live or dead cells. In vitro assays can also include cell-free assays, which cannot use intact cells.

[0051] Dendritic cell progenitor cells

[0052] Disclosed herein are artificially synthesized partially differentiated dendritic cell progenitors derived from host progenitor cells. As disclosed herein, the host progenitor cells include progenitor cells from a host that are capable of partial or complete differentiation. In some cases, the host progenitor cells can be isolated from a host, such as a mammal. In certain embodiments, the progenitor cells can be progenitor cells isolated from bone marrow or blood, such as hematopoietic stem or progenitor cells. Examples of hematopoietic progenitor cells include hematopoietic stem cells, multipotent progenitor cells, myeloid progenitor cells, and lymphoid progenitor cells. In certain embodiments, the progenitor cells can be dedifferentiated cells, such as induced pluripotent stem cells or neural progenitor cells.

[0053] As disclosed herein, artificially synthesized partially differentiated dendritic cell progenitors can be prepared from host progenitor cells by contacting the host progenitor cells with a synthetic culture medium to induce partial differentiation. In certain embodiments, the resulting artificially synthesized partially differentiated dendritic cell progenitors are capable of further differentiating into dendritic cells (i.e., the dendritic cell progenitors are not fully differentiated).

[0054] Also disclosed herein are host progenitor cells comprising human progenitor cells. In certain embodiments, the human progenitor cells can be isolated from bone marrow or blood, such as umbilical cord blood CD34 + In some embodiments, the human progenitor cells are capable of partially differentiating into dendritic cell progenitor cells. Thus, in some embodiments, the dendritic cell progenitor cells can be differentiated into dendritic cell progenitor cells by making human progenitor cells (e.g., CD34 + In some embodiments, the dendritic cell progenitor cells derived from human progenitor cells are capable of differentiating into antigen presenting cells (APCs), cDC2s, monocytes, immature dendritic cells, or a combination thereof in vitro.

[0055] The synthetic medium for differentiation may include an effective amount of the effector sufficient to induce partial differentiation of the host progenitor cells. In certain embodiments, the synthetic medium comprises 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, ng / mL, 30ng / mL, 31ng / mL, 32ng / mL, 33ng / mL, 34ng / mL, 35ng / mL, 36ng / mL, 37ng / mL, 38ng / mL, 39ng / mL, 40ng / mL, 41ng / mL, 42ng / mL, 43ng / mL, 44 ng / mL, 45ng / mL, 46ng / mL, 47ng / mL, 48ng / mL, 49ng / mL, 50ng / mL, 51ng / mL, 52ng / mL, 53ng / mL, 54ng / mL, 55ng / mL, 56ng / mL, 57ng / mL, 58ng / mL, 59 ng / mL, 60ng / mL, 61ng / mL, 62ng / mL, 63ng / mL, 64ng / mL, 65ng / mL, 66ng / mL, 67ng / mL, 68ng / mL, 69ng / mL, 70ng / mL, 71ng / mL, 72ng / mL, 73ng / mL, 7 4ng / mL, 75ng / mL, 76ng / mL, 77ng / mL, 78ng / mL, 79ng / mL, 80ng / mL, 81ng / mL, 82ng / mL, 83ng / mL, 84ng / mL, 85ng / mL, 86ng / mL, 87ng / mL, 88ng / mL, 8 9ng / mL, 90ng / mL, 91ng / mL, 92ng / mL, 93ng / mL, 94ng / mL, 95ng / mL, 96ng / mL, 97ng / mL, 98ng / mL, 99ng / mL, 100ng / mL, 101ng / mL, 102ng / mL, 103ng / mL, 104ng / mL, 105ng / mL, 106ng / mL, 107ng / mL, 108ng / mL, 109ng / mL, 110ng / mL, 111ng / mL, 112ng / mL, 113ng / mL, 114ng / mL, 115ng / mL, 116ng / mL,117ng / mL、118ng / mL、119ng / mL、120ng / mL、121ng / mL、122ng / mL、123ng / mL、124ng / mL、125ng / mL、126ng / mL、127ng / mL、128ng / mL、129ng / mL、130ng / mL、131ng / mL、132ng / mL、133ng / mL、134ng / mL、135ng / mL、136ng / mL、137ng / mL、138ng / mL、139ng / mL、140ng / mL、141ng / mL、142ng / mL、143ng / mL、144ng / mL、145ng / mL、146ng / mL、147ng / mL、148ng / mL、149ng / mL、150ng / mL、151ng / mL、152ng / mL、153ng / mL、154ng / mL、155ng / mL、156ng / mL、157ng / mL、158ng / mL、159ng / mL、160ng / mL、161ng / mL、162ng / mL、163ng / mL、164ng / mL、165ng / mL、166ng / mL、167ng / mL、168ng / mL、169ng / mL、170ng / mL、171ng / mL、172ng / mL、173ng / mL、174ng / mL、175ng / mL、176ng / mL、177ng / mL、178ng / mL、179ng / mL、180ng / mL、181ng / mL、182ng / mL、183ng / mL、184ng / mL、185ng / mL、186ng / mL、187ng / mL、188ng / mL、189ng / mL、190ng / mL、191ng / mL、192ng / mL、193ng / mL、194ng / mL、195ng / mL、196ng / mL、197ng / mL、198ng / mL、199ng / mL、200ng / mL、205ng / mL、210ng / mL、215ng / mL、220ng / mL、225ng / mL、230ng / mL、235ng / mL、240ng / mL、245ng / mL、250ng / mL、255ng / mL、260ng / mL、265ng / mL、270ng / mL、275ng / mL、280ng / mL、285ng / mL、290ng / mL、295ng / mL、300ng / mL、305ng / mL、310ng / mL、315ng / mL、320ng / mL、325ng / mL、330ng / mL、335ng / mL、340ng / mL、345ng / mL、350ng / mL、355ng / mL、360ng / mL、365ng / mL、370ng / mL、375ng / mL、380ng / mL、385ng / mL、390ng / mL、395ng / mL、400ng / mL、405ng / mL、410ng / mL、415ng / mL、420ng / mL、425ng / mL、430ng / mL、435ng / mL、440ng / mL、445ng / mL、450ng / mL、455ng / mL、460ng / mL、465ng / mL、470ng / mL、475ng / mL、480ng / mL、485ng / mL、490ng / mL、495ng / mL、500ng / mL、505ng / mL、510ng / mL、515ng / mL、520ng / mL、525ng / mL、530ng / mL、535ng / mL、540ng / mL、545ng / mL、550ng / mL、555ng / mL、560ng / mL、565ng / mL、570ng / mL、575ng / mL、580ng / mL、585ng / mL、590ng / mL、595ng / mL、600ng / mL、605ng / mL、610ng / mL、615ng / mL、620ng / mL、625ng / mL、630ng / mL、635ng / mL、640ng / mL、645ng / mL、650ng / mL、655ng / mL、660ng / mL、665ng / mL、670ng / mL、675ng / mL、680ng / mL、685ng / mL、690ng / mL、695ng / mL、700ng / mL、705ng / mL、710ng / mL、715ng / mL、720ng / mL、725ng / mL、730ng / mL、735ng / mL、740ng / mL、745ng / mL、750ng / mL、755ng / mL、760ng / mL、765ng / mL、770ng / mL、775ng / mL、780ng / mL、785ng / mL、790ng / mL、795ng / mL、800ng / mL、805ng / mL、810ng / mL、815ng / mL、820ng / mL、825ng / mL、830ng / mL、835ng / mL、840ng / mL、845ng / mL、850ng / mL、855ng / mL、860ng / mL、865ng / mL、870ng / mL、875ng / mL、880ng / mL、885ng / mL、890ng / 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 of an effector.

[0056] In certain embodiments, the synthetic medium may include a mixture of effectors. For example, the synthetic medium may include at least 2, 3, 4, 5, 6, 7, 8, 9 or more effectors. In some cases, the effector may be a cytokine. Examples of the cytokines include IL-1, TNF-α, TPO, SCF, IL-3, IL-6, IL-12, IL-4, CXCL8, FLT3L, GM-CSF, IFNa, PGE2, retroviral infection and T cell activation reagents, UM729 and G-CSF. In certain embodiments, the synthetic medium includes a mixture of GM-CSF and FLT3L. In certain embodiments, the IFNa is IFN2b. In certain embodiments, the synthetic medium includes a mixture of GM-CSF, FLT3L, SCF and IFN2b. In certain embodiments, the synthetic medium does not include UM729.

[0057] The artificially synthesized partially differentiated dendritic cell progenitor cells differentiated from host progenitor cells using synthetic culture medium as described herein are different from naturally occurring dendritic cell progenitor cells or mature dendritic cells. For example, under conditions where naturally occurring dendritic cells cannot differentiate, the artificially synthesized partially differentiated dendritic cell progenitor cells can differentiate into mature dendritic cells (such as cDC1 or cDC2) or immature dendritic cells. For example, in the presence of inflammatory or immunosuppressive cytokines (such as cytokines secreted by tumors), the artificially synthesized partially differentiated dendritic cell progenitor cells can differentiate into mature dendritic cells. For example, the artificially synthesized partially differentiated dendritic cell progenitor cells derived from human progenitor cells can differentiate into antigen presenting cells (APCs), cDC2s, monocytes, immature dendritic cells, or a combination thereof. Therefore, in certain embodiments, the artificially synthesized partially differentiated dendritic cell progenitor cells can differentiate into APCs, cDC2s, monocytes, immature dendritic cells, or a combination thereof in the presence of inflammatory or immunosuppressive cytokines (such as cytokines secreted by tumors).

[0058] As disclosed herein, the artificially synthesized partially differentiated dendritic cell progenitor cells may exhibit a specific phenotype different from naturally occurring dendritic cell progenitor cells. For example, the artificially synthesized partially differentiated dendritic cell progenitor cells may have CD115 + , CD11b negative, CD11c negative, MHCII negative, CD45R / B220 negative, cKIT negative / low and Clec9A negative. Alternatively, in certain embodiments, the artificially synthesized partially differentiated dendritic cell progenitor cells may have one or more flow cytometry phenotypes. - 、CD19 - 、CD335 - 、CD66b - 、CD10 - 、CD14 - 、CD34 + and CD115 + One or more flow cytometric phenotypes in the

[0059] In certain embodiments, the artificially synthesized partially differentiated dendritic cell progenitors can be engineered dendritic cell progenitors. For example, the artificially synthesized partially differentiated dendritic cell progenitors can be engineered to co-express a transgene that, when expressed, acts synergistically with the artificially synthesized partially differentiated dendritic cell progenitors to activate the subject's immune system. For example, the engineered dendritic cell progenitors described herein can be engineered to co-express an interleukin, an effector, or a combination thereof. Interleukins that can be co-expressed in dendritic cell progenitors can 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 certain embodiments, the interleukins described herein include interleukins derived from the same species as the host. For example, interleukin as described herein can include human interleukin, wherein dendritic cell progenitor cells derive from human cells.The effector that can be co-expressed in engineering dendritic cell progenitor cells can include internalization receptors, such as extracellular vesicle internalization receptors (EVIR);Or cytokines, such as IL-1, TNF-α, IL-6, IL-12, IL-2, CXCL8, FLT3L, GM-CSF, IFNa, PGE2, retroviral infection and T cell activation reagents and G-CSF.In certain embodiments, the effector includes an effector from the same species as dendritic cell progenitor cells.For example, effector as described herein includes human effector, wherein dendritic cell progenitor cells derive from human cells.

[0060] In vitro cell compositions can include artificially synthesized partially differentiated dendritic cell progenitor cells. In some cases, the in vitro cell compositions can be used to prepare functional mature dendritic cells (e.g., antigen presenting cells (APCs), monocytes, immature dendritic cells, cDC1 or cDC2) used as therapeutic agents in vitro. In some cases, the in vitro cell compositions can be included in a pharmaceutical composition further comprising a pharmaceutically acceptable excipient, diluent or carrier. In certain embodiments, the pharmaceutical preparation can include an excipient. The excipient includes an excipient described in the American Pharmaceutical Association's Handbook of Pharmaceutical Excipients (1986). In certain embodiments, the excipient can include a buffer, a preservative, a stabilizer, an adhesive, a compacting agent, a lubricant, a chelating agent, a dispersion enhancer, a disintegrating agent, a flavoring agent, a sweetener, or a coloring agent. The diluent may include water, glycerol, methanol, ethanol, aqueous solutions (such as acetic acid, citric acid, maleic acid, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid or the like), 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, glucose, magnesium oxide, dextrin, palmitic acid glycerin, calcium base, lactose, maltose, simethicone, sorbitol, starch, pregelatinized starch, talc, lactitol, xylitol), and / or anhydrous compounds, hydrates and / or pharmaceutically acceptable derivatives thereof or combinations thereof.

[0061] The subject can be administered a pharmaceutical composition comprising an in vitro cell composition as described herein to treat a disease as described herein. In certain embodiments, the pharmaceutical composition can also include an interleukin, an effector, or a combination thereof. The interleukin that can be included in the pharmaceutical composition can 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 certain embodiments, interleukin as described herein includes interleukin derived from the same species as host.For example, interleukin as described herein can include human interleukin, wherein dendritic cell progenitor cell derives from human cells.The effector that can be included in the pharmaceutical composition can include internalization receptor, such as extracellular vesicle internalization receptor (EVIR) or cytokine, such as IL-1, TNF-α, IL-6, IL-12, IL-2, CXCL8, FLT3L, IFNa and GM-CSF.In certain embodiments, the effector includes the effector from the same species as dendritic cell progenitor cell.For example, effector as described herein includes human effector, wherein dendritic cell progenitor cell derives from human cells.

[0062] Method for preparing dendritic cell progenitor cells

[0063] Also disclosed herein is a method for preparing artificially synthesized partially differentiated dendritic cell progenitor cells. As disclosed herein, the artificially synthesized partially differentiated dendritic cell progenitor cells can be prepared by contacting host progenitor cells with a synthetic culture medium as described herein. Amplification can include culturing host progenitor cells isolated from a host sample (e.g., bone marrow or blood) in an amplification culture medium. The amplification culture medium can include 10% FBS, 1% L-glutamine, 100ng / ml SCF, 40ng / ml TPO, 50ng / ml FLT3L, 30ng / ml IL-3, 30ng / ml IL-6, and 30ng / ml IL-1b. Similarly, human progenitor cells isolated from a human sample (e.g., umbilical cord blood CD34 + Expansion of human progenitor cells may comprise culturing the human progenitor cells in an expansion medium comprising FLT3L, SCF, IL3, IL6, and TPO.

[0064] The partial differentiation of host progenitor cells (such as amplified hematopoietic stem / progenitor cells) to artificially synthesized partially differentiated dendritic cell progenitor cells can be achieved by culturing host progenitor cells with synthetic culture medium as described herein (such as culture medium containing cytokines or effectors). In some cases, the partial differentiation can be carried out for 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, cells can be selected according to phenotype and separated to produce purified artificially synthesized partially differentiated dendritic cell progenitor cells.

