Dendritic cell tumor vaccine and its use

Abstract

The present disclosure provides a dendritic cell tumor vaccine comprising a chimeric antigen receptor for activating dendritic cells and a tumor antigen. The present disclosure also provides compositions and methods of making the dendritic cell tumor vaccine, as well as methods of treating cancer using the dendritic cell tumor vaccine.

Description

[Technical Field]

[0001] This disclosure generally relates to the field of cancer treatment. In particular, this disclosure relates to dendritic cell tumor vaccines, compositions of dendritic cell tumor vaccines and methods for producing them, and methods for using dendritic cell tumor vaccines to treat cancer. [Background technology]

[0002] Dendritic cells (DCs) play a crucial role in the immune system as an important link between innate and adaptive immunity (Non-Patent Literature 1 and Non-Patent Literature 2). DCs are major antigen-presenting cells (APCs) that activate T-cell-dependent immunity, and are particularly effective in inducing tumor-specific immune responses (Non-Patent Literature 3). In recent years, dendritic cell adoptive cell therapy has made significant progress and has become an important method of tumor immunotherapy.

[0003] Oncology vaccines are vaccines that use tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs) to induce a specific antitumor effect in the body through active immunity, stimulating the body's own immune defense mechanisms to achieve the treatment or prevention of tumor recurrence. Oncology vaccines can be divided into therapeutic vaccines and prophylactic vaccines depending on their purpose. Oncology vaccines can be divided into protein peptide vaccines, gene vaccines, viral vaccines, and dendritic cell vaccines (DC vaccines) depending on their components and manufacturing methods. Provenge™ (Sipuleucel-T) dendritic cell vaccine is the world's first and only therapeutic cancer vaccine approved by the US FDA. Currently, most DC vaccines in clinical trials are induced and differentiated in vitro from monocytes in the peripheral blood of patients. These are loaded with TAAs or TSAs using pulsed protein peptides or gene transfer, and then reinjected into the patient to stimulate tumor-specific T cells that can kill tumor cells.

[0004] Recent studies have revealed that tumor-infiltrating dendritic cells (TIDCs) typically exhibit immature or dysfunctional phenotypes in the immunosuppressive tumor microenvironment or tumor immunosuppressive microenvironment (TIME), suppressing T cell infiltration and activation (Non-Patent Literature 4). As a result, DC vaccines face limitations in stimulating tumor-specific T cells in TIME. Therefore, there is a need to develop novel DC vaccines that enhance infiltration and activation effects in TIME. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] RM 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] JM Tran Janco et al., Tumor-infiltrating dendritic cells in cancer pathogenesis. J. Immunol. 194, 2985-2991 (2015) [Overview of the project]

[0006] In one aspect, the present disclosure provides a composition comprising one or more vectors. In some embodiments, the one or more vectors comprise (a) a first polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating dendritic cells, wherein the CAR comprises (1) an extracellular antigen binding domain, (2) a transmembrane domain and (3) an intracellular signaling domain, and (b) a second polynucleotide encoding a tumor antigen.

[0007] In some embodiments, when expressed in dendritic cells, the CAR and / or the tumor antigen is capable of activating dendritic cells within an immunosuppressive tumor microenvironment. In some embodiments, the immunosuppressive tumor microenvironment comprises tumors and / or tumor-infiltrating immune cells that 1) express an immune inhibitory molecule, and / or 2) have an insufficiency of immunostimulatory cytokines. In some embodiments, the immune inhibitory 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), SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), HLA class I, sialoglycoproteins, CD112, CD113, galectin-9, CD24, and CD47. In some embodiments, the immune inhibitory molecule is CTLA-4 and / or PD-L1. In some embodiments, 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.

[0008] In some embodiments, the intracellular signaling domain comprises a cytoplasmic domain of a dendritic cell activating 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 combinations thereof. In some embodiments, the intracellular signaling domain comprises the cytoplasmic domain of dectin-1 and the cytoplasmic domain of FcγR. In some embodiments, the cytoplasmic domain of dectin-1 comprises the amino acid sequence set forth in SEQ ID NO: 1, or any functional variant thereof. In some embodiments, the cytoplasmic domain of FcγR comprises the amino acid sequence set forth in SEQ ID NO: 2, or any functional variant thereof. In some embodiments, the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 3, or any functional variant thereof. In some embodiments, the intracellular signaling domain comprises the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 4, or any functional variant thereof.

[0009] In some embodiments, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv is specific for a tumor surface marker. In some embodiments, the tumor surface marker is selected from the group consisting of EphA2, CD19, CD70, CD133, CD147, CD171, DLL3, EGFRvIII, mesothelin, ganglioside GD2, FAP (fibroblast activation protein), FBP (folate binding protein), Lewis Y, claudin 18.2, IL13Rα2, HER2, MDC1, PMSA (prostate membrane specific antigen), ROR1, B7-H3, CAIX, CD133, CD171, CEA, GPC3, MUC1, and NKG2D.

[0010] In some embodiments, 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 shown in SEQ ID NO: 5, or any functional form thereof.

[0011] In some embodiments, the transmembrane domain includes the transmembrane domain of CD8α. In some embodiments, the transmembrane domain of CD8α includes the sequence shown in Sequence ID No. 6, or any functional form thereof.

[0012] In some embodiments, the extracellular antigen-binding domain is linked to the transmembrane domain by a hinge region. In some embodiments, the hinge region includes the hinge region of CD8α. In some embodiments, the hinge region of CD8α includes the sequence shown in Sequence ID No. 7, or any functional form thereof.

[0013] In some embodiments, the tumor antigen is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). In some embodiments, the tumor antigen is encoded by a mutant oncogene or a fragment thereof. In some embodiments, the oncogene is selected from p53, ras, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, and TGF-βRII. In some embodiments, the tumor antigen is selected from CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride ion channel 2, cyclin-B1, 9D7, Ep-Cam, EphA3, GPC3, Her2 / neu, telomerase, mesothelin, SAP-2, and surviving. In some embodiments, the tumor antigen is selected from the p53R273H mutant peptide, the KRAS G12V mutant peptide, and the KRAS G12C mutant peptide. In some embodiments, the p53 R273H mutant peptide has the sequence shown in SEQ ID NO: 29, the KRAS G12V mutant peptide has the sequence shown in SEQ ID NO: 31, and the KRAS G12C mutant peptide has the sequence shown in SEQ ID NO: 30.

[0014] In some embodiments, the tumor antigen is linked to a DC-LAMP sorting signal. In some embodiments, the DC-LAMP sorting signal has the sequence shown in SEQ ID NO: 32, or any functional form thereof.

[0015] In some embodiments, one or more vectors are DNA vectors or RNA vectors.

[0016] In some embodiments, the first polynucleotide and / or the second polynucleotide are operably linked to at least one regulatory polynucleotide element for the expression of CAR and / or tumor antigen.

[0017] In some embodiments, the first polynucleotide and the second polynucleotide are contained in a single vector. In some embodiments, the first polynucleotide is functionally linked to the second polynucleotide via an IRES. In some embodiments, the IRES has the sequence shown in SEQ ID NO: 36, or any functional form thereof.

[0018] In some embodiments, the vector is a plasmid vector, a viral vector, a transposon, a site-directed insertion vector, or a suicide expression vector. In some embodiments, the viral vector is a lentiviral vector, a retroviral vector, or an AAV vector. In some embodiments, the viral vector is a lentiviral vector.

[0019] In another embodiment, the Disclosure provides engineered cells comprising one or more vectors disclosed herein. In some embodiments, the engineered cells are dendritic cells or their precursors or progenitor cells. In some embodiments, 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.

[0020] In yet another embodiment, the present invention provides a method for producing manipulated cells, comprising introducing one or more vectors disclosed herein into starting cells under conditions suitable for the expression of CARs and tumor antigens. In some embodiments, the starting cells are dendritic cells or their precursors or progenitor cells. In some embodiments, 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.

[0021] In another embodiment, the disclosure provides a population of cells produced ex vivo by a method for producing manipulated cells disclosed herein. In some embodiments, at least 60% of the cell population expresses detectable levels of CAR polypeptide.

[0022] In another embodiment, the Disclosure provides a pharmaceutical composition comprising (i) one or more vectors disclosed herein, or a population of manipulated cells disclosed herein, or a population of cells disclosed herein, and (ii) a pharmaceutically acceptable medium.

[0023] In another embodiment, the Disclosure provides a method for treating cancer in a subject requiring treatment for cancer. In some embodiments, the method comprises administering a pharmaceutical composition disclosed herein to a subject. In some embodiments, the subject has tumor cells containing a mutated gene.

[0024] In some embodiments, 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.

[0025] 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).

[0026] In some embodiments, a method for treating cancer further includes administering a population of modified immune cells to a subject. In some embodiments, the modified immune cells have the expression of synthetic receptors (e.g., CAR or TCR) on their cell surface. In some embodiments, the immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, eosinophils, or neutrophils. In some 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 some embodiments, the immune cells are autologous or allogeneic.

[0027] 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. In some embodiments, the method involves bringing an immunosuppressive microenvironment into contact with the manipulated cells disclosed herein. In some embodiments, the immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, eosinophils, or neutrophils. In some 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 some embodiments, the immune cells are autologous or allogeneic.

