Methods and compositions for using recombinant dendritic cells for cancer treatment

Abstract

Disclosed herein are methods and compositions for treating cancer by administering dendritic cells expressing heterologous proteins to cause an immune response. In some embodiments, the dendritic cells include one or more heterologous nucleic acid molecules encoding CD40L and CXCL13. In some embodiments, the dendritic cells further include heterologous nucleic acid molecules encoding CD93. In yet additional embodiments, the dendritic cells expressing heterologous proteins are activated.

Description

[0001] Priority claim

[0002] This application claims priority to U.S. Provisional Application No. 62 / 698,254, filed on July 15, 2018, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on July 3, 2019, is named 146616_00302_SL.txt, and is 14,051 bytes in size. Background Art

[0005] Cancer treatment includes a wide range of therapeutic methods, including surgery, radiation, chemotherapy, and cell-based immunotherapy. Although these various treatment methods provide a wide range of treatment options, existing therapeutic agents suffer from many shortcomings, including lack of targeted cell selectivity relative to healthy cells, toxicity, and cancer resistance to treatment. Recent newer approaches using targeted therapeutic agents that preferentially interfere with key cellular processes of cancer cells over normal cells have produced chemotherapy regimens with fewer side effects than non-targeted treatments such as radiation therapy.

[0006] Cancer immunotherapy has also emerged as a promising treatment approach to enhance and supplement existing standards of care. See, e.g., Miller et al. Cancer Cell; 27:439-449 (2015). These approaches involve the use of antibodies to modulate the immune system to kill cancer cells. Anti-tumor immune responses have been enhanced in some patients with solid tumors by anti-PD1 therapy. However, only a small fraction of patients respond to such treatments, highlighting the need for additional approaches and further cancer treatments to enhance and supplement existing standards of care. Summary of the invention

[0007] Provided herein are dendritic cells comprising one or more heterologous nucleic acid molecules encoding CD40L and / or CXCL13.

[0008] In some embodiments, the one or more heterologous nucleic acid molecules encode CD93.

[0009] Further provided herein are dendritic cells comprising a heterologous CD40L protein and a heterologous CXCL13 protein.

[0010] In some embodiments, the cells further comprise a heterologous CD93 protein.

[0011] Further provided herein are antigen-activated dendritic cells, wherein the dendritic cells comprise one or more heterologous nucleic acid molecules encoding CD40L and / or CXCL13. In some embodiments, the one or more heterologous nucleic acid molecules encode CD93.

[0012] In some embodiments, the antigen-activated dendritic cells are activated by exposure to one or more antigens.

[0013] In some embodiments, the antigen is a tumor antigen.

[0014] In some embodiments, the antigen is a viral antigen.

[0015] In some embodiments, the antigen is a cell lysate.

[0016] In some embodiments, the cell lysate is allogeneic or autologous with respect to the antigen-activated dendritic cells.

[0017] In some embodiments, the cell lysate is a lysate comprising one or a combination of allogeneic melanoma cell lysates.

[0018] In some embodiments, the allogeneic melanoma cell lysate is DDM-1.7 cell lysate, DDM-1.13 cell lysate, or a combination thereof.

[0019] In some embodiments, the cell lysate is a whole cell lysate.

[0020] In some embodiments, the cell lysate is a tumor cell lysate.

[0021] Further provided herein are compositions comprising the cells described herein (recombinant dendritic cells or activated cells or related compositions), wherein the composition does not comprise a heterologous antigen.

[0022] Further provided herein are pharmaceutical compositions comprising the cells provided herein.

[0023] Further provided herein are methods of treating a solid tumor, cancer, or malignancy in a subject, the method comprising administering to the subject any one of the cells or compositions provided herein.

[0024] Further provided herein are methods of treating a solid tumor, cancer, or malignancy in a subject, the method comprising administering to the subject any one of the cells or compositions provided herein.

[0025] In some embodiments, the method further comprises:

[0026] Prior to administration, screening the protein expression profile of a resected tumor or biopsy sample from the subject to cross-match the protein expression profile of an allogeneic tumor lysate; and

[0027] If at least three segments of the protein expression profile of the resected tumor or biopsy sample cross-match the protein expression profile of the allogeneic tumor lysate, then the allogeneic tumor lysate-activated dendritic cells or activated dendritic cell composition are administered.

[0028] In some embodiments, the allogeneic tumor lysate is derived from an allogeneic melanoma cell line selected from the group consisting of DDM-1.7, DDM-1.13, or a combination thereof.

[0029] In some embodiments, the dendritic cells do not have the same HLA type as the subject.

[0030] In some embodiments, the cancer is a solid tumor selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon / colorectal cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, thyroid carcinoma, thyroid gland ... The present invention relates to a group of tumors of the CNS, including lung cancer, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors such as gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, Schwannomas, craniopharyngiomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases.

[0031] In some embodiments, the administration route is via intratumoral, peritumoral, intradermal, subcutaneous, intramuscular, or intraperitoneal injection.

[0032] In some embodiments, the administering is via subcutaneous, intratumoral, or intradermal injection.

[0033] In some embodiments, the dendritic cells or composition are frozen or cryopreserved and thawed prior to administration.

[0034] In some embodiments, the dendritic cells have not been pulsed with antigen prior to administration.

[0035] Further provided herein are kits comprising any of the dendritic cells or compositions provided herein.In some embodiments, the dendritic cells or compositions are frozen or cryopreserved in a container and optionally comprise an allogeneic tumor lysate frozen or cryopreserved in a separate container.

[0036] Further provided herein is a kit comprising any of the dendritic cells or compositions described herein, wherein the dendritic cells or compositions are frozen or cryopreserved in a container, and optionally comprise an autologous tumor lysate frozen or cryopreserved in a separate container.

[0037] Further provided herein is a kit comprising any of the dendritic cells or compositions described herein, wherein the activated dendritic cells or compositions are frozen or cryopreserved in a container.

[0038] In some embodiments, the kit further comprises a separate container comprising one or more buffers and, optionally, one or more activators.

[0039] In some embodiments, the kit further comprises instructions for incubating and / or processing the dendritic cells or composition.

[0040] Further provided herein is a method of treating cancer in a subject, the method comprising:

[0041] Prior to administration, screening a protein expression profile of a resected tumor or biopsy sample from the subject to cross-match with a protein expression profile of an allogeneic tumor lysate;

[0042] If at least three segments of the protein expression profile of the resected tumor or biopsy sample match the protein expression profile of the allogeneic tumor lysate, administering any one of the dendritic cells or compositions provided herein to the subject; or

[0043] If the at least three segments of the protein expression profile of the resected tumor or biopsy sample do not match the allogeneic tumor lysate protein expression profile, then

[0044] (a) administering to the subject a composition comprising any of the dendritic cells or compositions described herein pulsed with autologous tumor lysate, or

[0045] (b) administering to the subject a cell or composition described herein that has not been pulsed with an antigen.

[0046] Further provided herein is a method of treating cancer in a subject, the method comprising administering to the subject any one of the dendritic cells or compositions provided herein, wherein the dendritic cells or composition have not been pulsed with a tumor antigen.

[0047] Further provided herein is a method of treating cancer in a subject, the method comprising administering to the subject any one of the dendritic cells or compositions provided herein, wherein the dendritic cells have been pulsed with a tumor antigen or lysate.

[0048] Further provided herein is a method of activating the immune system, the method comprising administering to a subject in need thereof an effective amount of any of the dendritic cells or compositions described herein.

[0049] The present invention further provides a method for producing immature dendritic cells, the method comprising:

[0050] CD14+ and / or CD1a+ monocytes heterologously expressing CD40L, CXCL13 and optionally CD93 are cultured in vitro into immature dendritic cells.

[0051] In some embodiments, the method comprises contacting the immature dendritic cells with a composition comprising one or more of: TNF-α, IL-1β, IFN-α, IFN-γ, and pIC.

[0052] In some embodiments, the method further comprises generating CD14+ monocytes heterologously expressing CD40L, CXCL13, and optionally CD93.

[0053] In some embodiments, the generating comprises contacting CD14+ monocytes with one or more vectors encoding CD40L, CXCL13, and optionally CD93.

[0054] In some embodiments, the vector is a recombinant adenoviral vector, a recombinant retroviral vector, or a recombinant lentiviral vector, or a combination thereof.

[0055] In some embodiments, the generating CD14+ monocytes heterologously expressing CD40L, CXCL13, and optionally CD93 comprises contacting the monocytes with one or more CRISPR constructs to generate CD14+ monocytes heterologously expressing CD40L, CXCL13, and optionally CD93.

[0056] In some embodiments, the CRISPR construct is CAS9.

[0057] In some embodiments, the contacting comprises transfection, transduction, electroporation, infection, or any combination thereof.

[0058] In some embodiments, the method further comprises freezing the immature dendritic cells.

[0059] In some embodiments, the method further comprises contacting the immature dendritic cells with autologous tumor lysate or allogeneic tumor lysate prior to maturation.

[0060] In some embodiments, the method further comprises freezing the dendritic cells after maturation.

[0061] In some embodiments, the CD14+ monocytes are further modified to heterologously express CD93.

[0062] In some embodiments, the allogeneic tumor lysate is derived from an allogeneic melanoma cell line selected from the group consisting of DDM-1.7, DDM-1.13, or a combination thereof.

[0063] In some embodiments, the lysate is a whole cell lysate.

[0064] In some embodiments, a graft-versus-tumor (GVT) response is increased in said patient.

[0065] In some embodiments, within a time range of 4-14 days after administration of the dendritic cells or composition, the patient is further administered an immune checkpoint inhibitor, including any one or a combination of two immune checkpoint inhibitors comprising: an inhibitor of PD-1 or PD-L1 (B7-H1), such as an anti-PD-1 antibody, including nivolumab (nivolumab from Bristol-Myers Squibb), pembrolizumab / pembrolizumab, also known as MK-3475 (Keytruda from Merck), pidilizumab (Curetech), AMP-224 (Amplimmune), or an anti-PD-L1 antibody, including MPDL3280A (Roche), MDX-1105 (Bristol Myer Squibb), MEDI-4736 (AstraZeneca), and MSB-0010718C (Merck), an antagonist of CTLA-4, such as an anti-CTLA-4 antibody, including the anti-CTLA4 antibody Yervoy TM(ipilimumab, Bristol-Myers Squibb), tremelimumab, Pfizer, decilimumab, or AMGP-224, or the tumor-specific antibodies trastuzumab (Herceptin) for breast cancer, or rituximab (MabThera) or cetuximab (Erbitux) for lymphoma.

[0066] In some embodiments, the method further comprises administering one or more additional agents commonly used to treat cancer.

[0067] In some embodiments, the additional agent is selected from radiation, chemotherapy, an antibody drug conjugate, and an immunomodulatory antibody.

[0068] In some embodiments, the chemotherapy is cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, vinblastine, irinotecan, etoposide, or pemetrexed, or a combination thereof, or a pharmaceutically acceptable salt thereof.

[0069] In some embodiments, the immunomodulatory antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD40 antibody, an anti-CTLA-4 antibody, or an anti-OX40 antibody, or any combination thereof.

[0070] In some embodiments, the antibody drug conjugate targets c-Met kinase, LRRC15, EGFR, or CS1, or any combination thereof.

[0071] In some embodiments, the treatment or increasing the immune response is repeated periodically as a maintenance therapy within the following time frame: from every 5 days, once a week, once every 10 days, once every 14 days, once every 21 days, once a month to once every two months, to once every 3 months, to once every 4 months, to once every 5 months, to once every 6 months, or once every 7 months, or once every 8 months, or once every 9 months, or once every 10 months, or once every 11 months, or once a year, as long as the patient shows improvement or stable / non-progressive disease.

[0072] Disclosed herein are methods and compositions for treating cancer by administering dendritic cells expressing heterologous proteins to induce an immune response. In some embodiments, the dendritic cells include one or more heterologous nucleic acid molecules encoding CD40L and CXCL13. In some embodiments, the dendritic cells further include heterologous nucleic acid molecules encoding CD93. In further embodiments, the dendritic cells include one or more heterologous nucleic acid molecules encoding CD40L and CD93. In further embodiments, the dendritic cells include one or more heterologous nucleic acid molecules encoding CXCL13 and CD93.

[0073] In some embodiments, the dendritic cells overexpressing CD40L and CXCL13 and optionally CD93 can be antigen-activated dendritic cells. In some embodiments, the antigen can be a tumor antigen or a viral antigen. In some embodiments, the dendritic cells are allogeneic or autologous to the subject.

