Dendritic cell-activated chimeric antigen receptor and its use

Abstract

This invention provides a composition and method for activating dendritic cells (DCs) in an immunosuppressive tumor microenvironment. [Solution] A chimeric antigen receptor (CAR) for activating dendritic cells (DCs) in an immunosuppressive tumor environment is provided. A composition containing the CAR, a polynucleotide encoding the CAR, a vector containing the polynucleotide encoding the CAR, manipulated cells containing the CAR, and a method using the CAR are also provided.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority to Chinese Patent Application No. 202110022268.5, filed on 8 January 2021, and the entire disclosure of this Chinese Patent Application constitutes part of this specification by reference.

[0002] Field of Invention This disclosure generally relates to the field of cell therapy. In particular, this disclosure relates to compositions and methods for activating dendritic cells (DCs) in an immunosuppressive tumor microenvironment. [Background technology]

[0003] As a crucial link between the innate and adaptive immune systems, dendritic cells (DCs) are the primary antigen-presenting cells (APCs) that activate T-cell-dependent immunity (Non-Patent Literature 1 and Non-Patent Literature 2), particularly in tumor-specific immune responses (Non-Patent Literature 3). Previous studies have revealed that tumor-infiltrating dendritic cells (TIDCs) typically exhibit immature or dysfunctional phenotypes in immunosuppressive tumor microenvironments or tumor immunosuppressive microenvironments (TIME) that suppress T-cell infiltration and activation (Non-Patent Literature 4).

[0004] To rescue the abnormal behavior of TIDCs, many signaling pathways, such as siRNA silencing of PD-L1 and PD-L2 on dendritic cells, have been identified, but significant progress has not been made in their clinical application (Non-Patent Documents 5, 6, and 7).

[0005] Therefore, it is necessary to develop novel methods for activating dendritic cells (e.g., tumor-infiltrating dendritic cells) in TIME. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] R. M. Steinman, Decisions about dendritic cells: past, present, and future. Annu. Rev. Immunol. 30, 1-22 (2012) [Non-Patent Document 2] S. Puhr et al., Dendritic cell development-History, advances, and open questions. Semin. Immunol. 27, 388-396 (2015) [Non-Patent Document 3] M. Hansen et al., The role of dendritic cells in cancer. Semin. Immunopathol. 39, 307-316 (2017) [Non-Patent Document 4] J. M. Tran Janco et al., Tumor-infiltrating dendritic cells in cancer pathogenesis. J. Immunol. 194, 2985-2991 (2015) [Non-Patent Document 5] W. Hobo et al., siRNA silencing of PD-L1 and PD-L2 on dendritic cells augments expansion and function of minor istocompatibility antigen-specific CD8+ T cells. Blood 116, 4501-4511 (2010) [Non-Patent Document 6] A. Harari et al., Antitumour dendritic cell vaccination in a priming and boosting approach. Nat. Rev. Drug Discovery 19, 635-652 (2020) [Non-Patent Document 7] Y. Ma et al., Dendritic Cells in the Cancer Microenvironment. J. Cancer 4, 36-44 (2013) [Overview of the project]

[0007] In one embodiment, the disclosure provides a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (1) an extracellular antigen-binding domain, (2) a transmembrane domain, and (3) an intracellular signaling domain, and the CAR is capable of activating dendritic cells in an immunosuppressive tumor microenvironment.

[0008] In a particular embodiment, the immunosuppressive tumor microenvironment comprises tumor and / or tumor-infiltrating immune cells that 1) express immunoinhibitory molecules and / or have insufficient immunostimulatory cytokines.

[0009] In a particular embodiment, the immunoinhibitory molecule is selected from the group consisting of PD-1, TIM-3, TIGIT, LAG-3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155), and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), HLA class I, sialycoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47.

[0010] In a particular embodiment, the immunoinhibitory molecule is CTLA-4 and / or PD-L1.

[0011] In a particular embodiment, the immunostimulatory cytokine is selected from TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, granulocyte-macrophage colony-stimulating factor, and combinations thereof.

[0012] In a particular embodiment, the tumor comprises cells expressing CTLA4-Ig and / or PD-L1.

[0013] In certain embodiments, the immunosuppressive tumor microenvironment includes tumors that are less responsive to adoptive cell therapy monotherapy (e.g., CAR-T monotherapy).

[0014] In a particular embodiment, the intracellular signaling domain includes the cytoplasmic domain of a dendritic cell activation receptor selected from the group consisting of RIG-1, NLRP10, DEC-205, BDCA-2, CD86, 4-1BBL, OX40L, CD40, IFNAR, TLR4, TNFR (e.g., TNFR2), CD80, CD40L, CD367 (DCIR), CD207 (Langerin), CD371 (DCAL-2, CLEC12a), CD204, CD36, IFNγR, Dectin-1, and FcγR, or a combination thereof.

[0015] In a particular embodiment, the intracellular signaling domain includes the cytoplasmic domain of Dectin-1 and the cytoplasmic domain of FcγR.

[0016] In a particular embodiment, the cytoplasmic domain of Dectin-1 and the cytoplasmic domain of FcγR are linked in tandem.

[0017] In a particular embodiment, the cytoplasmic domain of dectin-1 includes the amino acid sequence shown in SEQ ID NO: 1, or any functional form thereof.

[0018] In a particular embodiment, the cytoplasmic domain of FcγR comprises the amino acid sequence shown in SEQ ID NO: 2, or any functional form thereof.

[0019] In a particular embodiment, the intracellular signaling domain includes the amino acid sequence shown in SEQ ID NO: 3, or any functional form thereof.

[0020] In a particular embodiment, the intracellular signaling domain includes an amino acid sequence encoded by the nucleic acid sequence shown in Sequence ID No. 4, or any functional form thereof.

[0021] In a particular embodiment, the extracellular antigen-binding domain includes a single-stranded variable fragment (scFv).

[0022] In a particular embodiment, scFv is specific to a tumor surface marker (e.g., a solid tumor surface marker).

[0023] In a particular embodiment, the tumor surface marker is selected from the group consisting of EphA2, CD19, CD70, CD133, CD147, CD171, DLL3, EGFRvIII, mesothelin, ganglioside GD2, FAP (fibroblast-activating protein), FBP (folate-binding protein), Lewis Y, claudin 18.2, IL13Rα2, HER2, MDC1, PMSA (prostatic membrane-specific antigen), ROR1, B7-H3, CAIX, CD133, CD171, CEA, GPC3, MUC1, and NKG2D.

[0024] In a particular embodiment, the CAR further comprises a signal peptide.

[0025] In a particular embodiment, the signal peptide includes the CD8α signal peptide.

[0026] In a particular embodiment, the signal peptide of CD8α includes the sequence shown in SEQ ID NO: 5, or any functional form thereof.

[0027] In a particular embodiment, the transmembrane domain includes the transmembrane domain of CD8α.

[0028] In a particular embodiment, the transmembrane domain of CD8α includes the sequence shown in Sequence ID No. 6, or any functional form thereof.

[0029] In a particular embodiment, the extracellular antigen-binding domain is linked to the transmembrane domain by a hinge region.

[0030] In a particular embodiment, the hinge region includes the hinge region of CD8α.

[0031] In a particular embodiment, the hinge region of CD8α includes the arrangement shown in Sequence ID No. 7, or any functional type thereof.

[0032] In certain embodiments, the polynucleotides provided herein are DNA or RNA.

[0033] In another embodiment, the disclosure provides polypeptides encoded by polynucleotides provided herein.

[0034] In another embodiment, the Disclosure provides a vector comprising a polynucleotide provided herein, wherein a polynucleotide encoding a CAR is functionally linked to at least one regulatory polynucleotide element for the expression of the CAR.

[0035] In certain embodiments, the vector is a plasmid vector, a viral vector, a transposon, a site-directed insertion vector, or a suicide expression vector.

[0036] In a particular embodiment, the viral vector is a lentiviral vector, a retroviral vector, or an AAV vector.

[0037] In a particular embodiment, the viral vector is a lentiviral vector.

[0038] In another embodiment, the present disclosure provides manipulated cells comprising polypeptides provided herein.

[0039] In a particular embodiment, the engineered cell is a dendritic cell or its precursor or progenitor cell.

[0040] In a particular embodiment, the dendritic cell or its precursor or progenitor cell is derived from peripheral blood cells, bone marrow cells, embryonic stem cells, or induced pluripotent stem cells.

[0041] In another embodiment, the Disclosure provides a method for producing manipulated cells provided herein, comprising introducing a vector provided herein into starting cells under conditions suitable for the expression of a polynucleotide provided herein.

[0042] In a particular embodiment, the starting cell is a dendritic cell or its precursor or progenitor cell.

[0043] In a particular embodiment, the dendritic cell or its precursor or progenitor cell is derived from peripheral blood cells, bone marrow cells, embryonic stem cells, or induced pluripotent stem cells.

[0044] In another embodiment, the present disclosure provides a population of cells produced ex vivo by a method provided herein.

[0045] In a particular embodiment, at least 70% of the cell population expresses the polypeptide provided herein at a detectable level.

[0046] In another embodiment, the Disclosure provides a pharmaceutical composition comprising (i) a polynucleotide, a polypeptide, a vector, a population of manipulated cells, or a population of cells provided herein, and (ii) a pharmaceutically acceptable medium.

[0047] In another embodiment, the present disclosure provides a method for improving the efficacy of adoptive cell therapy in treating cancer in a subject in need of treatment, the method comprising administering a therapeutically effective amount of a pharmaceutical composition provided herein.

[0048] In certain embodiments, adoptive cell therapy includes adoptive transfer of modified immune cells.

[0049] In a particular embodiment, the pharmaceutical composition further comprises a population of modified immune cells.

[0050] In a particular embodiment, the method further includes administering a pharmaceutical composition comprising a population of modified immune cells.

[0051] In a particular embodiment, the modified immune cells have the expression of synthetic receptors (e.g., CAR or TCR) on their cell surface.

[0052] In a particular embodiment, the immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, eosinophils, or neutrophils.

[0053] In a particular embodiment, the immune cells are T cells selected from the group consisting of CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γδ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes.

[0054] In certain embodiments, the immune cells are self or allogeneic.

[0055] In a particular embodiment, cancer is a solid tumor selected from the group consisting of adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.

[0056] In certain embodiments, cancer is a hematological malignancy selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, HHV8-associated primary exudative lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Waldenström macroglobulinemia, and multiple myeloma (MM).

[0057] In another embodiment, the Disclosure provides a method for inducing the proliferation of immune cells, extending the survival of immune cells, and / or increasing the expression and / or secretion of immunostimulatory cytokines from immune cells in an immunosuppressive microenvironment, the method comprising bringing the immunosuppressive microenvironment into contact with manipulated cells provided herein.

[0058] In a particular embodiment, the immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, eosinophils, or neutrophils.

[0059] In a particular embodiment, the immune cells are T cells selected from the group consisting of CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γδ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes.

[0060] In certain embodiments, the immune cells are self or allogeneic.

[0061] In a particular embodiment, the immunosuppressive microenvironment is an immunosuppressive tumor microenvironment.

[0062] In a particular embodiment, the immunosuppressive tumor microenvironment comprises tumor and / or tumor-infiltrating immune cells that express immunosuppressive molecules.

[0063] In a particular embodiment, the immunoinhibitory molecule is selected from the group consisting of PD-1, TIM-3, TIGIT, LAG-3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155), and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), sialycoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47.

[0064] In a particular embodiment, the immunoinhibitory molecule is CTLA-4 and / or PD-L1.

[0065] In a particular embodiment, the tumor comprises cells expressing CTLA4-Ig and / or PD-L1.

[0066] In a particular embodiment, the immunostimulatory cytokine is one or more of TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and granulocyte-macrophage colony-stimulating factor.

[0067] In another embodiment, the present disclosure provides a method for treating a disease or pathological condition in a subject requiring treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition provided herein.

[0068] In a particular embodiment, the method provided herein further includes administering a second active substance.

[0069] In a particular embodiment, the second therapy is a population of modified immune cells.

[0070] In a particular embodiment, the second therapy is CAR-T therapy.

[0071] In certain embodiments, the disease includes cancer.

[0072] In another embodiment, the present disclosure relates to a method for selecting CARs capable of activating dendritic cells, (a) Prepare a non-human animal containing an immunosuppressive tumor microenvironment, (b) administering dendritic cells expressing candidate CARs to non-human animals, (c) To detect markers related to dendritic cell activation selected from improvements in infiltration into the immunosuppressive tumor microenvironment, improved survival rate, and enhanced function in inducing immune cell activation, compared to reference dendritic cells. (d) Selecting candidate CARs that can activate dendritic cells, This provides a method that includes [something].

[0073] In certain embodiments, the immunosuppressive tumor microenvironment is clinically relevant.

[0074] In certain embodiments, the non-human animal includes a human embryonic spleen and autologous human hematopoietic stem cells (e.g., human CD34+ hematopoietic stem cells).

[0075] In a particular embodiment, the immunosuppressive tumor microenvironment includes tumor and / or tumor-infiltrating immune cells that express immunosuppressive molecules.

[0076] In a particular embodiment, the immunoinhibitory molecule is selected from the group consisting of PD-1, TIM-3, TIGIT, LAG-3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155), and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), HLA class I, sialycoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47.

[0077] In a particular embodiment, the immunoinhibitory molecule is CTLA-4 and / or PD-L1.

[0078] In a particular embodiment, the tumor comprises cells expressing CTLA4-Ig and / or PD-L1.

[0079] In a particular embodiment, the immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, eosinophils, or neutrophils.

[0080] In a particular embodiment, the immune cells are T cells selected from the group consisting of CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γδ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes.

[0081] In certain embodiments, the immune cells are self or allogeneic.

[0082] In certain embodiments, the immune cells are modified immune cells (e.g., CAR-T cells) or innate immune cells.

[0083] In a particular embodiment, modified immune cells (e.g., CAR-T cells) are administered in combination with dendritic cells expressing a candidate CAR.

[0084] In certain embodiments, the non-human animal is a rodent, such as a rat or mouse.

[0085] The accompanying drawings incorporated herein form part of the specification. Together with this written specification, the drawings further serve to illustrate the principles of this disclosure and to enable a person skilled in the art to prepare and use this disclosure in relation to the relevant art(s). [Brief explanation of the drawing]