[0028] In some embodiments, the immunosuppressive microenvironment is an immunosuppressive tumor microenvironment.

[0029] 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]

[0030] [Figure 1-1] Figures 1A to 1J show the expression of chimeric antigen receptors (CARDFs) on the surface of cells transduced with lentiviral vectors for co-expression of CARDFs and tumor antigens. Figure 1A shows the structural diagram of the lentiviral vector. Figure 1B shows the expression of CARDFs on the cell surface detected by flow cytometry after transduction of lentivirus into 293FT cells. [Figure 1-2] Figure 1C shows the sequencing results of the p53 gene in H460 tumor cells. Figure 1D shows the sequencing results of the p53 gene in SW480 tumor cells. Figure 1E shows the sequencing results of the KRAS gene in SW837 tumor cells. Figure 1F shows the sequencing results of the KRAS gene in SW480 tumor cells. [Figure 1-3] Figure 1G shows the expression level of EphA2 on the surface of H460 tumor cells. Figure 1H shows the expression level of EphA2 on the surface of SW480 tumor cells. Figure 1I shows the expression level of EphA2 on the surface of SW837 tumor cells. [Figure 1-4] Figure 1J shows the mRNA levels of the p53R273H mutant polypeptide expressed in H460 tumor cells overexpressing p53R273H (hereinafter referred to as H460-p53R273Hov). [Figure 2-1] Figures 2A to 2D show the expression of CARDF-p53R273H in human dendritic cells (DCs) differentiated from humanized mouse myeloid cells and in transduced DCs. Figure 2A shows DCs differentiated from humanized mouse myeloid cells. Figure 2B shows the mRNA levels of p53R273H expressed in DCs. [Figure 2-2]Figure 2C shows dendritic cells (DCs) differentiated from humanized mouse myeloid cells, and the expression of CARDF-KRAS G12C and KRAS G12V in the DCs. Figure 2D shows the mRNA levels of KRAS G12C and KRAS G12V expressed in the DCs. [Figure 3-1] Figures 3A to 3G show tumor growth in a humanized mouse (Hu-mouse) tumor model after treatment with a dendritic cell tumor vaccine (hereinafter referred to as CARDF-DC vaccine). Figure 3A shows a schematic diagram of the treatment process using the p53R273H vaccine. Figure 3B shows a schematic diagram of the treatment process using the KRAS G12C vaccine and the KRAS G12C vaccine. Figure 3C shows the growth curves of H460 tumor tissue during treatment in different treatment groups using the p53R273H vaccine. [Figure 3-2] Figure 3D shows the growth curves of H460-p53R273Hov (overexpressing R273H) tumor tissue during treatment in different treatment groups using the p53R273H vaccine. Figure 3E shows the growth curves of SW480 tumor tissue during treatment in different treatment groups using the p53R273H vaccine. [Figure 3-3] Figure 3F shows the growth curves of SW480 tumor tissue during treatment in different treatment groups using the KRAS vaccine. Figure 3G shows the growth curves of SW837 tumor tissue during treatment in different treatment groups using the KRAS vaccine. [Figure 3-4] Figure 3H shows the growth curves of SK-BR-3 tumor tissue (expressing the p53 R175H mutant) treated in different treatment groups using the CAR-DC vaccine combo (p53-R175H-R248Q-R249S-R273H vaccine). Figure 3I shows the growth curves of SW480 tumor tissue (expressing the p53 R273H mutant) treated in different treatment groups using the CAR-DC vaccine combo. Figure 3J shows the growth curves of OVCAR3 tumor tissue (expressing the p53 R248Q mutant) treated in different treatment groups using the CAR-DC vaccine combo. [Figure 4-1]Figures 4A to 4H show the flow cytometry analysis results of humanized mouse (Hu-mouse) tumor models after treatment with dendritic cell tumor vaccines. Figure 4A shows the proportion of T cells in the spleen of different treatment groups with the p53R273H vaccine. Figure 4B shows the proportion of PD-1+ T cells in the spleen of different treatment groups with the p53R273H vaccine, and also shows the proportion of T cells in the spleen of different treatment groups with the p53R273H vaccine. [Figure 4-2] Figure 4C shows the proportion of T cells in the spleen of different KRAS vaccine treatment groups. Figure 4D shows the proportion of DC cells in the spleen of different KRAS vaccine treatment groups. [Figure 4-3] Figure 4E shows the mean fluorescence intensity of CD80 expression in spleen dendritic cells (DCs) from different p53R273H vaccine treatment groups. Figure 4F shows the percentage of B cells in peripheral blood from different p53R273H vaccine treatment groups. [Figure 4-4] Figure 4G shows the proportion of macrophages in peripheral blood of different treatment groups with the p53R273H vaccine. Figure 4H shows the proportion of B cells in peripheral blood of different treatment groups with the KRAS vaccine. [Figure 5-1] Figures 5A to 5D show the results of tumor mRNA analysis in humanized mouse (Hu-mouse) tumor models after treatment with dendritic cell tumor vaccines. Figure 5A shows the TNF-α gene mRNA expression levels in sw480 tumor tissue from different treatment groups with the p53R273H vaccine. Figure 5B shows the CARDF scFv gene mRNA expression levels in sw480 tumor tissue from different treatment groups with the p53R273H vaccine. [Figure 5-2] Figure 5C shows the CD3 gene mRNA expression levels in SW480 tumor tissue from different KRAS vaccine treatment groups. Figure 5D shows the TNF-α gene mRNA expression levels in SW837 tumor tissue from different KRAS vaccine treatment groups. Figure 5E shows the primer sequences used in this disclosure. [Modes for carrying out the invention]

[0031] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described and is therefore naturally modifiable. It should also be understood that 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] As used herein, the singular forms "a," "an," and "the" include multiple references unless otherwise explicitly indicated by the context.

[0037] 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.

[0038] In all cases of this application where there is a set of enumerated numbers, it is understood that any of the enumerated numbers may be the upper or lower limit of a numerical range. It is further understood that the present invention includes all such numerical ranges, i.e., ranges having combinations of upper and lower numerical limits, where each of the upper and lower limit numbers may be any of the numbers enumerated herein. A range presented 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, non-integer values. Similarly, a range delimited by “at least” is understood to include the lower limit and all higher numerical values ​​as presented.

[0039] 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.

[0040] In this specification, the terms “CAR” or “CARDF” are to be used interchangeably with the term “chimeric antigen receptor” and refer 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 a ζ signal. 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).

[0041] 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.

[0042] 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.

[0043] As used herein, the term “neoantigen” refers to an antigen having at least one modification that distinguishes it from the corresponding wild-type parent antigen. Modifications may occur, for example, through mutations in tumor cells or tumor-cell-specific post-translational modifications. Mutations may include changes in splice sites, splice variants, frameshifts or non-frameshift indels, genomic rearrangements or gene fusions, missense or nonsense substitutions, or any genomic or expression changes that result in neoORFs. Post-translational modifications may include abnormal phosphorylation and splice antigens generated by proteasomes. Neoantigens may include polypeptide sequences or nucleotide sequences.

[0044] 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.

[0045] 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.

[0046] "Self" cells refer to any cells derived from the same subject that are later reintroduced.

[0047] "Allogeneic" cells refer to any cells derived from different subjects of the same species.

[0048] In the context of immune cells, the term "effector cell" refers to a cell that is activated in response to a stimulus and capable of performing effector functions. Effector cells may, without limitation, include NK cells, cytotoxic T cells, and helper T cells.

[0049] "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.

[0050] 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.

[0051] 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.

[0052] The term "functional form," as used herein, refers to a different form of a parent molecule (e.g., variants, fragments, fusions, derivatives, and mimics) 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 variant forms and unnaturally occurring forms, such as those obtained by recombinant or chemical synthesis. Functional forms may contain unnatural amino acid residues.

[0053] 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 being correctly positioned and oriented relative to the coding sequence to regulate transcription.

[0054] 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)).

[0055] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. These terms also apply 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.

[0056] As used herein, the term "single-chain variable fragment" is used without distinction from 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)).

[0057] As used herein, the term "TCR" may be used interchangeably with the terms "T cell receptor" or "TCR complex" and refers to either 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 the recognition of antigen fragments as peptides bound to MHC molecules.

[0058] 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 phages or M13 phages, 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.

[0059] When used herein, the term “host cell” refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0060] 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.

[0061] 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.

[0062] As used herein, the terms “to treat” or “treatment” include preventing or reducing a condition, slowing the onset or progression of a condition, reducing the risk of progression of a condition, preventing or delaying the progression of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing complete or partial regression of a condition, curing a condition, or any combination thereof.

[0063] Dendritic cell (DC) tumor vaccine This disclosure provides a novel DC vaccine platform capable of stimulating immune responses even under immunosuppressive conditions. For example, the DC vaccine platform provided herein exhibits improved antitumor activity compared to conventional DC vaccines, particularly against solid tumors commonly associated with a tumor immunosuppressive microenvironment. The DC vaccine platform provided herein can harbor one or more (e.g., one, two, three, four, five, or more) of various known or unknown tumor antigens or non-tumor antigens. Therefore, the DC vaccine platform provided herein can be used to treat a wide range of diseases, such as cancer or infectious diseases with specific mutations. The DC vaccine platform can also be used to improve the efficacy of personalized neoantigen vaccines.

