Dendritic cell tumor vaccine and uses thereof

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] The present disclosure relates generally to the field of cancer therapy. In particular, the present disclosure relates to dendritic cell tumor vaccines, compositions and methods of making dendritic cell tumor vaccines, and methods of using dendritic cell tumor vaccines to treat cancer. [Background technology]

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

[0003] Tumor vaccines use tumor-associated antigens (TAA) or tumor-specific antigens (TSA) to provide specific antitumor effects to the body through active immunity, stimulating the body's own immune defense mechanism to achieve the effect of treating tumors or preventing recurrence. Tumor vaccines can be divided into therapeutic vaccines and preventive vaccines according to their purpose. Tumor vaccines can be divided into protein peptide vaccines, gene vaccines, virus vaccines, and dendritic cell vaccines (DC vaccines) according to their ingredients 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 patient's peripheral blood. These are loaded with TAA or TSA using pulsed protein peptide 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) usually exhibit immature or dysfunctional phenotypes in immunosuppressive tumor microenvironments or tumor immunosuppressive microenvironments (TIMEs), suppressing the infiltration and activation of T cells (Non-Patent Document 4). As a result, DC vaccines face obstacles in stimulating tumor-specific T cells in TIMEs. Therefore, it is necessary to develop new DC vaccines with enhanced infiltration and activation effects in TIMEs. [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) Summary of the Invention

[0006] In one aspect, the 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 a dendritic cell, the CAR comprising (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 tumor antigen can activate dendritic cells within an immunosuppressive tumor microenvironment, which in some embodiments includes tumors and / or tumor-infiltrating immune cells that 1) express immune inhibitory molecules and / or 2) are deficient in immune stimulatory 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) and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), HLA class I, sialoglycoprotein, 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 immune stimulatory 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 a combination thereof. In some embodiments, the intracellular signaling domain comprises a cytoplasmic domain of Dectin-1 and a 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 form thereof. In some embodiments, the cytoplasmic domain of FcγR comprises the amino acid sequence set forth in SEQ ID NO:2, or any functional form thereof. In some embodiments, the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 3, or any functional form 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 form 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, NKG2D.

[0010] In some embodiments, the CAR further comprises a signal peptide. In some embodiments, the signal peptide comprises the signal peptide of CD8α. In some embodiments, the signal peptide of CD8α comprises the sequence set forth in SEQ ID NO:5, or any functional form thereof.

[0011] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8α. In some embodiments, the transmembrane domain of CD8α comprises the sequence set forth in SEQ 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 comprises the hinge region of CD8α. In some embodiments, the hinge region of CD8α comprises the sequence set forth in SEQ ID NO:7, or any functional form thereof.

[0013] In some embodiments, the tumor antigen is a tumor associated antigen (TAA) or a tumor specific antigen (TSA). In some embodiments, the tumor antigen is encoded by a mutated 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 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 a p53 R273H mutant peptide, a KRAS G12V mutant peptide, and a KRAS G12C mutant peptide. In some embodiments, the p53 R273H mutant peptide has a sequence set forth in SEQ ID NO:29, the KRAS G12V mutant peptide has a sequence set forth in SEQ ID NO:31, and the KRAS G12C mutant peptide has a sequence set forth 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 set forth in SEQ ID NO: 32, or any functional form thereof.

[0015] In some embodiments, the one or more vectors is a DNA vector or an RNA vector.

[0016] In some embodiments, the first polynucleotide and / or the second polynucleotide are operably linked to at least one regulatory polynucleotide element for expression of the CAR and / or the 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 operably linked to the second polynucleotide via an IRES. In some embodiments, the IRES has the sequence set forth 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-specific 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 aspect, the disclosure provides an engineered cell comprising one or more vectors disclosed herein. In some embodiments, the engineered cell is a dendritic cell or a precursor or progenitor cell thereof. In some embodiments, the dendritic cell or a precursor or progenitor cell thereof is derived from a peripheral blood cell, a bone marrow cell, an embryonic stem cell, or an induced pluripotent stem cell.

[0020] In yet another aspect, the invention provides a method of producing an engineered cell, comprising introducing one or more vectors disclosed herein into a starting cell under conditions suitable for expression of a CAR and a tumor antigen. In some embodiments, the starting cell is a dendritic cell or a precursor or progenitor thereof. In some embodiments, the dendritic cell or a precursor or progenitor thereof is derived from a peripheral blood cell, a bone marrow cell, an embryonic stem cell, or an induced pluripotent stem cell.

[0021] In another aspect, the disclosure provides a population of cells produced ex vivo by the methods of producing engineered cells disclosed herein, in some embodiments, at least 60% of the cell population expresses a detectable level of a CAR polypeptide.

[0022] In another aspect, the disclosure provides a pharmaceutical composition comprising (i) one or more vectors disclosed herein, or a population of engineered cells disclosed herein, or a population of cells disclosed herein, and (ii) a pharma- ceutically acceptable vehicle.

[0023] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof. In some embodiments, the method comprises administering to the subject a pharmaceutical composition disclosed herein. In some embodiments, the subject has tumor cells that harbor a mutated gene.

[0024] In some embodiments, the cancer is a solid cancer selected from the group consisting of adrenal gland 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, the cancer is a hematological malignancy selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, and multiple myeloma (MM).

[0026] In some embodiments, the method of treating cancer further comprises administering to the subject a population of engineered immune cells. In some embodiments, the engineered immune cells have expression of a synthetic receptor (e.g., a CAR or a TCR) on the cell surface. In some embodiments, the immune cells are T cells, natural killer (NK) cells, NKT cells, B cells, macrophage cells, 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, gamma delta 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 aspect, the disclosure provides a method of inducing immune cell proliferation, prolonging immune cell survival, and / or increasing expression and / or secretion of immunostimulatory cytokines from immune cells in an immunosuppressive microenvironment. In some embodiments, the method comprises contacting the immunosuppressive microenvironment with an engineered cell as disclosed herein. In some embodiments, the immune cell is a T cell, a natural killer (NK) cell, a NKT cell, a B cell, a macrophage cell, an eosinophil, or a neutrophil. In some embodiments, the immune cell is a T cell selected from the group consisting of a CD4+ T cell, a CD8+ T cell, a cytotoxic T cell, a terminal effector T cell, a memory T cell, a naive T cell, a natural killer T cell, a gamma delta T cell, a cytokine-induced killer (CIK) T cell, and a tumor-infiltrating lymphocyte. In some embodiments, the immune cell is autologous or allogeneic.

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

[0029] The accompanying drawings, which are incorporated herein and form a part of the specification, together with this written specification, further serve to explain the principles of the present disclosure and to enable one skilled in the relevant art(s) to make and use the present disclosure. [Brief description of the drawings]

[0030] [Figure 1-1] Figure 1A-1J show the expression of chimeric antigen receptor (CARDF) on the surface of cells transduced with lentiviral vectors for co-expressing CARDF and tumor antigens. Figure 1A shows a structural diagram of the lentiviral vector. Figure 1B shows the expression of CARDF on the cell surface detected by flow cytometry after transduction of 293FT cells with lentivirus. [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, and Figure 1I shows the expression level of EphA2 on the surface of SW837 tumor cells. [Figure 1-4] FIG. 1J shows the mRNA levels of p53R273H mutant polypeptide expressed in H460 tumor cells overexpressing p53R273H (hereinafter referred to as H460-p53R273Hov). [Figure 2-1] Figures 2A-2D show the expression of CARDF-p53R273H in human DCs differentiated from humanized mouse bone marrow cells and in DCs after transduction. Figure 2A shows DCs differentiated from humanized mouse bone marrow cells. Figure 2B shows the mRNA levels of p53R273H expressed in DCs. [Figure 2-2]Figure 2C shows DCs differentiated from humanized mouse bone marrow cells, and the expression of CARDF-KRAS G12C and KRAS G12V in DCs. Figure 2D shows the mRNA levels of KRAS G12C and KRAS G12V expressed in DCs. [Figure 3-1] 3A-3G show tumor growth in humanized mouse (Hu-mouse) tumor models after treatment with dendritic cell tumor vaccine (hereinafter referred to as CARDF-DC vaccine). FIG. 3A shows a schematic diagram of the treatment process using p53R273H vaccine. FIG. 3B shows a schematic diagram of the treatment process using KRAS G12C vaccine and KRAS G12C vaccine. FIG. 3C shows the growth curves of H460 tumor tissues during treatment in different treatment groups using p53R273H vaccine. [Figure 3-2] Figure 3D shows the growth curve of H460-p53R273Hov (overexpressing R273H) tumor tissue during treatment in different treatment groups using p53R273H vaccine. Figure 3E shows the growth curve of SW480 tumor tissue during treatment in different treatment groups using p53R273H vaccine. [Figure 3-3] Figure 3F shows the growth curves of SW480 tumor tissues during treatment in different treatment groups using KRAS vaccines. Figure 3G shows the growth curves of SW837 tumor tissues during treatment in different treatment groups using KRAS vaccines. [Diagram 3-4] Figure 3H shows the growth curves of SK-BR-3 tumor (expressing p53 R175H mutant) tissues during treatment in different treatment groups with CAR-DC vaccine combo (p53-R175H-R248Q-R249S-R273H vaccine). Figure 3I shows the growth curves of SW480 tumor (expressing p53 R273H mutant) tissues during treatment in different treatment groups with CAR-DC vaccine combo. Figure 3J shows the growth curves of OVCAR3 tumor (expressing p53 R248Q mutant) tissues during treatment in different treatment groups with CAR-DC vaccine combo. [Figure 4-1]Figure 4A-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 ratio of T cells in the spleen of different treatment groups of p53R273H vaccine. Figure 4B shows the ratio of PD-1+ T cells in the spleen of different treatment groups, and Figure 4C shows the ratio of T cells in the spleen of different treatment groups of p53R273H vaccine. [Figure 4-2] Figure 4C shows the ratio of T cells in the spleen of different KRAS vaccine treatment groups, and Figure 4D shows the ratio 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 splenic DCs from different p53R273H vaccine treatment groups, and Figure 4F shows the ratio of B cells in peripheral blood from different p53R273H vaccine treatment groups. [Figure 4-4] Figure 4G shows the ratio of macrophages in peripheral blood in different p53R273H vaccine treatment groups, and Figure 4H shows the ratio of B cells in peripheral blood in different KRAS vaccine treatment groups. [Figure 5-1] Figure 5A-5D show the results of tumor mRNA analysis in a humanized mouse (Hu-mouse) tumor model after treatment with a dendritic cell tumor vaccine. Figure 5A shows the TNF-α gene mRNA expression levels in sw480 tumor tissues of different treatment groups of p53R273H vaccine. Figure 5B shows the CARDF scFv gene mRNA expression levels in sw480 tumor tissues of different treatment groups of p53R273H vaccine. [Figure 5-2] Figure 5C shows the CD3 gene mRNA expression level in SW480 tumor tissues of different KRAS vaccine treatment groups. Figure 5D shows the TNF-α gene mRNA expression level in SW837 tumor tissues of different KRAS vaccine treatment groups. Figure 5E shows the primer sequence used in the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Before describing the present disclosure in more detail, it is to be understood that the present disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure shall be limited only by the appended claims.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described herein.