[0065] Treatment

[0066] Also disclosed herein are methods for treating a disease in a subject in need and pharmaceutical compositions for treating a disease. In certain embodiments, the method for treatment may include administering to the subject artificially synthesized partially differentiated dendritic cell progenitor cells as described herein, an in vitro cell composition containing artificially synthesized partially differentiated dendritic cell progenitor cells as shown herein, or a pharmaceutical composition comprising artificially synthesized partially differentiated dendritic cell progenitor cells as described herein. In certain embodiments, the method for treating may include administering to the subject mature dendritic cells (e.g., differentiated in vitro) differentiated from artificially synthesized partially differentiated dendritic cell progenitor cells as described herein, or an in vitro cell composition or pharmaceutical composition comprising mature dendritic cells differentiated from artificially synthesized partially differentiated dendritic cell progenitor cells as shown herein.

[0067] Administration to a subject can include administration by inhalation, otic, oral, conjunctival, intradental, intracervical, intraantral, intratracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, intrastitial, intraperitoneal, intraamniotic, intraarterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardial, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebroventricular, intracerebral, intracorneal, intracoronary, intracoronary, intracoronary, intracavernous, intracorporeal, intradermal, intradiscal, intraductal, intraduodenal, intradural, intradermal, intraesophageal, intragastric, intragingival, intrahippocampal, intraileal, intra

[0014] The invention also includes the use of intrathecal, intrathecal, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathoracic, intraductal, intratumoral, intratympanic, intrauterine, intravascular, intravenous bolus, intravenous drip, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, transcutaneous, periarticular, epidural, perineural, periodontal, rectal, retrobulbar, subarachnoid, subcutaneous, sublingual, submucosal, topical, transcutaneous, transmucosal, transplacental, transtracheal, transtympanic, ureteral, vaginal, infraorbital administration, or any combination thereof.

[0068] In certain embodiments, the method of treatment may include treating a cancer in a subject. Examples of the cancer may include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone tumor, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, 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's sarcoma, intraocular melanoma, retinoblastoma, gallbladder cancer, cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, glioma, visual pathway and hypothalamus in children, Hodgkin lymphoma, melanoma, islet cell carcinoma, Kaposi's 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 tumor, plasmacytoma, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer, and uterine cancer.

[0069] In certain embodiments, the administration is sufficient to reduce the number and / or size of cancer cells. For example, where the cancer is a solid tumor cancer, the administration can result in a reduction in tumor size and / or inhibition of tumorigenesis.

[0070] Methods for treating cancer do not require knowledge of tumor antigens. In certain embodiments, administration of a composition comprising synthetic partially differentiated dendritic cells or mature dendritic cells differentiated therefrom does not require administration or co-expression of an antigen expressed on a particular cell of the cancer. Conversely, administration of a composition comprising synthetic partially differentiated dendritic cells or mature dendritic cells differentiated therefrom enables treatment of cancers where the target is unknown. In certain embodiments, the effector is not expressed on cells of the cancer. Example

[0071] In order to better understand the present disclosure and its many advantages, the following examples are given for illustration only and are not intended to limit the scope of the present disclosure.

[0072] Example 1: Generation of dendritic cell progenitors (DCPs) from mouse hematopoietic cells

[0073] Dendritic progenitor cells (DCPs) were prepared from mouse hematopoietic stem / progenitor cells using a two-step method ( Figure 1A ).

[0074] Step 1: Expansion of hematopoietic stem / progenitor cells

[0075] The expansion step uses a medium that supports the maintenance and expansion of hematopoietic stem / progenitor cells (HSPCs). The medium includes RPMI 1640 medium containing 10% FBS, 1% L-glutamine, and 1% Penstrep (referred to as complete RPMI medium), supplemented 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-1b.

[0076] Whole mouse bone marrow (BM) cells were isolated from the long bones of C57BL / 6 mice and incubated in 5-10 ml of RBC lysis buffer (Cat. No. R7767-100ML) for 5 minutes to remove red blood cells (RBCs). BM cells were then passed through a 70 μm cell strainer (Cat. No. 352350), washed in complete RPMI medium, and resuspended and plated in the above-mentioned HSPC medium (1-3 x 10 6 Cells were cultured in HSPC medium for 2 days. The suspended cells were then harvested and seeded in HSPC medium at the same density for another day to further remove the remaining adherent cells.

[0077] Step 2: Differentiation of HSCPs into DCPs

[0078] The suspended HSPCs (at the end of day 3 of step 1) were collected and washed once in complete RPMI medium. The collected cells were then cultured in a medium that supports cDC1 differentiation. The medium consisted of complete RPMI medium supplemented with 200 ng / ml FLT3L and 5 ng / ml GM-CSF. The cells were plated at 1-3 x 10 6 After 3 days of differentiation in cDC1 medium, an equal volume of cDC1 medium was added to each well. After another 2 days of culture (end of day 5 of step 2), the cell culture contained 20-50% of cells, which were called DCPs. Then, negative selection was performed to deplete the major lineage positive cells (CD5 + 、CD45R / B220 + 、CD11b + 、CD19 + 、Ly6C / G + TER119 + ), DCPs can be further enriched. At the end of the process (at the end of day 8 of steps 1 and 2), the final purity of DCPs after negative selection is 60-90% ( Figure 1B ).

[0079] After enrichment, DCPs showed the following phenotypes by flow cytometry analysis: CD115+, CD11b-negative, CD11c-negative, MHCII-negative, CD45R / B220-negative, cKIT-negative / low, and Clec9A-negative ( Figure 1C These DCPs are distinct from common dendritic cell progenitors (CDPs), as CDPs express Clec9A, and from pre-cDC1s, as pre-cDC1s express Clec9A and CD11c.

[0080] Thus, this example demonstrates a simple protocol for the efficient generation and enrichment of DCPs from mouse bone marrow.

[0081] Example 2: Better in vivo differentiation of DCPs into conventional type I and type II DCs (cDC1 and cDC2)

[0082] The ability of different types of DCs to form cDC1 in tumor-free mice was then investigated ( Figures 2A-2K ).

[0083] BM cells were isolated from CD45.1 mice to trace donor-derived cells in congenic CD45.2 C57BL / 6 mice. Different types of DCs were generated by differentiating mouse BM cells in different differentiation media ( Figure 2A ). Three types of DC are compared:

[0084] (i) Monocyte-derived dendritic cells (moDCs) were generated using an established protocol involving incubation of BM cells in complete RPMI medium supplemented with 100 ng / ml GM-CSF and 40 ng / ml IL-4 (referred to as moDC medium). The BM cells were cultured in moDC medium at a rate of 2-3 × 10 6 The cells were cultured at a density of 10 cells / ml for 2 days. Non-adherent and loosely adherent cells were then harvested and plated again at a density of 2-3 × 10 6 The cells were seeded at a density of 1 / ml and cultured in moDC medium for another 6 days (fresh moDC culture medium was added once). Figure 2B and 2K Shown are the phenotypes of moDC cultures at the end of the differentiation protocol (end of day 8; note that moDC cultures may contain some macrophages) and before injection into recipient CD45.2 mice. Figure 2B and 2K Two gating strategies are shown; Figure 2K identified high MHCII moDCs, and Figure 2B MHCII+ moDCs are shown as MHCII levels increase from low to high.

[0085] (ii) Conventional type I dendritic cells (cDC1) are professional antigen-presenting cells whose important role in anti-tumor immunity has recently been confirmed. Therefore, cDC1 cells can be used as a new type of DC for vaccination applications. cDC1 cells were generated. Briefly, BM cells were cultured at 2×10 6 Cells were directly seeded in cDC1 medium at a density of 10 cells / ml. 1 ml of fresh cDC1 medium was added every 3-4 days for a total of 16 days. Figure 2C Shown are the phenotypes of cDC1 cells at the end of the differentiation protocol (end of day 16) and before injection into recipient CD45.2 mice.

[0086] (iii) DCPs were generated as described in Example 1. Figure 2D Shown are the phenotypes of DCPs at the end of the protocol (end of day 8) and before injection into recipient CD45.2 mice.

[0087] Each type of DC prepared as described above was systemically infused (via tail vein) into congenic CD45.2 mice without any prior treatment. 6 Two DC doses of cells were administered 3 days apart (in 200 μl PBS) and recipient mice (4 mice per group) were sacrificed 4 days after the second DC injection to analyze the CD45.1 + Cell phenotype ( Figure 2A ). Control mice (3 mice) received PBS. Spleens were thoroughly triturated on a 70 μm cell strainer and depleted of RBCs using RBS lysis buffer; cells were then washed in PBS containing 10% FBS and 2 mM EDTA before analysis. The data showed that in mice that received DCPs, there was a high amount of chimerism between cDCs (cDC1 and cDC2; more than 15% of donor-derived cells were cDC1), while the amount of donor-derived cDCs was negligible in mice that received moDCs or cDC1 cells ( Figures 2E-2F In addition to the large number of cDC chimeras that appeared after DCPs injection, the incidence of donor-derived cells in DCP-treated mice was higher ( Figure 2G ), which suggests that the injected DCPs have a longer lifespan compared with other types of DCs.

[0088] CD45.1 + Analysis of splenocytes showed that moDC-derived cells did not contain cDCs or double negative (DN; CD11b negative, CD11c + , MHC-II + , CD8a negative) immature DCs ( Figure 2HMoreover, only a few of the cells derived from cDC1 are cDC1, cDC2 or DN DCs ( Figure 2I Importantly, the vast majority of cells derived from DCPs are cDC1, cDC2, or DN DCs ( Figure 2J ).

[0089] Statistical analysis: e.g., one-way ANOVA with Tukey's multiple comparison test. P values ​​are indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0090] Thus, this example demonstrates that DCPs efficiently generate cDC1, cDC2, and immature DCs in mice, whereas conventional moDCs or mature cDC1 cannot.

[0091] Example 3: Differentiation of DCPs into intratumoral cDC1 and cDC2 after systemic injection

[0092] Example 2 demonstrates that, unlike moDCs and mature cDC1s, DCPs can efficiently differentiate into cDC1s and cDC2s following systemic injection in tumor-free mice. DCPs demonstrate differentiation capacity in tumor-bearing mice in the presence of upregulated inflammatory and immunosuppressive cytokines. DCPs were subsequently infused into tumor-bearing mice to investigate the migration and phenotype of donor-derived cells within the spleen and tumor.

[0093] MC38 cancer cells (5×10 5 The DCPs were generated and enriched according to the two-step method described in Example 1 and then infused into the tumor-bearing mice. On the 5th and 8th days after tumor injection, the enriched DCPs (2×10 in 200 μl PBS) were injected systemically through the tail vein. 6 cells). A control group of mice (8 mice) received PBS. Mice were sacrificed 4 days after the second injection of DCPs. The spleen and tumor of each mouse were analyzed for the presence of donor-derived (CD45.1 + )cell. Figure 3A The experimental procedure is shown. At the end of the experiment, spleens were thoroughly triturated on a 70 μm cell strainer and depleted of RBCs using RBS lysis buffer. Cells were then washed with PBS containing 10% FBS and 2 mM EDTA before analysis. Single tumor cells were isolated using standard enzymatic digestion methods.

[0094] Figure 3B The gating strategy used to identify intratumoral donor-derived cDC1 and cDC2 is shown. Flow cytometric analysis of tumor-derived cells and splenocytes showed that donor CD45.1 +Cells infiltrate tumors and spleen after systemic injection ( Figure 3C ) and effectively differentiated into cDCs ( Figures 3D-3E and 3H-3I). Almost half of the cDC1s in both tumors and spleens were donor-derived (CD45.1 + )( Figures 3F-3G ), indicating that after infusion of DCPs in non-diseased mice, there was substantial chimerism in the most professional antigen-presenting cells. Chimerism in cDC2s was less pronounced but was well detected in all mice.

[0095] Thus, this example demonstrates that DCPs can efficiently generate cDC1 and cDC2 in tumors and spleens of tumor-bearing mice without prior treatment.

[0096] Example 4: Identification of cytokines that can differentiate DCPs into cDC1s with co-stimulatory capacity

[0097] In vitro screening studies were performed to select cytokines that could maintain the differentiation potential of DCPs into cDC1s and at the same time enable the resulting cDC1s to have T cell co-stimulatory ability when co-cultured with T cells.

[0098] To study the differentiation, DCPs were cultured in cDC1 medium supplemented with different concentrations of selected interleukins (ILs). To study T cell co-stimulation, the same cytokines were used in co-cultures of antigen-loaded cDC1-like cells and antigen-specific T cells.

[0099] DCPs were generated as described in Example 1. Enriched DCPs were 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 specified concentrations, ranging from 2 to 20 ng / ml. 1 ml of cDC1 medium supplemented with the ILs was added to the DCPs every 3-4 days for 15 days ( Figure 4A ). After 15 days, cDC1 cells (identified as CD11c + CD103 + ) and compared them with DCPs cultured in cDC1 medium without IL. Flow cytometric analysis showed that IL-18 and IL-21 induced premature activation of DCPs (by CD86 + The increase in the proportion of CD103 negative cells indicates that it is not conducive to the differentiation of DCP into cDC1 (indicated by CD103 + The proportion of CD86-negative cells decreased, while other ILs (IL-2, IL-12, IL-15, IL-23 and IL-27) did not weaken the differentiation of DCPs to cDC1 ( Figures 4B-4C To investigate the effects of the above ILs on T cell polarization, OVA-loaded cDC1-like cells were then incubated with OVA-specific CD8 + (OT-I) or CD4 + (OT-II) T cell co-culture (3 independent replicates for each condition). Briefly, 50,000 cDC1 were seeded with 0.5 mg of OVA protein (vacstova) or OVA peptide SIINFEKL in U-bottomed 96-well plates 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. T cell activation was determined by intracellular staining with an anti-interferon gamma (IFNγ) antibody using the BD Golgi Stop kit (Cat. No. 554715) according to the manufacturer's instructions. The results showed that IL-12 can induce robust production of IFNγ by OT-I and OT-II T cells ( Figures 4D-4F ).

[0100] Statistical analysis: One-way ANOVA with Tukey's multiple comparison test. All samples were compared with the "untreated" sample. P values ​​are coded as *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0101] Therefore, this example shows that IL-12 does not impair the in vitro differentiation of DCPs into cDC1s, while also enhancing the co-stimulatory capacity of cDC1 progeny in vitro.