[0064] While most conventional DC vaccines need to be used in combination with other cancer therapies to achieve the desired therapeutic effect, the DC vaccine platform provided herein can be used as a monotherapy to treat tumors and can even be used to treat solid tumors that are commonly associated with a tumor immunosuppressive microenvironment. This makes the DC vaccine platform provided herein a promising cancer therapy for treating a variety of cancers, particularly solid tumors, that are known to be difficult to treat with conventional cancer therapies such as CAR-T monotherapy or DC vaccine monotherapy.

[0065] In one embodiment, the disclosure provides a dendritic cell (DC) tumor vaccine that can stimulate tumor-specific T cells in an immunosuppressive tumor microenvironment or tumor immunosuppressive microenvironment (TIME). In one embodiment, the DC tumor vaccine comprises dendritic cells (DCs) and dendritic cells expressing a chimeric antigen receptor (CAR or CARDF) that can activate tumor antigens, or its precursor or progenitor cells.

[0066] Tumor immunosuppressive microenvironment The terms "immunosuppressive tumor microenvironment" and "tumor immunosuppressive microenvironment (TIME)" can be used interchangeably 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., Tumor microenvironment: Sanctuary of the devil. Cancer Lett. 368, 7-13 (2015)).

[0067] 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” with respect to immunoinhibitory molecules mean that the immunoinhibitory molecule is expressed at a level at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than the reference level. The term “reference level” with respect to the expression of immunoinhibitory molecules refers to the level of expression of immunoinhibitory molecules in tumors formed by wild-type tumor cells (e.g., wild-type A549 cells) in immunodeficient animal models (e.g., NSG mice).

[0068] "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.

[0069] 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.).

[0070] 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 “expressing” or “expressing” CTLA4-Ig mean expressing CTLA4-Ig at a level at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than a 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 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% 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).

[0071] 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.

[0072] 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 efficacy of the therapy (e.g., CAR-T therapy) compared to a control therapy known to be ineffective (e.g., a better therapeutic efficacy of less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, or less than 2%).

[0073] Dendritic cell-activated chimeric antigen receptor 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), 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-8, IL-10, IL-12). This can be indicated by the expression (and / or secretion) of 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.

[0074] 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) compared to a reference state (e.g., inactivated state) of the 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).

[0075] 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.

[0076] (1) Extracellular antigen-binding domain The extracellular antigen-binding domain can be any domain capable of recognizing and binding to a marker specifically expressed on a target cell, thereby delivering the CAR-DC vaccine provided herein, including the DC-activated CAR, in close proximity to the target cell. In certain embodiments, the extracellular antigen-binding domain is an antigen such as a tumor marker expressed on the surface of cancer cells (i.e., a tumor surface marker), or an antigen expressed on infected cells. The design of extracellular antigen-binding domains is well known in the art. In certain embodiments, the extracellular antigen-binding domain is the antigen-binding domain of an antibody. In some embodiments, the antigen-binding domain includes a human or humanized antibody or an antibody fragment thereof. The term “human antibody” refers to an antibody whose entire molecule is of human origin, or an antibody consisting of the same amino acid sequence as a human type of antibody or immunoglobulin. The term “humanized antibody” refers to an antibody containing 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 genetically modified to express antibodies derived from human heavy chain and / or light chain coding genes with antigens of interest.

[0077] 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, 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. scFv may be specific to tumor surface markers, such as solid tumor surface markers. As used herein, the term “tumor surface marker” refers to a molecule differentially expressed on the surface of tumor cells that is used as a target for recognizing tumor cells. 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 highly 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.

[0078] In some embodiments, scFv is specific to EphA2. 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 conserved 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%, 90% identical to SEQ ID NO: 11 while retaining substantial biological activity of SEQ ID NO: 11. 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 therefor, or any functional form thereof, and CDR3 comprises 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 therefor, 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 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 Sequence ID No. 15, 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 the substantial biological activity of Sequence ID No. 15.

[0079] 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.

[0080] 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 sequence number 16 and a VL containing the sequence shown in sequence number 17.

[0081] 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: 27 (GGGGSGGGGSGGGGS).

[0082] In some embodiments, scFv comprises the amino acid sequence shown in SEQ ID NO: 18.

[0083] Those skilled in the art will recognize that, depending on the disease of interest, appropriate extracellular antigen-binding domains specific to any disease marker may be selected, taking into account the current knowledge of identified markers for a variety of diseases, such as cancer, infectious diseases, or immunological diseases, to construct the CARs provided herein. The variety of disease markers include, but are not limited to, those described above.

[0084] (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.

[0085] In one embodiment, the CAR described herein comprises a 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.

[0086] 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.

[0087] In some embodiments, the CAR described herein further includes a hinge region that forms a linkage 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.

[0088] 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.

[0089] In some embodiments, the hinge region includes a CD8α hinge region, a human immunoglobulin (Ig) hinge region, or a glycine-serine rich sequence.

[0090] 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.

[0091] (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 an immune receptor tyrosine-based activation motif (ITAM) in their cytoplasmic domain, which triggers 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.

[0092] 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.

[0093] 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.

[0094] 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 the 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 thereto, or any functional form thereof.

[0095] 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 thereto, or any functional form thereof.

[0096] 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.

[0097] 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.

[0098] (4) Co-stimulatory signaling domain In some embodiments, the intracellular signaling domain further comprises a co-stimulatory signaling domain.

[0099] In some embodiments, the co-stimulatory signaling domain is derived from the intracellular domain of the co-stimulatory molecule.

[0100] 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 (Tacti le), CD100(SEMA4D), CD103, CD127, CD137(4-1BB), CD150(SLAM, SLAMF1, IPO-3), CD160(BY55), C D162 (SELPLG), CD226 (DNAM1), CD229 (Ly9), CD244 (SLAMF4, 2B4), CEACAM1, CRTAM, CDS, OX40, PD-1 Examples include ICOS, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, LAT, LFA-1, 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 ability, and any combination thereof.

[0101] In some embodiments, the CAR co-stimulatory signaling domain described herein comprises an intracellular domain of the co-stimulatory molecule CD137(4-1BB), CD28, OX40, or ICOS. In some embodiments, the CAR co-stimulatory signaling domain described herein has the sequence of SEQ ID NO: 58, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 58.

[0102] 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.

[0103] Tumor antigen Tumor antigens are antigenic substances produced within tumor cells; that is, tumor antigens trigger an immune response in the host. Normal proteins in the body are non-antigenic due to self-tolerance, which is the process by which cytotoxic T lymphocytes (CTLs) and B lymphocytes that produce autoantibodies are "centrally" recruited in primary lymphoid tissues (BM) and "peripherally" recruited in secondary lymphoid tissues (mainly the thymus for T cells and mainly the spleen / lymph nodes for B cells). Therefore, any protein not exposed to the immune system will trigger an immune response. This may include normal proteins that are sufficiently isolated from the immune system, proteins that are usually produced in very small amounts, proteins that are usually produced only at specific developmental stages, or proteins whose structure has been altered by mutation.

[0104] Tumor antigens can be broadly classified into two categories based on their expression patterns. Tumor-specific antigens (TSAs) are present only in tumor cells and not in any other cells, while tumor-associated antigens (TAAs) are present in some tumor cells and some normal cells. More precisely, tumor antigens include products of mutated oncogenes and tumor suppressor genes, products of other mutated genes, overexpressed or abnormally expressed cellular proteins, tumor antigens produced by oncoviruses, carcinoembryonic antigens, altered cell surface glycolipids and glycoproteins, and cell type-specific differentiation antigens.

[0105] Any protein produced within tumor cells that has an abnormal structure due to mutation may act as a tumor antigen. Such abnormal proteins are produced by mutations in the relevant genes. Since mutations in oncogenes and tumor suppressor factors that lead to the production of abnormal proteins are the cause of tumors, such abnormal proteins are called tumor-specific antigens. Examples of tumor-specific antigens include abnormal products of the ras gene and the p53 gene. In contrast, mutations in other genes unrelated to tumorigenesis may lead to the synthesis of abnormal proteins that are tumor-associated antigens. Therefore, in some embodiments, the tumor antigens described herein are peptides or polypeptides, or fragments thereof, encoded by mutated genes, i.e., oncogenes. In some embodiments, the oncogene is selected from p53, ras, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, and TGF-βRII.

[0106] In human tumors, p53 and RAS are the most frequently mutated genes. More than 50% of highly invasive tumors have p53 mutations. p53 mutations may prompt the body to produce cytotoxic T cells specific to the mutation site, making them ideal targets for tumor vaccines. Among the various types of p53 mutations, R175H, R248W, and R273H are the three most common hotspot mutations. Other mutations, though not limited to them, include R248Q and R249S. Of KRAS gene mutations, 97% are mutations at the 12th or 13th amino acid. The most important are G12D, G12C, G12V, and G13D. Structural studies have shown that most of these gene mutations impair the GTP hydrolysis ability of KRAS.