[0074] In a further embodiment, a method of treating cancer in a subject comprises administering to the subject a composition comprising dendritic cells, wherein the dendritic cells overexpress one or more proteins selected from CD40L, CXCL13 and CD93. In some embodiments, the method further comprises:

[0075] (a) obtaining a protein expression profile of a resected tumor or biopsy sample from a subject;

[0076] (b) comparing the protein expression profile of the excised tumor or biopsy sample with the protein expression profile of a melanoma cell lysate; and

[0077] (c) if at least three markers in the protein expression profile of the resected tumor or biopsy sample match the protein expression profile of the melanoma cell lysate, co-culturing the dendritic cells with the melanoma cell lysate to activate the dendritic cells and administering to the subject a composition comprising the activated dendritic cells;

[0078] or

[0079] (d) if at least three of the markers in the protein expression profile of the resected tumor or biopsy sample do not match the protein expression profile of the melanoma cell lysate, co-culturing the dendritic cells with the autologous tumor or biopsy lysate to activate the dendritic cells and administering to the subject a composition comprising autologous activated dendritic cells.

[0080] In additional embodiments, a method of generating immature dendritic cells comprises:

[0081] (a) isolating CD14+ monocytes from a subject;

[0082] (b) overexpressing CD40L, CXCL13 and CD93 in the isolated CD14+ monocytes; and

[0083] (c) differentiating the CD14+ monocytes into immature dendritic cells in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1A -D is a diagram and schematic diagram showing various expression and activation processes in certain immune cells. Figure 1A It is a diagram showing the mechanism of action of CD40L expression. Figure 1B It is a diagram showing the immune recruitment process of CXCL13 expression. Figure 1C is a schematic diagram showing the cancer-immunity cycle. Figure 1D is a schematic diagram showing the various stages of dendritic cell maturation.

[0085] Figure 2A -F is a flow cytometric image of CliniMACS-isolated CD14+ monocytes for phenotyping and purity assessment.

[0086] Figure 3A -P is a flow cytometry image showing the phenotyping of an immature allogeneic dendritic cell (alloDC) sample. This indicates that the sample has been analyzed for the following markers: Negative lineage: (CD3-, CD56-, CD19-, CD66b-), CD45+, CD14-, CD40L+, CXL13+, CD1c+, CD11b+, CD11c+, HLA-DR+, CD86+, CD80low, CD83-, CD16low, CD33+, CD163-, CD206+, CD209, CD40L.

[0087] Figure 4A - B is a flow cytometry image showing that immature recombinant alloDCs exhibit phagocytic ability.

[0088] Figure 5A -D is an image of a fluorescent indicator of allogeneic T cell proliferation stimulated by alloDCs shown by staining with dilute carboxyfluorescein succinimidyl ester (CFSE).

[0089] Fig. 6A -B is before treatment with autologous tumor-loaded alloDC CD40L+ vaccine ( Fig. 6A ) and six months after treatment ( Figure 6B ) of a patient with colorectal tumor.

[0090] Fig. 7A-D is before treatment with autologous tumor-loaded alloDC CD40L+ vaccine ( Fig. 7A , Figure 7C ) and six months after treatment ( Figure 7B and Fig.7D ) with multiple liver metastases caused by colorectal tumors.

[0091] Fig. 8A -D is before treatment with autologous tumor-loaded alloDC CD40L+CXCL13+ vaccine ( Fig. 8A , Figure 8C ) and six months after treatment ( Figure 8B and Fig.8D ) in a patient with a metastatic invasive ductal breast tumor.

[0092] Fig. 9 is a schematic diagram showing an exemplary overview for administering the recombinant alloDC vaccines described herein.

[0093] Fig.10 is a graph showing T cell proliferation in co-culture of recombinant DCs expressing CD40L, CD40L+CXCL13, or CD40L+CXCL13+CD93 triple expression, wherein triple expression showed the greatest amount of T cell proliferation.

[0094] Fig.11 is a graph showing NK cell proliferation in co-culture of recombinant DCs expressing CD40L, CD40L+CXCL13, or CD40L+CXCL13+CD93 triple expression, wherein triple expression showed the greatest amount of NK cell proliferation.

[0095] Fig.12 This is an image of cell sorting on day 4 to determine B cell activation by determining CD69 expression levels in CD19+ cells by flow cytometry. DETAILED DESCRIPTION

[0096] This embodiment part relates to methods and compositions related to dendritic cells transduced with CD40L, CXCL13 and / or CD93. CD40L, CXCL13 and / or CD93 can be human. In some embodiments, CD40L, CXCL13 and / or CD93 are rodents (mouse or rat) or pigs. Cells comprising heterologous expressed proteins can be referred to as recombinant cells. The composition can be an independent recombinant DC (dendritic cell) composition, or in alternative embodiments, the recombinant DC cells can be activated by antigens by contacting the cells with tumor lysates, which can also be referred to as "loading", or alternatively, with allogeneic ("general") tumor lysate loading, as a vaccine and / or immune adjuvant for subjects with solid tumors and malignancies. In some embodiments, the tumor lysate is allogeneic relative to the source of the dendritic cells. In some embodiments, the tumor lysate is autologous relative to the source of the dendritic cells.

[0097] Dendritic cells (DCs) are professional antigen-processing cells. They have many receptors that enhance antigen uptake, and they are specialized in converting these antigens into MHC-peptide complexes that can be recognized by lymphocytes.

[0098] Although not wishing to be bound by theory, the compositions and methods of the present invention are based, at least in part, on the ability of recombinant (or transgenic) dendritic cells to establish a mixed leukocyte reaction (MLR) at the injection site. When tested in the well-known clinical assay - MLR, recombinant alloDC as described herein are primary stimulators and exceptionally potent. In some embodiments, the DC may be allogeneic to the subject. In such embodiments, the DC may be referred to as alloDC. AlloDC are allogeneic cells relative to the subject to which they are administered.

[0099] In some embodiments, DCs are transduced with CD40L and / or CXCL13 to maximize the attraction of the patient's T cells and B cells and generate a cascade of anti-tumor cellular and humoral immune responses. In some embodiments, DCs are transduced with CD40L and CXCL13. In some embodiments, DCs are transduced with CD40L, CXCL13, and CD93. In some embodiments, DCs are transduced with CD40L and CD93. In some embodiments, DCs are transduced with CXCL13 and CD93. In some embodiments, cells are not transduced with CD93. In some embodiments, DCs do not heterologously express CD93.

[0100] In an optional further embodiment, transduction of CD40L+ and CXCL13+ alloDCs with CD93 promotes cross-talk between allogeneic DCs and host DCs, thereby generating stable and sustained host anti-tumor immunogenicity.

[0101] In some embodiments, human CD14+ monocytes are transfected with a recombinant vector encoding CD40L and CXCL13 (and optionally CD93) or any combination thereof as described herein to stably express them on human monocytes. Positively transduced monocytes can be selected, and then the monocytes can be differentiated into immature and / or mature DCs in vitro. For patients with malignant tumors that have been resected and biopsied, autologous tumor lysates can be produced, and the tumor lysates can be presented to immature recombinant alloDCs (recombinant im alloDCs) for processing autologous tumor lysates by recombinant im alloDCs. In some embodiments, the protein expression profiles of resected tumor samples and biopsy samples are screened. If the expression profile shows that at least 3 fragments are shared with GMP-MCV, recombinant imDCs are loaded with GMP-MCV before DC maturation in vitro. For any patient who cannot be resected and cannot be biopsied, recombinant CD40L+CXCL13+alloDCs (or optionally further including CD93+) can be administered to patients as immune adjuvants without antigen loading. Using this CD40L+CXCL13+(optional)CD93 AlloDC approach, patients with advanced cancer can mount new anti-tumor cellular and humoral immune responses against tumor antigens and, without being constrained by any specific mechanism, enhance immune responses.

[0102] Another tumor lysate that can be used to activate recombinant DCs includes DDM-1.7 and / or DDM-1.13. Descriptions of DDM-1.7 and DDM-1.13 can be found in U.S. Pat. Nos. 7,771,998 and 7,723,107 (including publicly available deposit information: ECACC 01112339 and ECACC 01112338, respectively), each of which is incorporated herein by reference in its entirety.

[0103] As used herein, immature dendritic cells are those having the following marker characteristics: CD1a positive, CD14 negative, and CD83 negative / low.

[0104] In some embodiments, the source of dendritic cells can be from donor peripheral blood, and include dendritic cells derived from CD14+ monocytes or CD34+ cells. In some embodiments, the donor is different from the subject treated with the dendritic cells and compositions described herein.

[0105] In recent years, it has been recognized that an effective way to deliver antigens to T cells, particularly to naive T cells, is through dendritic cells. Dendritic cells (DCs) are the most effective antigen presenting cells, and DC-based immunotherapy has been used in different settings to treat cancer ((Kugler et al., 2000, Nat. Med., v.6, pp.332-336; Nestle et al., 1998, Nature (Med.), v.4, pp.328-332; Thurner et al., 1999, J. Exp. Med., v.190, pp.1669-1678)) demonstrated the high efficacy of this immune approach.

[0106] One of the unique properties of DCs is their ability to take up exogenous proteins by endocytosis, which are then processed and presented on their surface as peptide epitopes together with MHC class I antigens. Antigen-presenting dendritic cells can be recognized by cytotoxic T cells. This property is important when tumor cell antigens are applied in the form of exogenously added tumor lysates or apoptotic bodies. Based on a comparison of immature DCs with mature DCs, it is believed that high endocytic activity is associated with the immature state of DC differentiation (Sallusto et al., 1995, J. Exp. Med., v. 182, pp. 389-400).

[0107] In some embodiments, the recombinant dendritic cells are treated with interferon-α (IFN-α) and / or 5'-aza-2'deoxycytidine (Aza) before or during loading of the recombinant dendritic cells with tumor cell lysate and / or antigen. See, https: / / doi.org / 10.1101 / 531616.

[0108] In some embodiments, purified human CD14+ monocytes are transduced with a recombinant vector, such as an adenoviral or lentiviral vector comprising human CD40L and CXCL13 and optionally a CD93 construct, allowing the recombinant molecules to be expressed on purified human monocytes. Positively transduced monocytes are then selected and then differentiated in vitro into immature DCs (imDCs) and are referred to as recombinant allogeneic DCs (recombinant alloDCs). In some embodiments, expression is stable expression. In some embodiments, expression is overexpression. "Overexpression" refers to a level that exceeds the natural expression level by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500%. The natural expression level refers to the expression level of cells that have not been transduced by a vector to express one or more heterologous proteins of interest, such as CD40L, CXCL13, and / or CD93.

[0109] The various stages and maturation / activation factors of dendritic cells are described in Figure 1D (see Front Immunol. 2013; 4: 438, which is incorporated herein by reference in its entirety).

[0110] In some embodiments, the recombinant DC is matured by contacting the cells with maturation factors after loading tumor cell lysates and / or antigens. These maturation factors include at least: IL-1β, IL-6, TNF-α and PGE2 or any combination thereof. In some embodiments, the recombinant DC is matured by contacting the cells with maturation factors before contacting with tumor cell lysates and / or antigens. In some embodiments, DC is matured with maturation factors and is not contacted with tumor cell lysates and / or antigens. In some embodiments, recombinant dendritic cells are cultured or incubated with cytokines selected from the group consisting of: IL-4, GM-CSF, IL-13, IFN-γ, Flt-31, SCF and TNF-α.

[0111] abbreviation

[0112] WBC: White blood cells

[0113] IPA: 70% Isopropyl Alcohol

[0114] QA: Quality Assurance

[0115] QC: Quality Control

[0116] Vol.

[0117] RT: Room temperature

[0118] DC: Dendritic cell

[0119] imDC: immature dendritic cell

[0120] mDC: mature dendritic cell

[0121] AlloDC: allogeneic dendritic cells

[0122] MCV: MalCancerVac

[0123] GM-CSF: Granulocyte-macrophage colony-stimulating factor

[0124] IL-4: interleukin 4

[0125] IL-1β: interleukin 1β

[0126] IL-15: Interleukin 15

[0127] TNFα: tumor necrosis factor alpha

[0128] pIC: polyinosinic:polycytidylic acid

[0129] IFN-α: interferon alpha

[0130] IFN-γ: interferon gamma

[0131] CXCL13: CXC motif chemokine ligand 13

[0132] CD40L: CD40 ligand

[0133] MAGE: melanoma-associated antigen

[0134] MOI: Multiplicity of Infection

[0135] As used in this application and the claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise. In addition, the term "include" means "comprise."