[0102] Example 5: DCPs engineered to express IL-12 and IL-2 differentiate into cDCs in mice

[0103] As shown in Example 4, exogenous IL-12 and IL-2 did not interfere with the in vitro differentiation of DCPs into cDC1; in addition, IL-12 enhanced the co-stimulatory capacity of cDC1-like cells. Therefore, the ability of transgenic expression of IL-12 or IL-2 to support the in vivo differentiation of DCPs into cDC1 and cDC2 was studied. To this end, lentiviral vectors (LVs) expressing IL-12 or IL-2 and the marker gene green fluorescent protein (GFP, SEQID NO: 1) were prepared, referred to as IL12-P2A-GFP (SEQ ID NO: 5) and IL2-P2A-GFP (SEQID NO: 3) LVs, respectively. Monocistronic LVs expressing only GFP were also prepared, referred to as GFP LVs. Flow cytometric analysis of mouse BM cells transduced with LVs at a multiplicity of infection (MOI) of 350 in HSPC culture medium and differentiated in cDC1 culture medium showed strong expression of GFP on day 5 after transduction (the time point when the transduced cells mainly contained DCPs). Figure 5A The difference in GFP expression between LVs can be attributed to their different infectivity, which is affected by the size of the transgene. In addition, at 2 weeks post-transduction (a time point when the transduced cells primarily comprised cDC1s), cells transduced with IL12-P2A-GFP and IL2-P2A-GFP-LVs secreted IL-12 and IL-2, respectively, in the cell culture medium as assessed by ELISA. Figure 5B and 5H ; "C" indicates concentration). Thus, LVs were able to achieve robust transduction of DC precursors and sustained expression of IL-12 or IL-2 in cDC progeny.

[0104] Next, the in vivo differentiation potential of untransduced DCPs (UT) and DCPs transduced with IL12-P2A-GFP, IL2-P2A-GFP, or GFP LV was investigated. DCPs were generated from the BM of CD45.1 mice as described in Example 1. Enriched DCPs were cultured at 1.5×10 6 The cells were transferred into cDC1 culture medium at a concentration of 10 cells / ml and transduced with LVs at an MOI of 350. Transduced DCPs were harvested 12 hours after transduction and infused into recipient mice (5 mice per group). Each mouse received 2×10 6 DCPs; 4 days after the infusion of DCPs, recipient mice were sacrificed and spleen cells (isolated by crushing the spleen as described in Example 2) were analyzed for donor-derived (CD45.1 + ) cells Figure 5C ).

[0105] Flow cytometric analysis of splenocytes showed that most CD45.1 + Cells expressing GFP ( Figures 5D-5E), indicating efficient LV transduction, transgene expression, and lack of counterselection of transduced cells in vivo (the differences in GFP expression between LVs can be attributed to their different infectivity, which is affected by the size of the transgene). Importantly, consistent with the in vitro studies shown in Example 4, transduction of DCPs with IL-12 or IL-2 did not hinder their differentiation into cDC1 and cDC2 ( Figures 5F-5G ). However, IL-2 slightly enhanced the generation of cDC1, while IL-12 slightly enhanced the generation of cDC2.

[0106] Statistical analysis: f) One-way ANOVA with Tukey's multiple comparison test. P values ​​are coded as *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0107] Therefore, this example demonstrates that transgenic expression of IL-12 does not impair the in vivo differentiation of DCPs into cDC1s.

[0108] Example 6: DCPs engineered to express IL-12 and tumor antigens promote the expansion of tumor antigen-specific T cells in mice

[0109] As shown in Examples 4 and 5, IL-2 and IL-12 were unable to prevent the differentiation of DCPs into cDC1 in vitro and in vivo. The in vitro data in Example 4 also showed that IL-12 could activate CD4 + and CD8 + To investigate whether IL-12 and IL-2 can activate antigen-specific T cells in vivo, these two cytokines were co-expressed with a truncated intracellular OVA (dOVA, SEQ ID NO: 7) sequence in DCPs by LV transduction. For transduction, enriched DCPs were injected at 1.5 × 10 6 Cells were transferred to cDC1 culture medium at a concentration of 10 cells / ml and transduced with LVs encoding GFP, dOVA, IL2-P2A-dOVA (SEQ ID NO: 9), or IL12-P2A-dOVA (SEQ ID ID: 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 A schematic diagram of the experiment is shown.

[0110] Transduced DCPs were infused into tumor-free mice (5 mice per group). Recipient mice were sacrificed 10 days after vaccination, and splenocytes (isolated by crushing the spleen as described in Example 2) were stained with OVA257-264-bound dextramer according to the manufacturer's instructions (Immunex) to identify OVA-reactive T cells. Flow cytometry data showed that IL-12 expression significantly increased OVA-reactive T cells. After a single DCP vaccination, approximately 12.8% of T cells in the spleen were OVA-reactive ( Figure 6B ). It is noteworthy that DCPs expressing dOVA alone and DCPs expressing dOVA and IL-2 induced much weaker OVA-specific responses. To investigate the phenotype of OVA-reactive T cells in vaccinated mice, splenocytes were also stained with anti-CD44 and anti-CD62L antibodies. Flow cytometric analysis showed a significant enhancement effect of IL12 (CD44 + Expansion of CD62L-negative OVA-reactive T cells ( Figure 6C ).

[0111] Statistical analysis: b, c) One-way ANOVA with Tukey's multiple comparison test. P values ​​are coded as *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0112] Thus, this example demonstrates that DCPs co-expressing IL-12 and tumor antigens stimulate tumor antigen-specific T cells in vivo.

[0113] Example 7: DCPs engineered to co-express IL-12 and tumor antigens block tumorigenesis in mice

[0114] A preventive vaccination study was performed to investigate the antitumor potential of DCP_IL2-P2A-dOVA and DCP_IL12-P2A-dOVA cells.

[0115] Transduced DCPs were prepared as described in Example 6. Transduced and non-transduced DCPs were then injected intravenously into tumor-free mice (0.7×10 6 cells; 6 mice per condition). Then, one week after inoculation, B16-OVA cancer cells (5×10 5 Mice were subcutaneously inoculated with Figure 7A ).

[0116] Blood samples were collected from mice on day 4 after tumor invasion and circulating T cells were analyzed by flow cytometry. Co-expression of IL-12 and dOVA increased CD4 + and CD8 +Effector T cells (CD44 + CD62L negative) Figures 7B-7C Staining of T cells with OVA257-264-bound dextramer showed that almost one-third of circulating CD8 + T cells are OVA-reactive ( Figure 7D ). In contrast, IL-2 stimulated a weaker immune response than OVA.

[0117] Inoculation of mice with DCP_IL12-P2A-dOVA cells completely blocked tumor growth ( Figure 7E In contrast, DCP_IL2-P2A-dOVA cells were unable to completely block tumor growth. Spleens from mice were isolated and purified 26 days after vaccination (19 days after tumor invasion). Flow cytometric analysis demonstrated robust expansion of OVA-reactive effector T cells in mice vaccinated with DCP_IL12-P2A-dOVA cells ( Figures 7F-7G ), which is consistent with the results obtained in tumor-free mice (see Example 6 above). In contrast, IL-2 stimulated a weaker immune response than OVA.

[0118] Statistical analysis: One-way ANOVA with Tukey's multiple comparison test was used in B and G. The # symbol in F and G indicates that the data of interest between the two groups were compared using an unpaired Student's t-test. P values ​​are coded as *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0119] Therefore, this example shows that DCPs co-expressing IL-12 and tumor antigens can stimulate tumor antigen-specific T cells and protect mice from tumor invasion.

[0120] Example 8: DCPs engineered to co-express IL-12 and tumor antigens inhibit tumor growth

[0121] Due to their superior efficacy, IL12-expressing DCP cells were prepared and tested as DCP vaccines in a therapeutic setting involving vaccination of tumor-bearing mice.

[0122] B16-OVA cancer cells (5×10 5 Mice were subcutaneously inoculated with transduced or non-transduced DCPs (0.7×10 6 cells; 6 mice per condition) were intravenously infused into mice ( Figure 8A ).

[0123] DCP_IL12-P2A-dOVA cells significantly inhibited tumor growth ( Figure 8B Furthermore, as shown by staining of immune cells with OVA257-264-bound dextramer, DCP_IL12-P2A-dOVA cells promoted the proliferation of OVA-reactive T cells in the blood, spleen, tumor-draining lymph nodes (tdLN), and tumors of vaccinated mice ( Figures 8C-8F ). Notably, in the tumors of mice inoculated with DCP_IL12-P2A-dOVA cells, an average of about half of the CD8 + T cells were OVA-reactive, consistent with the magnitude of the anti-tumor response observed.

[0124] Statistical analysis: b) Two-way ANOVA with Sidak's multiple comparison test. c) Unpaired Student's t-test. P values ​​are coded as *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0125] Therefore, this example demonstrates that DCPs co-expressing IL-12 and tumor antigens can stimulate tumor antigen-specific T cells and inhibit tumor growth.

[0126] Example 9: DCPs engineered to co-express IL-12 and EVIR enable vaccination against unknown tumor antigens and inhibit tumor growth

[0127] The studies presented in Examples 7-8 above vaccinated mice bearing OVA-expressing tumors with DCPs transduced with IL-12 and a specific surrogate tumor antigen (dOVA). Next, the ability of DCPs transduced with IL-12 to elicit anti-tumor immunity in a tumor antigen-agnostic manner was investigated, i.e., without forcing the DCPs to express any tumor antigen.

[0128] The extracellular vesicle (EV) internalization receptor (EVIR) was used with the extracellular scFv domain of GD2, a disialogangloside expressed on the plasma membrane of mouse and human melanomas and in the EVs they secrete. Unlike traditional DC vaccination against specific tumor antigens, which involves in vitro antigen loading of DCs and elicits T cell responses only against the target (loaded) antigen, vaccination with DCs expressing EVIR can elicit T cell responses that potentially target any EV-associated tumor antigen. Notably, vaccination with DCs expressing EVIR does not require prior knowledge of the tumor antigen, with the exception of molecules (such as GD2) used to capture tumor EVs in vivo.

[0129] Bicistronic IL12-P2A-EVIR (SEQ ID NO: 17) LVs were prepared to express IL-12 and EVIR in DCPs. IL12-P2A-dLNGFR (SEQ ID NO: 15) LVs were also prepared, which express a control non-signaling receptor (truncated low-affinity nerve growth factor receptor, dLNGFR, SEQ ID NO: 13) lacking the extracellular scFv domains of IL-12 and EVIR. Vaccination studies were then performed in tumor-bearing mice ( Figure 9A The generation and transduction of DCPs (DCP_IL12-P2A-EVIR or DCP_IL12-12-P2A-dLNGFR) were performed using the same protocol as described in Examples 5-8 above. B16-OVA-GD2 cancer cells (5×10 5 Mice (8 mice per condition) were inoculated subcutaneously and intravenously infused with transduced DCPs (1×10 6 cells, 7 and 9 days after tumor challenge) or PBS (as mock treatment). Mice were also treated with anti-PD1 antibody to Figure 9A The effector functions of T cells were released at the indicated time points.

[0130] Vaccination with DCP_IL12-12-P2A-EVIR cells resulted in significantly better tumor control compared to DCP_IL12-P2A-dLNGFR cells lacking functional EVIR ( Figure 9B ); Notably, this result was achieved in the absence of OVA vaccination. Mice were subsequently sacrificed and tumors analyzed by flow cytometry. Flow cytometric analysis showed that vaccination with DCP_IL12-12-P2A-EVIR cells activated CD4 T cells in the tumor microenvironment, compared with vaccination with DCP_IL12-P2A-dLNGFR cells lacking functional EVIR. + and CD8 + T cells lead to increased tumor infiltration ( Figures 9C-9F ).

[0131] Use B16 (1×10 5 cells) and B16-OVA-GD2 (3×10 5 Mice were subcutaneously inoculated with a mixture of melanoma cells (1×10 cells) and inoculated with DCP_IL12-P2A-EVIR (8 mice) or DCP_IL12-12-P2A-dOVA cells (1×10 6 cells, 3 and 6 days after tumor invasion; 9 mice), or mock treatment (PBS; 5 mice). All mice were treated with anti-PD1 antibody to release the effector function of T cells ( Figure 9G ).

[0132] Vaccination with DCP_IL12-P2A-EVIR cells resulted in better tumor control than vaccination with DCP_IL12-P2A-dOVA cells ( Figure 9H ), indicating that the EVIR can also elicit antitumor immunity against B16 melanoma cells lacking OVA or GD2. Thus, DCP_IL12-P2A-EVIR vaccination induced an increase in effector T cells in tdLN ( Figure 9I Furthermore, the DCP_IL12-P2A-EVIR vaccine induced OVA-specific T cells in tdLN and spleen (see comparison with PBS-treated mice), although their frequencies were expectedly lower than those in mice vaccinated with DCP_IL12-12-P2A-dOVA cells ( Figures 9J-9K Notably, analysis of tdLN and spleens from mice vaccinated with DCP_IL12-P2A-EVIR cells revealed expansion of non-OVA-reactive effector T cells compared to mice vaccinated with DCP_IL12-P2A-dOVA cells ( Figures 9L-9M ), which may explain its more significant tumor control against B16 tumors containing OVA-negative clones.

[0133] Statistical analysis: b, h) Two-way ANOVA with Tukey's multiple comparison test. c, im) One-way ANOVA with Tukey's multiple comparison test. The # symbol in jk indicates that the data of interest between the two groups were compared by unpaired Student's t-test. P values ​​are coded as *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0134] Thus, this example demonstrates that DCPs co-expressing IL-12 and EVIR inhibit tumor growth in a tumor antigen-agnostic manner and are superior to vaccination against specific tumor antigens.

[0135] Example 10: DCPs engineered to express IL-12 and FLT3L enable efficient vaccination against unknown tumor antigens

[0136] DCPs expressing IL-12 or FLT3L were generated by LV transduction, and vaccination studies were performed in mice by mixing these two DCP populations. To track the transduced DCPs and their progeny, IL-12 was conjugated to dLNGFR and FLT3L was conjugated to GFP (both dLNGFR and GFP are neutral marker proteins).

[0137] To express FLT3L in DCPs, a FLT3L-P2A-GFP (SEQ ID NO: 19) LV was constructed and validated for use as LVs encoding IL-12 and IL-2 (see Examples 5a-b above). Briefly, flow cytometric analysis of mouse BM cells transduced with LVs (at a multiplicity of infection (MOI) of 350) in HSPC culture medium and then differentiated in the absence of exogenous FLT3L showed robust GFP expression at day 5 post-transduction, a time point when the transduced cells primarily comprised DCPs. Figure 10A Furthermore, cells transduced with FLT3L-P2A-GFP LV secreted FLT3L in the cell culture medium, as assessed by ELISA at 2 weeks post-transduction (a time point when the transduced cells primarily contained cDC1s). Figure 10B and 10J ). Thus, LVs enable robust transduction of DC precursors and sustained expression of FLT3L in cDC progeny.