[0107] Other examples of tumor antigens include tissue differentiation antigens, mutant protein antigens, oncoviral antigens, oncotesticular antigens, and vascular-specific or stromal-specific antigens. Tissue differentiation antigens are antigens specific to certain types of tissue. Mutant protein antigens may be highly specific to cancer cells because they should not be present in normal cells. Normal cells present normal protein antigens on MHC molecules, while cancer cells present mutant versions. Several viral proteins are thought to be involved in cancer formation (tumor formation), and some viral antigens are also cancer antigens. Oncotesticular antigens are antigens mainly expressed in germ cells of the testes, but are also expressed in the fetal ovaries and trophoblasts. Some cancer cells abnormally express these proteins, presenting these antigens and enabling attack by T cells specific to these antigens. Examples of this type of antigen include CTAG1B and MAGEA1.

[0108] Proteins that are normally produced in very small amounts but whose production dramatically increases in tumor cells can trigger an immune response. One example of such a protein is tyrosinase, an enzyme necessary for melanin production. Normally, only small amounts of tyrosinase are produced, but in melanoma cells, the levels are extremely high.

[0109] Carcinoembryonic antigens (CARs) are another important class of tumor antigens. Examples include alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA). These proteins are typically produced in the early stages of embryonic development and disappear by the time the immune system is fully developed. Therefore, self-tolerance to these antigens does not develop.

[0110] Cells infected with tumor viruses, such as EBV and HPV, also produce abnormal proteins. These virus-infected cells contain latent viral DNA, which is transcribed, and the resulting proteins trigger an immune response.

[0111] Accordingly, in some embodiments, the tumor antigens used in the DC vaccines disclosed herein are selected from CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride ion channel 2, cyclin B1, 9D7, Ep-Cam, EphA3, GPC3, Her2 / neu, telomerase, mesothelin, SAP-2, and surviving.

[0112] In addition to proteins, other substances such as glycolipids and glycoproteins on the cell surface may also have abnormal structures within tumor cells and can become targets for the immune system.

[0113] p53 The oncoprotein p53, also known as p53, cellular tumor antigen p53, phosphorylated protein p53, tumor suppressor p53, antigen NY-CO-13, or transformation-associated protein 53 (TRP53), is an isoform of a protein encoded by homologous genes in various organisms, such as TP53 (human) and Trp53 (mouse). This homolog (originally thought to be a single protein, and often referred to as such) is extremely important for multicellular organisms and functions as a tumor suppressor to prevent cancer formation. Therefore, p53 has been described as a "guardian of the genome" because it plays a role in maintaining stability by preventing genomic mutations. Consequently, TP53 is classified as a tumor suppressor gene.

[0114] The name p53 was given in 1979 to represent its apparent molecular mass, and SDS-PAGE analysis has shown it to be a 53 kilodalton (kDa) protein. However, the actual mass of the full-length p53 protein (p53α), based on the sum of the amino acid residue masses, is only 43.7 kDa. This difference arises because of the large number of proline residues in the protein, which slows its movement on SDS-PAGE and therefore makes it appear heavier than it actually is. In addition to the full-length protein, the human TP53 gene encodes at least 15 different protein isoforms ranging in size from 3.5 kDa to 43.7 kDa. All of these p53 proteins are called p53 isoforms. The TP53 gene is the most frequently mutated gene (over 50%) in human cancers, and it has been shown to play a crucial role in preventing cancer formation. The TP53 gene encodes a protein that binds to DNA, regulates gene expression, and prevents genomic mutations.

[0115] In humans, the TP53 gene is located on the short arm of chromosome 17 (17p13.1). This gene is 20kb long and contains a non-coding exon 1 and a very long initial intron of 10kb. The coding sequence contains five regions that show high conservation in vertebrates, primarily in exons 2, 5, 6, 7, and 8, although sequences found in invertebrates are only slightly similar to mammalian TP53. TP53 orthologs have been identified in most mammals for which complete genomic data are available.

[0116] In humans, a common polymorphism involves the substitution of arginine with proline at codon position 72. Many studies have investigated the genetic association between this variation and cancer susceptibility, but the results have been controversial. For example, a 2009 meta-analysis found no association with cervical cancer. A 2011 study found that TP53 proline mutations significantly impact the risk of pancreatic cancer in men. A study of Arab women found that homozygosity of proline at TP53 codon 72 was associated with a reduced risk of breast cancer. One study suggested that the TP53 codon 72 polymorphism, MDM2 SNP309, and A2164G may be collectively associated with non-oropharyngeal cancer susceptibility, and that the combination of MDM2 SNP309 with TP53 codon 72 may accelerate the development of non-oropharyngeal cancer in women. A 2011 study found that the TP53 codon 72 polymorphism was associated with an increased risk of lung cancer.

[0117] A 2011 meta-analysis found no significant association between the TP53 codon 72 polymorphism and the risk of colorectal cancer or endometrial cancer. A 2011 study of a Brazilian birth cohort found an association between unmutated arginine TP53 and individuals with no family history of cancer. Another 2011 study found that the p53 homozygous (Pro / Pro) genotype was associated with a significantly increased risk of renal cell carcinoma.

[0118] KRAS KRAS (Kirsten rat sarcoma virus), or K-Ras, is a protein in the RAS / MAPK pathway that relays signals from outside the cell to the cell nucleus. These signals instruct cells to grow and divide (proliferate) or to mature and perform specialized functions (differentiation). The K-Ras protein is a GTPase, converting a molecule called GTP into another molecule called GDP. In this way, the K-Ras protein functions like a switch that is turned on and off by GTP and GDP molecules. To transmit signals, it needs to attach to (bind to) a GTP molecule to be turned on. The K-Ras protein is turned off (inactivated) when it converts GTP to GDP. When the protein binds to GDP, it stops relaying signals to the cell nucleus. It is called KRAS because it was first identified as an oncogene of Kirsten rat sarcoma virus. Viral oncogenes are derived from the cell genome. Therefore, the KRAS gene in the cell genome is called a proto-oncogene.

[0119] The gene product of KRAS was initially discovered as p21 GTPase. Like other members of the ras subfamily, the KRAS protein is a GTPase and plays an early role in many signaling pathways. KRAS is normally tethered to the cell membrane due to the presence of an isoprene group at its C-terminus. Mammalian cells have two protein products of the KRAS gene, K-Ras4A and K-Ras4B, resulting from the use of alternative exon 4s (exon 4A and exon 4B, respectively). These proteins have different structures in their C-terminal region and use different mechanisms to localize to the cell membrane, including the plasma membrane.

[0120] Single amino acid substitutions, particularly single nucleotide substitutions, in the KRAS molecule can lead to activating mutations. The resulting transformed proteins are thought to be involved in a variety of malignancies, including lung adenocarcinoma, myxoid adenoma, pancreatic ductal carcinoma, and colorectal cancer. Several germline KRAS mutations have been found to be associated with Noonan syndrome and cardiac-facial-cutaneous syndrome. Somatic KRAS mutations are frequently observed in leukemia, colorectal cancer, pancreatic cancer, and lung cancer.

[0121] MUC1 Cell surface-associated mucin 1 (MUC1), also known as polymorphic epithelial mucin (PEM) or epithelial membrane antigen (EMA), is a mucin encoded by the MUC1 gene in humans. MUC1 is a glycoprotein with extensive O-linked glycosylation in its extracellular domain. Mucins cover the apical surface of epithelial cells in the lungs, stomach, intestines, eyes, and several other organs. Mucins protect the body from infection by preventing pathogens from reaching the cell surface by binding to oligosaccharides in their extracellular domain. Overexpression of MUC1 is often associated with colon cancer, breast cancer, ovarian cancer, lung cancer, and pancreatic cancer. Joyce Taylor-Papadimitriou identified and characterized this antigen during her research on breast and ovarian tumors. MUC1 is a member of the mucin family and encodes a membrane-bound glycosylated phosphorylated protein. MUC1 has a core protein mass of 120kDa to 225kDa, which increases to 250kDa to 500kDa upon glycosylation. MUC1 extends beyond 200nm to 500nm from the cell surface.

[0122] This protein is anchored to the apical surface of many epithelial cells by a transmembrane domain. Across the transmembrane domain is a SEA domain containing a cleavage site for releasing a large extracellular domain. Mucin release is mediated by schedase. The extracellular domain contains a 20-amino acid variable-number tandem repeat (VNTR) domain, with the number of repeats varying from 20 to 120 depending on the individual. These repeats are rich in serine, threonine, and proline residues, enabling severe O-glycosylation.

[0123] Several alternative splicing transcription variants encoding different isoforms of this gene have been reported, but the full-length properties of only some of them have been determined.

[0124] In the endoplasmic reticulum, MUC1 is cleaved into two parts: a cytoplasmic tail containing a transmembrane domain and an extracellular domain. These domains are tightly bound together in a non-covalent manner. This strong non-covalent bond is not broken by treatment with urea, low pH, high salinity, or boiling. Treatment with sodium dodecyl sulfate causes dissociation of the subunits. The cytoplasmic tail of MUC1 is 72 amino acids long and contains several phosphorylation sites.

[0188] This protein binds to pathogens to perform a defense function and also functions in cell signaling.