[0136] The terms "patient" and "subject" are interchangeable and can be understood to mean any living organism that can be treated with the compounds of the present invention. Thus, the terms "patient" and "subject" can include, but are not limited to, any non-human mammal, primate, or human. In some embodiments, the "patient" or "subject" is a mammal, such as a mouse, rat, other rodent, rabbit, dog, cat, pig, cattle, sheep, horse, primate, or human. In some embodiments, the patient or subject is an adult, child, or infant. In some embodiments, the patient or subject is a human.

[0137] Unless the context of the present disclosure dictates otherwise or is inconsistent with such an interpretation, the term "about" when immediately preceding a numerical value means a range of plus or minus 10% of the value, e.g., "about 50" means 45 to 55, "about 25,000" means 22,500 to 27,500, etc. For example, in a numerical list such as "about 49, about 50, about 55," "about 50" means a range extending to less than half of the interval between the preceding and succeeding values, e.g., greater than 49.5 to less than 52.5. In addition, the phrases "less than about" or "greater than about" should be understood in light of the definition of the term "about" provided herein. In addition, if a range is written as "about X to Y," "about" will modify both the X and Y values ​​unless the context dictates otherwise.

[0138] As used herein, the terms "administer," "administering," or "administration" refer to the direct administration of a compound, cell, composition, or pharmaceutical composition, which may also be referred to as an agent of interest. In some embodiments, the composition is sterilized or filtered to remove any viral or bacterial particles.

[0139] The terms "co-administration" and the like are meant to encompass administration of multiple therapeutic agents of choice to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different routes of administration, or at the same or different times.

[0140] A "therapeutically effective amount" of a composition is an amount sufficient to achieve the desired effect, i.e., to alleviate, prevent or improve an undesirable condition, disease or symptom in a patient. The activities contemplated by the present method may appropriately include therapeutic and / or prophylactic treatments. The specific dose may be determined by the specific circumstances surrounding the case, including, for example, the therapeutic agent to be administered, the route of administration, and the condition to be treated. The effective amount to be administered may be determined by a physician based on the relevant circumstances, including the condition to be treated, the choice of therapeutic agent to be administered, and the route of administration selected.

[0141] The term "inhibit" includes the administration of a therapeutic agent of the embodiments herein to prevent the onset of symptoms, alleviate symptoms, or eliminate the disease, disorder, or condition.

[0142] By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the therapeutic agent and not deleterious to the recipient thereof.

[0143] As used herein, the terms "treat," "treated," or "treating" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to inhibit, prevent, or slow (mitigate) an undesirable physiological condition, disorder, or disease, or to ameliorate, inhibit, or otherwise obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, improvement or alleviation of symptoms; reduction in the extent of a condition, disorder, or disease; stabilization (i.e., not worsening) of a condition, disorder, or disease state; delaying the onset of a condition, disorder, or disease or slowing its progression; alleviating a condition, disorder, or disease state; and remission (whether partial or complete), whether the condition, disorder, or disease is detectable or undetectable, or is enhanced or improved. Treatment includes inducing a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to the expected survival if not receiving treatment.

[0144] The transitional term "comprising," which is synonymous with "including," "comprising," or "characterized by," is inclusive or open-ended and does not exclude other unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any elements, steps, or ingredients not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. In embodiments or claims in which the term "comprising" is used as a transitional phrase, it is also contemplated that the term "comprising" may be replaced with the terms "consisting of" or "consisting essentially of" in such embodiments.

[0145] Disclosed herein are methods and compositions for treating cancer by administering dendritic cells expressing heterologous proteins to elicit an immune response. In some embodiments, the DC heterologously express CD40L and CXCL13. In some embodiments, the DC heterologously express CD40L, CXCL13, and CD93. In some embodiments, the DC heterologously express CD40L and CD93. In some embodiments, the DC heterologously express CXCL13 and CD93. In some embodiments, the DC does not heterologously express CD93.

[0146] In some embodiments, the dendritic cells will be allogeneic to the subject to which they are administered, and thus not restricted by HLA expression. The allogeneic nature of the cells relative to the subject is an additional advantage that enhances the improved anti-tumor effect.

[0147] In some embodiments, the dendritic cells can be autologous to the subject to which they are administered.

[0148] Thus, while not wishing to be bound by theory, aspects of the invention relate to providing dendritic cells expressing heterologous proteins (e.g., recombinant alloDC) to patients to elicit an improved immune response to cancer (tumor) in patients. The methods and compositions described herein have many advantages over previous compositions and methods, including no co-culture on feeder cells, and minimizing the risk of contamination of cell compositions due to the short in vitro culture / incubation time of recombinant alloDC and the optional loading of autologous or "general (off the shelf)" antigens, and easy and rapid preparation of cell compositions and their delivery to patients, while the side effects of the cell compositions are very small, typically only level 1 (or less side effects). These recombinant allogeneic DC cells and compositions provide additional benefits of eliciting cellular and humoral immune responses in patients. In addition, the host's allogeneic response to HLA-mismatched DCs will amplify tumor-specific immune responses. These methods can also be used to provide a "vaccine effect" to train host, patient immune cells to recognize and kill cancer targets in a similar manner, and enhance host cellular and humoral responses. In some embodiments, the subject receiving the cells sees the cells as foreign and will increase its immune response against the cells, which can be referred to as rejecting the cells. This "rejection" can amplify the subject's tumor-specific immune response.

[0149] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be a complete immunoglobulin derived from a natural source or a recombinant source, and can be an immunoreactive portion of a complete immunoglobulin. Antibodies can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0150] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0151] The term "antigen" as used herein is defined as a molecule that causes an immune response. This immune response may involve the production of antibodies, or the activation of specific immunological component cells, or both. Antigens can also be used in vitro to activate DCs, which is equivalent to an immune response. The technician will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. In addition, the antigen can be derived from recombinant or genomic DNA. The technician will understand that any DNA including a nucleotide sequence or a partial nucleotide sequence encoding a protein that causes an immune response thus encodes the term "antigen" used herein. In addition, the skilled person will understand that the antigen does not have to be encoded only by the full-length nucleotide sequence of the gene. It is obvious that embodiments include but are not limited to using partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to induce a desired immune response. Moreover, the technician will understand that the antigen does not need to be encoded by a "gene" at all. Obviously, the antigen can be produced synthetically or can be derived from a biological sample. Such a biological sample can include but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid. As described herein, the antigen can be a tumor cell lysate. Examples of tumor cell lysates include but are not limited to those prepared from tumor biopsies or tumor resections as described herein. Methods for preparing lysates are known, and any method can be used.

[0152] The term "cancer" as used herein is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the blood and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0153] Cancers that can be treated include tumors that are not vascularized or not substantially vascularized as well as vascularized tumors. Types of cancers that can be treated with the recombinant dendritic cells described herein include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0154] Solid tumors are abnormal tissue masses that usually do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial tumors, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, Liver cancer, bile duct cancer, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors such as gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme) astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, Schwannomas, craniopharyngiomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases.

[0155] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of recombinant cells and therapeutic compositions to prevent tumors from occurring in the first place.

[0156] As used herein, the term "autologous" means any material derived from the same individual that is subsequently reintroduced into that individual.

[0157] "Allogeneic" refers to different animals derived from the same species, and then the sample is introduced into the sample (graft, cell or cell group) of the same species. In some embodiments, the recombinant dendritic cell is completely HLA mismatched relative to the subject receiving the recombinant dendritic cell, which is different from the situation of kidney and bone marrow transplantation in which matching HLA-A, -B and -DR is beneficial to graft survival. In the method of the present invention for treating cancer and / or increasing and / or improving immune response, the recombinant dendritic cells expressing CD40L, CXCL13 and optionally CD93 can attract T cells, and for example generate humoral immune response, for targeting cancer cells and combining with them, and killing them, releasing tumor cell antigens. This cycle is repeated with allogeneic recombinant dendritic cells until they are killed by host cell immune response. For example, this can occur within about 4 to about 7 days (and may be within the extended range of about 4 to about 15 days). Therefore, for allogeneic recombinant DC with such a short life span, the risk of GVHD (graft versus host disease) is reduced or absent. However, in the process of recognizing and killing tumor cells and releasing antigens over a span of about 4 to about 7 days, in some embodiments, allogeneic cells can be used to immunize host dendritic cells and other immune cells to recognize and kill tumor cells. See also the article Cancer Sci. 2019 Jan; 110 (1): 16-22, which is incorporated herein by reference in its entirety. Inducing anti-tumor immunity is a cyclic process that can propagate itself. It can amplify and extend T cell responses against cancer cells. When the target cell (cancer cell) is eradicated, it itself also contains several inhibitory factors to stop the cycle. As Figure 1C As shown in Figure 2, the cycle can be divided into seven steps, starting with the release of cancer cell antigens by cancer cells and ending with the killing of cancer cells. APC, antigen presenting cell; DC, dendritic cell.

[0158] A "disease" is a state of health in a subject in which the subject is unable to maintain homeostasis and in which the animal's health will continue to deteriorate if the disease is not alleviated. In contrast, a "disorder" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's health is less favorable than it would be in the absence of the disorder. Without treatment, a disorder will not necessarily cause a further decline in the subject's health.

[0159] "Encoding" refers to a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA used as a template to synthesize other polymers and macromolecules with a determined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a determined amino acid sequence in a biological process, and the inherent characteristics and biological properties resulting therefrom. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes a protein. The coding strand whose nucleotide sequence is consistent with the mRNA sequence and is usually provided in the sequence table and the non-coding strand used as a transcription template for a gene or cDNA can both be referred to as encoding a protein or other product of the gene or cDNA. The vector encoding a protein of interest used herein refers to a vector comprising a nucleotide sequence encoding the one or more proteins.

[0160] The term "exogenous" as used herein refers to any material introduced from outside an organism, cell, tissue or system or produced outside an organism, cell, tissue or system. Proteins referred to as heterologous proteins are expressed by exogenous materials (vectors, nucleotide sequences, etc.) that have been introduced into an organism, cell, tissue or system. For the avoidance of doubt, heterologous proteins, heterologous vectors or heterologous nucleotide molecules are different from heterologous proteins, heterologous vectors or heterologous nucleotide molecules that may be present in the natural unmodified genome of an organism, cell, tissue or system. Heterologous proteins, vectors or nucleotide sequences that may have sequences identical or similar to sequences already present in an organism, cell, tissue or system are expressed by positions or sequences that are different from the natural sequences found in the genome of an organism, cell, tissue or system.

[0161] The term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0162] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other expression elements may be provided by the host cell or by an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0163] "Homology" or "homology" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position is occupied by the same base or amino acid monomer subunit in both comparison sequences, for example, if each position in the two DNA molecules is occupied by adenine, the molecules are homologous at that position. The homology percentage between the two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions in the two sequences are matched or homologous, the two sequences are 60% homologous. For example, the DNA sequence ATTGCC has 50% homology with TATGGC. Typically, the comparison is performed when the two sequences are aligned to give maximum homology.

[0164] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE.

[0165] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment, such as a host cell. An "isolated" biological component (such as a nucleic acid, protein, or cell) has been substantially separated or purified away from other biological components (such as cell debris, other proteins, nucleic acids, or cell types). "Isolated" biological components include those components purified by standard purification methods.

[0166] Prevent, treat, or alleviate a disease: "Preventing" a disease means inhibiting the full development of a disease. "Treatment" means therapeutic intervention to reduce the signs or symptoms of a disease or pathological condition after it has begun to develop. "Allow" means a decrease in the number or severity of the signs or symptoms of a disease.

[0167] Chemotherapy includes treatment with a therapeutically effective chemical agent (such as a cytotoxic agent) to treat diseases characterized by abnormal cell growth, such as tumors, neoplasms, cancer, and psoriasis. Examples of chemotherapy include, but are not limited to, those described herein.

[0168] Recombination as used herein generally refers to the following: a recombinant nucleic acid or protein is a nucleic acid or protein having a sequence that does not occur naturally or that is made by the artificial combination of two otherwise isolated sequence fragments. This artificial combination is usually achieved by chemical synthesis or by artificial manipulation of isolated nucleic acid fragments, such as by genetic engineering techniques.

[0169] As used herein, the following abbreviations for common nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" is uridine.

[0170] The term "leukocyte" or "white blood cell" as used herein refers to any immune cell, including monocytes, neutrophils, eosinophils, basophils and lymphocytes. The term "lymphocyte" as used herein refers to cells typically found in the lymph, and includes natural killer cells (NK cells), T cells and B cells. Those skilled in the art will recognize that the immune cell types listed above can be divided into further subgroups.