[0138] Vaccination studies were then conducted in tumor-bearing mice ( Figure 10C Mice (10 mice per condition) were subcutaneously inoculated with B16-OVA melanoma cells (5×10 5 cells) and injected intravenously twice (on days 3 and 5 after tumor invasion) 1x10 6 DCP_IL12-P2A-dLNGFR cells and 2x10 6 To study DCPs expressing only a specific cytokine (i.e., IL-12 or FLT3L), cells were mixed with an appropriate number of control DCPs expressing GFP or dLNGFR. Control mice received only DCPs expressing GFP and dLNGFR.

[0139] like Figure 10DAs shown, the combination of DCP_FLT3L-P2A-GFP and DCP_IL12-P2A-LNGFR cells achieved better tumor control than DCP_IL12-12-P2A-dLNGFR (plus control DCP_GFP) cells. Notably, DCP_FLT3L-P2A-GFP (plus control DCP.dLNGFR) cells were ineffective, indicating that the combination of DCP_FLT3-P2A-GFP and DCP_IL12-P2A-dLNGFR cells has synergistic activity. To measure systemic levels of transgenic cytokines in vaccinated mice, serum samples were collected from the tail vein on days 1 and 8 after the last DCP infusion. Serum IL-12 (BD 555256, BD Biosciences) and FLT3L (EMFLT3L, Invitrogen) ELISAs showed an early transient increase in transgenic cytokines in mice receiving DCP_FLT3L-P2A-GFP and DCP_IL12-P2A-dLNGFR, followed by a rapid decrease to near-baseline levels by day 8 post-inoculation ( Figure 10E Interestingly, flow cytometric analysis of intratumoral T cells revealed that after combined vaccination, CD8 + and CD4 + More T cell infiltration ( Figures 10F-10G Furthermore, the combination of DCP_FLT3L-P2A-GFP and DCP_IL12-P2A-LNGFR induced effector CD8 + T cells and CD4 + T cell expansion ( Figures 10H-10I ).

[0140] Statistical analysis: d) Two-way ANOVA with Tukey's multiple comparison test. eh) One-way ANOVA with Tukey's multiple comparison test. The # symbol in d indicates that the data of interest between the two groups were compared by two-way ANOVA with Sidak's multiple comparison test. The # symbol in e-f indicates that the data of interest between the two groups were compared by unpaired Student's t-test. P values ​​are coded as *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0141] Thus, this example demonstrates that DCPs expressing IL-12 and FL3TL inhibit tumor growth in a manner unknown to the tumor antigen.

[0142] Example 11: DCPs engineered to express IL-12 and FLT3L outperform moDCs and cDC1

[0143] We next investigated the ability of transgenic expression of FLT3L and IL12 to confer upon other DC populations, namely moDCs or mature cDC1 (see Example 2 above), to expand T cells and control tumor growth.

[0144] DCPs, moDCs, and cDC1s were generated from the BM of CD45.1 mice as described in Example 2 and transduced with LVs encoding FLT3L, IL-12, and / or marker genes as described in Example 10. Briefly, DCPs and moDCs were transduced at the end of day 8 of the differentiation protocol, while cDC1s were transduced at the end of day 16 of the differentiation protocol.

[0145] moDCs were cultured at a rate of 1.5 × 10 6 cDC1 and DCPs were seeded at a concentration of 1.5 × 10 cells / ml in moDCs culture medium and transduced with FLT3L-P2A-GFP or IL12-P2A-dLNGFR LVs (MOI of 100) for 12–14 h. 6 Cells were seeded at a concentration of 10 cells / ml in cDC1 medium and transduced with FLT3L-P2A-GFP or IL12-P2A-dLNGFR LVs (MOI of 350) for 12-14 hours.

[0146] Vaccination studies were then conducted in tumor-bearing mice ( Figure 11A Mice (7-8 mice per condition) were subcutaneously inoculated with B16-OVA melanoma cells (5x10 5 ), and IL12-P2A-dLNGFR LV-transduced cells (1×10 6 ) and cells transduced with FLT3L-P2A-GFP LV (2x10 6 The control group of mice received only PBS.

[0147] Regardless of the type of DC used, expression of IL12-P2A-dLNGFR and FLT3L-P2A-GFP inhibited tumor growth (compared to mock treatment). However, vaccination with DCPs achieved better tumor control than vaccination with cDC1 or moDCs. Figure 11B ). moDCs were unable to suppress tumor growth like cDC1 or DCPs.

[0148] Flow cytometric analysis of tumors and lymph nodes from mice treated with DCP-IL12-P2A-dLNGFR plus DCP-FLT3L-P2A-GFP cells showed that tumors were heavily infiltrated by T cells, the vast majority of which had an activated phenotype ( Figures 11C-11D); the magnitude of this effect of DCPs was much greater than that of cDC1 and moDCs. tdLN showed similar results ( Figure 11E Tumor single cell suspensions stimulated in vitro with puromycin and ionomycin showed significantly higher production of IFNγ, GZMB, and TNFA in samples from DCPs-vaccinated mice compared to the other groups ( Figures 11F-11H Overall, tumors in mice inoculated with DCP-IL12-P2A-dLNGFR plus DCP-FLT3L-P2A-GFP cells displayed an extensively remodeled tumor microenvironment characterized by residual epithelial (cancer cell) components and a remarkably abundant T cell infiltrate ( Figures 11I-11L ).

[0149] Statistical analysis: b) Two-way ANOVA with Tukey's multiple comparison test. c) One-way ANOVA with Tukey's multiple comparison test. The # symbol in b indicates comparisons between two groups of interest by two-way ANOVA with Sidak's multiple comparison test. P values ​​are coded as *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0150] Thus, this example demonstrates that DCPs expressing IL-12 and FL3TL outperform moDCs and mature cDC1s and extensively remodel the tumor microenvironment toward a pattern dominated by activated T cells.

[0151] Example 12: DCPs engineered to express IL-12 and FLT3L are effective in different cancer types

[0152] The main advantage of the DCP vaccination platform described here is that this strategy is independent of known tumor antigens. Therefore, DCPs have the potential to function as universal DC vaccines. To test the efficacy of DCP vaccination in different tumor models, MC38 cancer cells derived from colorectal cancer were used.

[0153] Mice (10 per condition) were subcutaneously inoculated with MC38 cancer cells (5x10 5 cells) and intravenously infused twice (on the 3rd and 5th days after tumor stimulation) 1x10 6 DCP-IL12-P2A-dLNGFR cells and 2x10 6 Control mice received an equal number of DCPs transduced with control LVs (expressing only dLNGFR or GFP). Figure 12A ).

[0154] DCP-IL12-P2A-dLNGFR plus DCP-FLT3L-P2A-GFP vaccination may represent a robust and efficient method for the control of MC38 tumors in both unknown and known tumor types ( Figure 12B ). Consistent with the results in the B16-OVA melanoma model, engineered DCPs significantly enhanced CD8 + T cell infiltration of tumors and, to a lesser extent, CD4 + T cell infiltration ( Figures 12C-12D In addition, effector CD4 + and CD8 + The proportion of T cells increased significantly ( Figures 12E-12F Overall, MC38 tumors in mice inoculated with DCP-IL12-P2A-dLNGFR plus DCP-FLT3L-P2A-GFP cells displayed a broadly remodeled immune microenvironment characterized by a reduction in myeloid cells including immunosuppressive macrophages, CD8 + T cells were significantly enhanced ( Figures 12G-12H ).

[0155] Statistical analysis: b) Two-way ANOVA with Tukey's multiple comparison test. c) Student's t-test not shown. P values ​​are coded as *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0156] Thus, this example demonstrates that DCPs expressing IL-12 and FL3TL extensively remodel the tumor microenvironment to one dominated by activated T cells in melanoma and colorectal cancer models.

[0157] Example 13: Generation of human DCPs from CD34+ progenitor cells

[0158] Based, at least in part, on the preclinical efficacy of cytokine-armed mouse DCPs, a protocol for generating corresponding human DCPs was developed. + Progenitor cells were collected at 5x10 4 The initial concentration of cells / mL was 400 μg / mL after adding StemSpan CD34 + The cells were cultured in U-bottom 96-well plates in StemSpan SFEMII medium (StemSpan Technologies; 09605) with expansion supplement (StemSpan Technologies; 02691). + The expansion complement contains FLT3L, SCF, IL3, IL6 and TPO ( Figure 13A On the 7th day ( Figure 13C ) obtained the greatest enrichment of human DCPs, defined as CD3 - 、CD19- 、CD335 - 、CD66b - 、CD10 - 、CD14 - 、CD34 + and CD115 + ( Figure 13B Surprisingly, the addition of the stem cell expansion enhancer UM729 (Stem Cell Technologies; 72332) to the culture medium had a negative impact on the yield of human DCPs ( Figures 13D-13E Since there is no mouse model that can support the differentiation of human DCPs into true cDC1 and cDC2, the ability of human DCPs to differentiate into antigen-presenting cells (APCs) (including cDC1, cDC2 and moDCs) was tested in vitro. To this end, DCPs on day 7 were sorted into CD34 + and CD115 + cell( Figures 13F-13G ) and were further differentiated for 7 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), 100 ng / ml FLT3L (Peprotech, 300-19,), 20 ng / ml SCF (Peprotich, 300-07) and 10 ng / ml (1000 IU / ml) IFN2b (Invivogen, rcyc-hifana). Human DCPs mainly generate APCs (cDC1s: CD66b - 、CD3 - 、CD19 - 、CD14 - 、CD141 + and CLEC9A + ;cDC2s:CD66b - 、CD3 - 、CD19 - 、CD14 - 、CD141 - 、CLEC9A - and CD1c + ; Monocytes: CD66b - , CD3 - , CD19 - and CD14 + ; Immature DCs: CD66b - 、CD3 - 、CD19 -、CD14 - 、CD141 + and CLECEA - ), whereas mock-sorted cells differentiated into a wider range of cell types (including granulocytes: CD66b + ; T cells: CD66b - and CD3 + ; B cells: CD66b - 、CD3 - and CD19 + ; Other cells: CD66b - 、CD3 - 、CD19 - 、CD14 - 、CD141 - 、CLEC9A - and CD1c - )( Figure 13H ), indicating that CD34 + and CD115 + Human DCPs are indeed dendritic cell progenitors.

[0159] Statistical analysis: h) Two-way ANOVA with Sidak's multiple comparison test. P values ​​are coded as ****: P < 0.0001.

[0160] Therefore, this example demonstrates that human DCPs with the ability to differentiate into bona fide antigen-presenting cells, including cDC1 and cDC2, can be derived from CD34 + Produced in human hematopoietic stem and progenitor cells.

[0161] Example 14: DCP-derived DCs have superior antigen presentation capabilities compared to moDCs

[0162] To further demonstrate that human DCPs are a source of professional APCs, the antigen-presenting capacity of their progeny (DCP progeny) obtained as described in Example 13 was evaluated and compared with that of conventional moDCs ( Figure 13F To generate human moDCs, blood from healthy human donors was obtained from a blood transfusion center (Lausanne, Switzerland), and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (Stem Cell Technologies; 07801). Subsequently, CD14 + Monocytes, and the CD14 + Monocytes are 10 6Cells were cultured at a concentration of 10 cells / mL in RPMI 1640 (Gibco; 21875-034) containing 10% FBS (Gibco, 10270106), 100 U / mL penicillin, 100 μg / mL streptomycin (Gibco), 2 mM glutamine (Gibco: 25030-024), 50 ng / mL GM-CSF (Peprotech; 300-03), and 50 ng / mL IL4 (Peprotich; 200-04).

[0163] Using the cytomegalovirus (CMV) protein pp65 (or its HLA-A2 restricted peptide, pp65 495-504 ) and HLA-A2 restricted, CMV-specific T cells to assess antigen presentation. Two antigen presentation pathways were studied: (i) loading pp65 495-504 HLA-A2 presentation of peptides that mimics direct presentation, and (ii) pp65 endogenously processed by native pp65 protein. 495-504 Cross-presentation of peptides. T cells previously exposed to pp65 495-504 DCP progeny or moDCs were co-cultured in the presence of A2 / CMV / pp65 protein (exposed at 1 μg / mL for 1 hour at 37°C) or pp65 protein (ab43041-Abcam, exposed at 10 μg / mL for 2 hours at 37°C) to measure direct presentation and cross-presentation, respectively. The stimulated DCP progeny and moDCs were washed in RPMI 1640 (Gibco; 21875-034) containing 10% FBS (Gibco, 10270106), 100 U / mL penicillin, 100 μg / mL streptomycin (15140-122, Gibco), and then co-cultured with A2 / CMV / pp65 protein at a ratio of 1:1. 495-504 Specific CD8 + Co-culture. Co-cultures were incubated at 37°C overnight and maintained in the presence of Brefeldin A (1:1000; BD Biosciences, GolgiPlug, 51-2301KZ) for 4 hours before staining for flow cytometry analysis. + T cells increased ( Figures 14A-14B ), and in each case, DCP progeny outperformed moDCs. Notably, moDCs lack cross-presentation capacity. Thus, these data suggest that human DCPs can produce progeny with antigen-presenting abilities superior to conventional moDCs.

[0164] Statistical analysis: a) Unpaired t-test. P values ​​are coded as ***: P < 0.001.

[0165] Therefore, this example shows that antigen-presenting cells derived from enriched human DCPs have superior antigen-presenting capacity than traditional human monocyte-derived DCs.

[0166] Example 15: Efficient Generation of Transduced Human DCPs

[0167] On day 1, human DCPs were transduced using concentrated lentiviral vectors (LVs). Figure 15A Prior to transduction, cells were transferred to wells coated with a retroviral infection and T cell activation reagent (Takara; T100A) and dmPGE2 (Stem Cell Technologies; 72372) was added to a final concentration of 10 μM. Two hours later, cells were transduced with a 300 MOI of control dLNGFR-encoding LV. This protocol resulted in >80% transgene-expressing DCPs ( Figure 15B The DCPs were then transduced with functional anti-GD2-EVIR. GD2 uptake by DCPs transduced with GD2-EVIR was assessed compared with that of CTRL-dLNGFR. + or GD2 - To investigate the ability of tumor EVs to function, we analyzed the function of GD2-EVIR-DCPs. Tumor EVs (tEVs) were isolated from a human melanoma cell line that was expanded in RPMI 1640 (Gibco; 21875-034) containing 10% FBS (Gibco, 10270106), 1.1 μM arginine (Sigma-Aldrich), 0.48 μM asparagine (Sigma-Ardrich), and 11.25 μM glutamine (Gibco), 10 mM Hepes (Gibco (15630-056), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco: 15140-122). To isolate EVs, the culture medium of cells at 20% confluence was replaced with the above medium modified to contain 5% EV-depleted FBS (standard FBS obtained by ultracentrifugation at 134,000 g for 16 hours at 4°C and then filtered through a 0.1 μm vacuum filter).