[0125] Overexpression, abnormal intracellular localization, and altered glycosylation of this protein are associated with carcinoma; for example, the CanAg tumor antigen is a novel glycoform of MUC1. In the cell nucleus, the protein MUC1 regulates the activity of a transcription factor complex that has been shown to play a role in tumor-induced host immune changes.

[0126] PSMA Glutamate carboxypeptidase II (GCPII), a prostate-specific membrane antigen (PSMA), is also known as N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I) or NAAG peptidase, and is an enzyme encoded in humans by the FOLH1 (folate hydrolase 1) gene. Human PSMA contains 750 amino acids and weighs approximately 84 kDa. PSMA is a membrane-based zinc metalloenzyme. The majority of the enzyme resides in the extracellular space. PSMA is a class II membrane glycoprotein. As shown in the reaction diagram on the right, it catalyzes the hydrolysis of N-acetylaspartylglutamate (NAAG) to glutamate and N-acetylaspartate (NAA).

[0127] Neuroscientists primarily use the term NAALADase in their research, while scientists studying folate metabolism use folate hydrolase, and scientists studying prostate cancer or oncology use PSMA; all of these refer to the same protein.

[0128] PSMA is primarily expressed in four tissues of the body, including the prostatic epithelium, the proximal tubules of the kidneys, the jejunal brush border of the small intestine, and the ganglia of the nervous system.

[0129] In fact, the first cloning of the cDNA encoding the gene expressing PSMA was achieved using RNA from the prostate tumor cell line LNCaP. PSMA is homologous to the transferrin receptor and undergoes endocytosis, but a ligand for inducing internal translocation has not been identified. PSMA was found to be the same as a membrane protein in the small intestine that plays a role in removing gamma-binding glutamate from polygamma-glutamate folate. This releases folate, which is then transported into the body and made available as a vitamin. As a result, the cloned genome of PSMA was named FOLH1, representing the folate hydrolase.

[0130] The three extracellular domains of PSMA (protease, apical, and C-terminal domains) work together in substrate recognition. The protease domain is a central seven-stranded mixed β-sheet. Ten α-helices are adjacent to the β-sheet. The apical domain is located between the first and second strands of the central β-sheet of the protease domain. The apical domain forms a pocket that facilitates substrate binding. The C-terminal domain is an up-down-up-down bundle of four helices.

[0131] The central pocket is approximately 2 nanometers deep and opens from the extracellular space to the active site. This active site contains two zinc ions, each acting as a ligand for oxygen or phosphate of 2-PMPA during inhibition. PSMA also has a single calcium ion coordinated to a site distant from the active site. It has been proposed that this calcium ion binds the protease domain to the apical domain. Furthermore, human PSMA has 10 potential glycosylation sites, many of which (including those far from the catalytic domain) affect PSMA's NAAG hydrolysis ability.

[0132] The FOLH1 gene has multiple potential start sites and splice forms, resulting in differences in the structure, localization, and carboxypeptidase activity of the membrane protein based on the parental tissue.

[0133] Human PSMA is highly expressed in the prostate gland, approximately 100 times more than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, increasing 8 to 12 times compared to levels in non-cancerous prostate cells. Due to this high expression, PSMA is being developed as a potential biomarker for the treatment and imaging of several cancers. In human prostate cancer, tumors with higher expression are associated with shorter time to progression and a higher percentage of patients experiencing recurrence. In vitro studies using prostate cancer and breast cancer cell lines with reduced PSMA levels have shown significant reductions in cell proliferation, migration, invasion, adhesion, and survival.

[0134] PSMA is a target for several nuclear medicine imaging agents for prostate cancer. Capromab pentide (marketed as PROSTASCINT) is conjugated to indium-111 for detection by gamma camera. Second-generation antibodies and low molecular weight ligands for imaging and therapeutic purposes are under development. PSMA can also be used experimentally to target therapeutics. Lutetium-177 is a beta-ray emitter that conjugates to PSMA and is delivered to prostate tumors to treat them. In addition to human prostate and prostate cancer, PSMA is highly expressed in the neovascularization of tumors but not in the corresponding normal vascular systems of all types of solid tumors, including kidney, breast, and colon.

[0135] S100P S100 calcium-binding protein P (S100P) is a protein encoded in humans by the S100P gene. The protein encoded by this gene is a member of the S100 protein family, which contains two EF-hand calcium-binding motifs. S100 proteins are localized in the cytoplasm and / or nucleus of various cells and are involved in regulating many cellular processes, such as cell cycle progression and differentiation. The S100 gene contains at least 13 members and is located as a cluster on chromosome 1q21, but this gene is located on 4p16. This protein is Ca 2+ In addition to bonding, Zn 2+ and Mg 2+ It also binds to [another protein]. This protein may be involved in the pathogenesis of prostate cancer. S100P has been shown to interact with EZR and RAGE. The interaction between S100P and RAGE is inhibited by chromolin and pentamidine.

[0136] Neoantigen Neoantigens can be identified using conventional techniques known in the art, such as whole-genome / exome sequencing and RNA sequencing. In particular, neoantigens can be identified by sequencing each patient's tumor DNA and normal DNA to identify tumor-specific mutations and determine the patient's HLA allotype. The tumor-specific neoantigens and their congener native antigens are then subjected to bioinformatics analysis using validated algorithms to predict which tumor-specific mutations will create epitopes that can bind to the patient's HLA allotype (e.g., which tumor-specific mutations will create epitopes that bind to the patient's HLA allotype more effectively than congener native antigens). A detailed description of neoantigen identification for cancer treatment can be found, for example, in U.S. Patent Application Publication 2016 / 0101170, the disclosure of which is incorporated herein by reference in its entirety. Based on this analysis, polynucleotides encoding one or more peptides corresponding to subsets of these mutations can be loaded into the CAR-DC vaccine platform provided herein to form personalized cancer vaccines.

[0137] vector In another embodiment, the Disclosure provides one or more vectors comprising a first polynucleotide encoding a CAR as described herein and a second polynucleotide encoding a tumor antigen as described herein. The polynucleotides encoding the CAR and / or tumor antigen 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.

[0138] In some embodiments, the vector is an expression DNA vector (e.g., plasmid, virus). When an expression DNA vector is transiently introduced into cells, CAR mRNA is transcribed in the host cells. Since the DNA vector and mRNA are diluted with cell division, CAR expression is not considered to be permanent. In one embodiment, the DNA vector may be introduced into cells as a form of transient CAR expression.

[0139] 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.

[0140] 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 transcribed RNA CAR may be introduced into cells in the form of transient expression.

[0141] 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.

[0142] 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 functionally 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., metallothonine 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.

[0143] 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.

[0144] 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.

[0145] 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."

[0146] 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.

[0147] How to produce DC tumor vaccine In another embodiment, this disclosure provides a method for producing the DC tumor vaccine described herein. Numerous means for generating CAR-T cells known in the art can also be applied to the production of the DC tumor vaccine. 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 involves introducing a vector containing a first polynucleotide encoding a CAR and a second polynucleotide encoding a tumor antigen into starting cells under conditions suitable for the expression of the CAR and tumor antigen. The method provided herein may include one or more steps selected from obtaining starting cells (i.e., cells from a source), culturing the starting cells (including expansion and optionally maturation), and genetically modifying the cells. The starting cells may be dendritic cells or their precursors or progenitor cells, as described above.

[0148] cell source The DC tumor vaccines provided herein may be obtained from any source. In certain embodiments, the DC tumor vaccines provided herein are derived from immune cells isolated from a subject, for example, a human subject. In some embodiments, the immune cells are obtained from a subject of interest, for example, 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, the immune cells are obtained from a cancer subject that is poorly responsive to immunotherapy, for example, CAR-T therapy.

[0149] 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.

[0150] 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.

[0151] 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).

[0152] 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.

[0153] Genetic modification Genetic modification of DCs or their precursors or progenitor cells can be achieved by transducing a substantially homogeneous population of DCs with the vectors provided herein. In certain embodiments, retroviral vectors (e.g., lentiviral vectors) are used to introduce polynucleotides 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 CARs and tumor antigens as disclosed herein may be carried out at the locus of any endogenous gene.

[0154] 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 can 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).

[0155] In certain embodiments, the DC tumor vaccines provided herein are prepared by transfecting DCs with a vector containing a polynucleotide encoding a CAR and a tumor antigen provided herein, prior to administration. In certain embodiments, the DC tumor vaccines provided herein may be prepared by transfecting DC precursor or progenitor cells with, for example, a viral vector, and then differentiating the transfected cells into DCs. The DC tumor vaccines provided herein exhibit improved expression of CAR and tumor antigen on the cell surface. The DC precursor 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 precursor or progenitor cells may also be embryonic stem cells or induced pluripotent stem cells (iPSCs).

[0156] In another embodiment, the Disclosure also provides cell populations produced ex vivo by the methods described above. In certain embodiments, 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 and tumor antigens provided herein at detectable levels. In certain embodiments, at least 85% of the cell population express the CAR polypeptides and tumor antigens provided herein at detectable levels.

[0157] Pharmaceutical composition In another embodiment, the Disclosure also provides a pharmaceutical composition comprising a population of DC tumor vaccines provided herein and a pharmaceutically acceptable medium. As used herein, the term “pharmaceutical composition” means a composition formulated for pharmaceutical use.