[0171] As used herein, the term "tumor infiltrating leukocytes" refers to leukocytes present in solid tumors.

[0172] The term "blood sample" as used herein refers to any sample prepared from blood, such as plasma, blood cells separated from blood, and the like.

[0173] As used herein, the term "purified sample" refers to any sample in which one or more cell subpopulations are enriched. A sample can be purified by removing or isolating cells based on characteristics, such as size, protein expression, etc.

[0174] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions and additional agents.

[0175] Generally, the property of suitable carrier or vehicle for sending will depend on the specific mode of administration adopted.For example, parenteral preparations generally include injectable fluids, and the injectable fluids include pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solution, dextrose aqueous solution, glycerol etc. as vehicles.For solid compositions (such as powder, pill, tablet or capsule form), conventional nontoxic solid carriers may include for example pharmaceutical grade mannitol, lactose, starch or magnesium stearate.Except biological neutral carriers, pharmaceutical compositions to be administered may include a small amount of nontoxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers etc., for example sodium acetate or sorbitan monolaurate.

[0176] In some embodiments, the compositions, whether they are solutions, suspensions or other similar forms, may include one or more of the following: DMSO, sterile diluents, such as water for injection, saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerol, propylene glycol or other solvents that may be used as a solvent or suspending medium; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose.

[0177] Immunodeficiency used herein means lacking at least one basic function of the immune system. "Immunodeficiency" subjects (such as people) used herein are subjects lacking a specific component of the immune system or lacking a specific component of the immune system (such as, for example, B cells, T cells, NK cells or macrophages). In some cases, immunodeficiency subjects include one or more genetic changes that prevent or suppress the development of functional immune cells (such as B cells, T cells or NK cells). In some instances, the genetic changes are in IL17 or IL17 receptors.

[0178] As used herein, immunosuppressive refers to reduced immune system activity or function. For example, a subject may be immunosuppressed due to treatment with an immunosuppressant compound or as a result of a disease or illness (e.g., immunosuppression due to HIV infection or due to a genetic defect). In some cases, immunosuppression occurs as a result of a genetic mutation that prevents or inhibits the development of functional immune cells, such as T cells.

[0179] Examples of cancers and proliferative disorders that can be treated with effective amounts of the recombinant dendritic cell compositions and related methods described herein include, but are not limited to, fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcomas, angiosarcomas, endotheliosarcomas, Ewing's tumors, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, liver cancer, Wilms' tumor, cervical cancer, uterine cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, oligodendroglioma, melanoma, neuroblastoma, retinoblastoma, dysplasia, and hyperplasia. Treatment and / or prevention of cancer include, but are not limited to, alleviating one or more symptoms associated with cancer, inhibiting or reducing the progression of cancer, promoting regression of cancer, and / or promoting an immune response.

[0180] In certain other embodiments, a "therapeutically effective amount" is an amount of a recombinant dendritic cell composition that results in a reduction of at least 2.5%, at least 5%, at least 10%, at least 15%, at least 25%, at least 35%, at least 45%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% in tumor growth or cancer spread in a patient or animal administered the compositions and cells described herein, relative to tumor growth or cancer spread in a patient (or animal) or group of patients (or animals) not administered the compositions and cells of the invention.

[0181] In some embodiments, the recombinant dendritic cell composition can be administered simultaneously with the antimicrobial, antiviral, and other therapeutic agents. Alternatively, the recombinant dendritic cell composition can be administered at a selected time prior to the time of administration of the antimicrobial, antiviral, and other therapeutic agents.

[0182] Combined with antibodies

[0183] In some embodiments, the recombinant dendritic cell composition can be administered simultaneously with an antibody specific to a selected cancer type. Alternatively, the recombinant dendritic cell composition can be administered at a selected time before the administration of an antibody specific to a selected cancer type. Antibodies specific to a selected cancer type include any antibody approved for the treatment of cancer. Examples include trastuzumab (Herceptin) for breast cancer, rituximab (Rituxan) for lymphoma, and cetuximab (Erbitux) for head and neck squamous cell carcinoma.

[0184] Additional examples of such antibody agents include inhibitors of PD-1 or PD-L1 (B7-H1), such as anti-PD-1 antibodies, including nivolumab (nivolumab from Bristol-Myers Squibb) and pembrolizumab / pembrolizumab, also known as MK-3475 (Keytruda from Merck), pidilizumab (Curetech), AMP-224 (Amplimmune), and anti-PD-L1 antibodies, including MPDL3280A (Roche), MDX-1105 (Bristol Myer Squibb), MEDI-4736 (AstraZeneca), and MSB-0010718C (Merck). Other checkpoint inhibitors include antagonists of CTLA-4, such as anti-CTLA-4 antibodies. Exemplary anti-CTLA4 antibodies are Yervoy®, commercially available from Bristol-Myers Squibb. TM(Ipilimumab). Other exemplary CTLA-4 antibodies include Tremelimumab (Pfizer), Ticilimumab (AstraZenec), or AMGP-224 (Glaxo Smith Kline). In some cases, a combination of any two of these antibodies may also be specified.

[0185] In some embodiments, dendritic cells are isolated from various sources, including peripheral blood mononuclear cells.

[0186] "Peripheral blood mononuclear cells", "PBMC" or "monocytes" refer to mononuclear cells isolated from peripheral blood and generally used for anti-cancer immunotherapy. Peripheral blood mononuclear cells can be obtained from human blood collected using known methods, such as the Ficoll-Hypaque density gradient method.

[0187] According to an exemplary embodiment, "peripheral blood mononuclear cells" can be obtained from any suitable person. In some embodiments, the source of donor cells, including the source of peripheral blood mononuclear cells as described herein, can be allogeneic relative to the recipient patient to isolate dendritic cells for use in the cancer treatment and immunostimulatory methods described herein. Therefore, in some embodiments, the donor inhibitory ligand is HLA-mismatched with the patient (e.g., host).

[0188] In alternative embodiments, the source of donor cells, including the source of peripheral blood mononuclear cells as described herein, is autologous to the recipient patient to isolate dendritic cells for use in the cancer treatment and immunostimulatory methods described herein.

[0189] Cell source

[0190] PBMCs can be isolated by Ficoll-Hypaque density gradient centrifugation of samples obtained from discarded, de-identified leukocyte reduction filters (American Red Cross) or from blood donations from healthy volunteers with informed consent. Descriptions of cell populations, sources, and methods for selecting or enriching desired cell types can be found in, for example, U.S. Patent No. 9,347,044. Cell populations used in the methods for treating mammals described herein must be species-matched, such as human cells. Cells can be obtained from animals, such as human patients. If cells are obtained from animals, they may first be established in culture, such as by transformation; or more preferably, they may have been subjected to a preliminary purification method. For example, cell populations can be manipulated by positive selection or negative selection based on the expression of cell surface markers; stimulated in vitro or in vivo with one or more antigens; treated in vitro or in vivo with one or more biological modifiers; or all or any combination of these. In an illustrative embodiment, negative selection is performed on the cell population to exclude non-T cells and / or specific T cell subsets. Negative selection can be performed based on cell surface expression of a variety of molecules, including B cell markers such as CD19 and CD20; monocyte marker CD14; NK cell marker CD56. Alternatively, cell populations can be negatively selected to exclude non-CD34 + Hematopoietic cells and / or specific hematopoietic cell subsets. Negative selection can be performed based on cell surface expression of a variety of molecules, such as a combination of antibodies (e.g., CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a) that can be used to separate other types of cells, for example, via MACS or columns.

[0191] Cell populations include peripheral blood mononuclear cells, whole blood or portions thereof containing mixed populations, spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells obtained by leukapheresis, biopsy tissues, lymph nodes, such as lymph nodes drained from lymphatic vessels. Suitable donors include immunized donors, non-immunized (natural) donors, processed or unprocessed donors. "Processed" donors are donors that have been exposed to one or more biological modifiers. "Unprocessed" donors have not been exposed to one or more biological modifiers.

[0192] Methods for obtaining cell populations including monocytes (to be matured into DCs) or dendritic cells are well known in the art. For example, peripheral blood mononuclear cells (PBMCs) can be obtained as described according to methods known in the art. Examples of such methods are described in, for example, Nair, Smita et al. Current protocols in immunology vol. Chapter 7 (2012): Unit 7.32. doi: 10.1002 / 0471142735.im0732s99.

[0193] Cell samples can also be obtained from the subject, and then enriched to obtain the desired cell type. For example, PBMC can be separated from blood, as described herein. Countercurrent centrifugation (elutriation) can be used to enrich monocytes or dendritic cells from PBMC. Cells can also be separated from other cells using the following multiple techniques: such as separation and / or activation of antibodies combined with epitopes on the cell surface of the desired cell type, for example, some T cell separation kits use antibody-conjugated beads to both activate cells and then allow column separation with the same beads. Another method that can be used includes using antibodies for cell surface markers to carry out negative selection, to selectively enrich specific cell types when cells are not activated by receptor engagement.

[0194] Bone marrow cells (BM cells) can be obtained from the ilium, femur, tibia, spine, ribs or other bone marrow cavities. Bone marrow can be removed from the patient and separated by various separation and washing procedures. Known procedures for separating bone marrow cells include the following steps: a) centrifuging the bone marrow suspension into three fractions and collecting the intermediate fraction or buffycoat; b) centrifuging the buffycoat fraction from step (a) in a separation fluid, usually Ficoll (a trademark of Pharmacia Fine Chemicals AB), and collecting the intermediate fraction containing bone marrow cells; c) washing the collected fractions from step (b) to recover re-infusible bone marrow cells. In addition, BM cells can be collected by mobilization followed by leukapheresis.

[0195] Dendritic cells (DCs) are sentinel antigen-presenting cells of the immune system. These cells have the ability to acquire antigenic material from their environment and subsequently elicit a vigorous immune response, and are ideal candidates for providing vaccines for cancer immunotherapy. Dendritic cells comprise a heterogeneous population of cells with distinct morphologies and a wide range of tissue distribution. DCs display cell surface markers such as CD1c + 、CD14 + 、CD141 + 、CD16 + or HLA-DR +.

[0196] In particular, dendritic cells act as antigen presenting cells by endocytosis of exogenous proteins, which are then processed and presented as epitopes on their surface together with MHC class I and class II antigens. Antigen presenting dendritic cells can be recognized by cytotoxic T cells and T helper cells. The maturity state of dendritic cells is crucial for their phagocytic / endocytic activity. Immature dendritic cells are generally the most effective cells for antigen loading.

[0197] Immature dendritic cells refer to dendritic cells in which the expression of certain cell markers CD1a, CD14 and CD83 is characterized by: high expression of CD1a (more than 50% of DCs in the population are CD1a positive), no expression or low expression of CD14 (less than 15% of DCs in the population are CD14 positive) and low expression of CD83 (less than 25% of DCs in the population are CD83 positive).

[0198] In some embodiments, DCs are obtained from any tissue in which they reside, including non-lymphoid tissues, such as the epidermis of the skin (Langerhans cells) and lymphoid tissues, such as the spleen, bone marrow, lymph nodes and thymus, and the circulatory system including blood and peripheral blood. Since DCs appear in low numbers in any tissue in which they reside, DCs can be enriched or separated for use. Any of a number of procedures including repeated density gradient separations, positive selections, negative selections or combinations thereof can be used to obtain enriched DC populations. Once DCs are obtained, they can be cultured in appropriate culture media to expand cell populations and / or to maintain DCs in a state of optimal antigen uptake, processing and presentation.

[0199] In some embodiments, the DCs can be autologous to the subject with cancer, i.e., the DCs administered to the subject can be isolated from the same subject. In other embodiments, the DCs can be allogeneic to the subject with cancer. In some embodiments, the DCs disclosed herein can be any subtype, such as myeloid DCs, plasmacytoid DCs, interstitial DCs, lymphoid tissue resident DCs, follicular DCs, CD14 + DC, etc.

[0200] In some embodiments, methods are provided for developing genetically engineered / recombinant dendritic cells expressing CD40L, CXCL13, and optionally CD93 that can be used for cancer treatment.

[0201] CD40L is a 35 kDa type II membrane protein and a member of the tumor necrosis factor (TNF) gene family that is expressed on the surface of T cells after antigen recognition. CD40L is critically involved in the activation of T cells necessary for inducing effective protective immunity against tumor self-antigens. (See, Front Immunol. 2011; 2:31, which is incorporated herein by reference in its entirety). The complete sequence information of human CD40L can be found in NCBI, Gene ID: 959.