[0086] [Figure 1A] Figure 1 shows that CARDF enhanced the activity of dendritic cells (DCs) derived from THP-1 cells. Figure 1A shows schematic diagrams of various anti-CD19 CAR molecules. [Figure 1B] Figure 1B shows the determination of CARDF expression on the surface of the THP-1 cell line by flow cytometry. CARDF was detected by binding to protein L. [Figure 1C] Figure 1C shows flow cytometry analysis of the differentiation efficiency of CARDF+ THP-1 cells into dendritic cells (DCs). [Figure 1D] Figure 1D shows the expression of the costimulatory molecules CD80 and CD86 in control DCs and CARDF-DCs after co-culture with CD19+ H460 tumor cells for 2 days. [Figure 1E] Figure 1E shows the analysis of CFSE-labeled CD3+ primary T cell proliferation by flow cytometry after co-culture with control DCs or CARDF-DCs for 3 days. DCs were activated with CD19+ H460 tumor cells for 2 days, as shown in Figure 1D. The histogram on the right shows the median fluorescence intensity (MFI) of CFSE in T cells. n=3. [Figure 1F] Figure 1F shows the analysis of CD19 expression on the surface of H460 cells by flow cytometry. [Figure 1G] Figure 1G shows the analysis of anti-CD19 CAR expression on CAR-T cells by protein L binding. n=3. [Figure 1H] Figure 1H shows the specific cytotoxic activity of CAR-T cells against CD19+ H460 tumor cells in the presence of control DCs or CARDF-DCs for 24 hours. n=3. [Figure 1I] Figures 1I and 1J show that IFN-γ levels (Figure 1I) were evaluated by ELISA and LDH (Figure 1J) was analyzed by the CytoTox 96™ assay in the co-culture supernatant of Figure 1H. n=3. Data are presented as mean ± SD. Statistics: Brown-Forsyth test including one-way ANOVA and Tukey's multiple comparison test. nd indicates not detected. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns indicates non-significant. [Figure 1J] Figures 1I and 1J show that IFN-γ levels (Figure 1I) were evaluated by ELISA and LDH (Figure 1J) was analyzed by the CytoTox 96™ assay in the co-culture supernatant of Figure 1H. n=3. Data are presented as mean ± SD. Statistics: Brown-Forsyth test including one-way ANOVA and Tukey's multiple comparison test. nd indicates not detected. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns indicates non-significant. [Figure 2A] Figure 2 shows that peripheral monocyte-derived CARDF-DCs exhibit robust T cell activation activity in vitro. Figure 2A shows the analysis of CARDF expression on the surface of monocyte-derived DCs (Mo-DCs) by flow cytometry. PseudoDCs transduced with empty vector lentivirus were used as a control. [Figure 2B] Figure 2B shows the expression of various DC-specific markers in Mo-DCs, pseudoDCs, and CARDF-DCs after stimulation with LPS and TNF-α. n=3. [Figure 2C]Figure 2C shows the analysis of CD3+ primary T cell proliferation, assessed by CellTrace-CFSE dilution, after co-culture with pseudoDCs or CARDF-DCs for 3 days. DCs were pre-exposed to EPHA2+ A549 for 48 hours. The histogram on the right shows the MFI of CFSE in T cells. n=3. Statistical analysis: One-way ANOVA, Brown-Forsyth test including Tukey's multiple comparison test. [Figure 2D] Figure 2D shows that PD-L1 expression on A549-CP was analyzed by flow cytometry, and CTLA4-Ig was evaluated by RT-qPCR. n=3. Statistical analysis: Independent two-tailed Student's t-test. [Figure 2E] Figure 2E shows the surface expression of the activation marker in pseudoDCs and CARDF-DCs before and after co-culture with A549-CP for 48 hours. n=3. Statistical analysis: Independent two-tailed Student's t-test. [Figure 2F] Figure 2F shows the analysis of CD3+ primary T cell proliferation, assessed by CellTrace-CFSE dilution, after co-culture with pseudoDCs or CARDF-DCs in the presence of A549-CP for 4 days. The histogram on the right shows the MFI of CFSE for all T cells. n=3. Data are presented as mean ± SD. Statistics: Independent two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 3A] Figure 3 shows that CARDF-DCs activate the cytotoxicity of CAR-T cells against A549CP cells in vitro. Figure 3A shows the expression of CAR(scFv: anti-EphA2) on CAR-T cells. [Figure 3B] Figure 3B shows the results of flow cytometry analysis of EphA2 expression on A549 and A549CP. [Figure 3C] Figure 3C shows the cytolytic ability of CAR-T cells against A549 and A549CP tumor cells in the presence of pseudoDCs or CARDF-DCs for 24 hours. n=3. [Figure 3D]Figure 3D shows RT-qPCR analysis of IFN-γ, IL-2, and TNF-α expression in CAR-T cells derived under A549CP conditions, as shown in (C). n=3. [Figure 3E] Figure 3E shows the flow cytometry analysis of IFN-γ+ cells in cultured CD8+ CAR-T cells as shown in Figure 3C. [Figure 3F] Figures 3F and 3G show that IFN-γ levels (Figure 3F) were evaluated by ELISA and LDH (Figure 3G) was analyzed by the CytoTox 96™ assay in the supernatant collected from the culture in Figure 3C. n=3. Data are shown as mean ± SD. n=3. Statistics: Brown-Forsyth test including one-way ANOVA and Tukey's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns indicates non-significant. [Figure 3G] Figures 3F and 3G show that IFN-γ levels (Figure 3F) were evaluated by ELISA and LDH (Figure 3G) was analyzed by the CytoTox 96™ assay in the supernatant collected from the culture in Figure 3C. n=3. Data are shown as mean ± SD. n=3. Statistics: Brown-Forsyth test including one-way ANOVA and Tukey's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns indicates non-significant. [Figure 4A] Figure 4 shows that CARDF-DCs activate Car-T cells to eliminate solid lung tumors, including TIME. Figure 4A shows an experimental design for treating A549WT and A549CP lung tumors formed in NSG mice with CARDF-DCs and Car-T cells. [Figure 4B] Figure 4B shows the expression of immunosuppressive genes in A549WT and A549CP tumors as assessed by RT-qPCR. Data are shown as mean ± SD. n=3. Statistical analysis: Independent two-sided Student's t-test. [Figure 4C]Figure 4C shows photographs of tumors retrieved 17 days after the treatment described in Figure 4A. Left: A549WT tumor, Right: A549CP tumor. [Figure 4D] Figure 4D shows the weight of the tumors shown in Figure 4C. Data are presented as mean ± SD. n=5. Statistics: One-way ANOVA, Brown-Forsyth test including Tukey's multiple comparison test. [Figure 4E] Figure 4E shows gene expression in the recovered A549CP tumors shown in Figure 4C, as determined by RT-qPCR. Data are presented as mean ± SD. n=5. [Figure 4F] Figure 4F shows the proportions of total T cells, CD8+ T cells, dendritic cells, CD80+ dendritic cells, and CD86+ dendritic cells in the spleen, analyzed by flow cytometry. Data are shown as mean ± SD. n=5. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns indicates non-significant values. [Figure 5A] Figure 5 shows that CARDF-DCs promote CAR-T cell-mediated regression of lung tumors formed in Hu- mice. Figure 5A shows an experimental design for treating A549 lung tumors formed in Hu- mice. [Figure 5B] Figure 5B shows gene expression in lung tumors derived from NSG and Hu- mice after CAR-T cell therapy, as assessed by RT-qPCR. Data are presented as mean ± SD. n=3. Statistical analysis: Independent two-tailed Student's t-test. [Figure 5C] Figure 5C shows tumor volumes after various treatments. Data are presented as mean ± SD. Statistics: Two-way ANOVA followed by Tukey's multiple comparison test. [Figure 5D]Figures 5D and 5F show photographs (Figure 5D) and tumor weights (Figure 5E) of tumors collected 16 days after vaccination. The treatment progress is shown in Figure 5A. Data are shown as mean ± SD. Statistics: One-way ANOVA, Brown-Forsyth test including Tukey's multiple comparison test. Normal T: n=4. CAR-T: n=6. Pseudo-DC: n=6. CARDF-DC: n=6. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 5E] Figures 5D and 5F show photographs (Figure 5D) and tumor weights (Figure 5E) of tumors collected 16 days after vaccination. The treatment progress is shown in Figure 5A. Data are shown as mean ± SD. Statistics: One-way ANOVA, Brown-Forsyth test including Tukey's multiple comparison test. Normal T: n=4. CAR-T: n=6. Pseudo-DC: n=6. CARDF-DC: n=6. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 6A] Figure 6 shows that CARDF-DCs reverse the TIME of lung tumor formation in Hu- mice toward a pro-inflammatory state. Figure 6A shows the percentage of IFN-γ+ T cells in spleen CD3+ T cells analyzed by flow cytometry. Data are shown as mean ± SD. Normal T, n=2. CAR-T, pseudoDC, CARDF-DC, n=3. [Figure 6B] Figure 6B shows the results of flow cytometry analysis of PD-1+ and TIM-3+ T cells in the spleen. [Figure 6C] Figure 6C shows the analysis of MFI of CD86 and MHC-II expressed by spleen-derived dendritic cells by flow cytometry. Data are shown as mean ± SD. Normal T cells, n=2. CAR-T cells, pseudoDC cells, CARDF-DC cells, n=3. [Figure 6D] Figure 6D shows the evaluation of TNF-α, IL-2, CD86, and IL-12B expression in disseminated lung tumors by RT-qPCR. Normalization is shown in the figure. Data are presented as mean ± SD. Normal T, n=2. CAR-T, pseudoDC, CARDF-DC, n=3. [Figure 6E] Figure 6E shows the proportion of PD-1+TIM-3+ T cells in spleen CD3+ T cells, as shown in Figure 6B. Data are presented as mean ± SD. Normal T cells, n=2. CAR-T cells, pseudoDCs, CARDF-DCs, n=3. [Figure 6F] Figures 6F and 6G show the evaluation of PD-1, TIM-3, TGF-β (Figure 6F), or CD206 and CD163 (Figure 6G) expression in disseminated lung tumors by RT-qPCR. Normalization is shown in the figures. Data are presented as mean ± SD. Normal T, n=2. CAR-T, pseudoDC, CARDF-DC, n=3. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 6G] Figures 6F and 6G show the evaluation of PD-1, TIM-3, TGF-β (Figure 6F), or CD206 and CD163 (Figure 6G) expression in disseminated lung tumors by RT-qPCR. Normalization is shown in the figures. Data are presented as mean ± SD. Normal T, n=2. CAR-T, pseudoDC, CARDF-DC, n=3. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 7A] Figure 7 shows that CARDF-DCs can activate CAR-T cells in resistance to TIME in separate lung tumors. Figure 7A shows the analysis of PD-L1 expression on A549 and H460 tumor cell lines by flow cytometry. [Figure 7B] Figure 7B shows the relative gene expression in A549 and H460 tumors formed in Hu-mice, evaluated by RT-qPCR. Data are shown as mean ± SD. n=3. [Figure 7C] Figure 7C shows that EphA2 expression on H460 lung tumor cells was detected by flow cytometry. [Figure 7D] Figure 7D shows a schematic design of CAR-T and DC combination therapy for H460 tumors formed in Hu- mice. [Figure 7E]Figure 7E shows CAR(scFv: anti-EphA2) expression on the surface of Hu-mouse-derived DCs and T cells, which are generated from the same embryonic tissue as tumor-carrying Hu-mouse. [Figure 7F] Figure 7F shows tumor growth curves after various treatments. Data are presented as mean ± SD. n=6. Statistics: Two-way ANOVA followed by Tukey's multiple comparison test. [Figure 7G] Figure 7G shows the analysis of MFI of CD80 and CD86 on DCs infiltrating the tumor by flow cytometry. Data are shown as mean ± SD. n=3. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 8A] Figures 8A and 8B show the screening of CARs that activate dendritic cells (DCs) derived from THP-1 cells. Figure 8A shows that the expression of CARs (scFv: anti-CD19 and second-generation T cell activating domain, or TLR4 activating domain, or TNFR2 activating domain) on the surface of THP-1 cells was determined by binding to protein L. [Figure 8B] Figure 8B shows the expression of the costimulatory molecules CD80 and CD86 on DCs after co-culture with H460-CD19 tumor cells for 2 days. [Figure 9] Figures 9A and 9B show schematic diagrams of anti-EphA2 CAR molecules. Figure 9A shows a lentiviral vector containing the anti-EphA2 CAR construct (CARDF) for dendritic cells (DCs). Figure 9B shows a lentiviral vector containing the anti-EphA2 CAR construct (second generation, B) for T cells. [Figure 10A] Figure 10A shows the antibodies used in this disclosure. [Figure 10B] Figure 10B shows the primer sequences for RT-qPCR used in this disclosure. [Figure 11A] Not specified [Figure 11B] Not specified [Modes for carrying out the invention]

[0087] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, and that such embodiments may naturally be diverse. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them, as the scope of this disclosure is limited only by the accompanying claims.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this disclosure, but preferred methods and materials are described herein.

[0089] All publications and patents cited herein are indicated to constitute part of this Specification by specific and individual reference, and constitute part of this Specification as if each individual publication or patent were to constitute part of this Specification by specific and individual reference, to disclose and describe methods and / or materials relating to those cited. Any reference to a publication is in relation to its disclosure prior to the filing date and does not acknowledge that this disclosure is not entitled to precede such publication by prior disclosure. Furthermore, the dates of publications provided may differ from the actual publication dates and may need to be independently verified.

[0090] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features, which are readily distinguishable from or can be combined with features of any of several other embodiments without departing from the scope or spirit of this disclosure. Any of the methods listed may be performed in the order of the events listed, or in any other logically possible order.

[0091] definition To assist the reader, the following definitions are provided. Unless otherwise defined, all terms, notations, and other scientific or medical terms or nomenclas used herein in the art are intended to have meanings generally understood by those skilled in the art. In some cases, terms with generally understood meanings are defined herein for clarity and / or for easy reference, and the inclusion of such definitions herein is not necessarily considered to represent a substantial difference beyond the definitions of terms generally understood in the art.

[0092] As used herein, the singular forms "a," "an," and "the" include multiple references unless otherwise explicitly indicated by the context.

[0093] In this disclosure, terms such as “comprises,” “comprised,” “comprising,” “contains,” and “containing” have meanings derived from U.S. patent law, and are inclusive, i.e., open-ended, and do not exclude further unlisted elements or process steps. Terms such as “consisting essentially of” and “consists essentially of” have meanings derived from U.S. patent law, and allow for the inclusion of further components or processes that do not substantially affect the fundamental and novel characteristics of the claimed invention. Terms “consists of” and “consisting of” have meanings derived from U.S. patent law, i.e., these terms are closed-ended.

[0094] In all cases of this application where there is a set of enumerated numbers, it is understood that any enumerated number may be an upper or lower limit of a numerical range. It is further understood that the invention includes all such numerical ranges, i.e., ranges having combinations of upper and lower numerical limits, where each of the upper and lower numerical limits may be any of the numbers enumerated herein. A range provided herein is understood to include all values ​​within the range. For example, 1 to 10 is understood to include all values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and, where appropriate, decimal values. Similarly, a range delimited by “at least” is understood to include the lower limit and all higher numerical values, as provided.

[0095] As used herein, “about” is understood to include within three standard deviations of the mean or within an acceptable standard range within a particular articulate scope. In a particular embodiment, “about” is understood to mean a variation of 0.5 or less.

[0096] In this specification, the term "CAR" is interchangeable with the term "chimeric antigen receptor" and refers to a manipulated or synthetic receptor or an encoding polynucleotide. A manipulated or synthetic receptor comprises an extracellular domain containing an antigen-binding domain, a transmembrane domain, and / or an intracellular signaling domain, and optionally a signal peptide, which are linked together or functionally linked to each other. The most common CARs are, for example, single-stranded variable fragments (scFv) derived from monoclonal antibodies fused to the CD3-ζ transmembrane and endodomains. Such CARs, in response to the specific binding of the scFv to its target, result in the transmission of ζ signals. Methods for preparing CARs are publicly available (see, for example, Grupp et al., N Engl J Med., 368:1509-1518, 2013; Park et al., Trends Biotechnol., 29:550-557, 2011; Haso et al., (2013) Blood, 121, 1165-1174; Han et al., J. Hematol Oncol. 6:47, 2013; International Publication No. 2012 / 079000; U.S. Patent Application Publication No. 2012 / 0213783; and International Publication No. 2013 / 059593, each of which is incorporated herein by whole reference).

[0097] The term "chimeric antigen receptor T cell" is used interchangeably with the term "CAR-T cell" and refers to T cells or populations thereof that have been engineered through biological methods (e.g., genetic engineering) to express CAR on their surface. CAR-T cells may also be T helper CD4+ and / or T effector CD8+ cells. CAR-T cells can identify surface antigens and initiate an immune response.

[0098] An "antigen" refers to a molecule that triggers an immune response. This immune response may be humoral, cell-mediated, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can act as an antigen. It will be readily apparent that this disclosure includes therapeutic antibodies that act as antigen-induced immune responses.

[0099] An "antibody" refers to a polypeptide of the immunoglobulin (Ig) family that binds to an antigen. For example, a naturally occurring IgG-type "antibody" is a tetramer containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) (light chain CDRs containing LCDR1, LCDR2, and LCDR3, and heavy chain CDRs containing HCDR1, HCDR2, and HCDR3), between which exist more conserved regions called framework regions (FRs).The CDR boundary for the antibodies disclosed herein may be defined or identified according to the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997), Chothia, C. et al., J Mol Biol. Dec 5;186(3):651-63 (1985), Chothia, C. and Lesk, AM, J.Mol.Biol., 196,901 (1987), Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989), Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991), Marie-Paule Lefranc et al. Developmental and Comparative Immunology, 27: 55-77 (2003), Marie-Paule Lefranc et al, Immunome Research, 1(3), (2005), Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0100] As used herein, “antigen-binding domain” refers to an antibody fragment formed from an intact antibody portion containing one or more CDRs, or any other antibody fragment capable of binding to an antigen but not containing an intact native antibody structure. Examples of antigen-binding domains include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), single-chain Fv-Fc antibodies (scFv-Fc), scFv dimers (bivalent diabodies), bispecific antibodies, multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding domains are capable of binding to the same antigen to which the parent antibody binds.

[0101] "Self" cells refer to any cells derived from the same subject that are later reintroduced.

[0102] "Allogeneic" cells refer to any cells derived from different subjects of the same species.

[0103] In the context of immune cells, "effector cells" refer to cells that are activated in response to stimuli and capable of performing effector functions. Effector cells may, without limitation, include NK cells, cytotoxic T cells, and helper T cells.

[0104] "Effective dose" or "therapeutic effective dose" refers to the amount of cells, compositions, formulations, or any material described herein that is effective in achieving the desired biological outcome. Such outcomes may include, without limitation, the removal of B cells expressing a particular BCR and the antibodies produced therefrom.

[0105] The percentage of "identity" or "sequence identity" in the context of polypeptides or polynucleotides is determined by comparing two optimally aligned sequences across a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) when compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where identical nucleic acid bases or amino acid residues exist to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0106] When the term “conservative substitution” is used herein in reference to amino acid sequences, it refers to the substitution of an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions may occur between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions usually do not cause a significant change in the protein conformational structure and can therefore preserve the biological activity of the protein.

[0107] The term "functional form," as used herein, refers to a different form of a parent molecule (e.g., mutants, fragments, fusions, derivatives, and mimetic compounds) that retains substantial biological activity of the parent molecule despite having differences in amino acid sequence or chemical structure. The expression "retains substantial biological activity," as used herein, means exhibiting at least some (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more) or all of the biological activity of the parent molecule. Functional forms of a parent polypeptide may include both naturally occurring mutant forms and unnatural forms, such as those obtained by recombinant or chemical synthesis. Functional forms may contain unnatural amino acid residues.

[0108] As used herein, the term “functionally linked” refers to a functional relationship between two or more polynucleotide sequences. In the context of polynucleotides encoding a fusion protein, such as the polypeptide chain of a CAR in this disclosure, the term means that two or more polynucleotide sequences are linked such that the amino acid sequence encoded by these segments remains in frame. In the context of transcription or translational regulation, the term refers to a functional relationship between a regulatory sequence and a coding sequence, such as a promoter in the correct position and orientation relative to the coding sequence that regulates transcription.

[0109] As used herein, the terms “polynucleotide” or “nucleic acid” refer to a nucleotide chain. These also refer to synthetic and / or non-naturally occurring nucleic acid molecules (including, for example, nucleotide analogs or modified backchain residues or linkages). The term also refers to single-stranded or double-stranded deoxyribonucleotides or ribonucleotide oligonucleotides. The term includes nucleic acids containing analogs of natural nucleotides. The term also includes nucleic acid-like structures with synthetic backchains. Unless otherwise indicated, a particular polynucleotide sequence also implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences. In particular, degenerate codon substitution may be achieved by generating sequences in which the third position of one or more (or all) selected codons is replaced with a mixed base and / or a deoxyinosine residue (see Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985), and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0110] The terms “polypeptide,” “peptide,” and “protein” are interchangeable herein and refer to polymers of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring and unnaturally occurring amino acid polymers. In certain embodiments, polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0111] As used herein, the term "single-chain variable fragment" is interchangeable with the term "scFv" and refers to a manipulated antibody consisting of a light-chain variable region and a heavy-chain variable region linked to each other directly or through a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879(1988)).

[0112] As used herein, the term "TCR" is interchangeable with the term "T cell receptor" or "TCR complex" and refers to a native (or endogenous) TCR or a modified TCR. A TCR refers to a protein complex on the surface of a T cell that is involved in recognizing antigen fragments as peptides that bind to MHC molecules.