[0158] 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.

[0159] "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.

[0160] 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, a DC tumor vaccine or population thereof is 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.

[0161] Exemplary pharmaceutically acceptable media include sugars (e.g., lactose, glucose, and sucrose), starches (e.g., corn starch and potato starch), cellulose and its derivatives (e.g., sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate), tragacanth powder, malt, gelatin, lubricants (e.g., magnesium stearate, sodium lauryl sulfate, and talc), excipients (e.g., cocoa butter and suppository wax), and oils (e.g., peanut oil, cottonseed oil). This includes safflower oil, sesame oil, olive oil, corn oil, and soybean oil), glycols (e.g., propylene glycol), polyols (e.g., glycerin, sorbitol, mannitol, and polyethylene glycol (PEG)), esters (e.g., ethyl oleate and ethyl laurate), agar, buffers (e.g., magnesium hydroxide and aluminum hydroxide), alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, pH buffer solution, polyester, polycarbonate, polyanhydride, bulking agents (e.g., polypeptides and amino acids), serum alcohol (e.g., ethanol), (sterile) phosphate-buffered saline, Ringer's solution, dextrose solution, and other non-toxic compatible substances used in pharmaceutical formulations.

[0162] The pharmaceutical compositions provided herein may be administered systemically or directly to a subject in order to induce and / or enhance an immune response to an antigen, and / or to treat and / or prevent a neoplasm, pathogen infection, or infectious disease. 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 (e.g., the tumor vascular system).

[0163] The pharmaceutical compositions provided herein contain at least about 1 × 10 5 , about 2×10 5 , about 3×10 5 , about 4×10 5 Or approximately 5 x 10 5may comprise a population of DC tumor vaccines (i.e., engineered cells). A person skilled in the art can readily determine the percentage of the DC tumor vaccine provided herein in a population using various known methods, for example, fluorescence-activated cell sorting (FACS). Suitable ranges for the percentage (also referred to as "purity") of the DC tumor vaccine provided herein in the population may be about 50% to about 55%, about 55% to about 60%, 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%.

[0164] In certain specific embodiments, the recipient receives at least 1×10 3 cells / kg body weight, at least 5×10 3 cells / kg body weight, at least 1×10 4 cells / kg body weight, at least 5×10 4 cells / kg body weight, at least 1×10 5 cells / kg body weight, at least 5×10 5 cells / kg body weight, at least 1×10 6 cells / kg body weight, at least 5×10 6 cells / kg body weight, at least 1×10 7 cells / kg body weight, at least 5×10 7 cells / kg body weight, at least 1×10 8 cells / kg body weight, at least 2×10 8 cells / kg body weight, at least 3×10 8 cells / kg body weight, at least 4×10 8 cells / kg body weight, at least 5×10 8 cells / kg body weight, or at least 6×10 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 DC tumor vaccines provided herein to be administered in the manner of the present disclosure, as well as the amounts of optional additives, vehicles, media and / or carriers. Typically, additives, if any, are present 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.

[0165] 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, a DC tumor vaccine may be obtained by modifying starting cells derived from one subject and 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 the DC tumor vaccine provided herein and another active agent, such as 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 the DC tumor vaccine provided herein and another active agent, such as a chemotherapeutic agent, another type of immunotherapy (e.g., CAR-T therapy), or radiotherapy.

[0166] Usage This disclosure also provides diverse uses of the DC tumor vaccines provided herein.

[0167] General use In one embodiment, the Disclosure provides a method for treating cancer in a patient, comprising administering a therapeutically effective amount of a DC tumor vaccine provided herein to the patient. In some embodiments, a 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 and tumor antigens provided herein to produce a DC tumor vaccine; and returning and injecting the DC tumor vaccine into a subject. In some embodiments, a method for treating cancer comprises preparing DC precursor or progenitor cells (e.g., peripheral blood cells, bone marrow cells, embryonic stem cells, or iPSCs); manipulating the precursor or progenitor cells to express CARs and tumor antigens provided herein; differentiating the manipulated precursor or progenitor cells into DCs expressing CARs and tumor antigens provided herein; and returning and injecting the DCs expressing CARs and tumor antigens provided herein (e.g., a DC tumor vaccine) into a subject.

[0168] In some embodiments, 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).

[0169] In some embodiments, subjects with cancer are less responsive to cancer therapies (e.g., immunotherapy).

[0170] 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.

[0171] Examples of immunotherapies include, without limitation, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0172] 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, without limitation, 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.

[0173] 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.

[0174] Destruction of TIME In one embodiment, the Disclosure provides a method for disrupting TIME (e.g., converting TIME into an inflammatory state) using a DC tumor vaccine provided herein.

[0175] 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 contacting an immunosuppressive microenvironment with a DC tumor vaccine provided herein. 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. The 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 as the DC tumor vaccine provided herein (e.g., peripheral blood of the subject).

[0176] In certain embodiments, the immunosuppressive microenvironment is an immunosuppressive tumor microenvironment, which has been described above. 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 immunoinhibitory molecule is CTLA-4 and / or PD-L1. In certain embodiments, the tumor comprises cells expressing CTLA4-Ig and / or PD-L1.

[0177] Combination therapy In another embodiment, the present disclosure provides a combination therapy using a DC tumor vaccine provided herein and a second active agent.

[0178] 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 as the DC tumor vaccine provided herein (e.g., the peripheral blood of the subject). In certain embodiments, the ratio of the DC tumor vaccine to the CAR-T cells provided in the combination therapy is in the range of about 1:1 to 1:10.

[0179] In certain embodiments, the DC tumor vaccine and CAR-T cells provided herein are in the same pharmaceutical composition. In certain embodiments, the DC tumor vaccine and CAR-T cells provided herein are in two separate pharmaceutical compositions. In certain embodiments, the DC tumor vaccine provided herein is administered to a subject in need of treatment before, simultaneously with, or after administration of CAR-T cells.

[0180] 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.

[0181] 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.

[0182] 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).

[0183] In certain embodiments, the DC tumor vaccine provided herein is 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 DC tumor vaccine.

[0184] In some embodiments, the treatment further includes administering an agent that reduces or mitigates side effects associated with the administration of the DC tumor vaccine. 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]

[0185] 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 various modifications may be made to the type and details without departing from the spirit and scope of this disclosure as disclosed herein.

[0186] Example 1 This example demonstrates the construction of a lentiviral vector expressing both CAR and oncogene mutant peptides, and the expression of the vector in 293FT cells.

[0187] Construction of lentiviral vectors All sequences were optimized and synthesized by GUANGZHOU IGE BIOTECHNOLOGY. The CAR gene (SEQ ID NO: 28), the p53 R273H vaccine gene (SEQ ID NO: 33), the CAR-DC vaccine combo gene (SEQ ID NO: 38), the KRAS G12C vaccine gene (SEQ ID NO: 34), or the KRAS G12V vaccine gene (SEQ ID NO: 35) were cloned into a lentiCas9 (Addgene) vector and Cas9 was replaced. The structural diagram of the vector is shown in Figure 1A.

[0188] Preparation of lentiviruses All plasmid DNA used for lentivirus packaging was extracted and purified using the NucleoBond Xtra Midi EF kit (Takara Bio). Lentiviruses were produced according to the general usage instructions on the Addgene website and packaged using polyethyleneimine (PEI) (Sigma Aldrich). The day before packaging the viruses, 293FT cells (ATCC) were passaged in a 1:3 ratio and divided into 15 cm petri dishes. The following day, when cell confluence reached 90%, the viruses were packaged. The medium was changed to fresh medium one hour before transfection. Two packaging plasmids, pSPAX2 (Addgene, catalog no. 12260) and pMD2.G (Addgene, catalog no. 12259), the target lentiviral vector, and 1 mg / ml PEI were diluted in Opti-MEM (purchased from Gibco) in a DNA:PEI ratio of 1:3 to 1:4. After incubation at room temperature for 20 minutes, the plasmid mixture was gently added dropwise to cell culture medium, and the medium was replaced with DMEM complete medium (Gibco) 8 hours after transfection. Lentivirus particles were collected 48 to 72 hours after transfection. The supernatant of the culture medium containing the virus was concentrated using Lenti-X virus concentrate (Takara Bio). The collected medium containing the virus particles was centrifuged at 1500 g for 15 minutes, and 1 / 3 of the Lenti-X virus concentrate was added to the separated supernatant. After mixing well, the mixture was left overnight at 4°C. The next day, the mixture was centrifuged at 4°C and 3000 rpm for 45 minutes. The virus particles at the bottom of the centrifuge tube were resuspended in 0.6 ml to 0.8 ml of pre-cooled PBS buffer, and stored in a refrigerator at -80°C in small portions for later use.

[0189] The results for lentivirus-infected 293FT cells are shown in Figure 1B. As shown in Figure 1B, after lentivirus infection, 93% of 293FT cells expressed CAR on the cell surface, as detected by protein L, demonstrating that a constructed lentivirus vector expressing both CAR and oncogene mutant peptides can effectively transduce cells and express the CAR structure on the cell surface after being packaged with a lentivirus.