[0168] After 4 days of culture, the culture medium was collected for EV isolation by continuous ultracentrifugation: 500 x g for 5 minutes, 200 x g for 10 minutes, and 10,000 x g for 30 minutes at 4°C to remove dead cells and debris. The culture medium was then ultracentrifuged at 134,000 x g for 70 minutes at 4°C using a Hitachi CP80NX ultracentrifuge. The pellet was washed with 35 mL of PBS and ultracentrifuged again at 134,000 x g for 70 minutes at 4°C and finally resuspended in PBS. GD2 was labeled with the membrane dye PKH26 (1:200, MIDI26-1KT, Sigma, PE) at room temperature. + or GD2 - EVs were cultured for 10 min, washed twice with PBS containing 0.1% BSA, washed once with PBS in Vivaspin 500 (300000 MWCO PES, VS0152 Sartorius), and then added to CTRL (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 by flow cytometry the next day indicated that the progeny of GD2-EVIR-transduced DCPs were more sensitive to GD2 compared with the progeny of CTRL-transduced DCPs. + Enhanced tumor EV uptake indicates that EVIR expression in human DCPs specifically enhances the uptake and internalization of GD2-positive tEVs ( Figure 15E ).

[0169] DCPs were then transduced with LVs encoding FLT3L and IL-12. DCPs were transduced with LVs encoding FLT3L-GFP, IL12-dLNGFR, or dLNGFR on day 1 and sorted on day 7 for further culture in FLT3L-deficient StemSpam-SFEMII medium (Stem Cell Technologies; 09605) for 7 days ( Figure 15F ), the FLT3L-deficient StemSpam-SFEMII medium was supplemented with 50 units / ml penicillin (Gibco), 50 μg / ml streptomycin (Gibko), 20 ng / ml GM-CSF (Peproteh; 300-03), 20 ng / ml SCF (Peprotech; 300-07) and 10 ng / ml (1000 IU / ml) IFN2b (Invivogen; rcyc-hifna2b). On day 7, the cells were identified as CD34 + and CD115 + The DCPs of cells robustly expressed GFP from FLT3L-GFP LV ( Figure 15GIn addition, ELISA analysis of the supernatant on day 14 of culture (EHFL3LG, Thermo Fisher Scientific) showed that FLT3L - Transduced cells or FLT3L - DCPs transduced with a mixture of IL-12 and IL-12 (2:1 ratio) efficiently produced FLT3L ( Figure 15H ).

[0170] The ability of IL12 to enhance T cell activation of human DCPs was evaluated ( Figure 15I dLNGFR analysis by flow cytometry revealed efficient transduction of IL12-dLNGFR LVs on day 7 ( Figure 15J In addition, IL12 was detected by ELISA (431701, Biolegend) in the culture supernatant of IL12-transduced DCP progeny after 7 days of differentiation in StemSpam-SFEMII medium (Stem Cell Technologies; 09605) supplemented with 50 units / ml penicillin (Gibco), 50 μg / ml streptomycin (Gibko), 20 ng / ml GM-CSF (Peproteh; 300-03), 100 ng / ml FLT3L (Peprotech; 300-19), 20 ng / ml SCF (Peprotich; 300-07), and 10 ng / ml (1000 IU / ml) IFN2b (Invivogen; rcyc-hifna2b) ( Figure 15K Notably, DCP progeny transduced with IL12 or a mixture of FLT3L and IL12 (2:1 ratio) induced antigen-independent IFNγ production by CMV-specific T cells co-cultured with DCP progeny. Figure 15L ).

[0171] In summary, the present disclosure demonstrates that, like the mouse DCPs in the aforementioned examples, human DCPs can be equipped with effectors (such as EVIR, FLT3L, or IL12) to generate DCP progeny that can recapitulate the anti-tumor immune function of mouse DCPs.

[0172] Sequence Listing

[0173] GFP

[0174] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT

[0175] GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGAT

[0176] GCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG

[0177] CCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTAC

[0178] CCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC

[0179] CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGT

[0180] GAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA

[0181] AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA

[0182] CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCC

[0183] GCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC

[0184] CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC

[0185] GCCCTGAGCAAAGACCCCAACGAGAAGGCGCGATCACATGGTCCTGCTGGAGTTCGT

[0186] GACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA(SEQ ID NO:1)

[0187] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP

[0188] TLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEG

[0189] DTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSV

[0190] QLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMD

[0191] ELYK(SEQ ID NO:2)

[0192] mIL2-P2A-GFP

[0193] ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAAC

[0194] AGCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCA

[0195] GCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGG

[0196] AGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCT

[0197] TCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAG

[0198] ATGAACTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAAT

[0199] TGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGG

[0200] GCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACT

[0201] TTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAAACCG

[0202] GTTCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAAC

[0203] CCCGGTCCTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCT

[0204] GGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCG

[0205] AGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAG

[0206] CTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTC

[0207] AGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGA

[0208] AGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCC

[0209] GCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGC

[0210] ATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAA

[0211] CAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTT

[0212] CAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGC

[0213] AGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGC

[0214] ACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCT

[0215] GGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAG

[0216] TCGAC(SEQ ID NO:3)

[0217] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQEL

[0218] LSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDA

[0219] ENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQTGSGATNFSL

[0220] LKQAGDVEENPGPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL

[0221] KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD

[0222] GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK

[0223] VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL

[0224] LEFVTAAGITLGMDELYK*(SEQ ID NO:4)

[0225] mIL12-P2A-GFP

[0226] ATGTGTCCTCAGAAGCTAACCATCTCCTGGTTTGCCATCGTTTTGCTGGTGTCTCCAC

[0227] TCATGGCCATGTGGGAGCTGGAGAAAGACGTTTATGTTGTAGAGGTGGACTGGACT

[0228] CCCGATGCCCCTGGAGAAACAGTGAACCTCACCTGTGACACGCCTGAAGAAGATGA

[0229] CATCACCTGGACCTCAGACCAGAGACATGGAGTCATAGGCTCTGGAAAGACCCTGA

[0230] CCATCACTGTCAAAGAGTTTCTAGATGCTGGCCAGTACACCTGCCACAAAGGAGGC

[0231] GAGACTCTGAGCCACTCACATCTGCTGCTCCACAAGAAGGAAAATGGAATTTGGTC

[0232] CACTGAAATTTTAAAAAATTTCAAAAACAAGACTTTCCTGAAGTGTGAAGCACCAA

[0233] ATTACTCCGGACGGTTCACGTGCTCATGGCTGGTGCAAAGAAACATGGACTTGAAGT

[0234] TCAACATCAAGAGCAGTAGCAGTTCCCCTGACTCTCGGGCAGTGACATGTGGAATG

[0235] GCGTCTCTGTCTGCAGAGAAGGTCACACTGGACCAAAGGGACTATGAGAAGTATTC

[0236] AGTGTCCTGCCAGGAGGATGTCACCTGCCCAACTGCCGAGGAGACCCTGCCCATTG

[0237] AACTGGCGTTGGAAGCACGGCAGCAGAATAAATATGAGAACTACAGCACCAGCTTC

[0238] TTCATCAGGGACATCATCAAACCAGACCCGCCCAAGAACTTGCAGATGAAGCCTTT

[0239] GAAGAACTCACAGGTGGAGGTCAGCTGGGAGTACCCTGACTCCTGGAGCACTCCCC

[0240] ATTCCTACTTCTCCCTCAAGTTCTTTGTTCGAATCCAGCGCAAGAAAGAAAAGATGA

[0241] AGGAGACAGAGGAGGGGTGTAACCAGAAAGGTGCGTTCCTCGTAGAGAAGACATCT

[0242] ACCGAAGTCCAATGCAAAGGCGGGAATGTCTGCGTGCAAGCTCAGGATCGCTATTA

[0243] CAATTCCTCATGCAGCAAGTGGGCATGTGTTCCCTGCAGGGTCCGATCCGGCGGCGG

[0244] CGGGAGTGGCGGCGGGGGTTCTGGCGGAGGCCTCGCTAGCGGTGGCTCCATGGTCA

[0245] GCGTTCCAACAGCCTCACCCTCGGCATCCAGCAGCTCCTCTCAGTGCCGGTCCAGCA

[0246] TGTGTCAATCACGCTACCTCCTCTTTTTGGCCACCCTTGCCCTCCTAAACCACCTCAG

[0247] TTTGGCCAGGGTCATTCCAGTCTCTGGACCTGCCAGGTGTCTTAGCCAGTCCCGAAA

[0248] CCTGCTGAAGACCACAGATGACATGGTGAAGACGGCCAGAGAAAAACTGAAACATT

[0249] ATTCCTGCACTGCTGAAGACATCGATCATGAAGACATCACACGGGACCAAACCAGC

[0250] ACATTGAAGACCTGTTTACCACTGGAACTACACAAGAACGAGAGTTGCCTGGCTACT

[0251] AGAGAGACTTCTTCCACAACAAGAGGGAGCTGCCTGCCCCCACAGAAGACGTCTTT

[0252] GATGATGACCCTGTGCCTTGGTAGCATCTATGAGGACTTGAAGATGTACCAGACAG

[0253] AGTTCCAGGCCATCAACGCAGCACTTCAGAAATCACAACCATCAGCAGATCATTCTAG

[0254] ACAAGGGCATGCTGGTGGCCATCGATGAGCTGATGCAGTCTCTGAATCATAATGGCG

[0255] AGACTCTGCGCCAGAAACCTCCTGTGGGAGAAGCAGACCCTTACAGAGTGAAAATG

[0256] AAGCTCTGCATCCTGCTTCACGCCTTCAGCACCCGCGTCGTGACCATCAACAGGGTG

[0257] ATGGGCTATCTGAGCTCCGCCACCGGTTCCGGAGCCACGAACTTCTCTCTGTTAAAG

[0258] CAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGGTAGCAAGGCGAGGAGC

[0259] TGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCAC

[0260] AAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCT

[0261] GAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCAC

[0262] CCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACG

[0263] ACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCA

[0264] AGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG

[0265] GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGG

[0266] GCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGC

[0267] AGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGC

[0268] GTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT

[0269] GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACG

[0270] AGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG

[0271] GCATGGACGAGCTGTACAAGTAA(SEQ ID NO:5)

[0272] MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDI

[0273] TWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILK

[0274] NFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKV

[0275] TLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKN

[0276] LQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLV

[0277] EKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGLASGG

[0278] SMVSVPTASPSASSSSSQCRSSMCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRN

[0279] LLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETS

[0280] STTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVA

[0281] IDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSATG

[0282] SGATNFSLLKQAGDVEENPGPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGD

[0283] ATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQ

[0284] ERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIM

[0285] ADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPPVLLPDNHYLSTQSALSKDPN

[0286] EKRDHMVLLEFVTAAGITLGMDELYK*(SEQ ID NO:6)

[0287] dOVA

[0288] ATGGACAGCACCAGGACACAAATAAATAAGGTTGTTCGCTTTGATAAACTTCCAGG

[0289] ATTCGGAGACAGTATTGAAGCTCAGTGTGGCACATCTGTAAACGTTCACTCTTCACT

[0290] TAGAGACATCCTCAACCAAATCACCAAACCAAATGTTTATTCGTTCAGCCTTGC

[0291] CAGTAGACTTTATGCTGAAGAGAGATACCCAATCCCTGCCAGAATACTTGCAGTGTGT

[0292] GAAGGAACTGTATAGAGGAGGCTTGGAACCTATCAACTTTCAAACAGCTGCAGATC

[0293] AAGCCAGAGAGCTCATCAATTCCTGGGTAGAAAGTCAGACAAATGGAATTATCAGA

[0294] AATGTCCTTCAGCCAAGCTCCGTGGATTCTCAAACTGCAATGGTTCTGGTTAATGCC

[0295] ATTGTCTTCAAAGGACTGTGGGAGAAAGCATTTAAGGATGAAGCACACAAGCAAT

[0296] GCCTTTCAGAGTGACTGAGCAAGAAAGCAAACCTGTGCAGATGATGTACCAGATTG

[0297] GTTTATTTAGAGTGGCATCAATGGCTTCTGAGAAAATGAAGATCCTGGAGCTTCCAT

[0298] TTGCCAGTGGGACAATGAGCATGTTGGTGCTGTTGCCTGATGAAGTCTCAGGCCTTG

[0299] AGCAGCTTGAGAGTATAATCAACTTTGAAAAACTGACTGAATGGACCAGTTCTAATG

[0300] TTATGGAAGAGAGGAAGATCAAAGTGTACTTACCTCGCATGAAGATGGAGGAAAAA

[0301] TACAACCTCACATCTGTCTTAATGGCTATGGGCATTACTGACGTGTTTAGCTCTTCAG

[0302] CCAATCTGTCTGGCATCTCCTCAGCAGAGAGCCTGAAGATATCTCAAGCTGTCCATG

[0303] CAGCACATGCAGAAATCAATGAAGCAGGCAGAGAGGTGGTAGGGTCAGCAGAGGC

[0304] TGGAGTGGATGCTGCAAGCGTCTCTGAAGAATTTAGGGCTGACCATCCATTCCTCTT

[0305] CTGTATCAAGCACATCGCAACCAACGCCGTTCTCTTCTTTGGCAGATGTGTTTCCCCT

[0306] GGCGGCGGCTGA(SEQ ID NO:7)

[0307] MDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRL

[0308] YAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSV

[0309] DSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMA

[0310] SECMCILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPR

[0311] MKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVG

[0312] SAEAGVDAASSVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGG*(SEQ ID NO:8)

[0313] mIL2-P2A-dOVA

[0314] ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAAC

[0315] AGCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCA

[0316] GCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGG

[0317] AGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACC

[0318] TTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAA

[0319] GATGAACTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAA

[0320] TTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAA

[0321] GGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGG

[0322] ACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAAA

[0323] CCGGTTCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAA

[0324] AACCCCGGTCCTACGCGTATGGACAGCACCAGGACACAAATAAATAAGGTTGTTCG

[0325] CTTTGATAAACTTCCAGGATTCGGAGACAGTATTGAAGCTCAGTGTGGCACATCTGT

[0326] AAACGTTCACTCTTCACTTAGAGACATCCTCAACCAAATCACCAAACCAAATGATGT

[0327] TTATTCGTTCAGCCTTGCCAGTAGACTTTATGCTGAAGAGAGATACCCAATCCTGCC

[0328] AGAATACTTGCAGTGTGTGAAGGAACTGTATAGAGGAGGCTTGGAACCTATCAACTT

[0329] TCAAACAGCTGCAGATCAAGCCAGAGAGCTCATCAATTCCTGGGTAGAAAGTCAGA

[0330] CAAATGGAATTATCAGAAATGTCCTTCAGCCAAGCTCCGTGGATTCTCAAACTGCAA

[0331] TGGTTCTGGTTAATGCCATTGTCTTCAAAGGACTGTGGGAGAAAGCATTTAAGGATG

[0332] AAGACACACAAGCAATGCCTTTCAGAGTGACTGAGCAAGAAAGCAAACCTGTGCAG

[0333] ATGATGTACCAGATTGGTTTATTTAGAGTGGCATCAATGGCTTCTGAGAAAATGAAG

[0334] ATCCTGGAGCTTCCATTTGCCAGTGGGACAATGAGCATGTTGGTGCTGTTGCCTGAT

[0335] GAAGTCTCAGGCCTTGAGCAGCTTGAGAGTATAATCAACTTTGAAAAACTGACTGA

[0336] ATGGACCAGTTCTAATGTTATGGAAGAGAGGAAGATCAAAGTGTACTTACCTCGCAT

[0337] GAAGATGGAGGAAAAATACAACCTCACATCTGTCTTAATGGCTATGGGCATTACTG

[0338] ACGTGTTTAGCTCTTCAGCCAATCTGTCTGGCATCTCCTCAGCAGAGAGCCTGAAGAT

[0339] ATCTCAAGCTGTCCATGCAGCACATGCAGAAATCAATGAAGCAGGCAGAGAGGTGG

[0340] TAGGGTCAGCAGAGGCTGGAGTGGATGCTGCAAGCGTCTCTGAAGAATTTAGGGCT

[0341] GACCATCCATTCCTCTTCTGTATCAAGCACATCGCAACCAACGCCGTTCTCTTCTTTG

[0342] GCAGATGTGTTTCCCCTGGCGGCGGCTGA(SEQ ID NO:9)