[0202] CXCL13 is a chemokine expressed in follicular stromal cells, macrophages in the peritoneum and pleural cavity of lymphoid organs and myeloid dendritic cells. It has been shown that it mainly binds to the G protein-coupled receptor CXCR5. CXCR5 is in certain subgroups of B cells and T cells (including subgroups of follicular helper T cells, circulating memory CD4 T cells, and other T cell groups that are not fully differentiated into T-helper 1 (Th1) or T-helper 2 (Th2). CXCL13 has been confirmed to have a physiological effect in B cells and T cells co-localization by: affecting the homing of self-reactive B1 cells to Peyer's patches and other inflammatory sites, and recruiting Th cells to secondary lymphoid organs for T-dependent antibody production and play a role. (See, Front Immunol.2016; 7:225, the full text of which is incorporated herein by reference). The complete sequence information of human CXCL13 can be found in NCBI, Gene ID:10563.

[0203] Human CD93 (hCD93) is a type 1 transmembrane glycoprotein located on chromosome 20, p11.21. This protein is involved in cell-cell interactions during B cell development and phagocytosis. CD93 is expressed in myeloid lineages, hematopoietic stem cells, NK cells, platelets, microglia, and endothelial cells. The complete sequence information of human CD93 can be found at NCBI, Gene ID: 22918.

[0204] In some embodiments, the dendritic cells include one or more heterologous nucleic acid molecules encoding CD40L and / or CXCL13. The heterologous nucleic acid molecules encoding CD40L can be from any source, such as human, mouse, rat or pig or any other mammal. The human CD40L nucleic acid sequence is disclosed as SEQ ID NO: 1. Similarly, the heterologous nucleic acid molecules encoding CXCL13 can be from any source, such as human, mouse, rat or pig or any other mammal. The human CXCL13 nucleic acid sequence is disclosed as SEQ ID NO: 2.

[0205] In some embodiments, the dendritic cells may further comprise a heterologous nucleic acid molecule encoding CD93. The heterologous nucleic acid molecule encoding CD93 may be from any source, such as human, mouse, rat or pig or any other mammal. The human CD93 nucleic acid sequence is disclosed as SEQ ID NO: 3.

[0206] However, due to the degenerate nature of the genetic code, the nucleic acid sequence encoding the protein may be other sequences. These nucleic acid sequences are non-limiting examples, and other nucleic acid sequences may be used.

[0207] The corresponding human amino acid sequences are as follows: human CD40L: SEQ ID NO: 4; human CXCL13: SEQ ID No: 5; and human CD93: SEQ ID NO: 6. In some embodiments, the protein includes conservative substitutions.

[0208] The following sequences may be used as reference throughout this application as appropriate:

[0209] In some embodiments, human CD40L is encoded by:

[0210] ATGATCGAAACATACAACCAAACTTCTCCCCGATCTGCGGCCACTGGACTGCCCATCAGCATGAAAATTTTTATGTATTTACTTACTGTTTTTCTTATCACCCAGATGATTGGGTCAGCACTTTTTGCTGTGTATCTTCATAGAAGGTTGGACAAGATAGAAGATGAAAGGAATCTTCATGAAGATTTTGTATTCATGAAAACGATACAGAGATGCAACACAGGAGAAAGATCCTTATCCTTACTGAACTGTGAGGAGATTAAAAGCCAGTTTGAAGGCTTTGTGAAGGATATAATGTTAAACAAAGAGGAGACGAAGAAAGAAAACAGCTTTGAAATGCAAAAAGGTGATCAGAATCCTCAAATTGCGGCACATGTCATAAGTGAGGCCAGCAGTAAAACAACATCTGTGTTACAGTGGGCTGAAAAAGGATACTACACCATGAGCAACAACTTGGTAACCCTGGAAAATGGGAAACAGCTGACCGTTAAAAGACAAGGACTCTATTATATCTATGCCCAAGTCACCTTCTGTTCCAATCGGGAAGCTTCGAGTCAAGCTCCATTTATAGCCAGCCTCTGCCTAAAGTCCCCCGGTAGATTCGAGAGAATCTTACTCAGAGCTGCAAATACCCACAGTTCCGCCAAACCTTGCGGGCAACAATCCATTCACTTGGGAGGAGTATTTGAATTGCAACCAGGTGCTTCGGTGTTTGTCAATGTGACTGATCCAAGCCAAGTGAGCCATGGCACTGGCTTCACGTCCTTTGGCTTACTCAAACTC(SEQ ID NO:1)。

[0211] In some embodiments, human CXCL13 is encoded by:

[0212] ATGAAGTTCATCTCGACATCTCTGCTTCTCATGCTGCTGGTCAGCAGCCTCTCTCCAGTCCAAGGTGTTCTGGAGGTCTATTACACAAGCTTGAGGTGTAGATGTGTCCAAGAGAGCTCAGTCTTTATCCCTAGACGCTTCATTGATCGAATTCAAATCTTGCCCCGTGGGAATGGTTGTCCAAGAAAAGAAATCATAGTCTGGAAGAAGAACAAGTCAATTGTGTGTGTGGACCCTCAAGCTGAATGGATACAAAGAATGATGGAAGTATTGAGAAAAAGAAGTTCTTCAACTCTACCAGTTCCAGTGTTTAAGAGAAAGATTCCC(SEQ ID NO:2)。

[0213] In some embodiments, human CD93 is encoded by:

[0214]

[0215] In some embodiments, human CD40L comprises the following amino acid sequence:

[0216] MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASS KTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL(SEQ ID NO:4).

[0217] In some embodiments, human CXCL13 comprises the following amino acid sequence:

[0218] MKFISTSLLLMLLVSSLSPVQGVLEVYYTSLRCRCVQESSVFIPRRFIDRIQILPRGNGCPRKEIIVWKKNKSIVCVDPQAEWIQRMMEVLRKRSSSTLPVPVFKRKIP (SEQ ID N: 5).

[0219] In some embodiments, human CD93 comprises the following amino acid sequence:

[0220] MATSMGLLLLLLLLLTQPGAGTGADTEAVVCVGTACYTAHSGKLSAAEAQNHCNQNGGNLATVKSKEEAQHVQRVLAQLLRREAALTARMSKFWIGLQREKGKCLDPSLPLKGFSWVGGGEDTPYSNWHKELRNSCISKRCVSLLLDLSQPLLPSRLPKWSEGPCGSPGSPGSNIEGFVCKFSFKGMCRPLALGGPGQVTYTTPFQTTSSSLEAVPFASAANVACGEGDKDETQSHYFLCKEKAPDVFDWGSSGPLCVSPKYGCNFNNGGCHQDCFEGGDGSFLCGCRPGFRLLDDLVTCASRNPCSSSPCRGGATCVLGPHGKNYTCRCPQGYQLDSSQLDCVDVDECQDSPCAQECVNTPGGFRCECWVGYEPGGPGEGACQDVDECALGRSPCAQGCTNTDGSFHCSCEEGYVLAGEDGTQCQDVDECVGPGGPLCDSLCFNTQGSFHCGCLPGWVLAPNGVSCTMGPVSLGPPSGPPDEEDKGEKEGSTVPRAATASPTRGPEGTPKATPTTSRPSLSSDAPITSAPLKMLAPSGSPGVWREPSIHHATAASGPQEPAGGDSSVATQNNDGTDGQKLLLFYILGTVVAILLLLALALGLLVYRKRRAKREEKKEKKPQNAADSYSWVPERAESRAMENQYSPTPGTDC(SEQ ID NO:6)。

[0221] In some embodiments, the protein is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous to the sequences provided herein. "Conservative amino acid substitutions" are amino acids in which the amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0222] Heterologous nucleic acid molecules can be introduced into dendritic cells by any known recombinant technology known in the art, such as liposome transfection, chemical transfection, transgenic DNA recombination, viral infection, transposon insertion, jumping gene insertion, microinjection, electroporation, gene gun penetration, and combinations thereof. In some embodiments, recombinant adenoviral vectors, recombinant adeno-associated vectors, recombinant retroviral vectors, recombinant lentiviral vectors, or combinations thereof can be used to introduce heterologous nucleic acid molecules.

[0223] In some embodiments, a method for expressing desired genes-CD40L, CXCL13 and CD93 can be used. Such a method includes delivering CRISPR / Cas systems, TALEN and zinc finger nucleases. For example, TALEN and CRISPR / Cas systems can be used to insert specific transcriptional regulatory elements upstream and downstream of natural genes. TALEN and CRISPR / Cas9 can generate single-stranded or double-stranded DNA breaks at specific sites. This can stimulate homology-directed repair (HDR) from an exogenous template, thereby allowing accurate insertion of transcriptional regulatory elements to activate natural genes. CRISPR / Cas nucleases can be delivered by using any gene delivery vector, such as adenoviral vectors, adeno-associated vectors, retroviral vectors, lentiviral vectors or combinations thereof.

[0224] In some embodiments, the genetically engineered DCs disclosed herein can be cultured in conventional nutrient media under environmental conditions (such as temperature, pH, etc.) and are apparent to those skilled in the art.

[0225] In some embodiments, DCs overexpressing CD40L, CXCL13, and CD93 can be activated or impacted by antigens. In some embodiments, the antigen can be a tumor antigen or a viral antigen. Non-limiting examples of tumor antigens include antigens expressed by colorectal cancer cells, breast cancer cells, ovarian cancer cells, pancreatic cancer cells, head and neck cancer cells, bladder cancer cells, liver cancer cells, renal cancer cells, melanoma cells, gastrointestinal cancer cells, prostate cancer cells, small cell lung cancer cells, non-small cell lung cancer cells, sarcoma cells, glioblastoma cells, T cells and B cell lymphoma cells, endometrial cancer cells, or cervical cancer cells.

[0226] In some embodiments, the antigen can be a cancer (tumor) cell lysate, and the cancer cell lysate can be allogeneic or autologous relative to the dendritic cells. In some embodiments, the cancer cell lysate can be a melanoma cancer cell lysate, such as a DDM-1.7 cell lysate, a DDM-1.13 cell lysate, or a combination thereof.

[0227] Many methods of impacting or activating dendritic cells with antigens are known in the art. In some embodiments, antigens can be added to the dendritic cells of culture under the conditions of promoting cell viability, then cells are allowed to have enough time to absorb and process antigens, and on the cell surface, antigen peptides are expressed in combination with Class I or Class II MHC, about a time period of 24 hours (about 18 to about 30 hours, preferably 24 hours). Dendritic cells can also be exposed to antigens by DNA transfection with encoded antigens. DNA is expressed, and antigens may be processed via cytosol / Class I pathways.

[0228] In some embodiments, the separated peptide can be used to impact or activate DC. Peptide is impacted by any of the following multiple methods: including incubation of peptide with dendritic cells, incubation of protein containing peptide with DC, transduction of DC (or monocyte group expanded by progenitor cells) with genes encoding peptides (or proteins containing peptides), etc. Typical antigens used as peptides are derived from those expressed in target cells, such as transformed cells, cancer cells, bacterial cells, parasite-infected cells or virus-infected cells, etc. Examples include, but are not limited to, carbohydrates, such as mucins, tumor antigens, peptides derived from proteins selected from the group consisting of: HIV Gag, HIV Env, HER-2, MART-1, gp-100, PSA, HBVc, HBVs, HPV E6, HPVE7, tyrosinase, MAGE-1, trp-1, mycobacterial antigens and CEA, and many other antigens. Tumor antigens suitable for presentation include, but are not limited to, c-erb-β-2 / HER2 / neu, PEM / MUC-1, Int-2, Hst, BRCA-1, BRCA-2, truncated EGFRvIII, CEA, p53, ras, RK, Myc, Myb, OB-1, OB-2, BCR / ABL, GIP, GSP, RET, ROS, FIS, SRC, TRC, WTI, DCC, NF1, FAP, MEN-1, ERB-B1, MAGE-antigens and idiotype immunoglobulins (e.g., B cells from non-Hodgkin's lymphoma patients). The antigen presenting activity of dendritic cells can be enhanced by co-culturing with certain cytokines, such as TNF-α or IL-1α or IL-1β.

[0229] The activated dendritic cells disclosed herein may be present in a composition comprising a physiologically acceptable carrier, excipient, adjuvant or diluent. Neutral buffered saline or saline mixed with serum albumin is an exemplary appropriate diluent. Suitable carriers include aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In some embodiments, the composition may include a tumor antigen or a viral antigen. In some embodiments, the composition does not contain any heterologous antigens. In some embodiments, the composition includes at least about 10 5 dendritic cells / mL, at least about 10 6 cells / mL, at least about 10 7 cells / mL or more.