[0113] The term “vector,” as used herein, refers to a vehicle into which a protein-coding polynucleotide can be functionally inserted to result in the expression of that protein. A vector can be used to transform, transduce, or transfect a host cell to result in the expression of the genetic elements contained within the vector. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes, e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, e.g., λ phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Furthermore, the vector may contain an origin of replication. The vector may also contain a substance that assists entry into the cell, which may include, but is not limited to, viral particles, liposomes, or protein coatings. The vector may be an expression vector or a cloning vector. This disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence provided herein that encodes a fusion polypeptide, at least one promoter functionally linked to this nucleic acid sequence (e.g., SV40, CMV, EF-1α), and at least one select marker.Examples of vectors, though not limited to them, include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), λ phages, and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, and pALTER. , pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pB ABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT(TM), pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0114] When used herein, the term “host cell” refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0115] The term "pharmaceutically acceptable" indicates that the indicated carrier, vehicle, diluent, excipient(s) and / or salt are generally chemically and / or physically compatible with the other components of the formulation and physiologically compatible with its recipient.

[0116] The terms “subject,” “individual,” “animal,” or “patient,” as used herein, refer to humans or non-human animals, including mammals or primates, who require diagnosis, prognosis, mitigation, prevention, and / or treatment for a disease or disorder. Mammal subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, etc.

[0117] As used herein, the terms “to treat” or “to cure” a condition include preventing or reducing the condition, slowing the onset or progression of the condition, reducing the risk of the condition progressing, preventing or delaying the progression of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, producing complete or partial regression of the condition, curing the condition, or any combination thereof.

[0118] Dendritic cell (DC)-activated chimeric antigen receptor (CAR) This disclosure provides polynucleotides (e.g., DNA or RNA) encoding chimeric antigen receptors (CARs) capable of activating dendritic cells (DCs) in an immunosuppressive tumor microenvironment or tumor immunosuppressive microenvironment (TIME). The terms "immunosuppressive tumor microenvironment" and "TIME" are interchangeable and refer to a microenvironment that, along with a high-density extracellular matrix, may suppress tumor immune surveillance and immunotherapy, such as tumor cells, tumor-infiltrating immune cells, tumor-associated fibroblasts, endothelial cells, and a diverse array of chemotactic and inflammatory or immunostimulatory cytokines (FR Balkwill et al., The tumor microenvironment at a glance. J. Cell Sci. 125, 5591-5596 (2012), M. Binnewies et al., Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat Med. 24, 541-550 (2018), MA-M. Alireza Labani-Motlagh et al., The Tumor Microenvironment: A Milieu Hindering and Obstructing Antitumor Immune Responses. Front. Immunol. 11, 940 (2020) and L. Hui et al. al., Tumor microenvironment: Sanctuary of the devil. Cancer Lett. 368, 7-13 (2015)).

[0119] In certain embodiments, the immunosuppressive tumor microenvironment or TIME comprises solid tumor and / or tumor-infiltrating immune cells expressing immunosuppressive molecules. The immunosuppressive molecules may be selected from the group consisting of PD-1, TIM3, TIGIT, LAG3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155), and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), HLA class I, sialycoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47. In certain embodiments, the immunosuppressive molecule is CTLA-4 and / or PD-L1. As used herein, the terms “expressed” or “expressing” an immunoinhibitory molecule mean that the immunoinhibitory molecule is expressed at a level at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 60 times, at least 80 times, at least 100 times, at least 120 times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, or at least 1000 times higher than the reference level. The term “reference level” in relation to the expression of an immunoinhibitory molecule means the level of expression of an immunoinhibitory molecule in a tumor formed by wild-type tumor cells (e.g., wild-type A549 cells) in an immunodeficient animal model (e.g., NSG mouse).

[0120] "CTLA-4" is an abbreviation for Cytotoxic T-Lymphocyte-Associated protein 4, also known as CD152. A more detailed explanation can be found, for example, in Kolar et al., (January 1, 2009) CTLA-4 (CD152) controls homeostasis and suppressive capacity of regulatory T cells in mice. Arthritis Rheum. 60 (1): 123-32. "PD-L1" is an abbreviation for programmed cell death ligand 1, also known as surface antigen classification 274 (CD274) or B7 homolog 1 (B7-H1). A more detailed explanation can be found, for example, in Dong H et al., B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nature Medicine. 5 (12): 1365-9, 1999.

[0121] CTLA-4 and PD-L1 are crucial immunosuppressive molecules in maintaining peripheral immune tolerance by limiting T cell activity. CTLA-4 binds to CD80 and CD86 with higher affinity than CD28, and these are the primary costimulatory pathways for T cell activation. PD-L1 binds to PD-1 expressed on the surface of T cells, inhibiting T cell activity. PD-L1 plays a central role in maintaining T cell anergy and preventing autoimmunity (Walker LSK et al., The enemy within: keeping self-reactive T cells at bay in the periphery. Nat Rev Immunol. 2002; 2:11-19., Fife BT et al., Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4 and PD-1 pathways. Immunological Reviews. 2008; 224:166-182., and Keir ME et al., PD-1 and Its Ligands in Tolerance and Immunity. Annual Review of Immunology. 2008; 26:677-704.).

[0122] In certain embodiments, tumors within TIME include cells expressing CTLA-4-immunoglobulin fusion protein (CTLA4-Ig) and / or PD-L1. CTLA4-Ig has been developed to inhibit T cell-mediated immune responses (Walker LSK et al., The enemy within: keeping self-reactive T cells at bay in the periphery. Nat Rev Immunol. 2002; 2:11-19.). As used herein, the terms “expressed” or “expressing” CTLA4-Ig mean that CTLA4-Ig is expressed at a level at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 60 times, at least 80 times, at least 100 times, at least 120 times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, or at least 1000 times higher than the reference level. The term “reference level” in relation to CTLA4-Ig expression means the level of CTLA4-Ig expression in wild-type tumor cells (e.g., wild-type A549 cells).As used herein, the terms “expressed” or “expressing” PD-L1 mean that PD-L1 is expressed at a level at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 60 times, at least 80 times, at least 100 times, at least 120 times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, or at least 1000 times higher than the reference level. The term “reference level” in relation to PD-L1 expression means the level of PD-L1 expression in wild-type tumor cells (e.g., wild-type A549 cells).

[0123] In certain embodiments, CTLA-4-Ig includes the amino acid sequence shown in SEQ ID NO: 8, or a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 8 while retaining substantial biological activity of SEQ ID NO: 8, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof. In certain embodiments, PD-L1 includes the amino acid sequence shown in SEQ ID NO: 9, or a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 9 while retaining substantial biological activity of SEQ ID NO: 9, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof.

[0124] In certain embodiments, the immunosuppressive tumor microenvironment includes tumors that are less responsive to adoptive cell therapy monotherapy (e.g., CAR-T monotherapy). As used herein and throughout this specification, the term “less responsive” means the absence or reduction of responsiveness that can be detected by a similar level of therapeutic effect of the therapy (e.g., CAR-T therapy) compared to a control therapy known to be ineffective (e.g., a better therapeutic effect of less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, or less than 2%, preferably a better therapeutic effect of less than 10%).

[0125] Dendritic cells are professional antigen-presenting cells capable of priming naive T cells and reactivating memory responses. In cancer, dendritic cells can induce a stronger antitumor response by activating T cells (e.g., cytotoxic CD8+ T cells) through cross-presentation of tumor-associated antigens (TAAs) or neoantigens. DC activation can be assayed by measuring a variety of parameters, without limitation, including the activation state of DCs and / or the activation state of immune cells (e.g., T cells, macrophages), which include the expression levels of DC activation markers (e.g., CD80, CD86 and MHC-II, CD83, CD54, CMRF-44, CMRF-56, type III INF, IL-12, CXCL9 / 10, IRF8), the survival and / or cytotoxicity of immune cells (e.g., T cells), and immunostimulatory cytokines from immune cells (e.g., T cells) (e.g., TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-- This can be indicated by the expression (and / or secretion) of 8, IL-10, IL-12, IL-18 and granulocyte-macrophage colony-stimulating factor, the expression levels of immunosuppressive molecules from immune cells (e.g., T cells) (e.g., PD-1, TIM-3, TIGIT, LAG3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155), and SIGLEC9 (CD329)), and / or the expression levels of markers related to anti-inflammatory macrophages (e.g., M2 macrophages), such as CD206 and CD163.

[0126] In certain embodiments, dendritic cell activation leads to increased expression levels of DC activation markers (e.g., CD80, CD86 and / or MHC-II, CD83, CD54, CMRF-44, CMRF-56, type III INF, IL-12, CXCL9 / 10, IRF8) compared to a reference state (e.g., inactivated state) of dendritic cells, and immune cells (e.g., T cells (e.g., CD8+)). This includes increased survival of T cells (DCs), increased expression (and / or secretion) of immunostimulatory cytokines (e.g., TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and / or granulocyte-macrophage colony-stimulating factor) from immune cells (e.g., T cells), decreased expression of immunoinhibitory molecules (e.g., PD-1, TIM-3, TIGIT, LAG3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155), and SIGLEC9 (CD329)) from immune cells (e.g., T cells), and / or decreased expression levels of markers related to anti-inflammatory macrophages (e.g., M2 macrophages) (e.g., CD206 and CD163).

[0127] In certain embodiments, the DC-activated CAR provided herein comprises (1) an extracellular antigen-binding domain, (2) a transmembrane domain, and (3) an intracellular signaling domain.

[0128] (1) Extracellular antigen-binding domain In some embodiments, the antigen-binding domain comprises a human or humanized antibody or a fragment thereof. The term "human antibody" refers to an antibody whose entire molecule is of human origin or which consists of an amino acid sequence identical to that of a human type of antibody or immunoglobulin. The term "humanized antibody" refers to an antibody that contains a sequence derived from a non-human immunoglobulin (e.g., a CDR sequence). Human or humanized antibodies or fragments thereof can be prepared in a variety of ways, for example, through recombinant methodologies or by immunization of mice of interest with an antigen of interest that have been genetically modified to express antibodies derived from human heavy chain and / or light chain coding genes.

[0129] In some embodiments, the extracellular antigen-binding domain of the CARs provided herein comprises a single-strand variable fragment (scFv), Fv, Fab, (Fab)2, scFv, nanobody, non-covalent or covalent ligand / receptor domain, or any other scaffold known in the art to function as an antigen-binding domain. In some embodiments, the extracellular antigen-binding domain of the CARs provided herein is scFv. The scFv may be specific to a tumor surface marker, such as a solid tumor surface marker. In certain embodiments, tumor surface markers are selected from the group consisting of EphA2, CD19, CD70, CD117, CD133, CD147, CD171, DLL3, EGFRvIII, VGFR2, mesothelin, ganglioside GD2, FAP (fibroblast-activating protein), FBP (folate-binding protein), LMP1, Lewis Y, claudin 18.2, IL13Rα2, HER2, MDC1, PMSA (prostatic membrane-specific antigen), ROR1, ROR2, B7-H3, CAIX, CD133, CD171, CEA, GPC3, MUC1, MUC16, MAGE-A1, MAGE-A4, TROP2, EpCAM, NKG2D, other proteins found to be more concentrated on the tumor cell surface than in important normal tissues, and combinations thereof. The extracellular antigen-binding domain may also be specific to non-tumor markers related to diseases that may benefit from converting TIME to a pro-inflammatory state, such as markers for infectious diseases.

[0130] In some embodiments, scFv is specific to EphA2. In certain embodiments, scFv contains a peptide linker between its VL and VH regions, comprising at least 0, 1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues. The linker sequence may contain any naturally occurring amino acids. In certain embodiments, the peptide linker contains an amino acid sequence comprising SEQ ID NO: 57 (GGGGSGGGGSGGGGS).

[0131] In some embodiments, scFv includes a variable heavy chain (VH) region and a variable light chain (VL) region. In some embodiments, VH is a heavy chain CDR1 (HCDR1) having the sequence shown in SEQ ID NO: 10, or a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 10 while retaining substantial biological activity of SEQ ID NO: 10, or a sequence having one, two, three, four, five, six, seven, eight, nine, or 10 conservative substitutions thereto, or any functional form thereof, and the sequence shown in SEQ ID NO: 11, or a sequence that is at least 75%, 80%, 85%, or 90% identical to SEQ ID NO: 11 while retaining substantial biological activity of SEQ ID NO: 11 The CDR2 comprises a sequence having 95% or 99% identity, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof, and a CDR3 comprising a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to the sequence shown in SEQ ID NO: 12, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof, while retaining substantial biological activity of SEQ ID NO: 12.In some embodiments, the VL region is a light chain CDR1 (LCDR1) having the sequence shown in SEQ ID NO: 13, or a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 13 while retaining substantial biological activity of SEQ ID NO: 13, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conservative substitutions thereto, or any functional form thereof, and the sequence shown in SEQ ID NO: 14, or a sequence that is at least 75%, 80%, 85%, or 90% identical to SEQ ID NO: 14 while retaining substantial biological activity of SEQ ID NO: 14. CDR2 comprises a sequence having 1%, 95%, or 99% identity, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therefor, or any functional form thereof, and CDR3 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to Sequence ID No. 15 while retaining substantial biological activity of Sequence ID No. 15, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therefor, or any functional form thereof.

[0132] In a particular embodiment, scFv includes 1) VH, which includes HCDR1 containing the sequence shown in SEQ ID NO: 10, HCDR2 containing the sequence shown in SEQ ID NO: 11, and HCDR3 containing the sequence shown in SEQ ID NO: 12; and 2) VL, which includes LCDR1 containing the sequence shown in SEQ ID NO: 13, LCDR2 containing the sequence shown in SEQ ID NO: 14, and LCDR3 containing the sequence shown in SEQ ID NO: 15.

[0133] In some embodiments, scFv includes VH and VL. In certain embodiments, VH includes the amino acid sequence shown in SEQ ID NO: 16, or a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 16 while retaining substantial biological activity of SEQ ID NO: 16, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof. In certain embodiments, VL includes the amino acid sequence shown in SEQ ID NO: 17, or a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17 while retaining substantial biological activity of SEQ ID NO: 17, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof. In some embodiments, scFv includes a VH containing the sequence shown in SEQ ID NO: 16 and a VL containing the sequence shown in SEQ ID NO: 17.

[0134] In some embodiments, scFv comprises the amino acid sequence shown in SEQ ID NO: 18.

[0135] This disclosure successfully demonstrates that CAR-DCs expressing EphA2-specific scFv can significantly reduce lung tumor volume in an immunosuppressive environment. However, it should not be understood that the CAR-DCs provided herein can be used only for the treatment of lung cancer. Those skilled in the art will recognize that, depending on the disease of interest, the CARs provided herein may be constructed by selecting appropriate extracellular antigen-binding domains specific to any disease marker, taking into account the current knowledge of identified markers for a variety of diseases, such as cancer, infectious diseases, or immunological diseases. The variety of disease markers include, but are not limited to, those described above.

[0136] (2) Transmembrane domain The transmembrane domains of CARs described herein include, but are not limited to, BAFFR, BLAME(SLAMF8), CD2, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA-l), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD49f, and CD6. 4, CD80, CD84, CD86, CD96(Tactile), CD100(SEMA4D), CD103, CD134, CD137(4-1BB), CD150(IPO-3, SLAMF1, SLAM), C D154, CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(Ly9), CD244(2B4, SLAMF4), CD278(ICOS), CEACAM1, CRT It may be derived from any membrane-bound or transmembrane protein, including AM, GITR, HYEM (LIGHTR), IA4, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR, LTBR, ​​OX40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, the α, β, or ζ chains of the T cell receptor, TNFR2, VLA1, and VLA-6.

[0137] In one embodiment, the CAR described herein comprises the transmembrane domain of CD8α. In a particular embodiment, the transmembrane domain of CD8α comprises the sequence of SEQ ID NO: 6, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 6 while retaining substantial biological activity of SEQ ID NO: 6, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof.

[0138] In certain embodiments, the transmembrane domain of the CAR described herein is synthetic and comprises, for example, primarily hydrophobic residues, such as leucine and valine. In certain embodiments, the transmembrane domain of the CAR described herein is modified or designed to avoid binding of the same or different surface membrane proteins to the transmembrane domain in order to minimize interaction with other members of the receptor complex.

[0139] In some embodiments, the CAR described herein further includes a hinge region that forms a link between the extracellular domain and the transmembrane domain of the CAR. The hinge and / or transmembrane domain provide cell surface presentation of the extracellular antigen-binding domain of the CAR.

[0140] The hinge region is not limited to BAFFR, BLAME (SLAMF8), CD2, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA-l), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD49f, CD64, CD80, CD84, CD86, CD96(Tactile), CD100(SEMA4D), CD103, CD134, CD137(4-1BB), CD150(IPO-3, SLAMF1, SLAM), CD15 4, CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (Ly9), CD244 (2B4, SLAMF4), CD278 (ICOS), CEACAM1, CRT It may be derived from any membrane-bound or transmembrane protein, including AM, GITR, HYEM (LIGHTR), IA4, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR, LTBR, ​​OX40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, the α, β, or ζ chains of the T cell receptor, TNFR2, VLA1, and VLA-6.

[0141] In some embodiments, the hinge region includes the hinge region of CD8α, the hinge region of human immunoglobulin (Ig), or a glycine-serine-rich sequence.

[0142] In some embodiments, CAR includes a hinge region of CD8α. In certain embodiments, the hinge region includes the sequence of SEQ ID NO: 7, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 7 while retaining substantial biological activity of SEQ ID NO: 7, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conservative substitutions thereto, or any functional form thereof.