[0033] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publication is cited. The citation of any publication is for its disclosure prior to the filing date and does not constitute an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0034] As will be apparent to one of ordinary skill 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 or combinable with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0035] definition To assist the reader, the following definitions are provided. Unless otherwise defined, all terms, notations, and other scientific or medical terms or nomenclature of the art used herein are intended to have the meanings commonly understood by those skilled in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein is not necessarily deemed to indicate a substantial difference beyond the definition of the term as commonly understood in the art.

[0036] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0037] In this disclosure, terms such as "comprises," "comprised," "comprising," "contains," "containing," and the like have the meaning ascribed to them in U.S. Patent Law, which are inclusive, i.e., open-ended, and do not exclude additional, unrecited elements or method steps. Terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent Law, which permit the inclusion of additional components or steps that do not materially affect the basic and novel characteristics of the claimed invention. The terms "consist of" and "consisting of" have the meaning ascribed to them in U.S. Patent Law, which are closed-ended.

[0038] In all instances in this application where there is a series of recited numerical values, it is understood that any recited numerical value 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 a combination of upper and lower numerical limits, each of which may be any numerical value recited herein. Ranges provided herein are 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, as well as non-integer values, where appropriate. Similarly, ranges delimited by "at least" are understood to include the lower limit as provided and all higher numerical values.

[0039] As used herein, "about" is understood to include within 3 standard deviations of the mean or within the accepted standard range in a particular technology. In certain embodiments, about is understood to mean a variation of 0.5 or less.

[0040] As used herein, the term "CAR" or "CARDF" can be used interchangeably with the term "chimeric antigen receptor" and refers to engineered or synthetic receptors or polynucleotides encoding them. Engineered or synthetic receptors comprise an extracellular domain including an antigen-binding domain, a transmembrane domain, and / or an intracellular signaling domain, optionally a signal peptide, linked to each other or operably linked to each other. The most common CARs are, for example, single chain variable fragments (scFv) from monoclonal antibodies fused to CD3-zeta transmembrane and endodomains. Such CARs result in the transmission of a zeta signal in response to specific binding of the scFv to its target. Methods for preparing CARs are publicly available (see, e.g., 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; WO 2012 / 079000; U.S. Patent Application Publication No. 2012 / 0213783; and WO 2013 / 059593, each of which is incorporated by reference in its entirety).

[0041] The term "chimeric antigen receptor T cells" is used interchangeably with the term "CAR-T cells" and refers to T cells or populations thereof that have been engineered through biological methods (e.g., genetic engineering) to express a CAR on the T cell surface. CAR-T cells may be T helper CD4+ and / or T effector CD8+ cells. CAR-Ts can identify surface antigens and initiate an immune response.

[0042] "Antigen" refers to a molecule that elicits an immune response. This immune response may be either a humoral or a cell-mediated response, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, may act as an antigen. It is readily apparent that the present disclosure includes therapeutic antibodies that act as antigen-eliciting immune responses.

[0043] As used herein, the term "neo-antigen" refers to an antigen that has at least one alteration that differs from the corresponding wild-type parent antigen. The alteration may be, for example, through a mutation in the tumor cell or a post-translational modification specific to the tumor cell. The alteration may include a splice site alteration, a splice variant, a frameshift or non-frameshift indel, a genomic rearrangement or gene fusion, a missense or nonsense substitution, or any genomic or expression alteration that results in a neoORF. The post-translational modification may include aberrant phosphorylation and splice antigens generated by the proteasome. The neo-antigen may include a polypeptide sequence or a nucleotide sequence.

[0044] "Antibody" refers to a polypeptide of the immunoglobulin (Ig) family that binds to an antigen. Naturally occurring "antibodies", for example of the IgG type, are tetramers that contain at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2 and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3), between which are more conserved regions called framework regions (FR).CDR boundaries for the antibodies disclosed herein may be defined or identified by the Kabat, IMGT, Chothia or Al-Lazikani conventions (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 is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy 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 antigens.

[0045] As used herein, "antigen-binding domain" refers to an antibody fragment formed from a portion of an intact antibody that includes one or more CDRs, or any other antibody fragment that is capable of binding to an antigen but does not include an intact native antibody structure. Examples of antigen-binding domains include, without limitation, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), 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. An antigen-binding domain is capable of binding to the same antigen that the parent antibody binds.

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

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

[0048] "Effector cells" as used in the context of immune cells refers to cells that are activated in response to a stimulus and capable of carrying out an effector function. Effector cells can include, without limitation, NK cells, cytotoxic T cells, and helper T cells.

[0049] "Effective amount" or "therapeutically effective amount" refers to an amount of cells, compositions, combinations, or any materials as described herein effective to achieve a desired biological result, which may include, without limitation, the elimination of B cells expressing a particular BCR and the antibodies produced therefrom.

[0050] The percentage of "identity" or "sequence identity" in the context of a polypeptide or polynucleotide is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) when compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues occur in both sequences 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] The term "conservative substitution", as used herein with respect to amino acid sequences, refers to the replacement of an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made 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 known in the art, conservative substitutions usually do not cause significant changes in the conformational structure of a protein, and thus may retain the biological activity of the protein.

[0052] The term "functional form" as used herein refers to different forms (e.g., variants, fragments, fusions, derivatives and mimetics) of a parent molecule that retain substantial biological activity of the parent molecule despite differences in amino acid sequence or chemical structure. The phrase "retain 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 can include both naturally occurring variant forms and non-naturally occurring forms, such as those obtained by recombinant methods or chemical synthesis. Functional forms may contain non-natural amino acid residues.

[0053] As used herein, the term "operably linked" refers to a functional relationship between two or more polynucleotide sequences. In the context of a polynucleotide encoding a fusion protein, such as a polypeptide chain of a CAR of the present disclosure, the term means that two or more polynucleotide sequences are linked such that the amino acid sequences encoded by these segments remain in frame. In the context of transcriptional or translational control, the term refers to the functional relationship of a control sequence to 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 term "polynucleotide" or "nucleic acid" refers to a chain of nucleotides. They also refer to synthetic and / or non-naturally occurring nucleic acid molecules, such as those containing nucleotide analogs or modified backbone residues or linkages. The term also refers to deoxyribonucleotide or ribonucleotide oligonucleotides in either single-stranded or double-stranded form. The term includes nucleic acids containing analogs of natural nucleotides. The term also includes nucleic acid-like structures with synthetic backbones. Unless otherwise indicated, a particular polynucleotide sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (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 to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. In certain embodiments, a polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0056] As used herein, the term "single-chain variable fragment" is used interchangeably with the term "scFv" and refers to an engineered 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" can be used interchangeably with the term "T cell receptor" or the term "TCR complex" and refers to a natural (or endogenous) TCR or an engineered TCR. The TCR refers to a protein complex on the surface of a T cell that is involved in recognizing fragments of antigens as peptides that bind to MHC molecules.

[0058] The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein can be functionally inserted to effect expression of the protein. A vector can be used to transform, transduce, or transfect a host cell to effect expression of the genetic elements carried by the vector in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (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. Additionally, the vector may contain an origin of replication. The vector may also contain a substance that aids in entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating. The vector may be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) that contains the nucleic acid sequence provided herein that encodes a fusion polypeptide, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda phage, and M13 phage, and the plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, and the like. , 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] The term "host cell," as used herein, refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0060] The term "pharmaceutical acceptable" indicates that the indicated carrier, vehicle, diluent, excipient(s) and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation, and physiologically compatible with the recipient thereof.

[0061] The terms "subject" or "individual" or "animal" or "patient" as used herein refer to a human or non-human animal, including a mammal or primate, in need of diagnosis, prognosis, mitigation, prevention and / or treatment of a disease or disorder. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and the like.

[0062] As used herein, the term "treating" a condition or "treatment" includes preventing or alleviating the condition, slowing the onset or rate of progression of the condition, reducing the risk of progression of the condition, preventing or slowing the progression of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0063] Dendritic cell (DC) tumor vaccine The present disclosure provides a novel DC vaccine platform that can stimulate immune responses even in an immunosuppressive environment. For example, the DC vaccine platform provided herein has improved antitumor activity compared to conventional DC vaccines, especially against solid tumors that are commonly associated with tumor immunosuppressive microenvironments. The DC vaccine platform provided herein can carry one or more (e.g., 1, 2, 3, 4, 5, or more) of various known or unknown tumor or non-tumor antigens. Thus, the DC vaccine platform provided herein can be used to treat a wide range of diseases, such as cancers or infectious diseases with specific mutations. The DC vaccine platform can also be used to improve the efficacy of personalized neo-antigen vaccines.

[0064] While most of the conventional DC vaccines need to be used in combination with another cancer therapy to achieve the desired therapeutic effect, the DC vaccine platform provided herein can be used as a monotherapy to treat tumors, even solid tumors that are commonly associated with tumor immunosuppressive microenvironments, making the DC vaccine platform provided herein a promising cancer therapy for treating a variety of cancers, especially 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 aspect, the present disclosure provides a dendritic cell (DC) tumor vaccine capable of stimulating tumor-specific T cells in an immunosuppressive tumor microenvironment or tumor immunosuppressive microenvironment (TIME). In one embodiment, the DC tumor vaccine comprises a dendritic cell (DC) and a dendritic cell expressing a chimeric antigen receptor (CAR or CARDF) capable of activating a tumor antigen, or a precursor or progenitor cell thereof.