[0230] In some embodiments, the activated DC can be frozen (cryopreserved) before administration to the subject. In some embodiments, the activated DC can be frozen by suspending the cells in a culture medium containing at least 30% human serum and / or plasma and lowering the temperature of the suspension to at least -80°C, thereby freezing the DC. In some embodiments, the freezing medium is about 30% human serum and / or plasma, and about 10% of an agent that prevents ice crystal formation during freezing, such as DMSO. In another embodiment, the DC suspension is kept at -80°C for at least 24 hours, and then transferred to liquid nitrogen during storage. In another embodiment, the DC suspension is thawed at a temperature ranging from 34°C to 41°C.

[0231] Also disclosed herein are methods for developing immature DCs from monocytes and activating immature DCs with allogeneic or autologous tumor cell lysates. In some embodiments, the method comprises: (a) isolating monocytes from a subject; (b) overexpressing one or more genes selected from CD40L, CXCL13, and CD93 in the isolated monocytes; and (c) differentiating the monocytes expressing one or more genes selected from CD40L, CXCL13, and CD93 into immature dendritic cells in vitro. In some embodiments, the monocytes are CD14 + In some embodiments, the method comprises: differentiating monocytes heterologously expressing CD40L, CXCL13 and / or CD93 into immature dendritic cells in vitro. In some embodiments, the monocytes are CD14 + Monocytes. In some embodiments, the cells are CD34+ cells that are transduced and differentiated or expanded into monocytes and then matured, as provided herein. Also described herein are different combinations of heterologously expressed proteins.

[0232] In some embodiments, monocytes are obtained from various sources, such as leukapheresis of peripheral blood mononuclear cells from a patient, followed by elutriation of the separated peripheral blood to provide isolated monocytes. The isolated monocytes can be genetically manipulated by techniques disclosed herein, such as liposome transfection, chemical transfection, transgenic DNA recombination, viral infection, transposon insertion, jumping gene insertion, microinjection, electroporation, gene gun penetration, and combinations thereof, to express any one or combination of heterologous CD40L, CXCL13, and CD93 proteins. In some embodiments, recombinant adenoviral vectors, recombinant adeno-associated vectors, recombinant retroviral vectors, recombinant lentiviral vectors, or combinations thereof can be used. Methods such as delivery of CRISPR / Cas systems, TALENs, and zinc finger nucleases can also be used to overexpress any one or combination of heterologous proteins CD40L, CXCL13, and CD93.

[0233] In some embodiments, recombinant monocytes expressing CD40L, CXCL13 and optionally CD93 are grown in the presence of IL-3, causing monocyte proliferation, producing an expanded monocyte population. The expanded population of monocytes can be differentiated into immature dendritic cells, for example, by culturing the expanded cell population with GM-CSF and IL-4 (to produce baseline or type I DC) and optionally TNF-α, IL-1β, IL-6, IFN-α, IFN-γ and PGE2.

[0234] In some embodiments, recombinant allogeneic immature dendritic cells can be activated or impacted by antigen. In some embodiments, the antigen can be a tumor antigen or a viral antigen. In some embodiments, the antigen can be a cancer cell lysate, and the cancer cell lysate can be allogeneic or autologous relative to immature dendritic cells. In some embodiments, the cancer cell lysate can be a melanoma cancer cell lysate, such as DDM-1.7 cell lysate, DDM-1.13 cell lysate or its combination. In some embodiments, the cell is not activated or impacted by antigen.

[0235] In some embodiments, the activated immature DC disclosed herein can be present in a composition comprising a physiologically acceptable carrier, excipient, adjuvant or diluent. Neutral buffered saline or saline mixed with serum albumin is an exemplary suitable diluent. In some embodiments, the activated immature DC can be frozen before administration to a subject. The cells can be thawed before administration to a subject.

[0236] Also disclosed herein are methods of treating cancer in a subject. In some embodiments, a method of treating cancer in a subject comprises administering to the subject a composition comprising recombinant dendritic cells, wherein the dendritic cells heterologously express one or more proteins selected from CD40L, CXCL13, and optionally CD93. As described herein, the cells may be allogeneic to the subject. In some embodiments, the cells may be autologous to the subject.

[0237] In some embodiments, a method of eliciting an immune response in a subject with cancer comprises administering to the subject a composition comprising dendritic cells, wherein the dendritic cells heterologously express one or more proteins selected from CD40L, CXCL13, and optionally CD93. In some embodiments, the DC heterologously expresses CD40L and CXCL13. In some embodiments, the DC heterologously expresses CD40L, CXCL13, and CD93. In some embodiments, the DC heterologously expresses CD40L and CD93. In some embodiments, the DC heterologously expresses CXCL13 and CD93. In some embodiments, the DC does not heterologously express CD93.

[0238] In some embodiments, a method of providing a cancer vaccine to a subject having cancer comprises administering to the subject a composition comprising dendritic cells, wherein the dendritic cells heterologously express one or more proteins selected from CD40L, CXCL13, and optionally CD93. In some embodiments, the DC heterologously expresses CD40L and CXCL13. In some embodiments, the DC heterologously expresses CD40L, CXCL13, and CD93. In some embodiments, the DC heterologously expresses CD40L and CD93. In some embodiments, the DC heterologously expresses CXCL13 and CD93. In some embodiments, the DC does not heterologously express CD93.

[0239] In some embodiments, the administered dendritic cells (heterogeneously expressing CD40L, CXCL13 and / or CD93) are allogeneic to the subject. In some embodiments, the administered dendritic cells (heterogeneous CD40L, CXCL13 and / or CD93) are autologous to the subject. In some embodiments, the administered dendritic cells are immature dendritic cells that heterologously express CD40L, CXCL13 and / or CD93. In some embodiments, DC heterologously express CD40L and CXCL13. In some embodiments, DC heterologously express CD40L, CXCL13 and CD93. In some embodiments, DC heterologously express CD40L and CD93. In some embodiments, DC heterologously express CXCL13 and CD93. In some embodiments, DC does not heterologously express CD93.

[0240] In some embodiments, the subject has a cancer selected from the group consisting of colon cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, spermatocarcinoma, Primordial cell tumor, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, Merkel cell carcinoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease and combinations thereof.

[0241] In additional embodiments, the cancer is a solid tumor selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchopulmonary carcinoma, pulmonary embolism ... carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors such as gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme) astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, Schwannomas, craniopharyngiomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases.

[0242] In additional embodiments, within a time frame of 4-14 days after administration of the recombinant dendritic cells, the patient is administered an immune checkpoint inhibitor, including any one or a combination of two immune checkpoint inhibitors comprising: an inhibitor of PD-1 or PD-L1 (B7-H1), such as an anti-PD-1 antibody, including nivolumab (nivolumab from Bristol-Myers Squibb), pembrolizumab / pembrolizumab, also known as MK-3475 (Keytruda from Merck), pidilizumab (Curetech), AMP-224 (Amplimmune), or an anti-PD-L1 antibody, including MPDL3280A (Roche), MDX-1105 (Bristol Myers Squibb), MEDI-4736 (AstraZeneca) and MSB-0010718C (Merck), antagonists of CTLA-4, such as anti-CTLA-4 antibodies, including the anti-CTLA4 antibodies Yervoy™ (ipilimumab, Bristol-Myers Squibb), tremelimumab (Pfizer), decilimumab (AstraZenec) or AMGP-224 (Glaxo SmithKline), or tumor-specific antibodies trastuzumab (Herceptin) for breast cancer, rituximab (Rituxan) or cetuximab (Erbitux) for lymphoma.

[0243] In additional embodiments, the treatment, administration or boosting of the immune response is repeated periodically, for as long as the patient exhibits improvement or stable / non-progressive disease, over a time frame such as those in Examples 1-3.

[0244] In additional embodiments, the treatment is repeated periodically within the following time frame, administering or increasing the immune response as a maintenance therapy, as long as the patient shows improvement or stable / non-progressive disease: from every 5 days, once a week, once every 14 days, to once a month, to once every two months, to once every 3 months, to once every 4 months, to once every 5 months, to once every 6 months, or once every 7 months, or once every 8 months, or once every 9 months, or once every 10 months, or once every 11 months, or once a year.

[0245] In some embodiments, prior to administration, the recombinant dendritic cells (heterologous CD40L, CXCL13 and / or CD93) administered to the subject are not activated or impacted by antigen. In some embodiments, dendritic cells can be administered with a composition including adjuvants, cytokines and interleukins, which can help induce an immune response. In some embodiments, DC heterologously expresses CD40L and CXCL13. In some embodiments, DC heterologously expresses CD40L, CXCL13 and CD93. In some embodiments, DC heterologously expresses CD40L and CD93. In some embodiments, DC heterologously expresses CXCL13 and CD93. In some embodiments, DC does not heterologously express CD93.

[0246] In some embodiments, the method further comprises: (a) obtaining a protein expression profile of a resected tumor or biopsy sample from the subject;

[0247] (b) comparing the protein expression profile of the excised tumor or biopsy sample with the protein expression profile of a melanoma cell lysate; and

[0248] (c) if at least three markers in the protein expression profile of the resected tumor or biopsy sample match the protein expression profile of the melanoma cell lysate, co-culturing the dendritic cells overexpressing CD40L, CXCL13, and optionally CD93 with the melanoma cell lysate to activate the dendritic cells and administering to the subject a composition comprising the activated dendritic cells; and

[0249] (d) if at least three of the markers in the protein expression profile of the resected tumor or biopsy sample do not match the protein expression profile of the melanoma cell lysate, co-culturing the dendritic cells overexpressing CD40L, CXCL13, and optionally CD93 with the tumor or biopsy sample to activate the dendritic cells and administering to the subject a composition comprising the activated dendritic cells.

[0250] In some embodiments, the methods provided herein further include, prior to administration, screening the protein expression profile of the excised tumor or biopsy sample from the subject to cross-match with the protein expression profile of the allogeneic tumor lysate; and administering allogeneic tumor lysate-activated dendritic cells or activated dendritic cell compositions if at least three fragments of the protein expression profile of the excised tumor or biopsy sample cross-match with the protein expression profile of the allogeneic tumor lysate. The term "cross-matching" as used herein refers to comparing the protein expression profile of one sample with another, such as comparing a tumor lysate with a biopsy or excised tumor sample. If a protein fragment is found in both the lysate and the sample, it is called a match.

[0251] In some embodiments of the above methods, the DC heterologously expresses CD40L and CXCL13. In some embodiments, the DC heterologously expresses CD40L, CXCL13, and CD93. In some embodiments, the DC heterologously expresses CD40L and CD93. In some embodiments, the DC heterologously expresses CXCL13 and CD93. In some embodiments, the DC does not heterologously express CD93.

[0252] In some embodiments, protein expression profiles are measured or compared by techniques well known in the art, such as protein arrays, proteomics, mass spectrometry (MALDI-MS), etc. In some embodiments, gene expression profiles can be used instead of protein expression profiles for comparison. Gene expression profiles can be obtained using techniques well known in the art, such as gene arrays, microarrays, RT-PCR, etc.

[0253] In some embodiments, the markers compared between the protein expression profiles of excised tumors or biopsy samples and the protein expression profiles of melanoma cell lysates are MAGE antigens. See, e.g., Scanlan, Matthew J. et al., Immunological Revs. 2002, 188: 22-32 and Weon JL, Potts PR. The MAGE protein family and cancer. Curr Opin Cell Biol. 2015; 37: 1–8. doi: 10.1016 / j.ceb.2015.08.002, each of which is incorporated herein by reference in its entirety.

[0254] In some embodiments, the melanoma cell lysate is derived from a cell line selected from the group consisting of: DDM-1.7, DDM-1.13, or a combination thereof. In some embodiments, the melanoma cell lysate is an MCV tumor lysate, or a lysate that matches some portion of the expression profile of an MCV tumor lysate.

[0255] In some embodiments, the administration route is via intratumoral, peritumoral, intradermal, subcutaneous, intramuscular, intraperitoneal injection. The composition is administered to stimulate an immune response and may be administered by bolus injection, continuous infusion, sustained release from an implant, or other suitable technique.

[0256] For illustration purposes only, the method may be practiced by obtaining and storing a blood sample from a subject prior to infusion for subsequent analysis and comparison. Typically, at least about 10 4 Up to 10 6 and typically in the range of 1×10 8 With 1×10 10 The cells are infused intravenously or intraperitoneally into a 70 kg patient.