[0343] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQEL

[0344] LSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDA

[0345] ENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQTGSGATNFSL

[0346] LKQAGDVEENPGPTRMDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQI

[0347] TKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVE

[0348] SQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQ

[0349] MMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTS

[0350] SNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVH

[0351] AAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGG

[0352] G*(SEQ ID NO:10)

[0353] mIL12-P2A-dOVA

[0354] ATGTGTCCTCAGAAGCTAACCATCTCCTGGTTTGCCATCGTTTTGCTGGTGTCTCCAC

[0355] TCATGGCCATGTGGGAGCTGGAGAAAGACGTTTATGTTGTAGAGGTGGACTGGACT

[0356] CCCGATGCCCCTGGAGAAACAGTGAACCTCACCTGTGACACGCCTGAAGAAGATGA

[0357] CATCACCTGGACCTCAGACCAGAGACATGGAGTCATAGGCTCTGGAAAGACCCTGA

[0358] CCATCACTGTCAAAGAGTTTCTAGATGCTGGCCAGTACACCTGCCACAAAGGAGGC

[0359] GAGACTCTGAGCCACTCACATCTGCTGCTCCACCAAGAAGGAAAATGGAATTTGGTC

[0360] CACTGAAATTTTAAAAATTTCAAAAAACAAGACTTTCCTGAAGTGTGAAGCACCAA

[0361] ATTACTCCGGACGGTTCACGTGCTCATGGCTGGTGCAAAGAAACATGGACTTGAAGT

[0362] TCAACATCAAGAGCAGTAGCAGTTCCCCTGACTCTCGGGCAGTGACATGTGGAATG

[0363] GCGTCTCTGTCTGCAGAGAAGGTCACACTGGACCAAAGGGACTATGAGAAGTATTC

[0364] AGTGTCCTGCCAGGAGGATGTCACCTGCCCAACTGCCGAGGAGACCCTGCCCATTG

[0365] AACTGGCGTTGGAAGCACGGCAGCAGAATAAATATGAGAACTACAGCACCAGCTTC

[0366] TTCATCAGGGACATCATCAAACCAGACCCGCCCAAGAACTTGCAGATGAAGCCTTT

[0367] GAAGAACTCACAGGTGGAGGTCAGCTGGGAGTACCCTGACTCCTGGAGCACTCCCC

[0368] ATTCCTACTTCTCCCCTCAAGTTCTTTGTTCGAATCCAGCGCAAGAAAGAAAAGATGA

[0369] AGGAGACAGAGGAGGGTGTAACCAGAAAGGTGCGTTCCTCGTAGAGAAGACATCT

[0370] ACCGAAGTCCAATGCAAAGGCGGGAATGTCTGCGTGCAAGCTCAGGATCGCTATTA

[0371] CAATTCCTCATGCAGCAAGTGGGCATGTGTTCCCTGCAGGTCCGATCCGGCGGCGG

[0372] CGGGAGTGGCGGCGGGGTTCTGGCGGAGGCCTCGCTAGCGGTGGCTCCATGGTCA

[0373] GCGTTCCAACAGCCTCACCCTCGGCATCCAGCAGCTCCTCTCAGTGCCGGTCCAGCA

[0374] TGTGTCAATCACGCTACCTCCTTTTTGGCCACCCTTGCCCTCCTAAACCACCTCAG

[0375] TTTGGCCAGGGTCATTCCAGTCTCTGGACCTGCCAGGTGTCTTAGCCAGTCCCGAAA

[0376] CCTGCTGAAGACCACAGATGACATGGTGAAGACGGCCAGAGAAAAACTGAAACATT

[0377] ATTCCTGCACTGCTGAAGACATCGATCATGAAGACATCACACGGGACCAAACCAGC

[0378] ACATTGAAGACCTGTTTACCACTGGAACTACACAAGAACGAGAGTTGCCTGGCTACT

[0379] AGAGAGACTTCTTCCACAACAAGAGGGAGCTGCCTGCCCCCACAGAAGACGTCTTT

[0380] GATGATGACCCTGTGCCTTGGTAGCATCTATGAGGACTTGAAGATGTACCAGACAG

[0381] AGTTCCAGGCCATCAACGCAGCACTTCAGAAATCACAACCATCAGCAGATCATTCTAG

[0382] ACAAGGGCATGCTGGTGGCCATCGATGAGCTGATGCAGTCTCTGAATCATAATGGCG

[0383] AGACTCTGCGCCAGAAACCTCCTGTGGGAGAAGCAGACCCTTACAGAGTGAAAATG

[0384] AAGCTCTGCATCCTGCTTCACGCCTTCAGCACCCGCGTCGTGACCATCAACAGGGTG

[0385] ATGGGCTATCTGAGCTCCGCCACCGGTTCCGGAGCCACGAACTTCTCTCTGTTAAAG

[0386] CAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTACGCGTATGGACAGCACCAGGAC

[0387] ACAAATAAATAAGGTTGTTCGCTTTGATAAACTTCCAGGATTCGGAGACAGTATTGA

[0388] AGCTCAGTGTGGCACATCTGTAAACGTTCACTCTTCACTTAGAGACATCCTCAACCA

[0389] AATCACCAAACCAAATGATGTTTATTCGTTCAGCCTTGCCAGTAGACTTTATGCTGAA

[0390] GAGAGATACCCAATCCTGCCAGAATACTTGCAGTGTGTGAAGGAACTGTATAGAGG

[0391] AGGCTTGGAACCTATCAACTTTCAAACAGCTGCAGATCAAGCCAGAGAGCTCATCA

[0392] ATTCCTGGGTAGAAAGTCAGACAAATGGAATTATCAGAAATGTCCTTCAGCCAAGCT

[0393] CCGTGGATTCTCAAACTGCAATGGTTCTGGTTAATGCCATTGTCTTCAAAGGACTGTG

[0394] GGAGAAAGCATTTAAGGATGAAGACACACAAGCAATGCCTTTCAGAGTGACTGAGC

[0395] AAGAAAGCAAACCTGTGCAGATGATGTACCAGATTGGTTTATTTAGAGTGGCATCAA

[0396] TGGCTTCTGAGAAAATGAAGATCCTGGAGCTTCCATTTGCCAGTGGGACAATGAGCA

[0397] TGTTGGTGCTGTTGCCTGATGAAGTCTCAGGCCTTGAGCAGCTTGAGAGTATAATCA

[0398] ACTTTGAAAAACTGACTGAATGGACCAGTTCTAATGTTATGGAAGAGAGGAAGATCA

[0399] AAGTGTACTTACCTCGCATGAAGATGGAGGAAAAATACAACCTCACATCTGTCTTAA

[0400] TGGCTATGGGCATTACTGACGTGTTTAGCTCTTCAGCCAATCTGTCTGGCATCTCCTC

[0401] AGCAGAGAGCCTGAAGATATCTCAAGCTGTCCATGCAGCACATGCAGAAATCAATG

[0402] AAGCAGGCAGAGAGGTGGTAGGGTCAGCAGAGGCTGGAGTGGATGCTGCAAGCGT

[0403] CTCTGAAGAATTTAGGGCTGACCATCCATTCCTCTTCTGTATCAAGCACATCGCAAC

[0404] CAACGCCGTTCTCTTCTTTGGCAGATGTGTTTCCCCTGGCGGCGGCTGA(SEQ ID

[0405] NO:11)

[0406] MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDI

[0407] TWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILK

[0408] NFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKV

[0409] TLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKN

[0410] LQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLV

[0411] EKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGLASGG

[0412] SMVSVPTASPSASSSSSQCRSSMCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRN

[0413] LLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETS

[0414] STTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVA

[0415] IDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSATG

[0416] SGATNFSLLKQAGDVEENPGPTRMDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHS

[0417] SLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQAR

[0418] ELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVT

[0419] EQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINF

[0420] EKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAES

[0421] LKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFF

[0422] GRCVSPGGG*(SEQ ID NO:12)

[0423] dLNGFR

[0424] ATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCC

[0425] AAGGAGGCATGCCCCAGAGGCCTGTACACACACAGCGGTGAGTGCTGCAAAGCCTG

[0426] CAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGTGAGC

[0427] CCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTGCAAG

[0428] CCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCTGGAGGCCGA

[0429] CGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCGCT

[0430] GCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGAC

[0431] AAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAA

[0432] CCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCC

[0433] GCGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATT

[0434] ACACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCC

[0435] TGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCA

[0436] CAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATC

[0437] CCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGCCTTGTGGCCTACATAGCCT

[0438] TCAAGAGGTGGAACAGGGGGATCCTCTAG(SEQ ID NO:13)

[0439] MDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPC

[0440] LDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEAC

[0441] RVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRW

[0442] ADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVV

[0443] TRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWNRGIL*(SEQ ID NO:14)

[0444] mIL12-P2A-dLNGFR

[0445] ATGTGCCCTCAGAAGCTCACTATCTCCTGGTTTGCTATTGTCCTCCTGGTCAGCCCCC

[0446] TCATGGCTATGTGGGAACTGGAAAAAGACGTGTACGTGGTCGAGGTGGACTGGACA

[0447] CCCGATGCCCCTGGCGAGACAGTGAACCTGACATGTGACACCCCCGAGGAGGACGA

[0448] TATCACCTGGACATCCGATCAGAGGCACGGAGTGATCGGATCTGGCAAGACCCTGAC

[0449] AATCACCGTGAAGGAGTTCCTGGACGCTGGCCAGTACACATGCCACAAGGGCGGAG

[0450] AGACACTGTCTCACAGCCACCTGCTGCTGCACAAGAAGGAGAACGGAATCTGGTCC

[0451] ACAGAGATCCTGAAGAACTTCAAGAACAAGACCTTTCTGAAGTGTGAGGCCCCCAA

[0452] CTACTCTGGCCGGTTCACCTGCAGCTGGCTGGTGCAGCGCAACATGGACCTGAAGTT

[0453] TAACATCAAGAGCTCCTCTAGCTCCCCTGATAGCAGGGCTGTGACATGTGGCATGGC

[0454] CTCCCTGTCTGCTGAGAAGGTGACCCTGGACCAGAGAGATTACGAGAAGTACAGCG

[0455] TGTCCTGTCAGGAGGACGTGACATGCCCAACCGCCGAGGAGACACTGCCAATCGAG

[0456] CTGGCCCTGGAGGCTCGGCAGCAGAACAAGTACGAGAACTACTCTACCAGCTTCTTT

[0457] ATCCGCGACATCATCAAGCCTGATCCCCCTAAGAACCTGCAGATGAAGCCACTGAA

[0458] GAACTCCCAGGTGGAGGTGTCTTGGGAGTACCCCGACTCCTGGTCTACACCTCACTC

[0459] TTACTTCAGCCTGAAGTTCTTTGTGCGGATCCAGCGCAAGAAGGAGAAGATGAAGG

[0460] AGACAGAGGAGGGATGTAACCAGAAGGGCGCCTTTCTGGTGGAGAAGACAAGCAC

[0461] CGAGGTGCAGTGTAAGGGCGGAAACGTGTGCGTGCAGGCCCAGGATCGGTACTACA

[0462] ACTCTAGCTGCTCCAAGTGGGCTTGCGTGCCTTGTAGGGTGAGAAGCGGAGGAGGA

[0463] GGATCCGGAGGAGGAGGATCTGGAGGAGGCCTGGCTTCCGGAGGATCTATGGTGAG

[0464] CGTGCCAACAGCTAGCCCTTCCGCTTCCTCTAGCTCCTCTCAGTGTCGGAGCTCCATG

[0465] TGCCAGTCCCGCTACCTGCTGTTCCTGGCCACCCTGGCTCTGCTGAACCACCTGAGC

[0466] CTGGCTCGCGTGATCCCAGTGTCCGGACCAGCTAGGTGTCTGTCTCAGAGCCGCAAC

[0467] CTGCTGAAGACCACAGACGATATGGTGAAGACAGCCCGGGAGAAGCTGAAGCACTA

[0468] CAGCTGCACCGCTGAGGACATCGATCACGAGGACATCACACGCGATCAGACATCTA

[0469] CCCTGAAGACCTGTCTGCCCTCTGGAGCTGCACAAGAACGAGAGCTGCCTGGCCACA

[0470] AGGGAGACAAGCAGCACCACAAAGAGGATCTTGTCTGCCACCCCAGAAGACAAGCCT

[0471] GATGATGACCCTGTGCCTGGGCTCCATCTACGAGGACCTGAAGATGTACCAGACCG

[0472] AGTTTCAGGCCATCAACGCCGCTCTGCAGAACCACAACCACCAGCAGATCATCCTG

[0473] GACAAGGGCATGCTGGTGGCTATCGATGAGCTGATGCAGTCCCTGAACCACAACGG

[0474] AGAGACACTGAGGCAGAAGCCTCCAGTGGGCGAGGCCGATCCATACAGAGTGAAG

[0475] ATGAAGCTGTGCATCCTGCTGCACGCTTTCTCTACAAGGGTGGTGACCATCAACAGA

[0476] GTGATGGGATACCTGTCCTCTGCCACAGGATCCGGCGCTACCAACTTTTCTCTGCTG

[0477] AAGCAGGCTGGCGACGTGGAGGAGAACCCTGGACCAATGGATGGACCTAGGCTCCT

[0478] CCTGCTGCTGCTGCTGGGCGTGTCCCTGGGAGGAGCTAAGGAGGCTTGCCCAACAG

[0479] GACTGTACACCCACTCTGGCGAGTGCTGTAAGGCCTGTAACCTGGGAGAGGGAGTG

[0480] GCTCAGCCATGCGGAGCTAACCAGACAGTGTGCGAGCCTTGTCTGGACTCCGTGACC

[0481] TTCTCTGACGTGGTGAGCGCCACAGAGCCATGCAAGCCCTGTACCGAGTGCGTGGG

[0482] CCTGCAGTCCATGTCTGCCCCATGCGTGGAGGCTGACGATGCCGTGTGCAGATGTGC

[0483] TTACGGATACTACCAGGACGAGACAACAGGCAGGTGTGAGGCTTGCAGGGTGTGCG

[0484] AGGCTGGAAGCGGCCTGGTGTTTTCCTGCCAGGATAAGCAGAACACAGTGTGCGAG

[0485] GAGTGTCCAGACGGAACCTACAGCGATGAGGCCAACCACGTGGACCCTTGCCTGCC

[0486] ATGTACAGTGTGCGAGGATACCGAGAGGCAGCTGAGAGAGTGTACCAGATGGGCTG

[0487] ACGCTGAGTGCGAGGAGATCCCAGGACGGTGGATCACACGCAGCACCCCACCTGAG

[0488] GGAAGCGACTCCACAGCTCCTTCCACCCAGGAGCCAGAGGCTCCACCAGAGCAGGA

[0489] TCTGATCGCCTCTACAGTGGCTGGAGTGGTGACCACAGTGATGGGCAGCTCCCAGCC

[0490] AGTGGTGACCAGAGGCACCACAGACAACCTGATCCCCGTGTACTGCAGCATCCTGG

[0491] CCGCTGTGGTGGTGGGGCTGGTGGCATACATTGCTTTCAAGAGATGGAATAGGGGAATCCTGTGA(SEQID NO:15)