[0066] Tumor immunosuppressive microenvironment The terms "immunosuppressive tumor microenvironment" and "tumor immune suppressive microenvironment (TIME)" can be used interchangeably and refer to a microenvironment that has, for example, tumor cells, tumor-infiltrating immune cells, tumor-associated fibroblasts, endothelial cells, and a variety of chemotactic and inflammatory or immune stimulatory cytokines, together with a dense extracellular matrix, that may suppress tumor immune surveillance and immunotherapy (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 tumors and / or tumor-infiltrating immune cells expressing immune inhibitory molecules. The immune inhibitory 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, sialoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47. In certain embodiments, the immune inhibitory molecules are CTLA-4 and / or PD-L1. As used herein, the term "expressing" or "expressing" with respect to an immune inhibitory molecule refers to expression of the immune inhibitory molecule at a level at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than a reference level. The term "reference level" with respect to expression of an immune inhibitory molecule refers to the expression level of the immune inhibitory molecule in a tumor formed by wild-type tumor cells (e.g., wild-type A549 cells) in an immune-deficient animal model (e.g., NSG mice).

[0068] "CTLA-4" is an abbreviation for Cytotoxic T-Lymphocyte-Associated protein 4, also known as CD152, a more detailed description 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 death-ligand 1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), a more detailed description 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 highly important immunosuppressive molecules that maintain peripheral immune tolerance by restricting T cell activity. CTLA-4 binds to CD80 and CD86 with higher affinity than CD28, which are the major costimulatory pathways for activating T cells. PD-L1 binds to PD-1 expressed on the surface of T cells and inhibits 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, the tumor in the TIME comprises 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 term "expressing" or "expressing" with respect to CTLA4-Ig refers to expression of CTLA4-Ig at a level at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% higher than a reference level. The term "reference level" with respect to expression of CTLA4-Ig refers to the expression level of CTLA4-Ig in wild-type tumor cells (e.g., wild-type A549 cells). As used herein, the terms "expressing" or "expressing" with respect to PD-L1 refer to expression of PD-L1 at a level that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% higher than a reference level. The term "reference level" with respect to PD-L1 expression refers to the expression level of PD-L1 in wild-type tumor cells (e.g. wild-type A549 cells).

[0071] In certain embodiments, CTLA-4-Ig comprises the amino acid sequence set forth in SEQ ID NO:8, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:8, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions thereto, while retaining substantial biological activity of SEQ ID NO:8, or any functional form thereof. In certain embodiments, PD-L1 comprises the amino acid sequence set forth in SEQ ID NO:9, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:9, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions thereto, while retaining substantial biological activity of SEQ ID NO:9, or any functional form thereof.

[0072] In certain embodiments, the immunosuppressive tumor microenvironment comprises a tumor that has poor responsiveness to adoptive cell therapy monotherapy (e.g., CAR-T monotherapy). As used herein and throughout the specification, the term "poor responsiveness" refers to the absence or reduction in responsiveness that can be detected by a comparable therapeutic effect (e.g., less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, or less than 2% better therapeutic effect) of a therapy (e.g., CAR-T therapy) when compared to a control treatment known to have no therapeutic effect.

[0073] Dendritic cell-activating chimeric antigen receptor Dendritic cells are professional antigen-presenting cells that can prime naive T cells and reactivate memory responses. In cancer, dendritic cells can activate T cells (e.g., cytotoxic CD8+ T cells) through cross-presentation of tumor-associated antigens (TAA) or neo-antigens to induce stronger anti-tumor responses. Activation of DCs can be assayed by measuring a variety of parameters, including, but not limited to, the activation status of DCs and / or the activation status of immune cells (e.g., T cells, macrophages), including expression levels of DC activation markers (e.g., CD80, CD86 and MHC-II, CD83, CD54, CMRF-44, CMRF-56), survival and / or cytotoxicity of immune cells (e.g., T cells), and the release of immune stimulatory cytokines (e.g., TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-20, IL-22, IL-24, IL-26, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49 ... This may be indicated by the expression (and / or secretion) of markers for immune cells (e.g., T cells), such as expression (and / or secretion) of immune inhibitory 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)), and / or expression levels of markers for anti-inflammatory macrophages (e.g., M2 macrophages), such as CD206 and CD163.

[0074] In certain embodiments, activation of dendritic cells can be characterized by increased expression levels of DC activation markers (e.g., CD80, CD86 and / or MHC-II, CD83, CD54, CMRF-44, CMRF-56), increased expression levels of immune cells (e.g., T cells (e.g., CD8+ increased expression (and / or secretion) of immune stimulatory 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 immune inhibitory 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 for anti-inflammatory macrophages (e.g., M2 macrophages) (e.g., CD206 and CD163).

[0075] In certain embodiments, a DC-activating 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, such that the CAR-DC vaccine provided herein comprising a DC-activating CAR is brought in proximity to the target cell. In certain embodiments, the extracellular antigen-binding domain is an antigen, such as a tumor marker (i.e., a tumor surface marker) expressed on the surface of a cancer cell, or an antigen expressed on an infected cell. The design of extracellular antigen-binding domains is well known in the art. In certain embodiments, the extracellular antigen-binding domain is an antigen-binding domain of an antibody. In some embodiments, the antigen-binding domain comprises a human or humanized antibody or antibody fragment thereof. The term "human antibody" refers to an antibody whose entire molecule is of human origin or consists of amino acid sequences identical to a human form of an antibody or immunoglobulin. The term "humanized antibody" refers to an antibody that contains sequences (e.g., CDR sequences) derived from a non-human immunoglobulin. Human or humanized antibodies or fragments thereof can be prepared in a variety of ways, such as through recombinant methodologies or through immunization with the antigen of interest of a mouse that has been genetically modified to express the antibody from human heavy and / or light chain encoding genes.

[0077] In some embodiments, the extracellular antigen-binding domain of the CAR provided herein comprises a single chain 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 CAR provided herein is an scFv. The scFv may be specific for a tumor surface marker, e.g., a solid tumor surface marker. As used herein, the term "tumor surface marker" refers to a molecule that is differentially expressed on the surface of tumor cells and is used as a target to recognize tumor cells. In certain embodiments, the tumor surface marker is selected from the group consisting of EphA2, CD19, CD70, CD117, CD133, CD147, CD171, DLL3, EGFRvIII, VGFR2, mesothelin, ganglioside GD2, FAP (fibroblast activation protein), FBP (folate binding protein), LMP1, Lewis Y, claudin 18.2, IL13Rα2, HER2, MDC1, PMSA (prostate 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 enriched on the tumor cell surface than on important normal tissues, and combinations thereof. The extracellular antigen binding domain may also be specific for a non-tumor marker for a disease that may benefit from converting TIME to a pro-inflammatory state, such as a marker for an infectious disease.

[0078] In some embodiments, the scFv is specific for EphA2. In some embodiments, the scFv comprises a variable heavy chain (VH) region and a variable light chain (VL) region. In some embodiments, the VH comprises a heavy chain CDR1 (HCDR1) having a sequence as set forth in SEQ ID NO: 10, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 10 while retaining substantial biological activity of SEQ ID NO: 10, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions thereto, or any functional form thereof, and a sequence as set forth in SEQ ID NO: 11, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 11 while retaining substantial biological activity of SEQ ID NO: 11. and a CDR2 having a sequence as shown in SEQ ID NO:12 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:12 or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereto, or any functional form thereof, while retaining substantial biological activity of SEQ ID NO:12.In some embodiments, the VL region comprises a light chain CDR1 (LCDR1) having a sequence as set forth in SEQ ID NO:13, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:13 while retaining substantial biological activity of SEQ ID NO:13, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions thereto, or any functional form thereof; and a sequence as set forth in SEQ ID NO:14, or a sequence having at least 75%, 80%, 85%, 90% identity to SEQ ID NO:14 while retaining substantial biological activity of SEQ ID NO:14. , 95%, or 99% identity to, or a sequence having one, two, three, four, five, six, seven, eight, nine or ten conservative substitutions thereto, or any functional form thereof; and a CDR3 having a sequence as shown in SEQ ID NO:15, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to, or a sequence having one, two, three, four, five, six, seven, eight, nine or ten conservative substitutions thereto, or any functional form thereof, while retaining substantial biological activity of SEQ ID NO:15.

[0079] In certain embodiments, the scFv comprises 1) a VH comprising an HCDR1 comprising the sequence set forth in SEQ ID NO: 10, an HCDR2 comprising the sequence set forth in SEQ ID NO: 11, and an HCDR3 comprising the sequence set forth in SEQ ID NO: 12, and 2) a VL comprising an LCDR1 comprising the sequence set forth in SEQ ID NO: 13, an LCDR2 comprising the sequence set forth in SEQ ID NO: 14, and an LCDR3 comprising the sequence set forth in SEQ ID NO: 15.

[0080] In some embodiments, the scFv comprises a VH and a VL. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 16, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity 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 conservative substitutions thereto, or any functional form thereof. In certain embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 17, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity 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 conservative substitutions thereto, or any functional form thereof. In some embodiments, the scFv comprises a VH comprising the sequence set forth in SEQ ID NO:16 and a VL comprising the sequence set forth in SEQ ID NO:17.

[0081] In certain embodiments, the scFv comprises a peptide linker of 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 between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In certain embodiments, the peptide linker comprises an amino acid sequence comprising SEQ ID NO: 27 (GGGGSGGGGSGGGGS).

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

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

[0084] (2) Transmembrane domain The transmembrane domains of the CARs described herein include, but are not limited to, BAFFR, BLAME (SLAMF8), CD2, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA-1), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD49f, 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 the transmembrane domain of CD8α. In certain embodiments, the transmembrane domain of CD8α has a 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 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions thereto, or any functional form thereof.

[0086] In certain embodiments, the transmembrane domain of the CAR described herein is synthetic, e.g., comprises primarily hydrophobic residues, e.g., leucine and valine. In certain embodiments, the transmembrane domain of the CAR described herein is modified or designed to avoid binding to the transmembrane domain of the same or a different surface membrane protein, to minimize interactions with other members of the receptor complex.

[0087] In some embodiments, the CAR described herein further comprises a hinge region that forms a link between the extracellular domain and the transmembrane domain of the CAR. The hinge and / or transmembrane domain provide for cell surface presentation of the extracellular antigen-binding domain of the CAR.

[0088] Hinge regions include, but are not limited to, BAFFR, BLAME (SLAMF8), CD2, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA-1), 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 comprises a CD8α hinge region, a human immunoglobulin (Ig) hinge region, or a glycine-serine rich sequence.

[0090] In some embodiments, the CAR comprises the hinge region of CD8α. In certain embodiments, the hinge region comprises 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 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions thereto, or any functional form thereof.