[0257] In some aspects, any of the methods of treatment described herein may further include administering one or more additional anticancer treatments to an individual. Various classes of anticancer agents may be used. Non-limiting examples include: radiation therapy, alkylating agents (e.g., cisplatin, carboplatin, or oxaliplatin), antimetabolites (e.g., thiopurine or mercaptopurine), anthracyclines, plant alkaloids (including, for example, vinca alkaloids (such as vincristine, vinblastine, vinorelbine, or vindesine) and taxanes (such as paclitaxel, taxol, docetaxel), topoisomerase inhibitors (e.g., camptothecin, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, or teniposide), podophyllotoxin (and derivatives thereof, such as etoposide and teniposide), antibodies (e.g., monoclonal or polyclonal), tyrosine kinase inhibitors (e.g., imatinib mesylate ( or )), hormone therapy, soluble receptors and other antineoplastic drugs (such as actinomycin D, doxorubicin, epirubicin, bleomycin, mechlorethamine, cyclophosphamide, chlorambucil, or ifosfamide).

[0258] In addition, in some embodiments, the cells and compositions provided herein can be used in conjunction with or in combination with other agents or treatments having anti-cancer properties (see, U.S. Pat. No. 9,914,783, which is incorporated herein by reference). When used in conjunction, the recombinant DC and related compositions and other agents can be formulated into a single combined pharmaceutical preparation, or can be formulated separately into a single dosing regimen or different dosing regimens. The agents administered in conjunction with or in conjunction with the recombinant DC and related compositions will generally have complementary activities to the recombinant DC and related compositions, so that the cells and other agents do not adversely affect each other.

[0259] Agents that can be used in conjunction with anti-PD-1 antibodies include, but are not limited to, alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic agents, antiproliferative agents, antiviral agents, Aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell engager) antibodies, antibody drug conjugates, biological response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase 2 inhibitors, DVDs, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, immunology , inhibitors of apoptosis protein (IAP) inhibitors, intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum-based chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid / carotenoid plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, etc., and combinations of one or more of these agents.

[0260] BiTE antibodies are bispecific antibodies that instruct T cells to attack cancer cells by binding to two cells at the same time. Then, the T cells attack the target cancer cells. Examples of BiTE antibodies include adelimumab (Micromet MT201), rantumaomab (Micromet MT103), etc. Without being limited by theory, one of the mechanisms by which T cells induce apoptosis of target cancer cells is through exocytosis of cytolytic granule components including perforin and granzyme B.

[0261] siRNA is a molecule with endogenous RNA base or chemically modified nucleotide.Modification will not eliminate cell activity, but give increased stability and / or increased cell efficacy.The example of chemical modification includes phosphorothioate group, 2'-deoxynucleotide, ribonucleotide containing 2'-OCH3, 2'-F-ribonucleotide, 2'-methoxyethyl ribonucleotide, its combination etc.siRNA can have variable length (such as 10-200bps) and structure (such as hairpin, single strand / double strand, protrusion, gap / space, mispairing), and is processed in cells to provide active gene silencing.Double-stranded siRNA (dsRNA) can have the same number of nucleotides on each chain (flat end) or asymmetric end (overhang).The overhang of 1-2 nucleotide can be present in sense and / or antisense strand, and be present in 5'-and / or 3'-end of given chain.

[0262] Multivalent binding protein is a binding protein including two or more antigen binding sites.Multivalent binding protein is engineered to have three or more antigen binding sites, and is generally not a naturally occurring antibody.The term "multispecific binding protein" refers to a binding protein capable of binding to two or more related or unrelated targets.Dual variable domain (DVD) binding protein is a tetravalent or multivalent binding protein binding protein including two or more antigen binding sites.Such DVD can be monospecific (i.e., capable of binding to an antigen) or multispecific (i.e., capable of binding to two or more antigens).DVD binding proteins including two heavy chain DVD polypeptides and two light chain DVD polypeptides are referred to as DVD Ig.Each half of DVD Ig includes a heavy chain DVD polypeptide, a light chain DVD polypeptide and two antigen binding sites.Each binding site comprises a heavy chain variable domain and a light chain variable domain, wherein each antigen binding site has a total of 6 CDRs involved in antigen binding.

[0263] Alkylating agents include, but are not limited to, hexamethylmelamine, AMD-473, AP-5280, apaziquone, bendamustine, brostallicin, busulfan, carboquinone, carmustine (BCNU), chlorambucil, (Laromustine, VNP 40101M), cyclophosphamide, dacarbazine, estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine (CCNU), mafosfamide, melphalan, dibromomannitol, dibromodulanol, nimustine, nitrogen mustard N-oxide, ranimustine, temozolomide, thiotepa, (bendamustine), trofosfan, and trofosfamide.

[0264] Angiogenesis inhibitors include, but are not limited to, endothelial-specific receptor tyrosine kinase (Tie-2) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, vascular endothelial growth factor receptor (VEGF) inhibitors, delta-like ligand 4 (DLL4) inhibitors, insulin growth factor 2 receptor (IGFR-2) inhibitors, matrix metalloproteinase-2 (MMP-2) inhibitors, matrix metalloproteinase 9 (MMP-9) inhibitors, platelet-derived growth factor receptor (PDGFR) inhibitors, thrombospondin analogs, and vascular endothelial growth factor receptor tyrosine kinase (VEGFR) inhibitors.

[0265] Antibody drug conjugates include, but are not limited to, those targeting c-Met kinase (e.g., the ADC described in U.S. Pat. No. 7,615,529), LRRC15, CD30 (e.g., (velutegravir)), CS1 (e.g., the ADC described in U.S. Publication No. 20160122430), DLL3 (e.g., rovalvituzumab tesirine (ROVA-T)), HER2 (e.g., (entezuzumab)), EGFR (e.g., the ADC described in U.S. Publication No. 20150337042), and prolactin receptor (e.g., the ADC described in U.S. Publication No. 20140227294).

[0266] Antimetabolites include but are not limited to (pemetrexed disodium, LY231514, MTA), 5-azacitidine, (capecitabine), carmofur, (cladribine), clofarabine, cytarabine, cytarabine ocfosfate, cytarabine, decitabine, deferoxamine, doxifluridine, eflornithine, EICAR (5-ethynyl-1-β-D-ribofuranosyl imidazole-4-carboxamide), enocitabine, ethnylcytidine, fludarabine, 5-fluorouracil alone or in combination with folinic acid, (gemcitabine), hydroxyurea, (Melphalan), mercaptopurine, 6-mercaptopurine riboside, methotrexate, mycophenolic acid, nelarabine, noratript, sodium octadecyl phosphate (ocfosfate), piritrexol, pentostatin, raltitrexed, ribavirin, triapine, trimetrexate, SI, thiazofurine, tegafur, TS-1, adenosine, UFT, etc.

[0267] Antiviral agents include, but are not limited to, ritonavir, acyclovir, cidofovir, ganciclovir, foscarnet, zidovudine, ribavirin, and hydroxychloroquine.

[0268] Aurora kinase inhibitors include, but are not limited to, ABT-348, AZD-1152, MLN-8054, VX-680, Aurora A-specific kinase inhibitors, Aurora B-specific kinase inhibitors, and pan-Aurora kinase inhibitors.

[0269] Bcl protein inhibitors include but are not limited to ABT-263 (Navicla), AT-101 ((-) gossypol), (G3139 or olimusan (Bcl-2-targeting antisense oligonucleotide)), IPI-194, IPI-565, N-(4-(4-((4'-chloro(1,1'-biphenyl)-2-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobenzenesulfonamide), N-(4-( 4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide, venetoclax, GX-070 (obatoclax), and the like.

[0270] Examples of Bcr-Abl kinase inhibitors include, but are not limited to (BMS-354825) and (imatinib) etc.

[0271] BTK inhibitors include, but are not limited to, ibrutinib and acalabrutinib.

[0272] CDK inhibitors include, but are not limited to, AZD-5438, BMI-1040, BMS-032, BMS-387, CVT-2584, flavopyridol, GPC-286199, MCS-5A, PD0332991, PHA-690509, cyliciclib (CYC-202, R-roscovitine), abemaciclib, palbociclib, and ZK-304709.

[0273] COX-2 inhibitors include but are not limited to ABT-963, (Etoricoxib), (Valdecoxib), BMS347070, (celecoxib), COX-189 (lumiracoxib), CT-3, (Dericoxib), JTE-522, 4-methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoylphenyl-1H-pyrrole), MK-663 (etoricoxib), NS-398, parecoxib, RS-57067, SC-58125, SD-8381, SVT-2016, S-2474, T-614, and (Rofecoxib). EGFR inhibitors include but are not limited to ABX-EGF, anti-EGFR immunoliposomes, EGF vaccine, EMD-7200, (cetuximab), HR3, IgA antibody, (gefitinib), (erlotinib or OSI-774), (osimertinib), TP-38, EGFR fusion protein and (lapatinib).

[0274] ErbB2 receptor inhibitors include but are not limited to CP-724-714, CI-1033 (canutinib), (Trastuzumab), (lapatinib), (2C4, pertuzumab), TAK-165, GW-572016 (ionafarnib), GW-282974, EKB-569, PI-166, dHER2 (HER2 vaccine), APC-8024 (HER-2 vaccine), anti-HER / 2neu bispecific antibody, B7.her2IgG3, AS HER2 trifunctional bispecific antibody, mAB AR-209, and mAB 2B-1.

[0275] Histone deacetylase inhibitors include, but are not limited to, depsipeptide, LAQ-824, MS-275, trapoxin, suberoylanilide hydroxamic acid (SAHA), TSA, and valproic acid.

[0276] HSP-90 inhibitors include but are not limited to 17-AAG-nab, 17-AAG, CNF-101, CNF-1010, CNF-2024, 17-DMAG, geldanamycin, IPI-504, KOS-953, (human recombinant antibody to HSP-90), NCS-683664, PU24FCl, PU-3, radicicol, SNX-2112, STA-9090 and VER49009.

[0277] Inhibitors of apoptotic proteins include, but are not limited to, HGS1029, GDC-0145, GDC-0152, LCL-161, and LBW-242.

[0278] Activators of the death receptor pathway include, but are not limited to, TRAIL, antibodies or other agents targeting TRAIL or death receptors (e.g., DR4 and DR5), such as Apomab, conatumumab, ETR2-ST01, GDC0145 (lexalimumab), HGS-1029, LBY-135, PRO-1762, and trastuzumab.

[0279] Kinesin inhibitors include, but are not limited to, Eg5 inhibitors, such as AZD4877, ARRY-520; and CENPE inhibitors, such as GSK923295A.

[0280] JAK-2 inhibitors include, but are not limited to, CEP-701 (lesaurtinib), XL019, and INCB018424.

[0281] MEK inhibitors include, but are not limited to, ARRY-142886, ARRY-438162, PD-325901, and PD-98059.

[0282] mTOR inhibitors include, but are not limited to, AP-23573, CCI-779, everolimus, RAD-001, rapamycin, temsirolimus, ATP-competitive TORC1 / TORC2 inhibitors including PI-103, PP242, PP30, and Torin 1.

[0283] Nonsteroidal anti-inflammatory drugs include but are not limited to (salsalate), (diflunisal), (Ibuprofen), (Ketoprofen), (nabumetone), (piroxicam), ibuprofen cream, (naproxen) and (naproxen), (Diclofenac), (indomethacin), (Sulindac), (Tolmetin), (Etodolac), (Ketorolac) and (Oxaprozin).

[0284] PDGFR inhibitors include, but are not limited to, C-451, CP-673, and CP-868596.

[0285] Platinum chemotherapeutic agents include but are not limited to cisplatin, (Oxaliplatin) Aplatin, Lobaplatin, Nedaplatin, (carboplatin), satraplatin, and picoplatin.

[0286] Polo kinase inhibitors include, but are not limited to, BI-2536.

[0287] Phosphoinositide-3 kinase (PI3K) inhibitors include, but are not limited to, wortmannin, LY294002, XL-147, CAL-120, ONC-21, AEZS-127, ETP-45658, PX-866, GDC-0941, BGT226, BEZ235, and XL765.

[0288] Thrombospondin analogs include, but are not limited to, ABT-510, ABT-567, ABT-898, and TSP-1.

[0289] VEGFR inhibitors include, but are not limited to, ABT-869, AEE-788, ANGIOZYME TM (a ribozyme that inhibits angiogenesis (ribozyme drug (Boulder, Colo.) and Chiron (Emeryville, CA.)), axitinib (AG-13736), AZD-2171, CP-547,632, (ramucirumab), IM-862, (pegaptamib), (sorafenib, BAY43-9006), pazotinib (GW-786034), vatalanib (PTK-787, ZK-222584), (sunitinib, SU-11248), (regorafenib), VEGF traps and ZACTIMA TM (Vandetanib, ZD-6474).