[0143] (3) Intracellular signal transduction domains The intracellular signaling domains of CARs described herein are involved in the activation of at least one normal effector function of the immune cell (e.g., dendritic cell) in which the CAR resides. In the context of immune cells, the term "effector function" refers to the cell's specialized function, such as phagocytic activity, cytolytic activity, or helper activity. In certain embodiments, the intracellular signaling domains of CARs described herein are capable of activating (including maturating) dendritic cells in an immunosuppressive tumor microenvironment.DC activation responds to diverse stimuli and activates many cell surface receptors, such as TLR4 (A. Iwasaki et al., Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5, 987-995 (2004)), TNFR (LM Sedger et al., From mediators of cell death and inflammation to therapeutic giants - past, present and future. Cytokine Growth Factor Rev. 25, 453-472 (2014)), IFNγR (MZ Jianping Pan et al., Interferon-γ is an autocrine mediator for dendritic cell maturation. Immunol. Lett. 94, 141-151 (2004)), and Dectin-1 (TS Helen S. et al., Differential utilization of CARD9 by Dectin-1 in macrophages and dendritic cells. J Immunol. 182). These DC-activating receptors may also be induced by FcγR (1146-1154 (2009)) and FcγR (M. Guilliams et al., The function of Fcγ receptors in dendritic cells and macrophages. Nat. Rev. Immunol. 14, 94-108 (2014)., TH Flinsenberg, Fc receptor antigen targeting potentiates cross-presentation by human blood and lymphoid tissue BDCA-3 dendritic cells. Blood 120, 26 (2012).). These DC-activating receptors have one or more immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains, which trigger an activation signaling cascade to activate DCs.As used herein, the term “cytoplasmic domain” means the full-length domain of a protein located within the cytoplasm, or any fragment thereof, for example, a fragment having a length of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the full-length domain.

[0144] The intracellular signaling domain of the CAR described herein may include the cytoplasmic domain of a dendritic cell activation receptor selected from the group consisting of RIG-1, NLRP10, DEC-205, BDCA-2, CD86, 4-1BBL, OX40L, CD40, IFNAR, TLR4, TNFR (e.g., TNFR2), IFNγR, Dectin-1, and FcγR, or combinations thereof. In a particular embodiment, the intracellular signaling domain of the CAR described herein includes the cytoplasmic domain of Dectin-1 and the cytoplasmic domain of FcγR.

[0145] In certain embodiments, the cytoplasmic domains of Dectin-1 and FcγR are linked in tandem. In certain embodiments, the polynucleotide encoding the cytoplasmic domain of Dectin-1 is upstream of the polynucleotide encoding the cytoplasmic domain of FcγR. In certain embodiments, the polynucleotide encoding the cytoplasmic domain of Dectin-1 is downstream of the polynucleotide encoding the cytoplasmic domain of FcγR.

[0146] The cytoplasmic domain of Dectin-1 may include the amino acid sequence shown in Sequence ID No. 1, or a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No. 1 while retaining substantial biological activity of Sequence ID No. 1, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof. In a particular embodiment, the cytoplasmic domain of Dectin-1 may include the amino acid sequence shown in Sequence ID No. 58, or a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to Sequence ID No. 58 while retaining substantial biological activity of Sequence ID No. 58, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof. In a particular embodiment, the cytoplasmic domain of dectin-1 contains the amino acid sequence shown in SEQ ID NO: 58.

[0147] The cytoplasmic domain of FcγR may include the amino acid sequence shown in SEQ ID NO: 2, or a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2 while retaining substantial biological activity of SEQ ID NO: 2, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof. In certain embodiments, the cytoplasmic domain of FcγR may include the amino acid sequence shown in SEQ ID NO: 59 and / or SEQ ID NO: 60, or a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 59 and / or SEQ ID NO: 60 while retaining substantial biological activity of SEQ ID NO: 59 and / or SEQ ID NO: 60, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof. In certain embodiments, the cytoplasmic domain of FcγR may include the amino acid sequence shown in SEQ ID NO: 59 and / or SEQ ID NO: 60.

[0148] In certain embodiments, the intracellular signaling domain of the CAR described herein includes the amino acid sequence shown in SEQ ID NO: 3, or a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3 while retaining substantial biological activity of SEQ ID NO: 3, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions therein, or any functional form thereof.

[0149] In certain embodiments, the intracellular signaling domain of the CAR described herein includes an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 4 while retaining substantial biological activity of SEQ ID NO: 4.

[0150] (4) Co-stimulatory signaling domain In some embodiments, the intracellular signaling domain further comprises a co-stimulatory signaling domain.

[0151] In some embodiments, the co-stimulatory signaling domain is derived from the intracellular domain of the co-stimulatory molecule.

[0152] Examples of co-stimulatory molecules include B7-H3, BAFFR, BLAME (SLAMF8), CD2, CD4, CD8α, CD8β, CD7, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD83, CD84, CD96 (Tactile ), CD100(SEMA4D), CD103, CD127, CD137(4-1BB), CD150(SLAM, SLAMF1, IPO-3), CD160(BY55), CD1 62 (SELPLG), CD226 (DNAM1), CD229 (Ly9), CD244 (SLAMF4, 2B4), CEACAM1, CRTAM, CDS, OX40, PD-l, This includes ICOS, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-l, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, LAT, LFA-l, LIGHT, LTBR, ​​NKG2C, NKG2D, NKp44, NKp30, NKp46, NKp80 (KLRF1), PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, any derivatives, variants or fragments thereof, any synthetic sequences of costimulatory molecules having the same functional capacity, and any combination thereof.

[0153] In some embodiments, the CAR co-stimulatory signaling domain described herein includes the intracellular domain of the co-stimulatory molecule CD137(4-1BB), CD28, OX40, or ICOS.

[0154] Other areas In some embodiments, the CAR further comprises a signal peptide. In some embodiments, the signal peptide comprises the CD8α signal peptide. In some embodiments, the CD8α signal peptide comprises the sequence of SEQ ID NO: 5, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 5 while retaining substantial biological activity of SEQ ID NO: 5, or a sequence having one, two, three, four, five, six, seven, eight, nine, or ten conserved substitutions thereto, or any functional form thereof.

[0155] Human solid tumors develop complex and heterogeneous time-induced inflammatory states (TIMEs) to evade immunotherapy. Current immunotherapies (e.g., CAR-T cell therapy) are ineffective against solid tumors. Tumor-infiltrating immunosuppressive dendritic cells (DCs) significantly contribute to TIME. DC-activated CARs, as described above, disrupt TIME, convert it into an inflammatory state, enhance the cytotoxicity and survival of manipulated immune cells (e.g., CAR-T cells), and significantly improve the effectiveness of manipulated immune cells (e.g., CAR-T cells) to eliminate solid tumors with TIME.

[0156] vector In another embodiment, the Disclosure provides a vector comprising a polynucleotide encoding a CAR as described herein. The polynucleotide encoding the CAR may be inserted into different types of vectors known in the art, such as plasmids, phagemids, phage derivatives, viral vectors derived from animal viruses, cosmids, transposons, site-directed insertion vectors (e.g., CRISPR, zinc finger nucleases, TALENs), in vitro transcription RNA, or suicide expression vectors. In some embodiments, the vector is DNA or RNA.

[0157] In some embodiments, the vector is an expression DNA vector (e.g., plasmid, virus). When an expression DNA vector is transiently introduced into cells, the mRNA of the CAR is transcribed in the host cells. Since the DNA vector and mRNA are diluted with cell division, the expression of the CAR is not considered to be permanent. In one embodiment, the DNA vector may be introduced into cells as a form of transient expression of the CAR.

[0158] In some embodiments, the vector is a viral vector. Viral vectors may be derived from, for example, retroviruses, adenoviruses, adeno-associated viruses (AAVs), herpesviruses, and lentiviruses. Useful viral vectors generally contain a replication origin, promoter, restriction endonuclease site, and one or more selectable markers that are functional in at least one organism. In some embodiments, the vector is a lentiviral vector. Lentiviral vectors are particularly useful for the long-term stable integration of polynucleotides encoding CARs into the genome of non-proliferating cells, resulting in stable expression of CARs in host cells, such as host T cells. In some embodiments, the vector is an Addgene lenti-Cas9 vector.

[0159] In some embodiments, the vector is RNA (e.g., mRNA). Since RNA is diluted with cell division, RNA expression is not considered to be permanent. In one embodiment, an in vitro transcription RNA CAR may be introduced into cells in the form of transient expression.

[0160] In some embodiments, the vector is a transposon-based expression vector. A transposon is a DNA sequence that can change its position within the genome. In a transposon system, the polynucleotide encoding the CAR is flanked by a terminal repeat sequence that is recognizable by a transposase that mediates the transposon's movement. The transposase may be encoded on the same vector as the CAR, or on a separate vector, and delivered simultaneously as a protein. Not limited examples of transposon systems include Sleeping Beauty, Piggyback, Frog Prince, and Prince Charming.

[0161] In some embodiments, a polynucleotide is functionally ligated to at least one regulatory polynucleotide element in a vector for CAR expression. A typical vector contains a variety of regulatory polynucleotide elements, such as elements that control the expression of the inserted polynucleotide (e.g., transcription and translation terminators, start sequences, and promoters), elements that control the replication of the vector in host cells (e.g., origin of replication), and elements that control the integration of the vector into the host genome (e.g., terminal repeat sequences of transposons). CAR expression may be achieved by operably ligating the polynucleotide encoding the CAR to a promoter and incorporating this construct into the vector. Both constitutive promoters (e.g., CMV promoter, SV40 promoter, and MMTV promoter) and inductive promoters (e.g., metallothionine promoter, glucocorticoid promoter, and progesterone promoter) are intended for this disclosure. In some embodiments, the vector is an expression vector, which contains sufficient cis-acting elements for expression, and other elements for expression may be supplied by host cells or in an in vitro expression system.

[0162] To evaluate CAR expression, the vector may also contain a selectable marker gene or a reporter gene, or both, for the identification and selection of cells into which the vector is introduced. Useful selectable markers include, for example, antibiotic resistance genes, such as Neo. Useful reporters include, for example, luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes.

[0163] Chemical structures that enhance stability and / or translation efficiency may also be used in RNA. Methods for generating RNA for transfection may involve specially designed primers, followed by in vitro transcription (IVT) of a template with poly-A addition, to produce a construct containing 3' and 5' untranslated sequences ("UTRs"), a 5' cap and / or an internal ribosome entry site (IRES), the nucleic acid to be expressed, and a poly-A tail typically 50 to 2000 bases in length. The RNA thus produced can efficiently transfect different types of cells.

[0164] RNA may be introduced into target cells using one of many different methods, for example, but are not limited to, electroporation or Gene Pulser II (BioRad, Denver, Colorado), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or particulate gun particle delivery systems, such as "gene guns."

[0165] Vectors may be introduced into host cells, such as mammalian cells, by any method known in the art, for example, by physical, chemical, or biological means. Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Biological methods include the use of viral vectors, particularly retroviral vectors, for inserting genes into mammalian cells, such as human cells. Chemical means include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0166] cell In one embodiment, this disclosure provides engineered cells containing or expressing CARs as described herein. In some embodiments, the engineered cells contain a polynucleotide encoding a CAR, or a vector containing a CAR polynucleotide. The engineered cells provided herein may contain or express one or more (e.g., one, two, three, or more) CARs. The one or more CARs may be the same or different. In certain embodiments, the engineered cells are dendritic cells or their precursors or progenitor cells. The term “dendritic cells or their precursors or progenitor cells” means, as used herein, natural or modified dendritic cells or their precursors or progenitor cells.

[0167] cell source The manipulated cells (e.g., CAR-DCs) provided herein may be obtained from any source. In certain embodiments, the manipulated cells (e.g., CAR-DCs) provided herein are derived from immune cells isolated from a subject, e.g., a human subject. In some embodiments, immune cells are obtained from a subject of interest, e.g., a subject suspected to have a particular disease or condition, a subject suspected to be predisposed to a particular disease or condition, a subject who is scheduled to receive, is receiving, or has received treatment for a particular disease or condition, a subject who is a healthy volunteer or healthy donor, or from a blood bank. In some embodiments, immune cells are obtained from a cancer subject that is poorly responsive to immunotherapy, e.g., CAR-T therapy.

[0168] The cells may be autologous or allogeneic to the subject of interest. Allogeneic donor cells do not need to be human leukocyte antigen (HLA) compatible, and therefore, allogeneic cells may be treated to reduce their immunogenicity.

[0169] Immune cells may be collected from any location present in the subject, but are not limited to, blood, umbilical cord blood, spleen, thymus, lymph nodes, pleural fluid, splenic tissue, tumors, and bone marrow. Isolated immune cells may be used directly, or they may be stored for a certain period of time, for example, by freezing.

[0170] In some embodiments, manipulated cells are obtained by manipulating dendritic cells or their precursors or progenitor cells. Dendritic cells or their precursors or progenitor cells may be obtained from blood collected from a subject using any of the techniques known to those skilled in the art, such as apheresis. In some embodiments, dendritic cells or their precursors or progenitor cells are derived from peripheral blood cells (e.g., peripheral blood mononuclear cells, e.g., monocytes), bone marrow cells, embryonic stem cells, or induced pluripotent stem cells (iPSCs).

[0171] The presence of dendritic cells may be checked using previously described methods. For example, dendritic cells may be identified by measuring the expression of CD11c, CD80, CD86, MHC / HLA molecules, and / or CCR7 molecules, which are detectable using techniques such as immunochemistry, immunophenotyping, flow cytometry, Elispots assay, classical tetramer staining, and intracellular cytokine staining.

[0172] Method for producing CAR-DC In another embodiment, the Disclosure provides a method for producing engineered cells expressing CARs as described herein. Many means of generating CAR-T cells known in the Art may also be applied to CAR-DCs. A method for generating CAR-T cells is described, for example, in Zhang et al., Engineering CAR-T cells, Biomarker Research (2017) 5:22. In some embodiments, the method includes introducing a vector containing a polynucleotide encoding a CAR provided herein into starting cells under conditions suitable for the expression of the polynucleotide. The method provided herein may include one or more steps selected from the steps of obtaining starting cells (i.e., source-derived cells), culturing these starting cells (including expansion and optionally maturation), and genetically modifying these cells. The starting cells may be dendritic cells or their precursors or progenitor cells as described above.

[0173] Genetic modification of DCs or their precursors or progenitor cells can be achieved by transducing a substantially homogeneous population of DCs with polynucleotides encoding CARs provided herein. In certain embodiments, a retroviral vector (e.g., a lentiviral vector) is used to introduce the polynucleotides provided herein into DCs. For example, the polynucleotides provided herein may be cloned into a lentiviral vector and expression may be driven from an endogenous promoter, from a lentiviral long-terminal repeat sequence, or from a promoter specific to the target cell type of interest. Common delivery methods for delivering viral vectors include, but are not limited to, electroporation, microinjection, gene guns, and magnetofection. The placement of the CARs disclosed herein may be carried out at the locus of any endogenous gene.

[0174] Nonviral approaches may be used for gene modification of DCs or their precursors or progenitor cells. For example, by administering nucleic acids in the presence of lipofection (Ono et al., Neuroscience Letters 17:259, 1990, Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987, Staubinger et al., Methods in Enzymology 101:512, 1983, Brigham et al., Am. J. Med. Sci. 298:278, 1989), sialorosomucoid polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988, Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990) Nucleic acid molecules may be introduced into DCs or their precursor or progenitor cells. Other nonviral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for the delivery of DNA into cells. Transplantation of normal genes into the affected tissue of a subject may also be achieved by ex vivo introducing normal nucleic acids into a cultureable cell type (e.g., autologous or xenogeneic primary cells or their offspring), and then injecting the cells (or their offspring) into the targeted tissue or systemically. Recombinant receptors may also be derived or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR).

[0175] In certain embodiments, the manipulated cells provided herein are prepared by transfecting DCs with a polynucleotide encoding a CAR provided herein before administration. In certain embodiments, the manipulated cells provided herein may be prepared by transfecting DC precursors or progenitor cells with a polynucleotide encoding a CAR provided herein, for example, via a viral vector, and then differentiating the transfected cells into DCs. The manipulated cells provided herein exhibit improved CAR expression on the cell surface. The DC precursors or progenitor cells may be derived from peripheral blood cells (e.g., peripheral blood mononuclear cells, e.g., monocytes, e.g., THP-1 cells, peripheral monocytes), bone marrow cells, etc. The DC precursors or progenitor cells may also be embryonic stem cells or induced pluripotent stem cells (iPSCs).

[0176] In another embodiment, the Disclosure also provides cell populations produced ex vivo by the methods described above. In a particular embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell population express the CAR polypeptides provided herein at a detectable level. In a particular embodiment, at least 85% of the cell population express the CAR polypeptides provided herein at a detectable level.

[0177] How to select a DC activated car In another embodiment, the disclosure also provides a method for selecting CARs capable of activating DCs. The method provided herein comprises preparing a non-human animal containing an immunosuppressive tumor microenvironment. In certain embodiments, the immunosuppressive tumor microenvironment is clinically appropriate. As used herein, the terms “clinically appropriate” with respect to immunosuppressive tumor microenvironment or TIME mean an immunosuppressive tumor microenvironment characterized by one or more of the following features: 1) Hypoxia and acidity, 2) enrichment of negative immune regulatory cells, e.g., regulatory T cells, immunosuppressive DC cells, tumor-associated macrophages and tumor-associated fibroblasts, 3) overexpression of immunosuppressive molecules, e.g., PD-1, TIM3, TIGIT, LAG3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155) and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), HLA class I, sialycoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47, and 4) suppression of the activity of tumor-infiltrating immune cells (e.g., immune effector cells).

[0178] In certain embodiments, a non-human animal (e.g., mouse) model includes human fetal thymus and autologous human hematopoietic stem cells (e.g., autologous human CD34+ hematopoietic stem cells, e.g., autologous human embryonic liver CD34+ hematopoietic stem cells). The term “autologous,” as used herein, may mean that the human hematopoietic stem cells and human embryonic thymus originate from the same embryonic source. In certain embodiments, a non-human animal (e.g., mouse) model includes approximately 1 × 10⁻⁶ cells. 5 ~Approx. 5×10 5 Autologous human hematopoietic stem cells (e.g., autologous human CD34+ hematopoietic stem cells, e.g., autologous human embryonic liver CD34+ hematopoietic stem cells) are injected. In certain embodiments, non-human animal (e.g., mouse) models are used, such as human lymphoid hematopoietic cells, e.g., T cells (e.g., CD3 + T cells, B cells (e.g., CD19) +The present invention comprises a persistent human immune system including B cells and, optionally, dendritic cells (DCs) that enable normal human T cell maturation in the presence of autologous human leukocyte antigens (HLA) within the human thymic environment. In certain embodiments, the non-human animal is a rodent, such as a rat or mouse.