[0091] (3) Intracellular signaling domain The intracellular signaling domain of the CAR described herein is involved in the activation of at least one of the normal effector functions of the immune cell (e.g., dendritic cell) in which the CAR is placed. The term "effector function" as used in the context of an immune cell refers to a specialized function of the cell, such as phagocytic activity, cytolytic activity, or helper activity. In certain embodiments, the intracellular signaling domain of the CAR described herein is capable of activating (including maturing) dendritic cells in an immunosuppressive tumor microenvironment.DC activation responds to a variety of stimuli and is mediated by 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, 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 immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domain, which trigger an activation signal cascade to activate DCs.As used herein, the term "cytoplasmic domain" refers to the full-length domain of a protein that is within the cytoplasm, or any fragment thereof, such as 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 comprise 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), IFNγR, Dectin-1, and FcγR, or a combination thereof. In certain embodiments, the intracellular signaling domain of the CAR described herein comprises the cytoplasmic domain of Dectin-1 and the cytoplasmic domain of FcγR.

[0093] In certain embodiments, the cytoplasmic domain of dectin-1 and the cytoplasmic domain of 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 comprise the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:1 while retaining substantial biological activity of SEQ ID NO:1, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions thereto, or any functional form thereof.

[0095] The cytoplasmic domain of an FcγR may comprise the amino acid sequence set forth in SEQ ID NO:2, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:2 while retaining substantial biological activity of SEQ ID NO:2, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions thereto, or any functional form thereof.

[0096] In certain embodiments, the intracellular signaling domain of a CAR described herein comprises the amino acid sequence set forth in SEQ ID NO:3, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:3 while retaining substantial biological activity of SEQ ID NO:3, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions thereto, or any functional form thereof.

[0097] In certain embodiments, the intracellular signaling domain of a CAR described herein comprises an amino acid sequence encoded by the nucleic acid sequence set forth 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) Costimulatory signaling domain In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain.

[0099] In some embodiments, the costimulatory signaling domain is derived from the intracellular domain of a costimulatory molecule.

[0100] Examples of costimulatory 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 , 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 derivative, variant or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0101] In some embodiments, the costimulatory signaling domain of a CAR described herein comprises the intracellular domain of the costimulatory molecule CD137 (4-1BB), CD28, OX40, or ICOS. In some embodiments, the costimulatory signaling domain of a CAR described herein has a sequence of SEQ ID NO:58 or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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 signal peptide of CD8α. In some embodiments, the signal peptide of CD8α 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 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions thereto, or any functional form thereof.

[0103] Tumor antigens Tumor antigens are antigenic substances produced in tumor cells, i.e., tumor antigens induce immune responses in the host. Normal proteins in the body are not antigenic due to self-tolerance, a process in which self-reactive cytotoxic T lymphocytes (CTLs) and autoantibody-producing B lymphocytes are called "centrally" in primary lymphoid tissues (BM) and "peripherally" in secondary lymphoid tissues (mainly the thymus for T cells and spleen / lymph nodes for B cells). Thus, any protein that is not exposed to the immune system will induce an immune response. This may include normal proteins that are well isolated from the immune system, proteins that are usually produced in very small amounts, proteins that are usually produced only at certain developmental stages, or proteins whose structure is 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, and tumor-associated antigens (TAAs) are present in some tumor cells and some normal cells. More precisely, tumor antigens include the products of mutated oncogenes and tumor suppressor genes, the products of other mutated genes, overexpressed or aberrantly expressed cellular proteins, tumor antigens produced by tumor viruses, carcinoembryonic antigens, altered cell surface glycolipids and glycoproteins, and cell type-specific differentiation antigens.

[0105] Any protein produced in tumor cells that has an abnormal structure due to mutations may act as a tumor antigen. Such abnormal proteins are produced due to mutations in the relevant genes. Such abnormal proteins are called tumor-specific antigens because mutations in proto-oncogenes and tumor suppressors that lead to the production of abnormal proteins are the cause of tumors. Examples of tumor-specific antigens include the abnormal products of the ras and p53 genes. In contrast, mutations in other genes that are not related to tumorigenesis may lead to the synthesis of abnormal proteins that are tumor-associated antigens. Thus, 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 oncogenes are selected from p53, ras, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, TGF-βRII.

[0106] In human tumors, p53 and RAS are the most frequently mutated genes. Over 50% of aggressive tumors harbor 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 multiple mutation types of p53, R175H, R248W, and R273H are the three most common hotspot mutations. Other mutations include, but are not limited to, R248Q and R249S. Among KRAS gene mutations, 97% are mutations at the 12th or 13th amino acid. The most important ones are G12D, G12C, G12V, and G13D. Structural studies have shown that most of these gene mutations interfere with the GTP hydrolysis ability of KRAS.

[0107] Other examples of tumor antigens include tissue differentiation antigens, mutant protein antigens, tumor viral antigens, cancer testis antigens, and vascular or stromal specific antigens. Tissue differentiation antigens are antigens specific to a particular type of tissue. Mutant protein antigens can be highly specific to cancer cells because they would not be present in normal cells. Normal cells present normal protein antigens on MHC molecules, but cancer cells present mutant versions. Some viral proteins have been implicated in the formation of cancer (tumorigenesis), and some viral antigens are also cancer antigens. Cancer testis antigens are antigens expressed primarily in germ cells of the testes, but also in the ovaries and trophoblasts of the fetus. Some cancer cells express these proteins abnormally, thus presenting these antigens and allowing attack by T cells specific for these antigens. Examples of this type of antigen are CTAG1B and MAGEA1.

[0108] Immune responses are triggered by proteins that are normally produced in very small amounts but whose production is dramatically increased in tumor cells. One such protein is tyrosinase, an enzyme required for melanin production. Normally, tyrosinase is produced in trace amounts, but its levels are extremely high in melanoma cells.

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

[0110] Abnormal proteins are also produced by cells infected with oncogenic viruses, such as EBV and HPV. Cells infected with these viruses contain latent viral DNA, which is transcribed and the resulting proteins produce an immune response.

[0111] Thus, in some embodiments, the tumor antigen used in the DC vaccines disclosed herein is selected from CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride 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 cell surface glycolipids and glycoproteins may also have abnormal structures in tumor cells and become targets of the immune system.

[0113] p53 Tumor protein p53, also known as p53, cellular tumor antigen p53, phosphoprotein p53, tumor suppressor p53, antigen NY-CO-13, or transformation-related protein 53 (TRP53), is any 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 crucial for multicellular organisms, where it acts as a tumor suppressor to prevent the formation of cancer. As such, p53 has been described as the "guardian of the genome" due to its role in maintaining genome stability by preventing mutations. TP53 is therefore classified as a tumor suppressor gene.

[0114] The name p53 was given in 1979 to describe 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 masses of the amino acid residues, is only 43.7 kDa. This difference arises from the high number of proline residues in the protein, which makes it migrate slower on SDS-PAGE and therefore appear heavier than it actually is. In addition to the full-length protein, the human TP53 gene encodes at least 15 protein isoforms that range in size from 3.5 kDa to 43.7 kDa. All these p53 proteins are referred to as p53 isoforms. The TP53 gene is the most frequently mutated gene in human cancers (>50%), and it has been shown that the TP53 gene plays an important role in preventing cancer formation. The TP53 gene encodes a protein that binds to DNA to regulate gene expression and prevent genome mutations.

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

[0116] In humans, a common polymorphism involves the substitution of arginine for 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 showed no association with cervical cancer. A 2011 study found that TP53 proline mutations have a significant effect on pancreatic cancer risk in men. A study in Arab women found that TP53 codon 72 proline homozygosity was associated with a reduced risk of breast cancer. One study suggested that TP53 codon 72 polymorphism, MDM2 SNP309, and A2164G may be collectively associated with non-oropharyngeal cancer susceptibility, and that MDM2 SNP309 in combination with TP53 codon 72 may accelerate the development of non-oropharyngeal cancer in women. A 2011 study found that 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 colorectal or endometrial cancer risk.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 tell cells to either grow and divide (proliferate) or mature and take on specialized functions (differentiate). The K-Ras protein is a GTPase, which converts a molecule called GTP into another molecule called GDP. In this way, the K-Ras protein acts like a switch that is turned on and off by GTP and GDP molecules. To transmit a signal, it must be turned on by attaching (binding) to a GTP molecule. The K-Ras protein is turned off (inactivated) when it converts GTP to GDP. Once the protein is bound to GDP, it no longer relays signals to the cell nucleus. It was first identified as an oncogene in the Kirsten rat sarcoma virus, hence the name KRAS. Viral oncogenes are derived from the cellular genome. Thus, the KRAS gene in the cellular genome is called a proto-oncogene.

[0119] The gene product of KRAS was first discovered as a 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. In mammalian cells, there are two protein products of the KRAS gene, K-Ras4A and K-Ras4B, which result from the usage of alternative exon 4 (exon 4A and exon 4B, respectively). These proteins have different structures in their C-terminal regions and use different mechanisms to localize to cell membranes, including the plasma membrane.

[0120] Single amino acid substitutions, especially single nucleotide substitutions, in KRAS can result in activating mutations. The resulting transforming proteins have been implicated in a variety of malignancies, including lung adenocarcinoma, mucinous adenoma, pancreatic ductal carcinoma, and colorectal cancer. Several germline KRAS mutations have been found to be associated with Noonan syndrome and cardiofaciocutaneous syndrome. Somatic KRAS mutations are frequently found in leukemia, colorectal cancer, pancreatic cancer, and lung cancer.

[0121] MUC1 Cell surface-associated mucin 1 (MUC1), also called polymorphic epithelial mucin (PEM) or epithelial membrane antigen or EMA, is a mucin encoded by the MUC1 gene in humans. MUC1 is a glycoprotein with extensive O-linked glycosylation in the extracellular domain. Mucins coat the apical surface of epithelial cells in the lung, stomach, intestine, eye, and several other organs. Mucins defend the body against infection by binding to oligosaccharides in the extracellular domain and preventing pathogens from reaching the cell surface. Overexpression of MUC1 is often associated with colon, breast, ovarian, lung, and pancreatic cancers. Joyce Taylor-Papadimitriou identified and characterized this antigen while studying breast and ovarian tumors. MUC1 is a member of the mucin family and encodes a membrane-associated, glycosylated, phosphorylated protein. MUC1 has a core protein mass of 120 kDa to 225 kDa, which increases to 250 kDa to 500 kDa upon glycosylation. MUC1 extends more than 200 nm to 500 nm from the surface of the cell.

[0122] The protein is anchored to the apical surface of many epithelial cells by a transmembrane domain. Beyond the transmembrane domain is the SEA domain, which contains a cleavage site for the release of the large extracellular domain. Release of the mucin is achieved by a sheddase. The extracellular domain contains a variable number of tandem repeat (VNTR) domain of 20 amino acids, with the number of repeats varying between 20 and 120 in different individuals. These repeats are rich in serine, threonine, and proline residues, allowing for extensive O-glycosylation.