[0190] As shown in Figures 1C and 1D, H460 cells have the wild-type p53 gene, while SW480 cells have a mutant p53 gene containing the G>A mutation, resulting in the R273H mutation in the p53 protein. Therefore, SW480 is a tumor cell line containing the p53R273H mutation.

[0191] As shown in Figures 1E and 1F, in SW837 cells, the nucleotide encoding the 12th amino acid residue of KRAS has a GGT>TGT mutation, and the encoded amino acid is mutated to G12C. In SW480 cells, the nucleotide encoding the 12th amino acid residue of KRAS has a GGT>GTT mutation, and the encoded amino acid is mutated to G12V. Therefore, SW480 is a tumor cell line with the KRAS G12V mutation, and SW837 is a tumor cell line with the KRAS G12C mutation.

[0192] As shown in Figures 1G to 1I, H460 cells, SW480 cells, and SW837 cells express high levels of EphA2 on their cell surface.

[0193] As shown in Figure 1J, the constructed H460-p53R273Hov cells expressed the p53R273H mutant polypeptide at high levels.

[0194] Example 2 This example demonstrates the preparation of DCs from humanized mouse bone marrow cells.

[0195] The femurs and tibias of humanized mice were removed with sterile scissors, immersed in 70% alcohol for 3 minutes, and rinsed twice with ice-cold PBS. PBS was then aspirated using a sterile syringe (26-gauge needle). Bone marrow cells were washed away by rinsing from one end of the bone marrow cavity. The bone marrow cells were dispersed by repeated aspiration using a 1 ml pipette tip and then filtered through a 70 μm nylon mesh. The filtered cells were collected and centrifuged. The red blood cells were then lysed with lysis buffer (BD Biosciences). The remaining cells were washed twice with PBS and counted. The cells were then divided into 1 × 10⁶ cells in differentiation medium (RPMI-1640 complete medium containing 20 ng / ml recombinant human GM-CSF and 5 ng / ml recombinant human IL-4). 6 The cells were cultured at / ml and replenished with fresh differentiation medium every two days.

[0196] On day 8 of differentiation, immature DCs were infected with lentivirus with an MOI of 100. A concentrated lentivirus stock solution of an appropriate titer was slowly thawed at 37°C. An appropriate amount of the virus stock solution was mixed with 6 μg / ml of protamine sulfate and added to the differentiation medium. After incubation at 37°C for 12 hours, 1 ml of differentiation medium was added to each well. 24 hours after transduction, cells were collected for centrifugation, the virus-containing medium was carefully discarded, the cells were washed twice with PBS, and further cultured in fresh differentiation medium until use on day 10.

[0197] As shown in Figure 2A and Tables 1 and 1A below, Hu-mouse bone marrow cells can be induced into human dendritic cells (DCs) with an efficiency of over 90%. After transduction, the efficiency of CARDF expression on the surface of CARDF-DCs and p53R273H vaccine cells was 82.1% and 75%, respectively. In CARDC vaccine combo experiments, the efficiency of CARDF expression on the surface of CARDF-DCs and CARDC vaccine combo cells was 56.5% and 63.4%, respectively.

[0198] [Table 1]

[0199] As shown in Figure 2B, qPCR analysis of the CARDF-DC vaccine and the p53R273H vaccine showed a significant increase in the mRNA expression level of p53R273H in the p53R273H vaccine.

[0200] As shown in Figure 2C and Table 2 below, Hu-mouse bone marrow cells can be induced into human dendritic cells (DCs) with an efficiency of over 80%. After transduction, the efficiency of CARDF expression on the surface of CARDF-DCs, KRAS G12C vaccine, and KRAS G12V vaccine was 46%, 68.2%, and 69.4%, respectively.

[0201] [Table 2]

[0202] As shown in Figure 2D, qPCR analysis of CARDF-DC, KRAS G12C vaccine, and KRAS G12V vaccine showed a significant increase in mRNA expression levels of the mutant peptide in the KRAS G12C vaccine and KRAS G12V vaccine.

[0203] The above data demonstrate that humanized mouse bone marrow cells can effectively differentiate into human DCs, that lentiviral transduction can enable high levels of CARDF expression on the surface of DCs, and that p53R273H can also be expressed at high levels in DCs.

[0204] Example 3 This example demonstrates the use of a DC tumor vaccine derived from humanized mouse bone marrow cells in the treatment of tumors in a Hu-mouse xenograft model.

[0205] 2 x 10 6 H460 cells, 2 × 10 6 individual H460-p53R273Hov cells, and 2 × 10⁶ 6 SW480 cells were resuspended in 100 μL of PBS and subcutaneously injected into both sides of the back of Hu-mice to create a xenograft Hu-mouse tumor model. The tumor-bearing Hu-mice were randomly divided into the following three groups: (1) Sham DC treatment group (2)CARDF-DC treatment group (3) The group was divided into two groups: one receiving the p53R273H vaccine and the other receiving treatment.

[0206] The treatment process is shown in Figure 3A.

[0207] 2 x 10 6 Individual SW480 cells and 2 × 10 6 SW837 cells were resuspended in 100 μL of PBS and subcutaneously injected into the backs of Hu-mice to create a xenograft Hu-mouse animal tumor model. The tumor-bearing Hu-mice were randomly divided into the following three groups: (1)CARDF-DC treatment group (2) KRAS G12C vaccine treatment group (3) The patients were divided into a KRAS G12V vaccine treatment group and a treatment group.

[0208] The treatment process is shown in Figure 3B.

[0209] The cell therapy drug was injected into mice via the tail vein, and the cells were resuspended in 400 μL of PBS. During treatment, the dose of the first injection shown in Figure 3A was 6 × 10⁶. 6 The mice are DC / mouse, and the dose for the second injection is 2 × 10⁻⁶ 6 The treatment was DC / mouse. Figure 3B shows the injection dose during treatment: 5 × 10 6 These were DC / mouse mice. During cell therapy, tumor size was measured every other day using calipers and statistically analyzed. After euthanasia of the mice, all tumors were collected, weighed, and photographed. Furthermore, the spleen, blood, and bone marrow of the mice were collected, isolated and processed into single cells, stained with fluorescently labeled flow cytometry antibodies, and analyzed by flow cytometry. RNA extracted from tumor tissue was analyzed by qPCR. The results are shown in Figures 3 to 5. The primer sequences are shown in Figure 5E.

[0210] As shown in Figure 3C, tumor tissue formed by H460 showed no significant difference in tumor tissue growth after different treatments. This suggests that CAR-DCs alone do not have antitumor activity, and that CAR-DC vaccines do not act on tumors if the antigen is not loaded onto the CAR-DC vaccine.

[0211] As shown in Figure 3D and Table 3, the growth of tumor tissue formed by H460-p53R273Hov was inhibited after treatment with the p53R273H vaccine. On day 15, the mean tumor volume of H460-p53R273Hov tumor tissue in the CARDF-DC treatment group was 1286.64 mm². 3 The mean tumor volume in the p53R273H vaccine treatment group was 591.789 mm². 3 That was the case.

[0212] [Table 3]

[0213] As shown in Figure 3E and Table 4, the growth of tumor tissue formed by SW480 was inhibited after treatment with the p53R273H vaccine. On day 15, the mean tumor volume of SW480 tumor tissue in the CARDF-DC treatment group was 1055.52 mm². 3 The mean tumor volume in the p53R273H vaccine treatment group was 342.587 mm². 3 That was the case.

[0214] [Table 4]

[0215] As shown in Figure 3F and Table 5, the growth of tumor tissue formed by SW480 was inhibited after treatment with the KRAS G12V vaccine. On day 15, the mean tumor volume of SW480 tumor tissue in the CARDF-DC treatment group was 723.254 mm². 3 The mean tumor volume of SW480 tumor tissue in the KRAS G12C treatment group was 542.616 mm². 3The mean tumor volume in the KRAS G12V vaccine treatment group was 312.747 mm². 3 That was the case.

[0216] [Table 5]

[0217] As shown in Figure 3G and Table 6, the growth of tumor tissue formed by SW837 was inhibited after treatment with the KRAS G12C vaccine. On day 8, the mean tumor volume of SW837 tumor tissue in the CARDF-DC treatment group was 167.727 mm². 3 The mean tumor volume of SW837 tumor tissue in the KRAS G12C treatment group was 41.3374 mm². 3 The mean tumor volume in the KRAS G12V vaccine treatment group was 122.71 mm². 3 That was the case.

[0218] [Table 6]

[0219] As shown in Figure 4A and Table 7, the percentage of T cells in the p53R273H vaccine treatment group (12.163%) was higher than in the CARDF-DC treatment group (4.786%) and the sham DC treatment group (6.024%), indicating that the DC vaccine stimulated T cell proliferation in the body.

[0220] [Table 7]

[0221] As shown in Figure 4B and Table 8, the percentage of PD-1+ T cells in the p53R273H vaccine treatment group (18.7%) was lower than in the sham DC treatment group (31.6%), indicating that the DC vaccine reversed T cell depletion in the tumor microenvironment.

[0222] [Table 8]

[0223] As shown in Figure 4C and Table 9, the percentage of T cells in the KRAS G12C vaccine treatment group (27.3%) and the KRAS G12V vaccine treatment group (17.825%) were higher than in the CARDF-DC treatment group (12.3%), indicating that the DC vaccine stimulated the proliferation of T cells in the body.