[0179] In certain embodiments, the non-human animal includes an immunosuppressive microenvironment, such as an immunosuppressive tumor microenvironment. In certain embodiments, the immunosuppressive tumor microenvironment includes tumor and / or tumor-infiltrating immune cells expressing immunosuppressive molecules. The immunosuppressive molecules may be selected from the group consisting of PD-1, TIM3, TIGIT, LAG3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155), and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), HLA class I, sialycoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47. In certain embodiments, the immunoinhibitory molecule is CTLA-4 and / or PD-L1. In certain embodiments, the tumor comprises cells expressing CTLA4-Ig and / or PD-L1.

[0180] The methods provided herein further include administering dendritic cells expressing a candidate CAR to the aforementioned non-human animals, detecting markers relating to dendritic cell activation, including, for example, improved infiltration into the immunosuppressive tumor microenvironment, improved survival rate, and / or enhanced function in inducing activation of immune cells (e.g., T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, eosinophils, or neutrophils) compared to reference DCs, and selecting a candidate CAR as a CAR capable of activating DCs. In certain embodiments, the immune cells are T cells selected from the group consisting of CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γδ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes. In certain embodiments, the immune cells are autologous or allogeneic. In certain embodiments, the immune cells are modified immune cells (e.g., CAR-T cells) or naive immune cells. In a particular embodiment, modified immune cells (e.g., CAR-T cells) are administered in combination with dendritic cells expressing a candidate CAR.

[0181] A method for selecting DC-activated CARs involved in non-human animals with clinically appropriate time (TIME) provides more clinically appropriate DC-activated CARs or CAR-DCs. In other words, DC-activated CARs or CAR-DCs selected by the method provided herein are not only capable of activating DCs in animal models, but are also expected to activate DCs under clinical settings that have been largely unattainable with conventional animal models, due to the increased complexity and heterogeneity of the tumor microenvironment in human patients compared to conventional animal models.

[0182] Pharmaceutical composition In another aspect, the Disclosure also provides a pharmaceutical composition comprising a polynucleotide encoding a CAR provided herein and a pharmaceutically acceptable medium. In another aspect, the Disclosure also provides a pharmaceutical composition comprising a CAR polypeptide provided herein and a pharmaceutically acceptable medium. In another aspect, the Disclosure also provides a pharmaceutical composition comprising a vector for delivering a polynucleotide encoding a CAR provided herein and a pharmaceutically acceptable medium. In another aspect, the Disclosure also provides a pharmaceutical composition comprising a population of manipulated cells (e.g., CAR-DCs) provided herein and a pharmaceutically acceptable medium. As used herein, the term “pharmaceutical composition” means a composition formulated for pharmaceutical use.

[0183] The term "pharmaceutically acceptable" means that the specified carrier, vehicle, diluent, excipient(s), and / or salt are generally chemically and / or physically compatible with the other components of the formulation and physiologically compatible with the recipient.

[0184] "Medically acceptable media" refers to components in a pharmaceutical formulation other than the active ingredient that are biologically acceptable and non-toxic to the subject. Medically acceptable media used in the pharmaceutical compositions disclosed herein may include, for example, medicamentally acceptable liquids, gels, or solid carriers, aqueous or non-aqueous vehicles, antimicrobial agents, buffers, antioxidants, isotonic agents, suspensions / dispersants, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, or various combinations thereof.

[0185] The pharmaceutical compositions of this disclosure may be prepared using a variety of techniques known in the art. See, for example, Remington, The Science and Practice of Pharmacy (21st ed. 2005). Briefly, manipulated cells or populations thereof are mixed with a suitable medium before use or storage. A suitable pharmaceutically acceptable medium generally includes an inert substance that assists 1) administration of the pharmaceutical composition to a subject, 2) processing of the pharmaceutical composition into a deliverable preparation, and / or 3) storage of the pharmaceutical composition before administration. In certain embodiments, the pharmaceutically acceptable medium includes an active agent that can stabilize, optimize, or modify the type, consistency, viscosity, pH, pharmacokinetics, and / or solubility of the formulation. These active ingredients include, without limitation, buffers, humectants, emulsifiers, diluents, encapsulants, and skin penetration enhancers, such as physiological saline, buffered physiological saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethylcellulose, and combinations thereof.

[0186] Exemplary pharmaceutically acceptable media include sugars (such as lactose, glucose and sucrose), starches (such as corn starch and potato starch), cellulose and its derivatives (such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate), powdered tragacanth, malt, gelatin, lubricants (such as magnesium stearate, sodium lauryl sulfate and talc), excipients (such as cocoa butter and suppository wax), oils (such as peanut oil, cottonseed oil, sesame oil, castor oil, olive oil, corn oil and soybean oil), glycols (such as propylene glycol), polyols (such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG)), esters (such as ethyl oleate and ethyl laurate), agar, buffering agents (such as magnesium hydroxide and aluminum hydroxide), alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, pH buffer solutions, polyesters, polycarbonates, polyanhydrides, bulking agents (such as polypeptides and amino acids), serum alcohols (such as ethanol), (sterile) phosphate buffered saline, Ringer's solution, dextrose solution and other non-toxic compatible substances used in pharmaceutical formulations are included.

[0187] To induce and / or enhance an immune response against an antigen and / or to treat and / or prevent a neoplasm, pathogen infection, or infectious disease, the pharmaceutical compositions provided herein may be administered systemically or directly to a subject. In certain embodiments, the pharmaceutical compositions provided herein are injected directly into a tumor or organ of interest. In other embodiments, the pharmaceutical compositions provided herein are administered indirectly to an organ of interest, for example, by administration into the circulatory system (such as the tumor vasculature).

[0188] The pharmaceutical compositions provided herein are at least about 1×10 5 , about 2×10 5 , about 3×10 5 , about 4×10 5 or about 5×10 5The population may include manipulated cells (e.g., CAR-DCs). Those skilled in the art can easily determine the proportion of the manipulated cells (e.g., CAR-DCs) provided herein in the population using a variety of known methods, such as fluorescence-activated cell sorting (FACS). A suitable range for the proportion (also referred to as "purity") of the manipulated cells (e.g., CAR-DCs) provided herein in the population may be about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0189] In a particular embodiment, the recipient receives at least 1 × 10 3 Cells / kg body weight, at least 5 × 10⁻⁶ 3 Cells / kg body weight, at least 1 × 10⁻⁶ 4 Cells / kg body weight, at least 5 × 10⁻⁶ 4 Cells / kg body weight, at least 1 × 10⁻⁶ 5 Cells / kg body weight, at least 5 × 10⁻⁶ 5 Cells / kg body weight, at least 1 × 10⁻⁶ 6 Cells / kg body weight, at least 5 × 10⁻⁶ 6 Cells / kg body weight, at least 1 × 10⁻⁶ 7 Cells / kg body weight, at least 5 × 10⁻⁶ 7 Cells / kg body weight, at least 1 × 10⁻⁶ 8 Cells / kg body weight, at least 2 × 10⁻⁶ 8 Cells / kg body weight, at least 3 × 10⁻⁶ 8 Cells / kg body weight, at least 4 × 10⁻⁶ 8 Cells / kg body weight, at least 5 × 10⁻⁶ 8 Cells / kg body weight, or at least 6 × 10⁶ 8Cells / kg body weight are administered. Those skilled in the art will understand that the dosage of the pharmaceutical compositions provided herein may be determined based on various factors of the recipient, such as size, age, sex, weight, and condition. The dosage can be readily determined by those skilled in the art from the present disclosure and knowledge in the art. Those skilled in the art can readily determine the number of manipulated cells provided herein to be administered in the method of the present disclosure, as well as the amounts of optional additives, vehicles, media and / or carriers. Typically, additives, if present, are in the form of a 0.001% to 50% (by weight) solution in phosphate-buffered saline, and the active ingredients (e.g., modified / recombinant cells provided herein) are present in the order of micrograms to milligrams, for example, about 0.0001 to about 5% by weight, preferably about 0.0001% to about 1% by weight, more preferably about 0.0001% to about 0.05% by weight or about 0.001% to about 20% by weight, preferably about 0.01% to about 10% by weight, and more preferably about 0.05% to about 5% by weight. It is preferable to determine the toxicity of a particular dosage by determining the lethal dose (LD) and LD50, for example, in a suitable animal model (e.g., mouse). It is also preferable to determine the timing of administration of the composition(s) to induce a suitable response. Such determinations are based on the knowledge of those skilled in the art and the present disclosure and do not require excessive experimentation.

[0190] The pharmaceutical compositions provided herein may be administered, for example, by injection (e.g., systemic injection, local injection, intravenous injection, intralymphatic injection) or by catheter. In certain embodiments, the pharmaceutical compositions provided herein may be administered subcutaneously, intradermally, intratumorally, intramedullarily, or intraperitoneally. In one embodiment, the cell compositions of this disclosure are preferably administered by intravenous injection. Administration may be autologous or heterologous. For example, modified starting cells derived from one subject may be used to obtain manipulated cells (e.g., CAR-DCs) which may then be administered to the same subject or a different subject. The pharmaceutical compositions provided herein may be formulated in a unit-dose injectable form for administration (e.g., solution, suspension, emulsion). Administration of the pharmaceutical compositions provided herein may be performed as a single event or over a course of treatment, for example, daily, weekly, every two weeks, or monthly. The pharmaceutical compositions provided herein may be administered in combination with other active agents, such as chemotherapeutic agents, other types of immunotherapy (e.g., CAR-T therapy), or radiotherapy (e.g., before, after, or concurrently with these). Simultaneous administration may be carried out through the administration of separate compositions, each containing manipulated cells (e.g., CAR-DCs) provided herein and another active agent, e.g., a chemotherapeutic agent, another type of immunotherapy (e.g., CAR-T therapy), or radiotherapy. Simultaneous administration may also be carried out through the administration of a single composition containing manipulated cells (e.g., CAR-DCs) and another active agent, e.g., a chemotherapeutic agent, another type of immunotherapy (e.g., CAR-T therapy), or radiotherapy.

[0191] kit In another embodiment, the Disclosure also provides a kit comprising engineered cells (e.g., CAR-DCs) provided herein. In another embodiment, the Disclosure also provides a kit comprising polypeptides, polynucleotides, or expression vectors provided herein used in generating CAR-DCs provided herein.

[0192] In some embodiments, the kits of this disclosure include written instructions for use of the kit. In certain embodiments, the instructions include at least one of clinical studies, precautions for use, warnings, and / or references. The instructions may be printed directly on the container (if any), or provided in the container, or with the container as a label applied to the container, or as a separate sheet, pamphlet, card, or folder. Suitable containers include, for example, bottles, syringes, vials, and test tubes. Containers may be formed from a variety of materials, such as plastic or glass. In certain embodiments, the container holds the pharmaceutical composition provided herein and has a sterile access port.

[0193] In certain embodiments, the kit further includes a second container containing the pharmaceutically acceptable medium as described above. In certain embodiments, the kit further includes other commercially desirable or user-friendly substances, such as other diluents, buffers, needles, filters, syringes, and accompanying documentation including instructions for use.

[0194] Usage This disclosure also provides diverse uses of the manipulated cells (e.g., CAR-DCs) provided herein.

[0195] general use In one embodiment, the Disclosure provides a method for treating a disease or pathological condition in a patient, comprising administering to the patient a therapeutically effective amount of the manipulated cells provided herein. In some embodiments, the method for treating a disease or pathological condition comprises preparing DCs isolated from or derived from cells isolated from a subject (e.g., peripheral blood cells, bone marrow cells, embryonic stem cells) or derived from iPSCs, manipulating the DCs to express CARs as provided herein, and returning the manipulated cells (e.g., CAR-DCs) to the subject and injecting them. In some embodiments, the method for treating a disease or pathological condition comprises preparing precursor or progenitor cells of DCs (e.g., peripheral blood cells, bone marrow cells, embryonic stem cells, or iPSCs), differentiating and manipulating these precursor or progenitor cells to express CARs as provided herein, and returning the differentiated and manipulated cells (e.g., CAR-DCs) to the subject and injecting them. In some embodiments, a method for treating a disease or pathological condition includes preparing DC precursor or progenitor cells (e.g., peripheral blood cells, bone marrow cells, embryonic stem cells, or iPSCs), manipulating these precursor or progenitor cells to express a CAR as provided herein, differentiating the manipulated precursor or progenitor cells into DCs expressing a CAR as provided herein, and injecting the DCs expressing a CAR as provided herein (e.g., CAR-DCs) back into a subject.

[0196] In some embodiments, the disease is cancer.

[0197] In some embodiments, the cancer is a solid tumor selected from the group consisting of adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer. In some embodiments, cancer is a hematological malignancy selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, HHV8-associated primary exudative lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Waldenström macroglobulinemia, and multiple myeloma (MM).

[0198] In some embodiments, subjects with cancer are less responsive to cancer therapies (e.g., immunotherapy).

[0199] The term “immunotherapy,” as used herein, refers to a type of therapy that stimulates or boosts the immune system in a general manner to fight diseases such as cancer. Immunotherapy includes passive immunotherapy (e.g., antibody therapy or CAR-T cell therapy) which involves delivering an active agent (e.g., effector cells) with established tumor immunoreactivity that directly or indirectly mediates an antitumor effect and does not necessarily depend on the intact host immune system. Immunotherapy may further include active immunotherapy, in which treatment relies on in vivo stimulation of the endogenous host immune system to respond to disease cells, involving the administration of an immunomodulator.

[0200] Examples of immunotherapies include, without limitation, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0201] Checkpoint modulators may interfere with cancer cells' ability to evade immune system attacks, potentially helping the immune system respond more strongly to tumors. Immune checkpoint molecules may either enhance immune responses through co-stimulatory signals or suppress immune responses through co-inhibitory signals. Examples of checkpoint modulators include, but are not limited to, modulators of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGF-β, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD47, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-7, IL-15, IL-21, CD3, CD16, and CD83. In certain embodiments, the immune checkpoint modulator includes a PD-1 / PD-L1 axis inhibitor.

[0202] Adoptive cell transfer is a treatment that attempts to boost the natural ability of T cells to fight cancer. In this treatment, T cells are collected from the patient, expanded and activated in vitro. In certain embodiments, the T cells are modified into CAR-T cells in vitro. The T cells or CAR-T cells that are most active against cancer are cultured in large quantities in vitro for 2 to 8 weeks. During this period, the patient receives treatments such as chemotherapy and radiation therapy to reduce the body's immune system. After these treatments, the in vitro cultured T cells or CAR-T cells are returned to the patient. In certain embodiments, the immunotherapy is CAR-T therapy.

[0203] Destruction of TIME In one embodiment, the Disclosure provides a method for disrupting TIME (e.g., converting TIME into an inflammatory state) using CAR-DCs or a group thereof provided herein.

[0204] In another embodiment, the disclosure also provides a method for inducing immune cell proliferation, extending immune cell survival, and / or increasing the expression and / or secretion of immunostimulatory cytokines from immune cells in an immunosuppressive microenvironment. The immunostimulatory cytokines may be one or more of TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and granulocyte-macrophage colony-stimulating factor. The method comprises bringing an immunosuppressive microenvironment into contact with manipulated cells provided herein (e.g., CAR-DCs). The immune cells may be T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, eosinophils, or neutrophils. In certain embodiments, the immune cells are T cells selected from the group consisting of CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γδ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes. In certain embodiments, the immune cells are unmodified immune cells. In certain embodiments, the immune cells are modified immune cells. Unmodified or modified immune cells may be autologous or allogeneic. In certain embodiments, the modified immune cells are CAR-T cells. In certain embodiments, the CAR-T cells are derived from the same source (e.g., peripheral blood of the subject) as the manipulated cells (e.g., CAR-DCs) provided herein.

[0205] In certain embodiments, the immunosuppressive microenvironment is an immunosuppressive tumor microenvironment. The immunosuppressive tumor microenvironment is described in the section titled “Dendritic Cell (DC)-Activated Chimeric Antigen Receptor (CAR)”. In certain embodiments, the immunosuppressive tumor microenvironment includes tumor and / or tumor-infiltrating immune cells expressing immunosuppressive molecules selected from the group consisting of, for example, PD-1, TIM3, TIGIT, LAG3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155), and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), sialycoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47. In certain embodiments, the immunosuppressive molecules are CTLA-4 and / or PD-L1. In a particular embodiment, the tumor comprises cells expressing CTLA4-Ig and / or PD-L1.

[0206] Improvement in the effectiveness of adoptive cell therapy (e.g., CAR-T therapy) In another embodiment, the Disclosure provides a method for improving the efficacy of adoptive cell therapy in treating cancer in subjects requiring treatment. The method comprises administering a therapeutically effective amount of a pharmaceutical composition provided herein. In certain specific embodiments, the method provided herein further comprises administering a pharmaceutical composition comprising a population of modified immune cells.

[0207] Adoptive cell therapy involves adoptive transfer of modified immune cells, such as immune cells expressing synthetic receptors (e.g., CAR or TCR) on their cell surface. Modified immune cells may be T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, eosinophils, or neutrophils. In certain embodiments, the immune cells are T cells selected from the group consisting of CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γδ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes. Modified immune cells may be autologous or allogeneic. In certain embodiments, the modified immune cells are CAR-T cells. In certain embodiments, the CAR-T cells are derived from the same source (e.g., peripheral blood of the subject) as the manipulated cells (e.g., CAR-DCs) provided herein.

[0208] In some embodiments, cancer is a solid tumor as described above, or a hematological malignancy.

[0209] In some embodiments, subjects with cancer are less responsive to the cancer therapies described above (e.g., immunotherapy).

[0210] Combination therapy In another embodiment, the present disclosure provides a combination therapy using manipulated cells (e.g., CAR-DCs) and a second active agent provided herein.

[0211] In certain embodiments, the second active agent is a population of modified immune cells, such as CAR-T cells, as described above. In certain embodiments, the CAR-T cells are derived from the same source (e.g., the subject's peripheral blood) as the manipulated cells (e.g., CAR-DCs) provided herein. In certain embodiments, the ratio of manipulated cells (e.g., CAR-DCs) to CAR-T cells provided in the combination therapy is in the range of about 1:1 to 1:10.