[0123] Multiple alternatively spliced ​​transcript variants encoding different isoforms of this gene have been reported, only some of which have been fully characterized.

[0124] MUC1 is cleaved in the endoplasmic reticulum into two parts, a cytoplasmic tail containing the transmembrane domain and an extracellular domain. These domains are tightly bound in a non-covalent manner. This tight non-covalent association is not disrupted by treatment with urea, low pH, high salt, 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. The protein functions in defense by binding to pathogens and also functions in cell signaling capacity.

[0125] Overexpression, aberrant subcellular localization, and altered glycosylation of this protein are associated with carcinomas, e.g., 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 with a documented role in tumor-induced alterations of host immunity.

[0126] PSMA Prostate-specific membrane antigen (PSMA), glutamate carboxypeptidase II (GCPII), also known as N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I) or NAAG peptidase, 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-resident zinc metalloenzyme. The majority of the enzyme is found in the extracellular space. PSMA is a class II membrane glycoprotein. It catalyzes the hydrolysis of N-acetyl-L-glutamate (NAAG) to glutamate and N-acetylaspartate (NAA) according to the reaction diagram on the right.

[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 which refer to the same protein.

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

[0129] Indeed, the first cloning of a cDNA encoding a gene expressing PSMA was achieved using RNA from the prostate tumor cell line LNCaP. PSMA shares homology with the transferrin receptor and undergoes endocytosis, but no ligand has been identified to induce internalization. PSMA was found to be identical to a membrane protein in the small intestine responsible for removing gamma-linked glutamate from poly-gamma-glutamic acid folates. This liberates folate, which can then be transported into the body and used as a vitamin. As a result, the PSMA clone genome was named FOLH1, which stands for folate hydrolase.

[0130] Three domains in the extracellular portion of PSMA (protease, apical, and C-terminal domains) cooperate in substrate recognition. The protease domain is a central seven-stranded mixed beta-sheet. The beta-sheet is flanked by ten alpha helices. The apical domain is located between the first and second strands of the central beta-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 four-helix bundle.

[0131] The central pocket is approximately 2 nanometers deep and opens from the extracellular space to the active site. The active site contains two zinc ions, each of which acts as a ligand for the oxygen or phosphate of 2-PMPA during inhibition. PSMA also contains one calcium ion coordinated to a site distal to the active site. Calcium has been proposed to link the protease and apical domains. Furthermore, human PSMA contains 10 potential glycosylation sites, and many of these sites, including those distal to the catalytic domain, affect PSMA's ability to hydrolyze NAAG.

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

[0133] Human PSMA is highly expressed in the prostate, where expression is approximately 100-fold higher than in most other tissues. In some prostate cancers, PSMA is the second most commonly upregulated gene product, increasing 8- to 12-fold compared to levels in noncancerous prostate cells. Because of this high expression, PSMA is being developed as a potential biomarker for the treatment and imaging of several cancers. In human prostate cancer, higher expression is associated with shorter time to progression and a higher percentage of patients who relapse. In vitro studies using prostate and breast cancer cell lines with reduced PSMA levels have shown a significant decrease in cell proliferation, migration, invasion, adhesion, and survival.

[0134] PSMA is the target of several nuclear medicine imaging agents for prostate cancer. Capromab pentide (sold as PROSTASCINT) is conjugated to indium-111 for detection by gamma camera. Second generation antibodies and small molecular weight ligands for imaging and therapy are under development. PSMA can also be used experimentally for therapeutic targeting. Lutetium-177 is a beta emitter that binds 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 neovasculature of tumors but not in the corresponding normal vasculature of all types of solid tumors, including kidney, breast, and colon.

[0135] S100P S100 calcium-binding protein P (S100P) is a protein that in humans is encoded by the S100P gene. The protein encoded by this gene is a member of the S100 protein family that contains two EF-hand calcium-binding motifs. S100 proteins are localized in the cytoplasm and / or nucleus of various cells and are involved in the regulation of many cellular processes, such as cell cycle progression and differentiation. The S100 genes contain at least 13 members and are arranged as a cluster on chromosome 1q21, although the gene is located at 4p16. The protein binds Ca 2+ In addition to binding to Zn 2+ and Mg 2+ It also binds to EZR and RAGE. 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 disrupted by cromolyn and pentamidine.

[0136] Neoantigens Neo-antigens may be identified using conventional techniques known in the art, such as whole genome / exome sequencing and RNA sequencing. In particular, neo-antigens may be identified by sequencing each patient's tumor and normal DNA to identify tumor-specific mutations and determine the patient's HLA allotype. The population of tumor-specific neo-antigens and their cognate native antigens are then subjected to bioinformatics analysis using a validated algorithm to predict which tumor-specific mutations create epitopes that can bind to the patient's HLA allotype (e.g., which tumor-specific mutations create epitopes that can bind to the patient's HLA allotype more effectively than the cognate native antigen). A detailed description of the identification of neo-antigens for cancer therapy can be found, for example, in U.S. Patent Application Publication No. 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 a subset of these mutations may be loaded into the CAR-DC vaccine platform provided herein to form a personalized cancer vaccine.

[0137] vector In another aspect, 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 the 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-specific insertion vectors (e.g., CRISPR, zinc finger nucleases, TALEN), in vitro transcribed 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 the expression DNA vector is transiently introduced into a cell, the mRNA of the CAR is transcribed in the host cell. Since the DNA vector and the mRNA are diluted with cell division, the expression of the CAR is not considered to be permanent. In one embodiment, the DNA vector may be introduced into a cell as a form of transient expression of the CAR.

[0139] In some embodiments, the vector is a viral vector. The viral vector may be derived from, for example, retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, and lentivirus. Useful viral vectors generally contain a replication origin, a promoter, a 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 long-term stable integration of a polynucleotide encoding a CAR into a non-proliferating cell genome, resulting in stable expression of the CAR in host cells, such as host T cells. In some embodiments, the vector is Addgene's lenti-Cas9 vector.

[0140] In some embodiments, the vector is RNA (e.g., mRNA). RNA is diluted with cell division, so RNA expression is not considered to be permanent. In one embodiment, in vitro transcribed RNA CAR can be introduced into cells as a 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 in a genome. In a transposon system, the polynucleotide encoding the CAR is flanked by terminal repeat sequences that can be recognized by a transposase that mediates the movement of the transposon. The transposase can be encoded on the same vector as the CAR or on a separate vector and delivered together as a protein. Non-limiting examples of transposon systems include Sleeping Beauty, Piggyback, Frog Prince, and Prince Charming.

[0142] In some embodiments, the polynucleotide is operably linked to at least one regulatory polynucleotide element in a vector for expression of the CAR. A typical vector contains various regulatory polynucleotide elements, such as elements that control expression of the inserted polynucleotide (e.g., transcription and translation terminators, initiation sequences, and promoters), elements that control replication of the vector in a host cell (e.g., origin of replication), and elements that control integration of the vector into the host genome (e.g., terminal repeats of transposons). Expression of the CAR may be achieved by operably linking the polynucleotide encoding the CAR to a promoter and incorporating this construct into a vector. Both constitutive promoters (e.g., CMV promoter, SV40 promoter, and MMTV promoter) or inducible promoters (e.g., metallothonine promoter, glucocorticoid promoter, and progesterone promoter) are contemplated 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 the host cell or in an in vitro expression system.

[0143] To assess expression of the CAR, the vector may also contain a selectable marker gene or a reporter gene, or both, for 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 capable of promoting stability and / or translation efficiency may also be used in the RNA. Methods for generating RNA for use in transfection may involve in vitro transcription (IVT) of a template with specially designed primers followed by polyA addition to produce a construct containing 3' and 5' untranslated sequences ("UTR"), a 5' cap and / or internal ribosome entry site (IRES), the nucleic acid to be expressed, and a polyA tail, typically between 50 and 2000 bases in length. RNA thus produced may efficiently transfect different types of cells.

[0145] RNA may be introduced into target cells using any of a number of different methods, for example available methods include, but are not limited to, electroporation or cationic liposome-mediated transfection using the Gene Pulser II (BioRad, Denver, Colorado), the Multiporator (Eppendort, Hamburg, Germany), lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems, e.g., "gene guns."

[0146] Vectors may be introduced into host cells, e.g., mammalian cells, by any method known in the art, e.g., by physical, chemical, or biological means. Physical methods of introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods include the use of viral vectors, particularly retroviral vectors, to insert genes into mammalian, e.g., human cells. Chemical means include colloidal dispersion systems, e.g., macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0147] Methods for producing DC tumor vaccines In another aspect, the present disclosure provides a method of making the DC tumor vaccine described herein. Numerous means for generating CAR-T cells known in the art can also be applied to the generation of DC tumor vaccines. Methods for generating CAR-T cells are described, for example, in Zhang et al., Engineering CAR-T cells, Biomarker Research (2017) 5:22. In some embodiments, the method includes introducing a vector comprising a first polynucleotide encoding a CAR and a second polynucleotide encoding a tumor antigen into a starting cell under conditions suitable for expression of the CAR and the tumor antigen. The methods provided herein may include one or more steps selected from obtaining a starting cell (i.e., a cell from a source), culturing the starting cell (including expansion, and optionally maturation), and genetically modifying the cell. The starting cell may be a dendritic cell or a precursor or progenitor cell thereof, as described above.

[0148] cell source The DC tumor vaccine provided herein can be obtained from any source.In certain embodiments, the DC tumor vaccine provided herein is 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 of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, a subject that is going to undergo, undergoes, or has undergone treatment for a particular disease or condition, a subject that is a healthy volunteer or a healthy donor, or from a blood bank.In some embodiments, the immune cells are obtained from a cancer subject that has poor responsiveness to immunotherapy, for example, CAR-T therapy.

[0149] The cells may be autologous or allogeneic to the subject of interest. Allogeneic donor cells may not be human leukocyte antigen (HLA) compatible, and therefore the allogeneic cells may be treated to reduce immunogenicity.

[0150] Immune cells may be collected from any location present in a subject, including, but not limited to, blood, umbilical cord blood, spleen, thymus, lymph nodes, pleural effusion, splenic tissue, tumor, and bone marrow. The isolated immune cells may be used directly or they may be stored for some period of time, for example by freezing.