[0492] MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDI

[0493] TWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILK

[0494] NFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKV

[0495] TLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKN

[0496] LQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLV

[0497] EKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGLASGG

[0498] SMVSVPTASPSASSSSSQCRSSMCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRN

[0499] LLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETS

[0500] STTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVA

[0501] IDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSATG

[0502] SGATNFSLLKQAGDVEENPGPMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKA

[0503] CNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADD

[0504] AVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHV

[0505] DPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWNRGIL*(SEQ ID NO:16)

[0506] mIL12-P2A-EVIR (anti-GD2)

[0507] ATGTGCCCTCAGAAACTGACTATCTCCTGGTTTGCTATTGTCCTCCTCGTGTCCCCTC

[0508] TCATGGCTATGTGGGAACTGGAAAAAGACGTGTACGTGGTCGAGGTGGACTGGACC

[0509] CCAGATGCTCCAGGAGAGACAGTGAACCTGACATGTGACACCCCTGAGGAGGACGA

[0510] TATCACCTGGACATCTGATCAGAGACACGGCGTGATCGGAAGCGGCAAGACCCTGAC

[0511] AATCACCGTGAAGGAGTTCCTGGACGCCGGACAGTACACCTGCCACAAGGGCGGAG

[0512] AGACACTGTCTCACAGCCACCTGCTGCTGCACAAGAAGGAGAACGGCATCTGGAGC

[0513] ACCGAGATCCTGAAGAACTTCAAGAACAAGACATTTCTGAAGTGTGAGGCCCCCAA

[0514] CTACAGCGGACGGTTCACCTGCTCCTGGCTGGTGCAGCGCAACATGGACCTGAAGTT

[0515] TAACATCAAGAGCTCCTCTAGCTCCCCTGATTCCAGGGCTGTGACCTGTGGCATGGC

[0516] CTCCCTGTCTGCTGAGAAGGTGACACTGGACCAGAGAGATTACGAGAAGTACAGCG

[0517] TGTCCTGTCAGGAGGACGTGACATGCCCTACCGCCGAGGAGACACTGCCAATCGAG

[0518] CTGGCCCTGGAGGCTCGGCAGCAGAACAAGTACGAGAACTACTCTACAAGCTTCTTT

[0519] ATCCGCGACATCATCAAGCCCGATCCCCCTAAGAACCTGCAGATGAAGCCTCTGAA

[0520] GAACTCTCAGGTGGAGGTGAGCTGGGAGTACCCAGACTCCTGGTCTACCCCCCACTC

[0521] TTACTTCAGCCTGAAGTTCTTTGTGCGGATCCAGCGCAAGAAGGAGAAGATGAAGG

[0522] AGACAGAGGAGGGCTGTAACCAGAAGGGAGCCTTTCTGGTGGAGAAGACAAGCAC

[0523] CGAGGTGCAGTGTAAGGGCGGAAACGTGTGCGTGCAGGCCCAGGATAGGTACTACA

[0524] ACTCTAGCTGCTCCAAGTGGGCTTGCGTGCCATGTAGGGTGAGATCCGGAGGAGGA

[0525] GGATCTGGAGGAGGAGGAAGCGGAGGAGGCCTGGCTTCCGGAGGCTCTATGGTGAG

[0526] CGTGCCAACCGCTAGCCCATCCGCTTCCTCTAGCTCCTCTCAGTGTCGGAGCTCCAT

[0527] GTGCCAGAGCCGCTACCTGCTGTTCCTGGCCACACTGGCTCTGCTGAACCACCTGTC

[0528] CCTGGCTAGAGTGATCCCCGTGAGCGGACCAGCTAGGTGTCTGTCTCAGAGCCGCA

[0529] ACCTGCTGAAGACCACAGACGATATGGTGAAGACCGCCCGGGAGAAGCTGAAGCAC

[0530] TACTCCTGCACAGCTGAGGACATCGATCACGAGGACATCACCCGCGATCAGACATC

[0531] CACCCTGAAGACATGTCTGCCCCTGGAGCTGCACAAGAACGAGTCTTGCCTGGCCAC

[0532] AAGGGAGACAAGCAGCACCACAAGAGGCAGCTGTCTGCCACCCCAGAAGACATCCC

[0533] TGATGATGACCCTGTGCCTGGGAAGCATCTACGAGGACCTGAAGATGTACCAGACC

[0534] GAGTTCCAGGCCATCAACGCCGCTCTGCAGAACCACAACCACCAGCAGATCATCCT

[0535] GGACAAGGGCATGCTGGTGGCTATCGATGAGCTGATGCAGAGCCTGAACCACAACG

[0536] GCGAGACACTGAGGCAGAAGCCTCCAGTGGGAGAGGCCGATCCTTACAGAGTGAAG

[0537] ATGAAGCTGTGCATCCTGCTGCACGCTTTTTCCACAAGGGTGGTGACCATCAACAGA

[0538] GTGATGGGCTACCTGTCCTCTGCCACAGGATCTGGCGCTCACCAACTTCAGCCTGCTG

[0539] AAGCAGGCTGGCGACGTGGAGGAGAACCCAGGACCTATGGATTTTCAGGTGCAGAT

[0540] CTTCTCCTTTCTGCTGATCTCCGCTTCTGTGATCATGTCCAGGGGAGAGGTGCAGCTG

[0541] CTGCAGTCTGGCCCTGAGCTGGAGAAGCCAGGAGCCTCCGTGATGATCTCTTGCAAG

[0542] GCTAGCGGCAGCTCCTTCACCGGATACAACATGAACTGGGTGAGGCAGAACATCGG

[0543] CAAGAGCCTGGAGTGGATCGGAGCCATCGACCCACTACTACGGAGGCACCTCCTACA

[0544] ACCAGAAGTTTAAGGGCAGAGCCACACTGACCGTGGACAAGTCTAGCTCCACCGCT

[0545] TACATGCACCTGAAGTCCCTGACATCTGAGGATAGCGCCGGTACTACTGCGTGAGC

[0546] GGAATGGAGTACTGGGGACAGGGCACATCCGTGACCGTGTCTAGCGGCGGAGGAGG

[0547] CTCTGGCGGCGGCGGCTCGGCGGAGGAGGAAGCGACGTGGTCATGACACAGACCC

[0548] CACTGTCCCTGCCCGTGAGCCTGGGCGATCAGGCCAGCATCTCCTGCAGGTCCTCTC

[0549] AGTCCCTGGTGCACAGAAACGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCA

[0550] GGACAGTCTCCCAAGCTGCTGATCCACAAGGTGAGCAACCGGTTCTCCGGCGTGCCCT

[0551] GACCGCTTTCTGGAAGCGGCTCCGGAACAGATTTCACCCTGAAGATCAGCAGGGT

[0552] GGAGGCTGAGGACCTGGGAGTGTACTTCTGTTCTCAGAGCACCCACGTGCCCCCCTCT

[0553] GACATTTGGCGCTGGAACCAAGCTGGAGCTGACAGGCCTGCTGGGAGTGTCTCTGG

[0554] GAGGAGCTAAGGAGGCTTGCCCAACAGGCCTGTACACCCACAGCGGAGAGTGCTGT

[0555] AAGGCCTGTAACCTGGGAGAGGGAGTGGCTCAGCCATGCGGAGCTAACCAGACCGT

[0556] GTGCGAGCCCTGTCTGGCACTCTGTGACATTCAGCGATGTGGTGTCCGCTACCGAGCC

[0557] TTGCAAGCCATGTACAGAGTGCGTGGGCCTGCAGTCCATGTCTGCCCCATGCGTGGA

[0558] GGCTGACGATGCCGTGTGCAGGTGTGCTTACGGCTACTACCAGGACGAGACAACAG

[0559] GAAGGTGTGAGGCTTGCAGGGTGTGCGAGGCTGGATCCGGACTGGTGTTTTCTTGCC

[0560] AGGATAAGCAGAACACCGTGTGCGAGGAGTGCAGAGTGTCCAGACGGCACATACAGCGATGA

[0561] GGCCAACCACGTGGACCCCTGCCTGCCTTGACCGTGTGCGAGGATACAGAGAGGC

[0562] AGCTGAGAGAGTGTACCAGATGGGCTGACGCTGAGTGCGAGGAGATCCCAGGCCGG

[0563] TGGATCACACGCTCTACCCCACCAGAGGGAAGCGACTCCACAGCTCCCAGCACCCA

[0564] GGAGCCTGAGGCTCCTCCAGAGCAGGATCTGATCGCCTCTACCGTGGCTGGCGTGGT

[0565] GACCACAGTGATGGGAAGCTCCCAGCCTGTGGTGACACGCGGCACCACAGATAACC

[0566] TGATCCCAGTGTACTGCTCCATCCTGGCCGCTGTGGTGGTGGGACTCGTGGCATACA

[0567] TTGCTTTCAAACGGTGGAATAGGGGAATCCTGTGA(SEQ ID NO:17)

[0568] MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDI

[0569] TWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILK

[0570] NFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKV

[0571] TLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKN

[0572] LQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLV

[0573] EKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGLASGG

[0574] SMVSVPTASPSASSSSSQCRSSMCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRN

[0575] LLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETS

[0576] STTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVA

[0577] IDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSATG

[0578] SGATNFSLLKQAGDVEENPGPMDFQVQIFSFLLISASVIMSRGEVQLLQSGPELEKPGAS

[0579] VMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKS

[0580] SSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVM

[0581] TQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVP

[0582] DRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELTGLLGVSLGGAK

[0583] EACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTE

[0584] CVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTV

[0585] CEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGS

[0586] DSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVG

[0587] LVAYIAFKRWNRGIL*(SEQ ID NO:18)

[0588] mFLT3L-P2A-GFP

[0589] ATGGATTTTCAGGTCCAGATTTTCTCCTTCCTCCTCATTTCAGCCAGCGTCATTATGT

[0590] CTCGGGGGACACCTGACTGTTACTTCAGCCACAGTCCCATCTCCTCCAACTTCAAAG

[0591] TGAAGTTTAGAGAGTTGACTGACCACCTGCTTAAAGATTACCCAGTCACTGTGGCCG

[0592] TCAATCTTCAGGACGAGAAGCACTGCAAGGCCTTGTGGAGCCTCTTCCTAGCCCAGC

[0593] GCTGGATAGAGCAACTGAAGACTGTGGCAGGGTCTAAGATGCAAACGCTTCTGGAG

[0594] GACGTCAACACCGAGATACATTTTGTCACCTCATGTACCTTCCAGCCCCTACCAGAA

[0595] TGTCTGCGATTCGTCCAGACCAACATCTCCCACCTCCTGAAGGACACCTGCACACAG

[0596] CTGCTTGCTCTGAAGCCCTGTATCGGGAAGGCCTGCCAGAATTTCTCTCGGTGCCTG

[0597] GAGGTGCAGTGCCAGCCGGACTCCTCCACCCTGCTGCCCCCAAGGAGTCCCATAGCC

[0598] CTAGAAGCCACGGAGCTCCCAGAGCCTCGGCCCAGGCAGACCGGTTCCGGAGCCAC

[0599] GAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGG

[0600] TGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC

[0601] GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCAC

[0602] CTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTG

[0603] GCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA

[0604] CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGG

[0605] AGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAG

[0606] TTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGA

[0607] GGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCT

[0608] ATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCAC

[0609] AACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCAT

[0610] CGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCT

[0611] GAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCG

[0612] CCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAG(SEQ ID NO:19)

[0613] MDFQVQIFSFLLISASVIMSRGTPDCYFSHSPISSNFKVKFRELTDHLLKDYPVTVAVNLQ

[0614] DEKHCKALWSLFLAQRWIEQLKTVAGSKMQTLLEDVNTEIHFVTSCTFQPLPECLRFVQ

[0615] TNISHLLKDTCTQLLALKPCIGKACQNFSRCLEVQCQPDSSTLLPPRSPIALEATELPEPRP

[0616] RQTGSGATNFSLLKQAGDVEENPGPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEG

[0617] EGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEG

[0618] YVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNV

[0619] YIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSK

[0620] DPNEKRDHMVLLEFVTAAGITLGMDELYK*(SEQ ID NO:20)