[0212] In certain embodiments, the manipulated cells (e.g., CAR-DCs) and CAR-T cells provided herein are in the same pharmaceutical composition. In certain embodiments, the manipulated cells (e.g., CAR-DCs) and CAR-T cells provided herein are in two separate pharmaceutical compositions. In certain embodiments, the manipulated cells (e.g., CAR-DCs) provided herein are administered to a subject in need of treatment before, simultaneously with, or after administration of CAR-T cells.

[0213] In certain embodiments, the second active agent is an active agent that inhibits an immunosuppressive pathway, and this includes, but is not limited to, TGF-β, interleukin-10 (IL-10), adenosine, VEGF, indoleamine 2,3-dioxygenase-1 (IDO1), indoleamine 2,3-dioxygenase-2 (IDO2), tryptophan 2-3-dioxygenase (TDO), lactate, hypoxia, arginase, and prostaglandin E2 inhibitors. The second active agent may also be a T-cell checkpoint inhibitor, which may include, but is not limited to, anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD1 antibodies (e.g., nivolumab, pembrolizumab, cemiprimab), anti-PD-L1 antibodies (e.g., atezolizumab, avelumab, durvalumab), anti-PD-L2 antibodies, anti-BTLA antibodies, anti-LAG3 antibodies, anti-TIM3 antibodies, anti-VISTA antibodies, anti-TIGIT antibodies, and anti-KIR antibodies.

[0214] In certain embodiments, the second active agent is a T-cell agonist, which includes, but is not limited to, antibodies that stimulate CD28, ICOS, OX-40, CD27, 4-1BB, CD137, GITR, and HVEM. In certain embodiments, the second active agent is a therapeutic oncolytic virus, which includes, but is not limited to, rhabdoviruses, retroviruses, paramyxoviruses, picornaviruses, reoviruses, parvoviruses, adenoviruses, herpesviruses, and poxviruses.

[0215] In certain embodiments, the second active agent is an immunostimulant, such as a Toll-like receptor agonist, which includes, but is not limited to, TLR3, TLR4, TLR7, and TLR9 agonists. In certain embodiments, the second active agent is an interferon gene stimulant (STING) agonist, such as a cyclic GMP-AMP synthase (cGAS).

[0216] In certain embodiments, the CAR-DCs or CAR-DC populations provided herein are administered to a subject in need of treatment, for example, before, concurrently with, or after such treatment, in combination with any number of suitable therapeutic methods, including, but not limited to, treatment with cytokines that enhance dendritic cell or T cell proliferation and persistence, and including, but not limited to, Flt3L, IL-2, IL-7, and IL-15 or analogues, or the expression of such cytokines from within the CAR-DCs.

[0217] In some embodiments, the treatment further includes administering an agent that reduces or mitigates side effects associated with the administration of manipulated cells. Exemplary side effects include cytokine release syndrome (CRS) and hemophagocytic lymphohistiocytosis (HLH, also known as macrophage activation syndrome (MAS)). In certain embodiments, the agent administered to treat the side effects includes an agent that neutralizes soluble factors, such as IFN-γ, IFN-α, IL-2, and IL-6. Exemplary agents include, without limitation, TNF-α inhibitors (e.g., etanercept) and IL-6 inhibitors (e.g., tocilizumab). [Examples]

[0218] While this disclosure has been shown and described in particular in relation to certain embodiments (some of which are preferred embodiments), those skilled in the art will understand that a variety of variations may be made in type and detail without departing from the spirit and scope of this disclosure as disclosed herein.

[0219] Example 1 Generation of CARs that specifically activate DCs Because the pathways involved in activating T cells and DCs are separate, the inventors hypothesized that typical CAR molecules of CAR-T cells cannot activate DCs (Figures 1A, 8A, and 8B). Therefore, the inventors evaluated novel CARs that incorporate DC activation pathways such as TLR4, TNFR2, Dectin-1, and FcγR. First, the inventors tested CAR structures consisting of anti-human CD19 scFv and the intracellular activation domains of TLR4, TNFR2, Dectin-1, and FcγR in DCs derived from THP-1 cells, a human monocytic leukemia cell line capable of differentiating into functional DCs with a cytokine cocktail (C. Berges et al., A cell line model for the differentiation of human dendritic cells. Biochem. Biophys. Res. Commun. 333, 896-907 (2005)). CARs with TLR4 or TNFR2 tails were unable to efficiently activate DCs, indicating that stimulatory signals delivered solely by TLR4 or TNFR tails were insufficient for DC activation (Figures 8A and 8B). Expression of a CAR consisting of anti-human CD19 scFv containing a tandem fusion of Dectin 1 and FcRγ cytoplasmic tails in THP-1 cells did not affect differentiation into DCs, referred to as CARDF-DCs (Figures 1B and 1C). When CARDF-DCs and control THP-1-derived DCs were exposed to H460-CD19 (Figure 1F), CARDF-DCs expressed higher levels of co-stimulatory molecules (CD80 and CD86) compared to control DCs (Figure 1D). Furthermore, CARDF-DCs were able to induce more robust proliferation of allogeneic T cells than control DCs (Figure 1E). To investigate whether CARDF-DCs can activate CAR-T cell function, second-generation anti-CD19 CAR-T cells were cultured with H460-CD19 tumor cells in the presence of CARDF-DCs or control DCs (Figures 1A and 1G). CAR-T cells exhibited higher cytotoxicity against CD19+ H460 tumor cells in the presence of CARDF-DCs than in the presence of control DCs (Figure 1H).Furthermore, CARDF-DCs induced higher levels of IFN-γ expression in CAR-T cells and increased lactate dehydrogenase (LDH) release by tumor cells compared to control DCs (Figures 1I and 1J). These data suggest that CARDF can enhance DC activity and activate CAR-T cells.

[0220] Example 2 CARDF can activate DCs derived from normal peripheral monocytes. To confirm our understanding of CARDF in THP-1 cell-derived dendritic cells (DCs), we investigated the effect of CARDF expression on normal dendritic cells (Mo-DCs) derived from peripheral monocytes, a common source of dendritic cells for clinical use (J. Constantino et al., Antitumor dendritic cell-based vaccines: lessons from 20 years of clinical trials and future perspectives. Transl. Res. 168, 74-95 (2016)). Monocytes purified from healthy donor PBMCs were transduced with a lentivirus expressing CARDF and induced to differentiate into dendritic cells (Figure 2A). CARDF expression did not affect the differentiation and maturation of Mo-DCs, and the surface expression levels of the dendritic cell markers CD11C, CD80, CD86, HLA-ABC, and HLA-DR were comparable to those of control dendritic cells (Figure 2B).Instead of using an anti-CD19 scFv CAR that is not specific to unmanipulated solid tumors, the inventors used an antibody scFv targeting EphA2, which is highly expressed by many types of solid tumors (Figure 9A) (J. Wykosky et al., The EphA2 receptor and ephrinA1 ligand in solid tumors: function and therapeutic targeting. Mol. Cancer Res. 6, 1795-1806 (2008), JM Brannan et al., EphA2 in the early pathogenesis and progression of non-small cell lung cancer. Cancer Prev. Res. 2, 1039-1049 (2009), VM Youngblood et al., The Ephrin-A1 / EPHA2 Signaling Axis Regulates Glutamine Metabolism in HER2-Positive Breast Cancer. Cancer Res. 76, 1825-1836 (2016), M. Tandon et al., Emerging strategies for EphA2 receptor targeting for cancer therapeutics. Expert Opin Ther Targets. 15, 31-51 (2011). To evaluate whether anti-EphA2 CARDF-DCs can enhance CD3+ T cell proliferation, the inventors cultured CFSF-labeled T cells with CARDF Mo-DCs or control Mo-DCs that had been pre-exposed for 48 hours to human lung cancer A549 cells expressing EphA2. CARDF-DCs were able to induce T cell proliferation more robustly than control Mo-DCs (Figure 2C). In summary, the inventors' findings indicate that CARDF can activate Mo-DCs in response to tumor antigen stimulation.

[0221] Previous findings suggest that immunosuppressive TIDCs within TIME can be induced by the expression of PD-L1 and CTLA4 on the surface of solid tumors, as they suppress effector T cells and promote tumor growth (Non-patent Literature 4, C. Fu et al., Dendritic Cells and CD8 T Cell Immunity in Tumor Microenvironment. Front Immunol. 9, 3059 (2018), C. Pfirschke et al., Tumor Microenvironment: No Effector T Cells without Dendritic Cells. Cancer cell 31, 614-615 (2017), RA Belderbos et al., Enhancing Dendritic Cell Therapy in Solid Tumors with Immunomodulating Conventional Treatment. Mol. Ther. Oncolytics 13, 67-81 (2019)). To evaluate the activation state of Mo-DCs in response to tumor cells expressing CTLA4-Ig and PD-L1, we constructed human lung cancer cells A549 (A549-CP) overexpressing CTLA4-Ig and PD-L1 by knocking in an expression cassette into the HPRT locus, as previously described (Rong Z et al., An Effective Approach to Prevent Immune Rejection of Human ESC-Derived Allografts. Cell Stem Cell 14, 121-130 (2014)). Compared to control A549 cells, CP expression was much higher in A549-CP tumor cells (Figure 2D). When CARDF-DCs or control Mo-DCs were co-cultured with A549-CP cells for 48 hours, CARDF-DCs expressed much higher levels of CD80, HLA-ABC, and HLA-DR than control Mo-DCs (Figure 2E).When CARDF-DCs and control Mo-DCs were pre-exposed to A549-CP for 48 hours, CARDF-DCs were more robust in activating T cells than control Mo-DCs (Figure 2F). Therefore, CARDF can effectively activate T cells by activating DCs to resist tumor cell-induced immunosuppression.

[0222] Example 3 CARDF-DCs activate the cytotoxicity of CAR-T cells in vitro. To investigate whether our CARDF-DCs increase the cytotoxicity of CAR-T cells against tumor cells, we produced anti-EphA2 CAR-T cells using T cells derived from the same donor as Mo-DCs. We confirmed the expression of CAR on the surface of CAR-T cells, as well as EphA2 on the surface of A549 and A549-CP tumor cells (Figures 3A, 3B, and 9B). To examine the cytotoxic activity of CAR-T cells activated by control Mo-DCs or CARDF-DCs, A549 and A549-CP cells were co-cultured with CAR-T cells and DCs. Compared to control Mo-DCs, CARDF-DCs significantly increased the cytotoxic activity of CAR-T cells against A549 cells (Figure 3C). The cytolytic activity of CAR-T cells directed toward A549-CP cells was decreased compared to that of A549 cells, indicating that CP expression suppressed the cytolytic activity of CAR-T cells. In contrast, the inhibition of CAR-T cell cytolytic activity by CP expression in tumor cells was reversed by CARDF-DCs (Figure 3C). Consistent with this finding, co-culture of CARDF-DCs with CAR-T cells increased the expression of IL-2, IFN-γ, and TNF-α by CAR-T cells compared to control Mo-DCs (Figure 3D), and increased the proportion of IFN-γ+CAR-T cells and the levels of IFN-γ and LDH in the supernatant (Figures 3E-3G). Therefore, CARDF-DCs can activate CAR-T cell cytolytic activity in resistance to CP-mediated immunosuppression.

[0223] Example 4 CARDF-DCs resist TIME in vivo and activate the antitumor activity of CAR-T cells. To investigate the effects of CARDF-DCs on CAR-T cells in vivo, the inventors subcutaneously injected A549-WT and A549-CP tumor cells, respectively, into immunodeficient NOD / SCID / IL-2γ- / -(NSG) mice. Once the tumors reached a palpable size, 1 × 10⁻¹⁶ cells were injected. 7 CAR-T cells and 5 × 10 6The control Mo-DCs or CARDF-DCs were each intravenously injected (Figure 4A). In contrast to A549 tumors, A549-CP tumors developed a clinically relevant TIME (Figure 4B). Consistent with previous findings that CAR-T cells rapidly exhausted in solid tumors with a TIME (J. L.-M. Chen et al., NR4A transcription factors limit CAR T cell function in solid tumours. Nature 567, 530-534 (2019), J. Li et al., Chimeric antigen receptor T cell (CAR-T) immunotherapy for solid tumors: lessons learned and strategies for moving forward. J Hematol Oncol. 11, 22 (2018)), CAR-T cells effectively eliminated A549-WT tumors but not A549-CP tumors (Figure C). CAR-T cells combined with CARDF-DCs efficiently reduced A549-CP tumor burden compared to CAR-T cell therapy alone (Figures 4C and 4D). Furthermore, CARDF-DCs significantly prolonged the survival of T cells, including CD8+ T cells, and promoted the survival and activation of the DCs themselves in vivo (Figures 4E and 4F). Additionally, the inventors also detected higher expression levels of CD11C and CD80 in the tumors of the CARDF-DC treatment group (Figure 4E), suggesting that CARDF-DCs may infiltrate better or survive longer in CP-overexpressing tumors than control Mo-DCs. Taken together, these data suggest that CARDF-DCs reverse the TIME, promote the survival and activity of CAR-T cells, and suppress solid tumors.

[0224] Example 5 CARDF-DCs reverse the TIME, activate CAR-T cells, and eliminate solid tumors in Hu-mice The interaction between the immune system and tumors plays an important role in the formation of the TIME (M. Binnewies et al., Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat Med. 24, 541-550 (2018)). Therefore, the inventors used the HuS model in which human solid tumors develop a clinically appropriate TIME in immune system humanized mice as previously described (Q. Li, et al., Developing Covalent Protein Drugs via Proximity-Enabled Reactive Therapeutics. Cell 182, 85-97.e16 (2020)) to further evaluate the activity of CARDF-DC in reversing the TIME of solid tumors (Figure 5A). CARDF-DC and CAR-T cells were derived from the bone marrow cells and T cells of Hu-mice established with the same donor tissue used to establish the HuS model and to inoculate human lung tumors. Therefore, CARDF-DC, CAR-T cells, and the immune system in HuS mice were all derived from the same donor. As expected, CAR-T cells with or without control Mo-DC were unable to suppress human lung tumors that developed a clinically appropriate TIME in HuS mice (Figures 5B-5E). In contrast, when CAR-T cells were combined with CARDF-DC, the growth of human lung tumors formed in the same batch of Hu-mice was efficiently suppressed (Figures 5C-5E). Therefore, CARDF-DC was able to activate the antitumor activity of CAR-T cells against clinically appropriate TIME.

[0225] To test the hypothesis that CARDF-DCs can convert the TIME of solid tumors toward a pro-inflammatory state by activating T cells, the inventors investigated the activation state of T cells in the periphery and tumors. CARDF-DCs increased the proportion of IFN-γ+ T cells in the spleen (Figure 6A) and decreased the expression of inhibitory surface receptors PD-1 and TIM-3 in splenic T cells (Figures 6B and 6E). Furthermore, CARDF-DCs increased the expression of the DC activation markers CD86 and MHC-II in splenic DCs (Figure 6C). Therefore, CARDF-DCs appeared to activate the systemic immune system. Supporting the finding that CARDF-DCs can convert TIME in solid tumors toward a pro-inflammatory state, CARDF-DCs increased the intratumoral expression of TNF-α, IL-2, CD86, and IL-12B (Figure 6D), while decreasing the expression of immune checkpoint molecules PD-1, TIM-3, and TGF-β (Figure 6F), and M2 macrophage markers CD206 and CD163 (Figure 6G). These data suggest that CARDF-DCs can reverse TIME toward a pro-inflammatory state and activate immunity against solid tumors.

[0226] Example 6 CARDF-DCs exhibit uniform T cell activation activity at different time points (TIMEs). Solid tumors exhibit heterogeneity in terms of TIME (V. Thorsson et al., The Immune Landscape of Cancer. Immunity 48, 812-830 e814 (2018)). To test the hypothesis that CARDF-DCs may reverse the TIME of different solid tumors toward a pro-inflammatory state, we used another human lung cancer cell line, H460, which expresses higher levels of PD-L1 and similarly forms clinically appropriate TIME in Hu- mice (Figures 7A and 7B). These tumor cells were found to express EphA2 (Figure 7C). In Hu- mice, consistent with our findings in lung tumors formed by A549, CARDF-DCs efficiently rescued the antitumor activity of CAR-T cells and suppressed solid tumors formed by H460 cells in HuS- mice (Figures 7D-7F). CARDF-DCs were able to infiltrate H460 tumors more efficiently or survive for longer distances than control DCs (Figure 7G). Therefore, these data reveal the homogeneous T-cell activation ability of CARDF-DCs, which reverses immunosuppression at heterogeneous time intervals in solid tumors.

[0227] Example 7 Consideration Despite the outstanding efficacy of CAR-T cell therapy for treating hematological malignancies, immunotherapy for solid tumors remains challenging due to the presence of an immunosuppressive microenvironment (TIME). Therefore, developing strategies to disrupt TIME is crucial to improving the efficacy of solid tumor immunotherapy. It is well established that immunosuppressive TIDCs play a vital role in TIME establishment by suppressing cytotoxic T cell function and promoting immunosuppressive regulatory T cells (Non-Patent Literature 4). To achieve this objective, we have developed a CAR-DC strategy that allows DCs to specifically target tumor cells and remain activated after encountering TIME. Against this backdrop, we demonstrate that standard T cell CARs are unable to activate DCs after they encounter TIME. To specifically activate DCs, we designed a DC-activating CAR molecule with an intracellular domain composed of diverse DC-activating domains. After testing CARs with various combinations of DC activation domains, the inventors discovered that tandem linkage of the cytoplasmic tails of Dectin 1 and FcRγ can effectively activate DCs after encounter with TIME.