[0151] In some embodiments, the engineered cells are obtained by engineering 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 several techniques known to those of skill 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 methods previously described. For example, dendritic cells may be identified by measuring the expression of CD11c, CD80, CD86, MHC / HLA molecules, and / or CCR7 molecules, which can be detected 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 progenitors can be achieved by transducing a substantially homogenous population of DCs with the vectors provided herein. In certain embodiments, a retroviral vector (e.g., lentiviral vector) is used to introduce polynucleotides into DCs. For example, the polynucleotides provided herein can be cloned into a lentiviral vector and expression can be driven from an endogenous promoter, from a lentiviral long terminal repeat, 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 gun, and magnetofection. The placement of the CAR and tumor antigens disclosed herein can be performed at the locus of any endogenous gene.

[0154] For the genetic modification of DCs or their precursor or progenitor cells, non-viral approaches may be used. For example, nucleic acids can be administered by 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), in the presence of sialo-orosomucoid 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 precursors or progenitors. Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially useful for delivery of DNA into cells. Transplantation of normal genes into diseased tissues of subjects may also be achieved by introducing normal nucleic acids ex vivo into culturable cell types (e.g., autologous or heterologous primary cells or their progeny) and then injecting the cells (or their progeny) into targeted tissues 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 a vector comprising a polynucleotide encoding a CAR and a tumor antigen provided herein into DCs prior to administration. In certain embodiments, the DC tumor vaccines provided herein can be produced by transfecting DC precursors or progenitor cells, for example, with a viral vector, and then differentiating the transfected cells into DCs. The DC tumor vaccines provided herein show improved expression of CAR and tumor antigens on the cell surface. The DC precursors or progenitor cells can 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. The DC precursors or progenitor cells can also be embryonic stem cells, or induced pluripotent stem cells (iPSCs).

[0156] In another aspect, the present disclosure also provides a cell population produced ex vivo by the method as 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 expresses the CAR polypeptide and tumor antigens provided herein at detectable levels. In certain embodiments, at least 85% of the cell population expresses the CAR polypeptide and tumor antigens provided herein at detectable levels.

[0157] Pharmaceutical Compositions In another aspect, the present disclosure also provides a pharmaceutical composition comprising the population of DC tumor vaccines provided herein and a pharma- ceutical acceptable vehicle.As used herein, the term "pharmaceutical composition" refers to a composition formulated for pharmaceutical use.

[0158] The term "pharmaceutical acceptable" indicates that the specified carrier, vehicle, diluent, excipient(s), and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation, and physiologically compatible with the recipient thereof.

[0159] "Pharmaceutically acceptable medium" refers to an ingredient in a pharmaceutical formulation other than an active ingredient that is biologically acceptable and non-toxic to a subject. Pharmaceutically acceptable mediums used in the pharmaceutical compositions disclosed herein may include, for example, pharma- ceutically acceptable liquid, gel, or solid carriers, aqueous or non-aqueous vehicles, antimicrobial agents, buffers, antioxidants, isotonicity agents, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, or various combinations thereof.

[0160] The pharmaceutical composition of the present disclosure can 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, the DC tumor vaccine or its population is mixed with a suitable medium before use or storage. A suitable pharmaceutically acceptable medium generally includes an inert material that assists in 1) administering the pharmaceutical composition to a subject, 2) processing the pharmaceutical composition into a deliverable preparation, and / or 3) storing the pharmaceutical composition before administration. In certain embodiments, the pharmaceutically acceptable medium includes an agent that can stabilize, optimize, or modify the formulation type, consistency, viscosity, pH, pharmacokinetics, and / or solubility. Such agents include, without limitation, buffers, wetting agents, emulsifiers, diluents, encapsulating agents, and skin penetration enhancers, such as saline, buffered saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethylcellulose, and combinations thereof.

[0161] Exemplary pharma- ceutically acceptable vehicles 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), powdered tragacanth, malt, gelatin, lubricants (e.g., magnesium stearate, sodium lauryl sulfate, and talc), excipients (e.g., cocoa butter and suppository waxes), oils (e.g., peanut oil, cottonseed oil, and the like), and mixtures thereof. , 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 buffered solutions, polyesters, polycarbonates, polyanhydrides, 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 to induce and / or enhance an immune response to an antigen and / or to treat and / or prevent a neoplasm, a pathogen infection, or an infectious disease. In certain embodiments, the pharmaceutical compositions provided herein are directly injected 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., tumor vasculature).

[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 about 5 x 10 5The population may include a DC tumor vaccine (i.e., engineered cell) population of 100%. One of skill in the art can readily determine the percentage of the DC tumor vaccine provided herein in the population using a variety of known methods, such as fluorescence-activated cell sorting (FACS). A suitable range for the percentage of the DC tumor vaccine provided herein in the population (also referred to as "purity") 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 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 x 10 4 Cells / kg body weight, at least 5 × 10 4 Cells / kg body weight, at least 1 x 10 5 Cells / kg body weight, at least 5 × 10 5 Cells / kg body weight, at least 1 x 10 6 Cells / kg body weight, at least 5 × 10 6 Cells / kg body weight, at least 1 x 10 7 Cells / kg body weight, at least 5 × 10 7 Cells / kg body weight, at least 1 x 10 8 At least 2 x 10 cells / kg body weight 8 Cells / kg body weight, at least 3 × 10 8 Cells / kg body weight, at least 4 x 10 8 Cells / kg body weight, at least 5 × 10 8 cells / kg body weight or at least 6 x 10 8Cells / kg body weight are administered. Those skilled in the art will understand that the dosage of the pharmaceutical composition provided herein can be determined based on various factors such as size, age, sex, weight and condition of the recipient. The dosage can be easily determined by those skilled in the art from the present disclosure and knowledge in the art. Those skilled in the art can easily determine the number of DC tumor vaccines provided herein in the composition to be administered in the method of the present disclosure, as well as the amount of optional additives, vehicles, media and / or carriers. Typically, the additives, if any, are present in an amount of 0.001% to 50% (by weight) solution in phosphate buffered saline, and the active ingredient (e.g., modified / recombinant cells provided herein) is present in the order of micrograms to milligrams, e.g., about 0.0001% to about 5% by weight, preferably about 0.0001% to about 1% by weight, even 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, even more preferably about 0.05% to about 5% by weight. It is preferred to determine the toxicity of a particular dosage, for example, by determining the lethal dose (LD) and LD50 in an appropriate animal model (e.g., mouse). It is also preferred to determine the timing of administration of the composition(s) that elicits an appropriate response. Such determinations are within the knowledge and present disclosure of the skilled artisan, without undue 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 catheter. In certain embodiments, the pharmaceutical compositions provided herein may be administered subcutaneously, intradermally, intratumorally, intramedullary, or intraperitoneally. In one embodiment, the cell composition of the present disclosure is preferably administered by intravenous injection. Administration may be autologous or heterologous. For example, a DC tumor vaccine may be obtained by modifying starting cells from one subject and administered to the same subject or a different subject. The pharmaceutical compositions provided herein may be formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) for administration. Administration of the pharmaceutical compositions provided herein may be performed as a single event or over a time course of treatment, for example, daily, weekly, biweekly, or monthly. The pharmaceutical compositions provided herein may be administered in combination with (e.g., before, after, or simultaneously with) another agent, such as a chemotherapeutic agent, another type of immunotherapy (e.g., CAR-T therapy), or radiation therapy. Concurrent administration may occur through administration of separate compositions, each of which contains a DC tumor vaccine provided herein and another agent, such as a chemotherapeutic agent, another type of immunotherapy (e.g., CAR-T therapy), or radiation therapy. Concurrent administration may occur through administration of a single composition that contains a DC tumor vaccine provided herein and another agent, such as a chemotherapeutic agent, another type of immunotherapy (e.g., CAR-T therapy), or radiation therapy.

[0166] Usage The present disclosure also provides various uses of the DC tumor vaccines provided herein.

[0167] Common Use In one aspect, the disclosure provides a method of treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of a DC tumor vaccine provided herein. In some embodiments, the method of treating a disease or pathological condition comprises providing DCs isolated or derived from cells isolated from a subject (e.g., peripheral blood cells, bone marrow cells, embryonic stem cells), or derived from iPSCs, engineering the DCs to express a CAR and tumor antigen provided herein to generate a DC tumor vaccine, and injecting the DC tumor vaccine back into the subject. In some embodiments, the method of treating cancer comprises providing precursor or progenitor cells of DCs (e.g., peripheral blood cells, bone marrow cells, embryonic stem cells, or iPSCs), engineering the precursor or progenitor cells to express a CAR and tumor antigen provided herein, differentiating the engineered precursor or progenitor cells into DCs expressing a CAR and tumor antigen provided herein, and injecting the DCs expressing a CAR and tumor antigen provided herein (e.g., DC tumor vaccine) back into the subject.

[0168] In some embodiments, the cancer is a solid cancer selected from the group consisting of adrenal gland 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, the cancer is a hematological malignancy selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, and multiple myeloma (MM).

[0169] In some embodiments, the subject with cancer is poorly responsive to cancer therapy (eg, immunotherapy).

[0170] The term "immunotherapy" as used herein refers to a type of therapy that stimulates the immune system to fight against disease, such as cancer, or boosts the immune system in a general manner. Immunotherapy includes passive immunotherapy (e.g., antibody therapy or CAR-T cell therapy) by delivering agents (e.g., effector cells) with established tumor immune reactivity that directly or indirectly mediate antitumor effects and do not necessarily rely on an intact host immune system. Immunotherapy may also include active immunotherapy, where treatment involves administration of immune response modifiers and relies on in vivo stimulation of the endogenous host immune system to react against diseased cells.

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

[0172] Checkpoint modulators may interfere with the ability of cancer cells to evade immune system attack and help the immune system respond more strongly to tumors. Immune checkpoint molecules may mediate costimulatory signals to enhance the immune response or co-inhibitory signals to suppress the immune response. Examples of checkpoint modulators include, without limitation, modulators of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGF beta, 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 comprises 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 taken from a patient, expanded in vitro, and activated. In certain embodiments, the T cells are engineered in vitro into CAR-T cells. The T cells or CAR-T cells that are most active against cancer are cultured in vitro in large volumes for 2 to 8 weeks. During this period, the patient undergoes treatments such as chemotherapy and radiation therapy that reduce the body's immunity. 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 of disrupting TIME (eg, converting TIME to an inflammatory state) using the DC tumor vaccines provided herein.