[0621] hFLT3L-P2A-GFP

[0622] ATGGATTTTCAGGTCCAGATCTTCTCCTTTCTGCTGATTTCCGCCTCTGTGATCAT

[0623] GTCCAGAGGCACCCAGGACTGCTCTTTCCAGCACAGCCCTATCAGCTCCGATTT

[0624] TGCCGTGAAGATCAGGGAGCTGTCTGACTACCTGCTGCAGGATTATCCAGTGAC

[0625] CGTGGCCAGCAATCTGCAGGACGAGGAGCTGTGCGGAGGCCTGTGGAGGCTGG

[0626] TGCTGGCACAGCGGTGGATGGAGAGACTGAAGACAGTGGCCGGCTCTAAGATG

[0627] CAGGGCCTGCTGGAGCGGGTGAACACCGAGATCCACTTCGTGACAAAGTGCGC

[0628] CTTTCAGCCCCCTCCAAGCTGTCTGCGGTTTGTGCAGACCAATATCAGCAGACT

[0629] GCTGCAGGAGACATCCGAGCAGCTGGTGGCCCTGAAGCCATGGATCACCAGGC

[0630] AGAACTTCAGCAGGTGCCTGGAGCTGCAGTGTCAGCCAGACTCTAGCACACTGC

[0631] CCCCTCCATGGTCCCCCCGCCCTCTGGAGGCAACCGCACCAACAGCCGGCTCCG

[0632] GCGCCACCAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCAG

[0633] GCCCCATGGTGAGCAAGGGCGAGGAGCTGTTCACAGGCGTGGTGCCCATCCTG

[0634] GTGGAGCTGGACGGCGATGTGAATGGCCACAAGTTTAGCGTGTCCGGAGAGGG

[0635] AGAGGGCGATGCAACCTACGGCAAGCTGACACTGAAGTTCATCTGCACCACAG

[0636] GCAAGCTGCCTGTGCCATGGCCCACCCTGGTGACCACACTGACATACGGCGTGC

[0637] AGTGTTTCTCCCGGTATCCCGACCACATGAAGCAGCACGATTTCTTTAAGTCTGC

[0638] CATGCCTGAGGGCTACGTGCAGGAGAGGACCATCTTCTTTAAGGACGATGGCAA

[0639] CTATAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACACTGGTGAACCGGA

[0640] TCGAGCTGAAGGGCATCGACTTTAAGGAGGATGGCAATATCCTGGGCCACAAG

[0641] CTGGAGTACAACTATAATTCCCACAACGTGTATATCATGGCCGATAAGCAGAAG

[0642] AACGGCATCAAGGTGAACTTCAAGATCAGACACAATATCGAGGACGGCTCTGT

[0643] GCAGCTGGCCGATCACTACCAGCAGAACACCCCAATCGGCGACGGACCTGTGCT

[0644] GCTGCCAGATAATCACTATCTGTCTACACAGAGCGCCCTGTCCAAGGACCCTAA

[0645] CGAGAAGAGGGATCACATGGTGCTGCTGGAGTTTGTCACTGCCGCTGGGATTAC

[0646] CCTGGGAATGGATGAGCTGTATAAATAA(SEQ ID NO:21)

[0647] hFLT3L-P2A-GFP

[0648] MDFQVQIFSFLLISASVIMSRGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVAS

[0649] NLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPP

[0650] SCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLE

[0651] ATAPTAGSGATNFSLLKQAGDVEENPGPMVSKGEELFTGVVPILVELDGDVNGHKF

[0652] SVSGEGEGDATYGKLTLKFICTTGKLPVPWPWPTLVTTLTYGVQCFSRYPDHMKQHDF

[0653] FKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH

[0654] KLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLL

[0655] PDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*(SEQ ID NO:22)

[0656] hIL-12-P2A-dLNGFR

[0657] ATGTGCCATCAGCAGCTGGTCATCTCTTGGTTTAGTCTGGTGTTTCTGGCTTCTC

[0658] CACTGGTCCGCAATCTGGGAACTGAAAAAGGACGTGTACGTGGTGGAGCTGGAC

[0659] TGGTATCCTGATGCCCCAGGCGAGATGGTGGTGCTGACCTGTGACACACCCCGAG

[0660] GAGGATGGCATCACCTGGACACTGGATCAGAGCTCCGAGGTGCTGGGAAGCGG

[0661] CAAGACCCTGACAATCCAGGTGAAGGAGTTCGGCGACGCAGGACAGTACACCT

[0662] GCCACAAGGGAGGCGAGGTGCTGTCTCACAGCCTGCTGCTGCTGCACAAGAAG

[0663] GAGGACGGCATCTGGTCCACAGACATCCTGAAGGATCAGAAGGAGCCCAAGAA

[0664] CAAGACCTTCCTGAGGTGTGAGGCCAAGAATTATTCTGGCCGCTTTACCTGCTG

[0665] GTGGCTGACCACAATCTCCACCGATCTGACATTTTCTGTGAAGTCTAGCAGGGG

[0666] ATCCTCTGACCCTCAGGGAGTGACATGCGGAGCAGCCACCCTGAGCGCCGAGC

[0667] GGGTGAGAGGCGATAACAAGGAGTACGAGTATTCTGTGGAGTGTCAGGAGGAC

[0668] AGCGCCTGCCCAGCAGCAGAGGAGTCCCTGCCCATCGAAGTGATGGTGGATGC

[0669] CGTGCACAAGCTGAAGTACGAGAATTATACCAGCTCCTTCTTTATCCGGGACAT

[0670] CATCAAGCCCGATCCCCCTAAGAACCTGCAGCTGAAGCCTCTGAAGAATAGCAG

[0671] ACAGGTGGAGGTGTCCTGGGAGTACCCCGACACCTGGTCCACACCTCACAGCTA

[0672] TTTCTCCCTGACCTTTTGCGTGCAGGTGCAGGGCAAGTCCAAGAGGGAGAAGAA

[0673] GGACCGCGTGTTCACCGATAAGACATCTGCCACCGTGATCTGCCGGAAGAACGC

[0674] CTCCATCTCTGTGAGGGCCCAGGATCGCTACTATTCTAGCTCCTGGTCTGAGTGG

[0675] GCCAGCGTGCCTTGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGG

[0676] AGGAGGCTCCAGAAATCTGCCAGTGGCAACCCCAGACCCTGGAATGTTCCCATG

[0677] TCTGCACCACTCCCAGAACCTGCTGCGGCCGTGTCTAATATGCTGCAGAAGGC

[0678] CAGACAGACACTGGAGTTTTACCCTTGCACCAGCGAGAGATCGACCACGAGG

[0679] ATATCACAAAGGATAAGACCTCCACAGTGGAGGCCTGTCTGCCACTGGAGCTGA

[0680] CCAAGAACGAGTCTTGCCTGAATAGCAGGGAGACATCCTTCATCACCAACGGCT

[0681] CTTGTCTGGCCAGCCGCAAGACATCCTTTATGATGGCCCTGTGCCTGTCTAGCAT

[0682] CTACGAGGACCTGAAGATGTATCAGGTGGAGTTCAAGACCATGAACGCCAAGC

[0683] TGCTGATGGACCCAAAGCGGCAGATCTTTCTGGATCAGAATATGCTGGCCGTGA

[0684] TCGACGAGCTGATGCAGGCCCTGAACTTCAATAGCGAGACAGTGCCTCAGAAGT

[0685] CCTCTCTGGAGGAGCCAGATTTCTACAAGACCAAGATCAAGCTGTGCATCCTGC

[0686] TGCACGCCTTTAGGATCCGCGCCGTGACAATCGACAGAGTGATGTCTTATCTGA

[0687] ACGCAAGCGGATCCGGAGCAACCAACTTCAGCCTGCTGAAGCAGGCCGGCGAT

[0688] GTGGAGGAGAATCCAGGACCAATGGACGGACCAAGGTTATTACTGCTGCTGCT

[0689] GCTGGGCGTGTCTCTGGGAGGAGCAAAGGAGGCATGCCCAACAGGCCTGTACA

[0690] CCCACAGCGGAGAGTGCTGTAAGGCATGTAACCTGGGAGAGGGAGTGGCACAG

[0691] CCTTGCGGAGCCAATCAGACAGTGTGCGAGCCATGTCTGGACTCTGTGACCTTC

[0692] AGCGATGTGGTGTCCGCCACAGAGCCCTGCAAGCCTTGTACCGAGTGCGTGGGC

[0693] CTGCAGTCTATGAGCGCCCCTTGCGTGGAGGCAGACGATGCCGTGTGCAGATGT

[0694] GCCTACGGCTACTATCAGGATGAGACAACAGGCCGGTGTGAGGCATGCCGGGT

[0695] GTGCGAGGCAGGATCTGGCCTGGTGTTTAGCTGCCAGGACAAGCAGAACACAG

[0696] TGTGCGAGGAGTGTCCAGACGGCACCTATTCCGATGAGGGCCAATCACGTGGATC

[0697] CATGCCTGCCCTGTACAGTGTGCGAGGACACCGAGCGGCAGCTGAGAGAGTGT

[0698] ACCAGATGGGCAGATGCAGAGTGCGAGGAGATCCCAGGCCGGTGGATCACAAG

[0699] AAGCACCCCACCCGAGGGCTCCGATTCTACAGCACCTTCCACCCAGGAGCCAGA

[0700] GGACCTCCAGAGCAGGACCTGATCGCCAGCACAGTGGCAGGAGTGGTGACCA

[0701] CAGTGATGGGAAGCTCCCAGCCAGTGGTGACCAGGGGCACCAGACAACCTG

[0702] ATCCCCGTGTACTGCTCCATCCTGGCAGCAGTGGTGGTGGGCCTGGTGGCCTATATCGCTTTCAAGAGGTGGAATCGGGGCATCCTGTAA(SEQ ID NO:23)

[0703] hIL-12-P2A-dLNGFR

[0704] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEE

[0705] DGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGI

[0706] WSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQG

[0707] VTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYEN

[0708] YTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQG

[0709] KSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGG

[0710] GGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEID

[0711] HEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIY

[0712] EDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSL

[0713] EEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGSGATNFSLLKQAGDVEENPG

[0714] PMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTV

[0715] CEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDET

[0716] TGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDT

[0717] ERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWNRGIL*(SEQ ID NO:24)

[0718] Although exemplary embodiments have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art. It should be understood that various alternatives to the described embodiments of the present invention may be employed. The following claims define the scope of the present disclosure, and methods and structures within the scope of these claims and their equivalents are intended to be included within the scope of this disclosure.

Claims

1. An in vitro cell composition comprising artificially synthesized partially differentiated dendritic cell progenitor cells, characterized in that: The artificially synthesized partially differentiated dendritic cell progenitor cells have a CD115+, CD11c- and Clec9A- phenotype, which is determined by flow cytometry.

2. The in vitro cell composition according to claim 1, characterized in that The phenotype of the artificially synthesized partially differentiated dendritic cell progenitor cells further includes one or more phenotypes selected from the following group: CD11b - , MHCII - 、CD45R / B220 - and cKIT - , the phenotype was determined by flow cytometry.

3. A differentiated cDC1 or cDC2 dendritic cell, wherein the differentiated cDC1 or cDC2 dendritic cell is differentiated from the artificially synthesized partially differentiated dendritic cell progenitor cell according to claim 1 or 2.

4. The differentiated cDC1 or cDC2 dendritic cell according to claim 3, characterized in that The artificially synthesized differentiated cDC1 or cDC2 is an engineered dendritic cell expressing interleukin or effector.

5. The differentiated cDC1 or cDC2 dendritic cell according to claim 4, characterized in that The engineered dendritic cells express an interleukin, which is IL12.

6. The differentiated cDC1 or cDC2 dendritic cell according to claim 4, characterized in that The engineered dendritic cells express effectors 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.

7. An in vitro cell composition comprising artificially synthesized partially differentiated dendritic cell progenitor cells, characterized in that: The artificially synthesized partially differentiated dendritic cell progenitor cells comprise one or more phenotypes selected from the group consisting of: CD115 + 、CD34 + 、CD3 - 、CD19 - 、CD335 - 、CD66b - 、CD10 - and CD14 - , the phenotype was determined by flow cytometry.

8. Antigen presenting cells (APCs) differentiated from the artificially synthesized partially differentiated dendritic cell progenitor cells according to claim 7.

9. The APC according to claim 8, wherein: The APCs are engineered dendritic cells expressing interleukins or effectors.

10. The APC according to claim 9, wherein: The engineered dendritic cells express an interleukin, which is IL12.

11. The APC according to claim 9, wherein: The engineered dendritic cells express effectors selected from the group consisting of extracellular vesicle internalizing receptor (EVIR), FMS-like tyrosine kinase 3 ligand (FLT3L), GM-CSF, IL-6, IL-12, IFNα2β, IFNγ, SCF and TNF-α.

12. A method for preparing artificially synthesized partially differentiated dendritic cell progenitor cells, the method comprising: (a) Obtaining transiently expanded hematopoietic stem / progenitor cells (HSPCs); and (b) contacting the transiently expanded HSPCs with a synthetic culture medium comprising FMS-like tyrosine kinase 3 ligand (FLT3L) and GM-CSF, including or excluding IL-1, IL-2, IL-4, IL-6, IL-12, CXCL8, G-CSF, TNF-α, IFNa, PGE2 or recombinant human fibronectin fragments, in an amount sufficient to differentiate the HSPCs into cells with CD115 + 、CD11c - and Clec9A - Synthetic partially differentiated dendritic cell progenitors with phenotypes determined by flow cytometry.

13. The method of claim 12, further comprising contacting the HSPCs with a culture medium comprising FBS, L-glutamine, SCF, TPO, FLT3L, IL-3, IL-6, and IL-1b, thereby preparing transiently expanded HSPCs prior to the contacting in step (b).

14. A method for preparing artificially synthesized partially differentiated dendritic cell progenitor cells, the method comprising: (a) Obtaining transient expansion of CD34 + Human hematopoietic stem and progenitor cells (human HSPCs); and (b) contacting the transiently expanded human HSPCs with a synthetic culture medium comprising FMS-like tyrosine kinase 3 ligand (FLT3L), IL-3, IL-6, TPO and SCF, and including or excluding IFNγ, IL-12, recombinant human fibronectin fragment, TNF-α or UM729, in an amount sufficient to differentiate the HSPCs into cells with a phenotype selected from the group consisting of CD115 + 、CD34 + 、CD3 - 、CD19 - 、CD335 - 、CD66b - 、CD10 - and CD14 - The invention relates to artificially synthesized partially differentiated dendritic cell progenitor cells having one or more phenotypes as determined by flow cytometry.

15. A pharmaceutical composition for treating a disease, comprising: (a) an in vitro cell composition according to any one of claims 1, 2 or 7, a differentiated cDC1 or cDC2 dendritic cell according to any one of claims 3-6, or an APC according to any one of claims 8-11; and (b) a pharmaceutically acceptable excipient, diluent or carrier.

16. The pharmaceutical composition according to claim 15, characterized in that The disease is cancer.

17. The pharmaceutical composition of claim 15 or 16, further comprising an interleukin or an effector.

18. The pharmaceutical composition according to claim 15 or 16, characterized in that The pharmaceutical composition comprises differentiated cDC1 or cDC2 dendritic cells, wherein the differentiated cDC1 or cDC2 dendritic cells are engineered dendritic cells expressing interleukins or effectors.

19. The pharmaceutical composition according to claim 15 or 16, characterized in that The pharmaceutical composition includes APCs, which are engineered dendritic cells expressing interleukins or effectors.

20. The pharmaceutical composition according to any one of claims 17 to 19, characterized in that The interleukin is IL-12.

21. The pharmaceutical composition according to any one of claims 17 to 19, characterized in that 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.

22. The pharmaceutical composition according to claim 17, characterized in that The effector is not expressed on cancer cells.

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