[0228] One of the major bottlenecks in tumor immunotherapy research is the lack of clinically appropriate in vivo models to evaluate the effectiveness of immunotherapy (PS Hegde et al., Top 10 Challenges in Cancer Immunotherapy. Immunity 52, 17-35 (2020)). For example, solid tumors established in immunodeficient mice cannot develop TIME, and in this model, efficient removal of solid tumors by CAR-T cells is possible. To overcome this bottleneck, we developed two humanized mouse models that produce human solid tumors with clinically appropriate TIME. First, solid tumor formation by CTLA4-Ig / PD-L1 overexpressing human tumor cells in immunodeficient mice developed an immunosuppressive microenvironment. Second, to replicate the heterogeneity of TIME in solid tumors, solid tumor formation by human tumor cells in immune system-humanized mice developed clinically appropriate TIME. Using these models, we demonstrate that human CAR-DCs promote CAR-T cell activity and suppress solid tumors harboring clinically appropriate TIME. Against this backdrop, this is the first report demonstrating that CAR-DCs can reverse TIME to a pro-inflammatory state, thereby activating the antitumor activity of CAR-T cells and suppressing solid tumors.

[0229] Given the heterogeneity of solid tumors, it would be important to investigate whether CAR-DCs can reverse the time lapse rate (TIME) across various types of human solid tumors. Furthermore, one potential limitation of this strategy is that, after multiple rounds of chemotherapy or radiotherapy, cancer patients may not have enough healthy DCs remaining. This problem can be mitigated by recent advances in obtaining functional dendritic cells (DCs) from induced pluripotent stem cells of patients (D. Todorova et al., hESC-derived immune suppressive dendritic cells induce immune tolerance of parental hESC-derived allografts. EBioMedicine 62, 103120 (2020), S. Senju et al., Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy. Gene Ther. 18, 874-883 (2011), S. Sontag et al., Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells. Stem cells 35, 898-908 (2017)). Based on the ability of CAR-DCs to reverse immunosuppressive TIME into a pro-inflammatory state, there will likely be interest in testing combinations of CAR-DCs with other immunotherapies to treat malignant solid tumors.For example, CAR-DCs can enhance the antitumor activity of natural killer cells and immune checkpoint inhibitors, such as anti-PD1 antibodies, which are only effective against a small fraction of solid tumors (T. Walzer et al., Natural-killer cells and dendritic cells: "l'union fait la force". Blood 106, 2252-2258 (2005), E. Mamessier et al., Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity. J. Clin. Invest. 121, 3609-3622 (2011), K. Foley et al., Current progress in immunotherapy for pancreatic cancer. Cancer lett. 381, 244-251 (2016), JS O'Donnell et al., Resistance to PD1 / PDL1 checkpoint inhibition. Cancer Treat. Rev. 52, 71-81). (2017) 67-70). The novel humanized solid tumor models with clinically appropriate time intervals used herein would provide an ideal platform for evaluating the efficacy of these combination immunotherapies. In summary, the CAR-DC approach represents a promising and potentially universal strategy for overcoming the time interval that determines the outcome of immunotherapy for malignant solid tumors.

[0230] Example 8 Materials and methods research design The results shown are mean values ​​with standard deviation. The number of independent experimental replicates is indicated in the legend of the figure. For in vivo experiments on tumor growth, animals were blinded into treatment groups before treatment and measurement, such as tumor weight and volume measurement, RT-qPCR assay, flow cytometry analysis, or ELISA measurement. Primary data are included in data file S1.

[0231] animal research NOD / SCID / IL-2γ- / -(NSG) mice were purchased from Nanjing Model Biology Company. The NSG and Hu- mice used in this study were maintained in a pathogen-free isolation animal facility. All animal studies were authorized by the Institutional Animal Care and Use Committee (IACUC).

[0232] Construction of lentiviral vectors containing chimeric antigen receptors (CARs) The structures of the second-generation CAR anti-CD19 and anti-EphA2 consist of a CD8 reader sequence and scFv, a CD8 transmembrane domain, and 4-1BB and CD3ζ intracellular domains. To generate DC CARs, the cytoplasmic sequences of TLR4 (NM_138554.5), TNFR2 (NM_001066.3), and Dectin 1 (NM_197947) and FcRγ (NM_004106) were amplified and substituted for the 4-1BB and CD3ζ intracellular domain regions in the second-generation CARs. All sequences were optimized and synthesized by IGene company (Guangzhou). The expression cassettes were then cloned into a lenti-Cas9 vector (Addgene) by substituting the Cas9 region.

[0233] Primary cell and cell line cultures DCs were generated from monocytes isolated from PBMCs (LDEBIO catalog number 1501). Briefly, monocytes were isolated using anti-CD14 microbeads (Miltenyi Biotech catalog number 130-050-201) and an autoMACS Pro separator. Subsequently, the monocytes were cultured for 5-6 days in RPMI1640 (Corning) supplemented with 10% FBS (Gibco), 100 units / ml penicillin, and 100 μg / ml streptomycin (Thermo Fisher Scientific), along with GM-CSF (100 ng / ml, PeproTech catalog number 300-03) and IL-4 (100 ng / ml, PeproTech catalog number 200-04) to generate immature DCs. Cytokines were supplemented every 2-3 days. DC maturation was performed for 24 hours using TNF-α (10 ng / ml, PeproTech catalog number 300-01A) and LPS (3 μg / ml, Sigma-Aldrich catalog number L4391).

[0234] Primary T cells were isolated from peripheral blood mononuclear cells using anti-CD3 microbeads (Miltenyi Biotech catalog number 130-050-101) and maintained in RPMI1640 supplemented with 10% FBS, 2 mM L-glutamine (Thermo Fisher Scientific), 1% penicillin-streptomycin, 2-mercaptoethanol (25 μM, Gibco), and 100 U / ml human IL-2 (PeproTech catalog number AF-200-02-500).

[0235] A549 (catalog number ATCC(trademark) CCL-185(trademark)) and H460 (catalog number ATCC(trademark) HTB-177(trademark)) were purchased from ATCC (Manassas, Virginia). As previously described by the inventors (60), A549-CP was constructed. H460-CD19 was constructed by overexpressing human CD19 on the surface of H460 using a lentivirus. The THP-1 cell line (catalog number ATCC(trademark) TIB-202(trademark)) is a leukemia cell line established from patients with chronic myeloid leukemia. All of the above cells were cultured in RPMI1640 supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin, and 25 μM 2-mercaptoethanol. 293FT cells (Thermo Scientific catalog number R70007) were cultured in Dulbecco's modified Eagle medium (DMEM, Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin-streptomycin. When the cells reached complete density, the cell line was subcultured using 0.25% trypsin-EDTA (Thermo Fisher Scientific) in an appropriate ratio. All cells were incubated in a dark, humidified 37°C incubator containing 5% CO2.

[0236] Mo-DC Transduction Human monocytes were cultured in 24-well ultra-low adhesion tissue culture plates, with 2-5 × 10⁶ cells per well, before transduction. 5Cells were transferred to 400 μL of differentiation medium (RPMI1640 complete medium supplemented with 100 ng / ml GM-CSF and 100 ng / ml IL-4). Lentiviral load was calculated using a qPCR lentiviral titer determination kit (ABM catalog number LV900-iC). Transduction was performed using an MOI of 100 by thawing the titrated lentiviral stock at 37°C. Appropriate volumes of virus concentrate and 6 μg / ml protamine sulfate (Sigma-Aldrich catalog number 1578612-2) were mixed in different media to achieve a total volume of 500 μL per well. After incubation at 37°C for 12 hours, an additional 500 μL of differentiation medium was added to each well. 24 hours after transduction, most of the medium was aspirated, cells were washed twice with PBS, and further cultured in differentiation medium. On day 5, Mo-DCs were collected for future co-culture experiments, or directly matured for 24-48 hours with TNF-α (10 ng / ml, PeproTech) and LPS (3 μg / ml, Sigma-Aldrich).

[0237] iPSC transduction The manipulated cells of this disclosure (e.g., CAR-DCs) may also be prepared by transfecting human induced pluripotent stem cells (hiPSCs) with a viral vector provided herein (e.g., a lentiviral vector) to prepare a stable CAR-expressing cell line (e.g., CARDF-hiPSCs). hiPSCs have the ability to proliferate immortally and differentiate into a variety of tissue cells, and thus have great potential in disease cell therapy. Preferably in this disclosure, the OP9 stromal cell nutrition method (Nat Protoc. 2011 March; 6(3): 296-313. doi: 10.1038 / nprot. 2010.184) is used to induce differentiation of hiPSCs into DCs. In this disclosure, the initial number of differentiated cells derived from hiPSCs is preferably 1 × 10⁶ 6 ~1.5×10 6The initial culture medium is preferably a complete medium of MEM-α supplemented with 20% fetal bovine serum and 1% penicillin-streptomycin. The entire process of DC cell differentiation preferably takes about 31 to 38 days. The CARDF-hiPSCs of this disclosure can be induced to differentiate on a large scale to produce homogeneous CARDF-DCs. As described above, hiPSC-derived CARDF-DCs are expected to have functions such as disrupting TIME, converting TIME into an inflammatory state, and enabling the activation of DCs in an immunosuppressive tumor microenvironment.

[0238] Preparation of CAR-T cells Primary CD3+ T cells were isolated from PBMCs and activated using a human T cell activation kit according to the manufacturer's instructions. Briefly, 12-well plates were coated overnight at 4°C with 3 μg / mL PBS-diluted anti-CD3 antibody (BD catalog number 555329, RRID: AB_395736). The following day, the plates were washed twice with PBS, and then the T cells were thawed and transferred to the plates in T cell medium with 1 μg / mL anti-CD28 antibody (BD catalog number 555725, RRID: AB_396068). Activation lasted for 2 days, and on the 3rd day, activated T cells were harvested and infected with a lentivirus expressing the T-CAR construct shown. Briefly, before transduction, 5 × 10⁶ T cells were induced per well. 5 Cells were transferred to a 24-well tissue culture plate at a density of 400 μL of T cell medium. Transduction was performed using an MOI of 10 by thawing titrated virus stock at 37°C. An appropriate volume of virus concentrate was mixed with 10 μg / ml polyblen (Sigma-Aldrich catalog number TR-1003-G) in the culture medium to achieve a total volume of 500 μL per well. After incubation at 37°C for 12 hours, an additional 500 μL of medium was added to each well. 24 hours after transduction, cells were collected, washed twice with PBS, resuspended in T cell medium, and cultured for growth. On the cytotoxicity assay day (approximately 10 days after activation), cells were collected and analyzed by flow cytometry, and cell counts were obtained using a hemocytometer.

[0239] In vitro T cell proliferation assay Primary CD3+ T cells were stained with CellTrace-CFSE (Life Technologies catalog number 65-0850-84) according to the manufacturer's instructions. In the experiment, DCs were pre-incubated with cancer targets (H460-CD19 cells, A549 cells or A549-CP cells) at a 1:1 ratio in 48-well plates for 48 hours, and then primary T cells (DC:T cell = 1:5) were added to the co-culture. In other experiments, DCs and T cells were incubated with cancer targets simultaneously (day 0). Unless otherwise indicated, a ratio of target:DC:T cell = 1:1:5 was used for each cell co-culture condition. Proliferation was analyzed by flow cytometry by gating on viable CD3+ T cells.

[0240] In vitro DC and tumor cell co-culture assay 1×10 6 of H460-CD19 cells, A549 cells or A549-CP cells were co-cultured with 1×10 6 of THP-1 or monocyte-derived mock DCs or CAR-DCs in 6-well plates. After 48 hours of co-culture, the cells were treated with 0.25% trypsin-EDTA at 37°C for 5 minutes, washed with PBS, and then the cells were directly stained with fluorescently conjugated antibodies CD11C, CD80, CD86, HLA-ABC, HLA-DR and analyzed by flow cytometry.

[0241] In vitro killing assay CD19 target Approximately 1×10 4 of H460 cells and 1×10 4 of H460-CD19 cells (target cells) were plated in each well of a 48-well plate in 200 μl of RPMI1640 complete medium, and 2×10 4 of WT-DCs or CARDF-DCs (stimulating cells) in 100 μl of RPMI1640 medium were added to the corresponding wells, and 10 5CAR-T cells (effector cells) were added to the corresponding wells. Subsequently, any missing wells were supplemented with culture medium up to 400 μl. After incubation for 24 hours, the remaining cells were collected for flow cytometry, and the culture supernatant was collected for subsequent assays. For each well, the specific cell lysis percentage was calculated as follows: Specific lysis % = (%CD19 (tumor cells only) - %CD19 (killed cells)) / CD19% (tumor cells only) × 100%.

[0242] EphA2 target Approximately 2 x 10 4 A549 cells or 2 × 10 4 A549-CP cells (target cells) were plated in 200 μl of RPMI1640 complete medium in each well of a 48-well plate, and 2 × 10⁶ cells were added to 100 μl of RPMI1640 medium. 4 Add the pseudo-DC or CAR-DC (stimulated cells) to the corresponding well and add 1 × 10⁶ cells in 100 μl of RPMI1640 medium. 5 CAR-T cells (effector cells) were added to the corresponding wells. Subsequently, any missing wells were supplemented with culture medium up to 400 μl. After incubation for 12 hours and 24 hours, the remaining cells were collected for flow cytometry, and the culture supernatant was collected for subsequent assays.

[0243] IFN-γ staining After an in vitro cytotoxicity assay of A549-CP tumor cells, the remaining cells were collected and stained using an intracellular staining kit (BD Biosciences) according to the manufacturer's instructions. Briefly, the cells were fixed and permeabilized in 200 μl of fixation / permeabilization buffer on ice for 20 minutes, and then washed twice with 1× wash buffer. The cells were stained with IFN-γ-BV650, CD3-V450, and CD8-PE in wash buffer at 4°C for 30 minutes, then washed twice with 1× wash buffer, and subsequently analyzed by flow cytometry.

[0244] IFN-γ and LDH assays The culture supernatant from the in vitro cytotoxicity assay was collected and tested for cytokine IFN-γ levels by an ELISA kit (Invitrogen catalog number 88-7316-76) and for LDH levels by the CytoTox96® non-radioactive cytotoxicity assay (Promega catalog number G1780), according to the manufacturer's instructions. The supernatant was diluted to 1:50 or 1:100 according to preliminary experiments.

[0245] Tumor xenograft model in NSG mouse research 1.5 × 10 in 100 μl of PBS 6 A549WT tumor models and A549-CP tumor models were generated by subcutaneous injection of cells into both flanks of 6-week-old NSG mice. In the experiment, T cells and DCs were divided into 500 μl of PBS at a rate of 5 × 10⁶ 6 DC and 1×10 7 CAR-T cells were injected intravenously 5 and 14 days after tumor exposure. Tumor volume was determined by caliper measurement and calculated using the following formula: Volume (mm³) 3 ) = 1 / 2 × D × d², where D is the longer tumor axis and d is the shorter tumor axis. Mice were euthanized, all tumors were collected, weighed, and photographed. In addition, mouse spleens and blood were collected, isolated, processed into single cells, stained with the indicated fluorescent dye conjugated antibody, and analyzed by flow cytometry.

[0246] Tumor xenograft model in Hu-mouse studies A detailed description of Hu-mouse generation can be found, for example, in Rong Z et al., An Effective Approach to Prevent Immune Rejection of Human ESC-Derived Allografts. Cell Stem Cell 14, 121-130 (2014). Hu-mouse-derived DCs were differentiated from bone marrow cells according to a published protocol. Briefly, Hu-mouse femurs and tibias were removed with sterile scissors, immersed in 70% alcohol for 3 minutes, and washed twice with ice-cold PBS. Bone marrow cells were then flushed using a sterile syringe (26-gauge needle). The bone marrow cells were resuspended, passed through a 70 μm nylon mesh, and the erythrocytes were lysed in Lyse buffer (BD Bioscience). The remaining cells were washed twice with PBS, counted, and 1 × 10⁶ cells were lysed in complete RPMI-1640 medium supplemented with 20 ng / ml human GM-CSF and 5 ng / ml human IL-4. 6 The cell suspension was adjusted to cells / ml. 3 ml of the cell suspension was transferred to each well of a 6-well plate. The medium was replaced every two days by gently rotating the plate, aspirating half of the medium, and adding fresh medium containing GM-CSF and IL-4. After 9 days of culture, cells were collected, washed, stained with anti-human CD11C antibody, and analyzed by flow cytometry. For CARDF transduction, immature BM-DCs were added to each well (50 × 10⁶ cells) before transduction. 5 ~10×10 5 Cells were transferred to a 6-well tissue culture plate at a cell / ml differentiation medium density (RPMI1640 complete medium supplemented with 20 ng / ml GM-CSF and 5 ng / ml IL-4). Transduction was performed using an MOI of 100 by thawing titrated lentivirus stocks at 37°C. In the differentiation medium, the virus concentrate was mixed with 6 μg / ml protamine sulfate. After incubation at 37°C for 12 hours, an additional 1 ml of differentiation medium was added to each well. 24 hours after transduction, most of the medium was aspirated, the cells were washed twice with PBS, and further cultured in the differentiation medium until ready for use.

[0247] T cells derived from Hu-mouse were isolated from spleen cells. Briefly, the spleen of Hu-mouse was removed with sterile forceps, immersed in ice-cold PBS for 3 minutes, and then ground on a 70 μm nylon mesh surface using the bottom of a syringe. Single cells were flushed through the mesh and washed with PBS. Red blood cells were then lysed. T cells were separated by anti-human CD3 magnetic microbeads and then maintained in RPMI1640 complete medium supplemented with 100 U / ml human IL-2. CAR-T cells were prepared as described above.

[0248] 1.5 × 10 in 100 μl of PBS supplemented with Matrigel 6 A549 cells were subcutaneously inoculated into both flanks of Hu- mice. Eight days later, tumor-bearing Hu- mice were randomly assigned to four cohorts. In the experiment, 3 × 10⁶ cells in 400 μl of PBS were administered. 6 DC and 1×10 7 DCs and T cells were injected via tail vein injection of CAR-T cells. 2 × 10⁶ cells in 100 μl of PBS supplemented with Matrigel. 6 H460 cells were subcutaneously inoculated into both flanks of Hu- mice. After 13 days, tumor-bearing Hu- mice were randomly assigned to four cohorts. In the experiment, 3 × 10⁶ cells in 400 μl of PBS were administered. 6 DC and 1×10 7 DCs and T cells were injected via tail vein injection of CAR-T cells. Tumor volume was determined by caliper measurement and calculated using the following formula: Volume (mm³) 3 ) = 1 / 2 × D × d², where D is the longer tumor axis and d is the shorter tumor axis. When mice were euthanized, tumors, spleens, bone marrow, and blood were collected for analysis.