[0175] In another aspect, the present disclosure also provides a method of inducing immune cell proliferation, prolonging immune cell survival, and / or increasing 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 includes contacting the immunosuppressive microenvironment with a DC tumor vaccine provided herein. The immune cell may be a T cell, a natural killer (NK) cell, a NKT cell, a B cell, a macrophage cell, an eosinophil, or a neutrophil. 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, gamma delta 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 (e.g., peripheral blood of a subject) as the DC tumor vaccines provided herein.

[0176] In certain embodiments, the immunosuppressive microenvironment is an immunosuppressive tumor microenvironment, as described above. In certain embodiments, the immunosuppressive tumor microenvironment comprises tumors and / or tumor-infiltrating immune cells expressing immune inhibitory molecules, for example, 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), sialoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47. In certain embodiments, the immune inhibitory molecule is CTLA-4 and / or PD-L1, hi certain embodiments, the tumor comprises cells that express CTLA4-Ig and / or PD-L1.

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

[0178] In certain embodiments, the second 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 a subject). In certain embodiments, the ratio of the DC tumor vaccine and the CAR-T cells provided in the combination therapy ranges from about 1:1 to 1:10.

[0179] In certain embodiments, the DC tumor vaccine provided herein and the CAR-T cells are in the same pharmaceutical composition. In certain embodiments, the DC tumor vaccine provided herein and the CAR-T cells 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 the CAR-T cells.

[0180] In certain embodiments, the second agent is an agent that inhibits an immunosuppressive pathway, including, but not limited to, inhibitors of 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. The second agent may also be a T cell checkpoint inhibitor, including, but not limited to, an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD1 antibody (e.g., nivolumab, pembrolizumab, cemiplimab), an anti-PD-L1 antibody (e.g., atezolizumab, avelumab, durvalumab), an anti-PD-L2 antibody, an anti-BTLA antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, an anti-VISTA antibody, an anti-TIGIT antibody, and an anti-KIR antibody.

[0181] In certain embodiments, the second agent is a T cell agonist, including but not limited to antibodies that stimulate CD28, ICOS, OX-40, CD27, 4-1BB, CD137, GITR, and HVEM. In certain embodiments, the second agent is a therapeutic oncolytic virus, including but not limited to rhabdovirus, retrovirus, paramyxovirus, picornavirus, reovirus, parvovirus, adenovirus, herpesvirus, and poxvirus.

[0182] In certain embodiments, the second agent is an immune stimulatory agent, such as a toll-like receptor agonist, including but not limited to, TLR3, TLR4, TLR7, and TLR9 agonists. In certain embodiments, the second agent is a stimulator of interferon genes (STING) agonist, such as cyclic GMP-AMP synthase (cGAS).

[0183] In certain embodiments, the DC tumor vaccines provided herein are administered to a subject in need of treatment in combination with any number of suitable therapeutic modalities, including treatment with cytokines, including, but not limited to, Flt3L, IL-2, IL-7, and IL-15 or analogs thereof, or expression of such cytokines from within the DC tumor vaccine, to a subject in need of treatment, e.g., prior to, concurrently with, or following such treatment.

[0184] In some embodiments, the method of treatment further comprises administering an agent that reduces or alleviates side effects associated with administration of the DC tumor vaccine. Exemplary side effects include cytokine release syndrome (CRS) and hemophagocytic lymphohistiocytosis (HLH, also called macrophage activation syndrome (MAS)). In certain embodiments, agents administered to treat side effects include agents that neutralize soluble factors, such as IFN-γ, IFN-α, IL-2, and IL-6. Exemplary agents include, without limitation, inhibitors of TNF-α (e.g., etanercept) and inhibitors of IL-6 (e.g., tocilizumab). EXAMPLES

[0185] While the present disclosure has been particularly shown and described with reference to certain embodiments, some of which are preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

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

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

[0188] Preparation of lentivirus All plasmid DNA used for lentivirus packaging was extracted and purified using the NucleoBond Xtra Midi EF kit (Takara Bio). Lentivirus was produced according to the general instructions on the Addgene website and packaged using polyethylenimine (PEI) (Sigma Aldrich). The day before packaging the virus, 293FT cells (ATCC) were passaged at a ratio of 1:3 and split into 15 cm Petri dishes. The next day, when the cells reached 90% confluence, the virus was packaged. The medium was replaced with fresh medium 1 hour before transfection. The two packaging plasmids pSPAX2 (Addgene, Cat. No. 12260) and pMD2.G (Addgene, Cat. No. 12259), the target lentivirus vector and 1 mg / ml PEI were diluted in Opti-MEM (purchased from Gibco) at a DNA:PEI ratio of 1:3 to 1:4. After incubation at room temperature for 20 min, the plasmid mixture was gently added dropwise to the cell culture medium, and the medium was replaced with DMEM complete medium (Gibco) 8 h after transfection. Lentiviral particles were collected 48 h–72 h after transfection. The culture medium supernatant 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 min, and 1 / 3 volume of Lenti-X virus concentrate was added to the separated supernatant, mixed well, and then left at 4 °C overnight. The next day, the mixture was centrifuged at 3000 rpm at 4 °C for 45 min. The virus particles at the bottom of the centrifuge tube were resuspended in 0.6 ml–0.8 ml of pre-chilled PBS buffer and stored in a refrigerator at -80 °C in small aliquots for later use.

[0189] The results of 293FT cells infected with lentivirus 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, indicating that the constructed lentiviral vector expressing both CAR and oncogene mutant peptides can effectively transduce cells and express the CAR construct on the cell surface after being packaged into lentivirus.

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

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

[0192] As shown in Figures 1G to 1I, H460 cells, SW480 cells and SW837 cells express high levels of EphA2 on the 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 min, and rinsed twice with ice-cold PBS. The PBS was then aspirated using a sterile syringe (26-gauge needle). One end of the bone marrow cavity was rinsed to wash out the bone marrow cells. The bone marrow cells were dispersed by repeated blowing and aspirating using a 1 ml pipette tip, and then filtered through a 70 μm nylon mesh. The filtered cells were collected and centrifuged. Red blood cells were then lysed with lysis buffer (BD Biosciences). The remaining cells were washed twice with PBS and counted. The cells were cultured at 1 × 10 6 / ml and replenished with fresh differentiation medium every 2 days.

[0196] On day 8 of differentiation, immature DCs were infected with lentivirus at MOI 100. Concentrated lentivirus stock solutions with appropriate titers were slowly thawed at 37°C. Appropriate amounts of virus stock solutions were mixed with 6 μg / ml protamine sulfate and added to the differentiation medium. After 12 h of incubation at 37°C, 1 ml of differentiation medium was added to each well. 24 h after transduction, cells were collected by centrifugation, the medium containing the virus 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 to human DCs with an efficiency of over 90%. After transduction, the efficiency of CARDF expression on the surface of CARDF-DC and p53R273H vaccine is 82.1% and 75%, respectively. In the CARDC vaccine combo experiment, the efficiency of CARDF expression on the surface of CARDF-DC and CARDC vaccine combo is 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 that the mRNA expression level of p53R273H in the p53R273H vaccine was significantly increased.

[0200] As shown in Figure 2C and Table 2 below, Hu-mouse bone marrow cells could be directed to human 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 that the mRNA expression levels of the mutant peptides in KRAS G12C vaccine and KRAS G12V vaccine were significantly increased.

[0203] The above data demonstrate that humanized mouse bone marrow cells can be efficiently differentiated into human DCs, that CARDF can be expressed at high levels on the surface of DCs by lentiviral transduction, 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 treating tumors in a Hu-mouse xenograft model.

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

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

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

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

[0209] Cell therapy drugs were injected into mice via the tail vein, and cells were resuspended in 400 μL of PBS. During treatment, the dose of the first injection in Figure 3A was 6 × 10 6 DC / mouse, and the dose for the second injection was 2 × 10 6 The injection dose during treatment in Figure 3B was 5 × 10 6 DC / mouse. During cell therapy, the size of the tumor was measured with a caliper every other day and statistical processing was performed. After the mice were euthanized, all tumors were collected, weighed, and photographed. In addition, the spleen, blood, and bone marrow of the mice were collected, dissociated and processed into single cells, stained with fluorescently labeled flow cytometry antibodies, and analyzed by flow cytometry. RNA extracted from the tumor tissues 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, the tumor tissues formed by H460 did not show significant differences in tumor tissue growth after different treatments, suggesting that CAR-DC alone had no antitumor activity and that the CAR-DC vaccine did not act on tumors if it was not loaded with antigens.

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

[0212] [Table 3]

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

[0214] [Table 4]

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

[0216] [Table 5]

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

[0218] [Table 6]

[0219] As shown in Figure 4A and Table 7, the percentage of T cells in the p53R273H vaccine-treated group (12.163%) was higher than that in the CARDF-DC-treated group (4.786%) and the sham DC-treated group (6.024%), indicating that the DC vaccine stimulated the proliferation of T cells 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-treated group (18.7%) was lower than that in the sham DC-treated group (31.6%), indicating that the DC vaccine reversed the exhaustion of T cells in the tumor microenvironment condition.

[0222] [Table 8]

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

[0224] [Table 9]

[0225] As shown in FIG. 4D and Table 10, the percentage of DC cells in the KRAS G12C vaccine treatment group (3.2425%) and the percentage of DC cells in 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-treated group and the p53R273H vaccine-treated group was significantly increased compared with the mock-DC-treated group, indicating that the presence of CARDF effectively activated 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 percentage of B cells and macrophages in peripheral blood in different treatment groups, indicating the absence of immune 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 levels of TNF-α gene in SW480 tumor tissues were significantly increased in the CARDF-DC-treated group and the p53R273H vaccine-treated group compared with the sham DC-treated 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, CARDF scFv gene expression in SW480 tumor tissues of the p53R273H vaccine-treated group was significantly increased compared with the sham DC-treated group and the CARDF-DC-treated group, indicating increased infiltration of the DC vaccine in tumor tissues.

[0236] [Table 16]

[0237] As shown in Figure 5C and Table 17, CD3 gene expression in SW480 tumor tissue 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 increased T cell infiltration in SW480 tumor tissue after KRAS G12V vaccine treatment.

[0238] [Table 17]

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

[0240] [Table 18]

[0241] The above data indicate that the DC vaccine stimulated effector T cell responses in Hu- mice and effectively inhibited the growth of tumor tissues expressing the mutant gene, without any immune cytotoxic side effects.