[0224] [Table 9]

[0225] As shown in Figure 4D and Table 10, the percentage of DC cells in the KRAS G12C vaccine treatment group (3.2425%) and the KRAS G12V vaccine treatment group (2.7%) were increased compared to the CARDF-DC treatment group (1.665%), indicating that the survival period of the DC vaccine in the body was extended.

[0226] [Table 10]

[0227] As shown in Figure 4E and Table 11, the mean fluorescence intensity of CD80 expression in DC cells in the CARDF-DC treatment group and the p53R273H vaccine treatment group was significantly increased compared to the sham DC treatment group, indicating that the presence of CARDF effectively activates DCs after contact with tumor targets.

[0228] [Table 11]

[0229] As shown in Figures 4F to 4H and Tables 12 to 14, there was no significant difference in the proportion of B cells and macrophages in the peripheral blood of the different treatment groups, indicating that there was no immune cell cytotoxicity response after DC vaccine treatment.

[0230] [Table 12]

[0231] [Table 13]

[0232] [Table 14]

[0233] As shown in Figure 5A and Table 15, the expression level of the TNF-α gene in SW480 tumor tissues was significantly increased in the CARDF-DC treatment group and the p53R273H vaccine treatment group compared with the mock DC treatment group, indicating that the presence of CARDF promotes the activation of DCs, thereby enhancing the expression of pro-inflammatory genes in tumors.

[0234] [Table 15]

[0235] As shown in Figure 5B and Table 16, compared with the mock DC treatment group and the CARDF-DC treatment group, the CARDF scFv gene expression in SW480 tumor tissues of the p53R273H vaccine treatment group was significantly increased, indicating that the infiltration of DC vaccines in tumor tissues was increased.

[0236] [Table 16]

[0237] As shown in Figure 5C and Table 17, the expression of the CD3 gene in SW480 tumor tissues was significantly increased in the KRAS G12V vaccine treatment group compared with the CARDF-DC treatment group and the KRAS G12C vaccine treatment group, indicating that T cell infiltration in SW480 tumor tissues was increased after KRAS G12V vaccine treatment.

[0238] [Table 17]

[0239] As shown in Figure 5D and Table 18, TNF-α gene expression in SW837 tumor tissue was significantly increased in the KRAS G12C vaccine treatment group compared to the CARDF-DC treatment group and the KRAS G12V vaccine treatment group, indicating increased T cell infiltration in SW837 tumor tissue after treatment with the KRAS G12C vaccine.

[0240] [Table 18]

[0241] The data above demonstrates that the DC vaccine stimulated the effector T cell response in Hu-mice, effectively suppressing the growth of tumor tissue expressing the mutant gene, without any immunocytotoxic side effects.

[0242] Example 4 This example demonstrates the use of DC tumor vaccines derived from humanized mouse myeloid cells in the treatment of diverse cancers with various mutations in a Hu-mouse xenograft model. A wide variety of tumor antigens can be loaded into a single CAR-DC vaccine combo. In this example, the CAR-DC vaccine combo contains P53 R175H ,P53 R248Q ,P53 R249S , and P53 R273H Multiple short peptides represented by [the formula] were loaded and combined to form a functional fusion.

[0243] 0.5 × 10 6 SK-BR-3 cells (breast cancer cells possessing the p53 R175H mutant), 1 × 10 6 1 x 10⁶ SW480 cells (colorectal cancer cells possessing the p53 R273H mutant), and 1 x 10⁶ 6OVCAR3 cells (ovarian cancer cells harboring the p53 R248Q mutant) were subcutaneously injected into Hu-mice to establish a xenograft Hu-mouse tumor model. Tumor-bearing Hu-mice were randomly divided into the following two groups, namely, (1) CARDF-DC treatment group, and (2) CARDC vaccine combination treatment group.

[0244] The treatment process is the same as that shown in Figure 3A.

[0245] 3×10 6 CARDF-DC cells or CARDF-DC vaccine cells were resuspended in 500 μL of DPBS and injected into mice via the tail vein. During cell therapy, tumor size was measured with a vernier caliper twice a week, and statistical processing was performed. After the mice were euthanized, all tumors were collected, weighed, and photographed. As shown in Figures 3H to 3J, the vaccine combination comprising the CARDF-DC vaccine successfully inhibited tumors expressing various p53 mutants, including breast cancer harboring the p53 R175H mutant, colorectal cancer harboring the p53 R273H mutant, and ovarian cancer harboring the p53 R248Q mutant. This suggests that the CARDF-DC vaccine provided herein can be used as a monotherapy for treating various tumors with various mutations.

[0246] [Table 19-1]

[0247] [Table 19-2]

[0248] [Table 19-3]

[0249] [Table 19-4]

Claims

1. A vector, (a) A first polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating dendritic cells, wherein the CAR comprises (1) an extracellular antigen-binding domain, (2) a transmembrane domain, and (3) an intracellular signaling domain, (b) A second polynucleotide encoding one or more tumor antigens, wherein the tumor antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA), The extracellular antigen-binding domain comprises a single-stranded variable fragment (scFv), and the scFv is specific to a tumor surface marker. The intracellular signaling domain comprises the cytoplasmic domain of Dectin-1 and the cytoplasmic domain of FcγR, The intracellular signaling domain comprises the amino acid sequence shown in Sequence ID No. 3, When the CAR and the tumor antigen are expressed in the dendritic cells, the dendritic cells can be activated within the immunosuppressive tumor microenvironment. The vector is a vector that is DNA or RNA.

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

3. The vector according to claim 1, wherein the intracellular signaling domain includes an amino acid sequence encoded by the nucleic acid sequence shown in Sequence ID No.

4.

4. The vector according to claim 1, 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, CEA, GPC3, MUC1, and NKG2D.

5. The vector according to claim 1, wherein the CAR further comprises a signal peptide, and the signal peptide comprises a CD8α signal peptide.

6. The vector according to claim 5, wherein the signal peptide of CD8α comprises the amino acid sequence shown in SEQ ID NO:

5.

7. The vector according to claim 1, wherein the transmembrane domain includes a transmembrane domain of CD8α.

8. The vector according to claim 7, wherein the transmembrane domain of CD8α comprises the amino acid sequence shown in SEQ ID NO:

6.

9. The vector according to claim 1, wherein the extracellular antigen-binding domain is linked to the transmembrane domain by a hinge region, and the hinge region includes a CD8α hinge region.

10. The vector according to claim 9, wherein the hinge region of CD8α includes the amino acid sequence shown in Sequence ID No.

7.

11. The second polynucleotide is a mutated oncogene or a fragment thereof, The vector according to claim 1, wherein the tumor gene is selected from p53, ras, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, and TGF-βRII.

12. The vector according to claim 1, wherein the tumor antigen is selected from CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride ion channel 2, cyclin-B1, 9D7, Ep-Cam, EphA3, GPC3, Her2 / neu, telomerase, mesothelin, SAP-2, and survivorbin.

13. The vector according to claim 1, wherein the tumor antigen is selected from p53 R273H mutant peptide, p53 R175H mutant peptide, p53 R248Q mutant peptide, p53 R249S mutant peptide, p53-R175H-R248Q-R249S-R273H mutant peptide, KRAS G12V mutant peptide, KRAS G12C mutant peptide, or a combination thereof.

14. The vector according to claim 13, wherein the p53 R273H mutant peptide has the amino acid sequence shown in SEQ ID NO: 29, the p53-R175H-R248Q-R249S-R273H mutant peptide has the amino acid sequence shown in SEQ ID NO: 37, the KRAS G12V mutant peptide has the amino acid sequence shown in SEQ ID NO: 31, or the KRAS G12C mutant peptide has the amino acid sequence shown in SEQ ID NO:

30.

15. A manipulated cell comprising the vector according to any one of claims 1 to 14.

16. A method for producing manipulated cells in vitro, comprising introducing a vector according to any one of claims 1 to 14 into starting cells under conditions suitable for the expression of the CAR and the tumor antigen.

17. The method according to claim 16, wherein the starting cell is a dendritic cell or a precursor or progenitor cell thereof.

18. The method according to claim 17, 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.

19. A population of cells produced in ex vivo by the method described in claim 16.

20. (i) a vector according to any one of claims 1 to 14, and (ii) a pharmaceutically acceptable medium.

21. (i) a population of manipulated cells as described in claim 15, and (ii) a pharmaceutically acceptable medium.

22. (i) a population of cells according to claim 19, and (ii) a pharmaceutically acceptable medium.

23. The pharmaceutical composition according to claim 20, for use in the treatment of cancer in a subject requiring cancer treatment.

24. The pharmaceutical composition according to claim 21, for use in the treatment of cancer in a subject requiring cancer treatment.

25. The pharmaceutical composition according to claim 22, for use in the treatment of cancer in a subject requiring cancer treatment.

26. The manipulated cells according to claim 15, used in an immunosuppressive microenvironment to induce the proliferation of immune cells, to extend the survival of immune cells, and / or to increase the expression and / or secretion of immunostimulatory cytokines from immune cells, wherein the immune cells are self or allogeneic.

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