[0249] Digestion and staining of tumor tissue The harvested pair of tumors, transplanted into one Hu-mouse, were mixed, cut into patches, and dissociated using a tissue digestive enzyme solution (100 Kunitz units of DNase I (STEM CELL catalog no. 07900), 8 Unsch units (8 U / mL) of Liberase™™ (Sigma catalog no. LIBTM-RO), and Liberase™ TH (Sigma catalog no. LIBTH-RO) (8 U / mL) in medium 199 (GIBCO) containing 20 μM HEPES (GIBCO). After shaking at 37°C and 150 rpm for 1.5 hours, digestion was stopped by adding 5 mL of RPMI-1640 containing 10% FBS. Subsequently, the suspension was filtered through a 40 μm cell strainer (Corning), and the obtained cells were subjected to antibody staining for flow cytometry analysis.

[0250] Flow cytometry analysis All flow cytometry analyses were performed using LSR Fortessa (BD Biosciences). Flow data was analyzed using FlowJo software (Tree Star, Ashland, Ohio). Appropriate sample gate processing is provided in the detailed data figures. Fluorescent dye-conjugated antibodies APC-CD45, PE-CD11C, FITC-CD80, BV605-CD86, PE-cy7-CD83, APC-HLA-ABC, BV510-HLA-DR, V450-CD3, PE-cy7-CD3, BV421-TIM-3, PE-PD-1, PE-CD8, BV650-IFNγ, BV421-IFNγ, FITC-PDL1, Percp-cy5.5-CD19, PE-cy5-streptavidin, and APC-streptavidin were purchased from BD Sciences; FITC-TIM-3 was purchased from Miltenyi Biotech; Biotin-Protein L was purchased from GenScript; and PE-EphA2 was purchased from BioLegend. For the dendritic cell staining assay, FcR blocking reagent (Miltenyi Biotech) was used according to the manufacturer's instructions. For surface marker staining, cells were centrifuged and stained with diluted antibody in FACS buffer (PBS + 1% FBS + 2 mM EDTA) at 4°C for 30 minutes, according to the manufacturer's instructions. The cells were then washed twice with PBS and immediately analyzed by flow cytometry. Protein L staining required secondary antibody staining, according to the manufacturer's instructions. See Figure 10A for antibody details.

[0251] statistical analysis Statistical analysis was performed using appropriate statistical comparisons, including independent two-tailed t-tests with Welch correction, one-way ANOVA with Tukey's multiple comparison test, and Tukey's multiple comparison test after two-way ANOVA, as required by Prism7 (GraphPad Software). Data are presented as mean ± SD. P ≤ 0.05 was considered statistically significant.

[0252] Example 9 Supplementary material Materials and methods: THP-1 cell transduction and differentiation into dendritic cells. THP-1 cells were divided into 5 × 10⁶ cells per well before transduction. 5 Cells were transferred to 24-well tissue culture plates at a density of 400 μL / 400 μL RPMI1640 complete medium. Transduction was performed using an MOI of 10 by thawing titrated lentivirus stock at 37°C. An appropriate volume of virus concentrate was mixed with 6 μg / ml protamine sulfate in RPMI1640 complete medium to achieve a total volume of 500 μL per well. After incubation at 37°C for 12 hours, an additional 500 μL of medium was added to each well. 24 hours after transduction, most of the culture medium was aspirated, cells were washed twice with PBS, and further cultured. On day 3, cells were collected and transduction efficiency was analyzed by flow cytometry.

[0253] Collect THP-1 or CAR+ THP-1 cells and measure 2 × 10⁻⁶ 5 The cells were resuspended in RPMI1640 complete medium at a density of cells / ml, and then 3 ml of each cell suspension was transferred to one well of a 6-well plate. Recombinant human GM-CSF (100 ng / ml) and recombinant human IL-4 (100 ng / ml) were added to the culture medium to stimulate DC differentiation. Every 2 or 3 days, the culture medium was replaced with fresh cytokine-supplemented medium. DC differentiation in the presence of cytokines continued for at least 7–10 days before further experiments.

[0254] lentivirus production Plasmid DNA for lentiviral packaging was purified using the NucleoBond Xtra Midi EF kit (Takara Bio catalog number 740420.50) according to the manufacturer's instructions. After slight modifications, PEI packaging was performed according to Addgene's lentiviral production protocol. Briefly, 293FT packaging cells were plated in a 15cm dish at a 1:3 dilution ratio. The following day, when the colony density reached 90%, the medium was changed 1 hour before transfection. Two packaging plasmids, psPAX2 (Addgene catalog number 12260) and pMD2.G (Addgene catalog number 12259), along with the target plasmids, were diluted in Opti-MEM (Gibco) containing 1 mg / ml PEI at a DNA:PEI ratio of 1:3 to 1:4. After incubation at room temperature for 20 minutes, the plasmid mixture was gently added to the cells, and 8 hours after transfection, the medium was replaced with complete DMEM medium. Following the manufacturer's instructions, lentivirus particles were collected 48–72 hours after transfection using a Lenti-X concentrator (Takara Bio catalog number 631232). Briefly, the collected medium was centrifuged at 1500 g for 15 minutes, and the supernatant was incubated overnight at 4°C with 1 / 3 volume of Lenti-X Concentrator. After centrifugation at 3000 rpm at 4°C for 45 minutes, the virus pellet was resuspended in 0.6–0.8 ml of cold PBS, aliquoted, and stored at -80°C.

[0255] Quantitative PCR analysis As previously described, total RNA was extracted from cells or tumor tissue using Trizol reagent (TaKaRa). Following the manufacturer's instructions, cDNA was synthesized from 1 μg of total RNA using the PrimeScript RT reagent kit (TaKaRa catalog number RR047A). Following the manufacturer's instructions, real-time PCR analysis was performed using the StepOnePlus real-time PCR system (Applied Biosystems) and the Roche system (Lifescience) together with TB Green reagent (TaKaRa catalog number RR820A). The primer sequences are shown in Figure 10B.

[0256] Table 1 Sequences referenced in this disclosure

[0257] [Table 1-1]

[0258] [Table 1-2]

Claims

1. A polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (1) an extracellular antigen-binding domain, (2) a transmembrane domain, and (3) an intracellular signaling domain, and the CAR is capable of activating dendritic cells in an immunosuppressive tumor microenvironment.

2. The polynucleotide according to claim 1, wherein the immunosuppressive tumor microenvironment comprises tumor and / or tumor-infiltrating immune cells that 1) express immunoinhibitory molecules and / or 2) have insufficient immunostimulatory cytokines.

3. The polynucleotide according to claim 2, wherein the immunoinhibitory molecule is selected from the group consisting of PD-1, TIM-3, TIGIT, LAG-3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155), and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), HLA class I, sialycoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47.

4. The polynucleotide according to claim 3, wherein the immunoinhibitory molecule is CTLA-4 and / or PD-L1.

5. The polynucleotide according to claim 2, wherein the immunostimulatory cytokine is selected from TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, granulocyte-macrophage colony-stimulating factor, and combinations thereof.

6. The polynucleotide according to claim 2, wherein the tumor comprises cells expressing CTLA4-Ig and / or PD-L1.

7. The polynucleotide according to claim 1, wherein the immunosuppressive tumor microenvironment comprises a tumor having poor responsiveness to adoptive cell therapy monotherapy (e.g., CAR-T monotherapy).

8. The polynucleotide according to claim 1, wherein the intracellular signaling domain comprises the cytoplasmic domain of a dendritic cell activation receptor selected from the group consisting of RIG-1, NLRP10, DEC-205, BDCA-2, CD86, 4-1BBL, OX40L, CD40, IFNAR, TLR4, TNFR (e.g., TNFR2), CD80, CD40L, CD367 (DCIR), CD207 (Langerin), CD371 (DCAL-2, CLEC12a), CD204, CD36, IFNγR, Dectin-1, and FcγR, or a combination thereof.

9. The polynucleotide according to claim 1, wherein the intracellular signaling domain comprises the cytoplasmic domain of Dectin-1 and the cytoplasmic domain of FcγR.

10. The polynucleotide according to claim 9, wherein the cytoplasmic domain of Dectin-1 and the cytoplasmic domain of FcγR are linked in tandem.

11. The polynucleotide according to claim 10, wherein the cytoplasmic domain of dectin-1 comprises the amino acid sequence shown in SEQ ID NO: 1, or any functional form thereof.

12. The polynucleotide according to claim 10 or 11, wherein the cytoplasmic domain of FcγR comprises the amino acid sequence shown in SEQ ID NO: 2, or any functional form thereof.

13. The polynucleotide according to any one of claims 1 to 12, wherein the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 3, or any functional form thereof.

14. The polynucleotide according to any one of claims 1 to 13, wherein the intracellular signaling domain comprises an amino acid sequence encoded by the nucleic acid sequence shown in Sequence ID No. 4, or any functional form thereof.

15. The polynucleotide according to any one of claims 1 to 14, wherein the extracellular antigen-binding domain comprises a single-stranded variable fragment (scFv).

16. The polynucleotide according to claim 15, wherein the scFv is specific to a tumor surface marker (e.g., a solid tumor surface marker).

17. The polynucleotide according to claim 16, wherein the tumor surface marker is selected from the group consisting of EphA2, CD19, CD70, CD133, CD147, CD171, DLL3, EGFRvIII, mesothelin, ganglioside GD2, FAP (fibroblast-activating protein), FBP (folate-binding protein), Lewis Y, claudin 18.2, IL13Rα2, HER2, MDC1, PMSA (prostatic membrane-specific antigen), ROR1, B7-H3, CAIX, CD133, CD171, CEA, GPC3, MUC1, and NKG2D.

18. The polynucleotide according to any one of claims 1 to 17, wherein the CAR further comprises a signal peptide.

19. The polynucleotide according to claim 18, wherein the signal peptide comprises the CD8α signal peptide.

20. The polynucleotide according to claim 19, wherein the signal peptide of CD8α comprises the sequence shown in Sequence ID No. 5, or any functional form thereof.

21. The polynucleotide according to any one of claims 1 to 20, wherein the transmembrane domain includes the transmembrane domain of CD8α.

22. The polynucleotide according to claim 21, wherein the transmembrane domain of CD8α includes the sequence shown in SEQ ID NO: 6, or any functional form thereof.

23. The polynucleotide according to any one of claims 1 to 22, wherein the extracellular antigen-binding domain is linked to the transmembrane domain by a hinge region.

24. The polynucleotide according to claim 23, wherein the hinge region includes the hinge region of CD8α.

25. The polynucleotide according to claim 24, wherein the hinge region of CD8α includes the sequence shown in Sequence ID No. 7, or any functional form thereof.

26. A polynucleotide according to any one of claims 1 to 25, which is DNA or RNA.

27. A polypeptide encoded by a polynucleotide according to any one of claims 1 to 26.

28. A vector comprising a polynucleotide according to any one of claims 1 to 26, wherein the polynucleotide encoding the CAR is operably linked to at least one control polynucleotide element for the expression of the CAR.

29. The vector according to claim 28, wherein the vector is a plasmid vector, a viral vector, a transposon, a site-directed insertion vector, or a suicide expression vector.

30. The vector according to claim 29, wherein the viral vector is a lentiviral vector, a retroviral vector, or an AAV vector.

31. The vector according to claim 30, wherein the viral vector is a lentiviral vector.

32. A modified cell comprising the polypeptide described in claim 27.

33. The manipulated cell according to claim 32, wherein the manipulated cell is a dendritic cell or a dendritic cell or precursor cell thereof.

34. The manipulated cell according to claim 32 or 33, wherein the dendritic cell or its precursor or progenitor cell is derived from peripheral blood cells, bone marrow cells, embryonic stem cells, or induced pluripotent stem cells.

35. A method for producing the manipulated cells according to any one of claims 32 to 34, comprising introducing the vector according to any one of claims 28 to 31 into starting cells under conditions suitable for the expression of the polynucleotide according to any one of claims 1 to 26.

36. The method according to claim 35, wherein the starting cell is a dendritic cell or a precursor or progenitor cell thereof.

37. The method according to claim 36, wherein the dendritic cells or their precursors or progenitor cells are derived from peripheral blood cells, bone marrow cells, embryonic stem cells, or induced pluripotent stem cells.

38. A population of cells produced in ex vivo by the method described in any one of claims 35 to 37.

39. The cell population according to claim 38, wherein at least 70% of the cell population expresses the polypeptide described in claim 27 at a detectable level.

40. (i) a polynucleotide according to any one of claims 1 to 26, or a polypeptide according to claim 27, or a vector according to any one of claims 28 to 31, or a population of manipulated cells according to any one of claims 32 to 34, or a population of cells according to claim 38 or 39, and (ii) a pharmaceutically acceptable medium.

41. A method for improving the effectiveness of adoptive cell therapy in treating cancer in a subject in need of treatment, comprising administering a therapeutically effective amount of the pharmaceutical composition described in claim 40.

42. The method according to claim 41, wherein the adoptive cell therapy includes adoptive transfer of modified immune cells.

43. The method according to claim 41 or 42, wherein the pharmaceutical composition further comprises a population of modified immune cells.

44. The method according to claim 41 or 42, further comprising administering a pharmaceutical composition containing a population of modified immune cells.

45. The method according to any one of claims 42 to 44, wherein the modified immune cells express synthetic receptors (e.g., CAR or TCR) on their cell surface.

46. The method according to any one of claims 42 to 45, wherein the immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, eosinophils, or neutrophils.

47. The method according to claim 46, wherein the immune cells are T cells selected from the group consisting of CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γδ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes.

48. The method according to any one of claims 42 to 47, wherein the immune cells are self or of the same species.

49. The method according to any one of claims 41 to 48, wherein the cancer is a solid tumor selected from the group consisting of adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.

50. The method according to any one of claims 41 to 48, wherein the cancer is a hematological malignancy selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, HHV8-associated primary exudative lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocytocyte-rich B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Waldenström macroglobulinemia, and multiple myeloma (MM).

51. A method for inducing the proliferation of immune cells, extending the survival of immune cells, and / or increasing the expression and / or secretion of immunostimulatory cytokines from immune cells in an immunosuppressive microenvironment, the method comprising contacting the immunosuppressive microenvironment with manipulated cells according to any one of claims 32 to 34.

52. The method according to claim 51, wherein the immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, eosinophils, or neutrophils.

53. The method according to claim 52, wherein the immune cells are T cells selected from the group consisting of CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γδ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes.

54. The method according to any one of claims 51 to 53, wherein the immune cells are self or of the same species.

55. The method according to any one of claims 51 to 54, wherein the immunosuppressive microenvironment is an immunosuppressive tumor microenvironment.

56. The method according to claim 55, wherein the immunosuppressive tumor microenvironment comprises tumor and / or tumor-infiltrating immune cells that express immunosuppressive molecules.

57. The method according to claim 56, wherein the immunoinhibitory molecule is selected from the group consisting of PD-1, TIM-3, TIGIT, LAG-3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155), and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), sialycoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47.

58. The method according to claim 57, wherein the immunoinhibitory molecule is CTLA-4 and / or PD-L1.

59. The method according to claim 56, wherein the tumor comprises cells expressing CTLA4-Ig and / or PD-L1.

60. The method according to any one of claims 51 to 59, wherein the immunostimulatory cytokine is one or more of TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and granulocyte-macrophage colony-stimulating factor.

61. A method for treating a disease or pathological condition in a subject requiring treatment, comprising administering a therapeutically effective amount of the pharmaceutical composition described in claim 40.

62. The method according to claim 61, further comprising administering a second active substance.

63. The method according to claim 62, wherein the second therapy is a population of modified immune cells.

64. The method according to claim 63, wherein the second therapy is CAR-T therapy.

65. The method according to claim 61, wherein the disease includes cancer.

66. A method for selecting a CAR capable of activating dendritic cells, (a) Prepare a non-human animal containing an immunosuppressive tumor microenvironment, (b) administering dendritic cells expressing candidate CARs to the non-human animals, (c) To detect markers related to dendritic cell activation selected from improvements in infiltration into the immunosuppressive tumor microenvironment, improved survival rate, and enhanced function in inducing immune cell activation, compared to reference dendritic cells, (d) Selecting the candidate CAR as a CAR capable of activating dendritic cells, Methods that include...

67. The method according to claim 66, wherein the immunosuppressive tumor microenvironment is clinically relevant.

68. The method according to claim 66 or 67, wherein the non-human animal comprises a human embryonic spleen and autologous human hematopoietic stem cells (e.g., human CD34+ hematopoietic stem cells).

69. The method according to claim 66, wherein the immunosuppressive tumor microenvironment comprises tumor and / or tumor-infiltrating immune cells that express immunosuppressive molecules.

70. The method according to claim 69, wherein the immunoinhibitory molecule is selected from the group consisting of PD-1, TIM-3, TIGIT, LAG-3, A2AR, BTLA (CD272), CTLA-4 (CD152), IDO1, IDO2, TDO, NOX2, VISTA, SIGLEC7 (CD328), PVR (CD155), and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), HLA class I, sialycoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47.

71. The method according to claim 70, wherein the immunoinhibitory molecule is CTLA-4 and / or PD-L1.

72. The method according to claim 69, wherein the tumor comprises cells expressing CTLA4-Ig and / or PD-L1.

73. The method according to any one of claims 66 to 72, wherein the immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, eosinophils, or neutrophils.

74. The method according to claim 73, wherein the immune cells are T cells selected from the group consisting of CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γδ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes.

75. The method according to any one of claims 66 to 74, wherein the immune cells are self or of the same species.

76. The method according to any one of claims 66 to 75, wherein the immune cells are modified immune cells (e.g., CAR-T cells) or innate immune cells.

77. The method according to claim 76, wherein the modified immune cells (e.g., CAR-T cells) are administered in combination with the dendritic cells expressing the candidate CAR.

78. The method according to claim 66 or 77, wherein the non-human animal is a rodent, such as a rat or a mouse.

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