[0242] Example 4 This example shows the use of DC tumor vaccine derived from humanized mouse bone marrow cells in the treatment of various cancers with various mutations in Hu-mouse xenograft models. A wide variety of tumor antigens can be loaded into the CAR-DC vaccine combo. In this example, the CAR-DC vaccine combo is loaded with DC tumor antigens such as P53 R175H ,P53 R248Q ,P53 R249S , and P53 R273H We loaded multiple short peptides represented by the following:

[0243] 0.5×10 6 SK-BR-3 cells (breast cancer cells harboring the p53 R175H mutant), 1 × 10 6 SW480 cells (colorectal cancer cells harboring the p53 R273H mutant), and 1 × 10 6OVCAR3 cells (ovarian cancer cells carrying p53 R248Q mutation) were subcutaneously injected into Hu-mice to generate xenograft Hu-mouse tumor models. The tumor-bearing Hu-mice were randomly divided into two groups: (1) CARDF-DC treatment group, and (2) They were divided into the CARDC vaccine combo treatment group.

[0244] The treatment process is similar to that shown in FIG. 3A.

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

[0246] [Table 19-1]

[0247] [Table 19-2]

[0248] [Table 19-3]

[0249] [Table 19-4]

Claims

1. One or more vectors, (a) a first polynucleotide encoding a chimeric antigen receptor (CAR) capable of activating a dendritic cell, the CAR comprising (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; and One or more vectors comprising:

2. 2. The one or more vectors of claim 1, wherein the CAR and / or the tumor antigen, when expressed in the dendritic cell, are capable of activating the dendritic cell within an immunosuppressive tumor microenvironment.

3. 3. The one or more vectors of claim 2, wherein the immunosuppressive tumor microenvironment comprises tumors and / or tumor-infiltrating immune cells that 1) express immune inhibitory molecules and / or 2) are deficient in immune stimulatory cytokines.

4. 4. The one or more vectors of claim 3, wherein 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) and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), HLA class I, sialoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47.

5. The one or more vectors of claim 3, wherein the immune inhibitory molecule is CTLA-4 and / or PD-L1.

6. 4. The one or more vectors of claim 3, 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.

7. 2. The one or more vectors of claim 1, wherein 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 a combination thereof.

8. The one or more vectors of claim 1, wherein the intracellular signaling domain comprises the cytoplasmic domain of Dectin-1 and the cytoplasmic domain of FcγR.

9. The one or more vectors of claim 8, wherein the cytoplasmic domain of Dectin-1 comprises the amino acid sequence set forth in SEQ ID NO: 1, or any functional form thereof.

10. The one or more vectors of claim 8, wherein the cytoplasmic domain of an FcγR comprises the amino acid sequence set forth in SEQ ID NO:2, or any functional form thereof.

11. 11. The one or more vectors of any one of claims 1 to 10, wherein the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:3, or any functional form thereof.

12. 12. The one or more vectors of any one of claims 1 to 11, wherein the intracellular signaling domain comprises an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO:4, or any functional form thereof.

13. 13. The one or more vectors according to any one of claims 1 to 12, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv).

14. The one or more vectors of claim 13, wherein the scFv is specific for a tumor surface marker.

15. 15. The one or more vectors of claim 14, wherein the tumor surface markers are 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.

16. The one or more vectors of any one of claims 1 to 15, wherein the CAR further comprises a signal peptide.

17. The one or more vectors described in claim 16, wherein the signal peptide comprises the signal peptide of CD8α.

18. 18. The one or more vectors according to claim 17, wherein the signal peptide of CD8α comprises the sequence shown in SEQ ID NO:5, or any functional form thereof.

19. The one or more vectors according to any one of claims 1 to 18, wherein the transmembrane domain comprises the transmembrane domain of CD8α.

20. 20. The one or more vectors described in claim 19, wherein the transmembrane domain of CD8α comprises the sequence shown in SEQ ID NO: 6, or any functional form thereof.

21. 21. The one or more vectors according to any one of claims 1 to 20, wherein the extracellular antigen binding domain is linked to the transmembrane domain by a hinge region.

22. The one or more vectors described in claim 21, wherein the hinge region comprises the hinge region of CD8α.

23. 23. The one or more vectors described in claim 22, wherein the hinge region of CD8α comprises the sequence shown in SEQ ID NO: 7, or any functional form thereof.

24. The one or more vectors according to any one of claims 1 to 23, wherein the tumor antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

25. 25. The one or more vectors of claim 24, wherein the second polynucleotide is a mutated oncogene or a fragment thereof.

26. 26. The one or more vectors of claim 25, wherein the oncogene is selected from p53, ras, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, TGF-βRII.

27. 25. The one or more vectors of claim 24, wherein the tumor antigen is selected from CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium activated chloride channel 2, cyclin-B1, 9D7, Ep-Cam, EphA3, GPC3, Her2 / neu, telomerase, mesothelin, SAP-2, and surviving.

28. 25. The one or more vectors of claim 24, wherein the tumor antigen is selected from a p53 R273H mutant peptide, a p53 R175H mutant peptide, a p53 R248Q mutant peptide, a p53 R249S mutant peptide, a p53-R175H-R248Q-R249S-R273H mutant peptide, a KRAS G12V mutant peptide, a KRAS G12C mutant peptide, or a combination thereof.

29. 29. The one or more vectors of claim 28, wherein the p53 R273H mutant peptide has the sequence set forth in SEQ ID NO:29, the p53-R175H-R248Q-R249S-R273H mutant peptide has the sequence set forth in SEQ ID NO:37, the KRAS G12V mutant peptide has the sequence set forth in SEQ ID NO:31, and the KRAS G12C mutant peptide has the sequence set forth in SEQ ID NO:

30.

30. The one or more vectors of claim 24, wherein the tumor antigen is linked to a DC-LAMP sorting signal having the sequence shown in SEQ ID NO:

32.

31. 31. One or more vectors according to any one of claims 1 to 30, which are DNA or RNA.

32. 32. The one or more vectors of claim 31 , wherein the first polynucleotide and / or the second polynucleotide are operably linked to at least one regulatory polynucleotide element for expression of the CAR and / or the tumor antigen.

33. 32. The one or more vectors of claim 31 , wherein the first polynucleotide and the second polynucleotide are comprised in a single vector.

34. 34. The one or more vectors of claim 33, wherein the first polynucleotide is operably linked to the second polynucleotide via an IRES having the sequence set forth in SEQ ID NO:

36.

35. 32. The one or more vectors of claim 31 , wherein the vector is a plasmid vector, a viral vector, a transposon, a site-specific insertion vector, or a suicide expression vector.

36. 36. The one or more vectors of claim 35, wherein the viral vector is a lentiviral vector, a retroviral vector, or an AAV vector.

37. 37. The one or more vectors of claim 36, wherein the viral vector is a lentiviral vector.

38. 38. An engineered cell comprising one or more vectors according to any one of claims 1 to 37.

39. 39. The engineered cell of claim 38, wherein the engineered cell is a dendritic cell or a precursor or progenitor cell thereof.

40. 40. The engineered cell of claim 39, wherein the dendritic cell or a precursor or progenitor thereof is derived from a peripheral blood cell, a bone marrow cell, an embryonic stem cell, or an induced pluripotent stem cell.

41. 38. A method for producing an engineered cell, comprising introducing one or more vectors according to any one of claims 1 to 37 into a starting cell under conditions suitable for expression of said CAR and said tumor antigen.

42. 42. The method of claim 41, wherein the starting cell is a dendritic cell or a precursor or progenitor cell thereof.

43. 42. The method of claim 41, 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.

44. A population of cells produced ex vivo by the method of any one of claims 41 to 43.

45. 45. The population of cells of claim 44, wherein at least 60% of the population of cells expresses the polypeptide of claim 27 at a detectable level.

46. 13. A pharmaceutical composition comprising (i) one or more vectors according to any one of claims 1 to 37, or a population of engineered cells according to any one of claims 38 to 40, or a population of cells according to claim 44 or 45, and (ii) a pharma- ceutically acceptable vehicle.

47. 47. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 46.

48. 48. The method of claim 47, wherein the subject has tumor cells that harbor a mutated gene.

49. 48. The method of claim 47, wherein the cancer is a solid cancer selected from the group consisting of adrenal gland cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.

50. 48. The method of claim 47, wherein the cancer is a hematological malignancy selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, and multiple myeloma (MM).

51. 48. The method of claim 47, further comprising administering to the subject a population of modified immune cells.

52. 52. The method of claim 51 , wherein the engineered immune cell has expression of a synthetic receptor (e.g., a CAR or a TCR) on the cell surface.

53. 52. The method of claim 51, wherein the immune cell is a T cell, a natural killer (NK) cell, an NKT cell, a B cell, a macrophage cell, an eosinophil, or a neutrophil.

54. 48. The method of claim 47, wherein the immune cell is a T cell selected from the group consisting of a CD4+ T cell, a CD8+ T cell, a cytotoxic T cell, a terminal effector T cell, a memory T cell, a naive T cell, a natural killer T cell, a γδ T cell, a cytokine-induced killer (CIK) T cell, and a tumor-infiltrating lymphocyte.

55. 48. The method of claim 47, wherein the immune cells are autologous or allogeneic.

56. 41. A method of inducing immune cell proliferation, prolonging immune cell survival, and / or increasing expression and / or secretion of immunostimulatory cytokines from immune cells in an immunosuppressive microenvironment, comprising contacting said immunosuppressive microenvironment with an engineered cell according to any one of claims 38 to 40.

57. 57. The method of claim 56, wherein the immune cell is a T cell, a natural killer (NK) cell, an NKT cell, a B cell, a macrophage cell, an eosinophil or a neutrophil.

58. 57. The method of claim 56, wherein the immune cell is a T cell selected from the group consisting of a CD4+ T cell, a CD8+ T cell, a cytotoxic T cell, a terminal effector T cell, a memory T cell, a naive T cell, a natural killer T cell, a γδ T cell, a cytokine-induced killer (CIK) T cell, and a tumor-infiltrating lymphocyte.

59. 57. The method of claim 56, wherein the immune cells are autologous or allogeneic.

60. 57. The method of claim 56, wherein the immunosuppressive microenvironment is an immunosuppressive tumor microenvironment.

61. 61. The method of claim 60, wherein the immunosuppressive tumor microenvironment comprises tumors and / or tumor-infiltrating immune cells that express immune inhibitory molecules.

62. 62. The method of claim 61, wherein 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) and SIGLEC9 (CD329), PD-L1, PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), PVR (CD155), sialoglycoprotein, CD112, CD113, galectin 9, CD24, and CD47.

63. The method of claim 61, wherein the immune inhibitory molecule is CTLA-4 and / or PD-L1.

64. 57. The method of claim 56, wherein the immunostimulatory cytokine is one or more of TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and granulocyte-macrophage colony-stimulating factor.

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