[0290] Antibiotics include, but are not limited to, intercalating antibiotics aclarubicin, actinomycin D, amrubicin, anthracycline (annamycin), doxorubicin, (bleomycin), daunorubicin, or (liposomal doxorubicin), elsamicin, epirubicin, glarbuicin, (idarubicin), mitomycin C, nelubicin, neocarcin, pelopycin, pirarubicin, butterfly mycin, stimalamer, streptozocin, (valrubicin) and ninstatin.

[0291] Topoisomerase inhibitors include, but are not limited to, aclarubicin, 9-aminocamptothecin, amifide, amsacrine, batkalin, belotecan, BN-80915, (irinotecan hydrochloride), camptothecin, (Dextrorotatory propanediol), diflutecan, etecarin, or (epirubicin), etoposide, exatecan, 10-hydroxycamptothecin, gimatecan, lutetotecan, mitoxantrone, Onivyde TM (liposomal irinotecan), pirarbucin, orathecin, pixantrone, rubitecan, sobuzoxane, SN-38, taflutoposide, and topotecan.

[0292] Antibodies include but are not limited to (bevacizumab), CD40-specific antibody, chTNT-1 / B, denosumab, (cetuximab), (Zamuximab), IGF1R-specific antibody, Lintuzumab, OX-40-specific antibody, (edrecolomab), (WX G250), (rituximab), ticilimumab, trastuzumab, pertuzumab, (panitumumab) and CD20 type I and II antibodies.

[0293] Hormone therapy includes but is not limited to (Anaroazole), (exemestane), arzoxifene, (Bicalutamide), (Cetrolix), Degarelix, Deslorelin, (Trilostane), dexamethasone, (Flutamide), (raloxifene), AFEMA TM (Farozole), (Toremifene), (Fulvestrant), (lerozole), formestane, glucocorticoids, (dodecyl ferulate), (Sevelamer carbonate), lasofoxifene, leuprolide acetate, (Megestrol), (mifepristone), NILANDRON TM (Nilutamide), (tamoxifen citrate), PLENAXIS TM (Abarelix), prednisone, (finasteride), rilostane, (Buserelin), (luteinizing hormone-releasing hormone (LHRH)), (Histrelin implant), (trilostane or modrastane), (fosrelin, goserelin).

[0294] deltoid and retinoids include but are not limited to ceocalcitol (EB1089, CB1093), lexacalcitol (KH1060), fenretinide, (Alitretinoin), (liposomal retinoic acid), (bexarotene) and LGD-1550.

[0295] PARP inhibitors include, but are not limited to, ABT-888 (veliparib), KU-59436, AZD-2281 (olaparib), AG-014699 (rucaparib), MK4827 (niraparib), BMN-673 (talazoparib), iniparib, BSI-201, BGP-15, INO-1001, and ONO-2231.

[0296] Plant alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0297] Proteasome inhibitors include but are not limited to (bortezomib), (carfilzomib), MG132, NPI-0052, and PR-171.

[0298] Examples of immunotherapies include, but are not limited to, interferons, immune checkpoint inhibitors, co-stimulators, and other immunoenhancers. Interferons include interferon α, interferon α-2a, interferon α-2b, interferon β, interferon γ-1a, (interferon gamma-1b) or interferon gamma-n1, combinations thereof, etc. Immune checkpoint inhibitors include antibodies targeting PD-L1 (e.g., durvalumab, atezolizumab, avelumab, MEDI4736, MSB0010718C, and MPDL3280A) and CTLA4 (cytotoxic lymphocyte antigen 4; e.g., ipilimumab, tremelimumab). Co-stimulatory agents include, but are not limited to, antibodies to CD3, CD40, CD40L, CD27, CD28, CSF1R, CD137 (e.g., usulerumab), B7H1, GITR, ICOS, CD80, CD86, OX40, OX40L, CD70, HLA-DR, LIGHT, LIGHT-R, TIM3, A2AR, NKG2A, KIR (e.g., lirilumab), TGF-β (e.g., fesutuzumab), and combinations thereof.

[0299] Other agents include but are not limited to (IFN-α), BAM-002 (oxidized glutathione), (Tasonamine), (Tositumomab), (alemtuzumab), dacarbazine, denileukin, epratuzumab, (Legulostim), Lentinan, leukocyte interferon alpha, imiquimod, melanoma vaccine, mitumomab, molaspomandib, MYLOTARG TM (gemtuzumab ozogamicin), (filgrastim), OncoVAC-CL, (Ogovolumab), pemtumomab (Y-muHMFG1), (sipuleucel-T), sargaramostim, sizofilan, teciloxil, (Bacillus Calmette-Guerin), Ubenimex, Z-100(Maruyama's specific substance (SSM)), WF-10(Tetrachlorodecaoxide (TCDO)), (Aldesleukin), (thymus alpha-sin), (Daclizumab High-yield Method) and (90Y-ibritumomab tiuxetan).

[0300] Biological response modifiers are agents that modulate the defense mechanisms or biological responses of living organisms, such as the survival, growth or differentiation of tissue cells) to direct them to have anti-tumor activity, and include but are not limited to krestin, lentinan, sizoran, picibanil PF-3512676 (CpG-8954) and ubenimex.

[0301] Pyrimidine analogs include, but are not limited to, cytosine arabinoside (ara C or arabinoside C), cytosine arabinoside, doxifluridine, (Fludarabine), 5-FU (5-fluorouracil), floxuridine, (gemcitabine), (ratitrexed) and TROXATYL TM (Triacetyl uridine troxacitabine).

[0302] Purine analogs include but are not limited to (thioguanine) and (Mercaptopurine).

[0303] Antimitotic agents include, but are not limited to, batabulin, epothilone D (KOS-862), N-(2-(((4-hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide, ixabepilone (BMS 247550), (paclitaxel), (docetaxel), PNU100940(109881), patupilone, XRP-9881(larotaxel), vinflunine and ZK-EPO (synthetic epothilone).

[0304] Ubiquitin ligase inhibitors include, but are not limited to, MDM2 inhibitors such as nutlins and NEDD8 inhibitors such as MLN4924.

[0305] Recombinant DC and related compositions can also be used to enhance the efficacy of radiation therapy.Examples of radiation therapy include external beam radiation therapy, internal radiation therapy (ie, brachytherapy), and whole body radiation therapy.

[0306] Recombinant DC and related compositions can be administered adjunctively or in combination with other chemotherapeutic agents such as ABRAXANE TM (ABI-007), ABT-100 (farnesyl transferase inhibitor), (Ad5CMV-p53 vaccine), or (lovastatin), (Poly I: Poly C12U, synthetic RNA), (Exisuline), (pamidronic acid), arglabin, L-asparaginase, atamestane (1-methyl-3,17-dione-androsta-1,4-diene), (tazarotene), AVE-8062 (combreastatin derivative), BEC2 (mitumomab), cachexin (tumor necrosis factor), canvaxin (vaccine), (Cancer Vaccines), (Simo interleukin), (histamine dihydrochloride), (Human Papillomavirus), (C: (cyclophosphamide); (hydroxydoxorubicin); O: vincristine P: prednisone), CYPAT TM (cyproterone acetate), combrestatin A4P, DAB(389)EGF (catalytic and translocation domains of diphtheria toxin fused to human epidermal growth factor via a His-Ala linker), or trans-MID-107R TM (diphtheria toxin), dacarbazine, actinomycin, 5,6-dimethylxanthone-4-acetic acid (DMXAA), eniluracil, EVIZON TM (Squalamine Lactate), (T4N5 liposome lotion), scutellariae, DX-8951f (exatecan mesylate), enzastaurin, EPO906 (epithilone B), (quadrivalent human papillomavirus (types 6, 11, 16, 18) recombinant vaccine), GMK (ganglioside conjugate vaccine), (prostate cancer vaccine), halofuginone, histrelin, hydroxyurea, ibandronic acid, IGN-101, IL-13-PE38, IL-13-PE38QQR (cintredekin besudotox), IL-13-Pseudomonas exotoxin, interferon-α, interferon-γ, JUNOVAN TM or MEPACT TM (mifamotide), lonafarnib, 5,10-methylenetetrahydrofolate, miltefosine (hexadecylphosphocholine), (AE-941), (trimetrexate glucuronate), (Pentastatin), (a ribonuclease), (Melanoma Vaccine Therapy), (IL-2 vaccine), ORATHECIN TM (Rubitecan), (Antibody-based Cellular Medicine), MAb (mouse monoclonal antibody), paclitaxel, PANDIMEX TM (aglycone saponins from ginseng, including 20(S) protopanaxadiol (aPPD) and 20(S) protopanaxadiol (aPPT)), panitumumab, (investigational cancer vaccine), pegaspargase, PEG interferon A, dehydroequol (phenoxodiol), procarbazine, remastat, (catumaxomab), (lenalidomide), RSR13 (efaxirole), LA (Lanreotide), (Acitretin), staurosporine (Streptomyces staurosporine), tabotoxetine (PT100), (bexarotene), (DHA-paclitaxel), (canfosfamide, TLK286), temilifene, (temozolomide), tesmilifen, thalidomide, (STn-KLH), thymitaq (2-amino-3,4-dihydro-6-methyl-4-oxo-5-(4-pyridylthio)quinazoline hydrochloride), TNFERADE TM (Adenovirus vector: a DNA vector containing the tumor necrosis factor-α gene), or (Bosentan), retinoic acid (vitamin A acid), tetrandrine, (arsenic trioxide), ukrain (a derivative of an alkaloid from the greater celandine plant), vitaxin (anti-αvβ3 antibody), (Motexafin gadolinium), XINLAY TM (atrasentan), XYOTAX TM (paclitaxel poliglumex), (trabectedin), ZD-6126, (Right Lei Zuosheng), (zolamdronic acid) and daunorubicin, and combinations of any of these agents.

[0307] Reagent test kit

[0308] Additionally, certain components or embodiments of these recombinant dendritic cell compositions can be provided in kits. For example, any of the recombinant dendritic cell compositions and autologous or other tumor cell lysate compositions can be provided frozen or packaged as a kit, either alone or in separate containers with any of the other reagents from the pre-conditioning or post-conditioning steps and instructions for use.

[0309] Some embodiments also relate to any of the aforementioned cell compositions in the kit. In some embodiments, the kit may include an ampoule, a disposable syringe, a capsule, a vial, a tube, etc. In some embodiments, the kit may include a single-dose container or a multi-dose container, and the single-dose container or the multi-dose container include the topical preparation of the embodiments herein. In some embodiments, each dose container may include one or more unit doses. In some embodiments, the kit may include an applicator. In some embodiments, the kit includes all components required for the conditioning / treatment stage. In some embodiments, the cell composition may have a preservative or be preservative-free (e.g., in a single-use container). In some embodiments, any one or more recombinant dendritic cell compositions expressing CD40L, CXCL13, or CD93 may be prepared and frozen in an immature stage, to be suitable for transport to a hospital or treatment center. In some embodiments, an antigen for loading autologous or, for example, derived from a cell line such as DDM-1.7, DDM-1.13, or a combination thereof of a melanoma cell lysate may be prepared, and standard methods may be used to separate the recombinant dendritic cell compositions from freezing, so that these compositions may be transported to a hospital or treatment center for further processing and administration to the patient. In yet further embodiments, recombinant dendritic cell compositions expressing any one or more of CD40L, CXCL13, or CD93 can be prepared and mixed with desired autologous or, for example, melanoma cell lysates derived from cell lines such as DDM-1.7, DDM-1.13, or a combination thereof, to facilitate loading of the dendritic cell compositions with tumor antigens, after which these mixtures can be frozen to allow shipment of these compositions to a hospital or treatment center for further processing and administration to patients.

[0310] In addition, in some patients, it is expected that any of the methods or treatment regimens will be repeated regularly to enhance the immune system's response to tumors or infectious agents. Such regular treatments may range from weekly, monthly, to once every two months, to once every 3 months, to once every 4 months, to once every 5 months, to once every 6 months, or once every 7 months, or once every 8 months, or once every 9 months, or once every 10 months, or once every 11 months, or once a year to maintain treatment as long as the patient requires.

[0311] Treatment Overview

[0312] In some embodiments, alloDCs are transduced with CD40L and CXCL13 to maximize the attraction of the patient's T cells and B cells and generate a cascade of anti-tumor cellular and humoral immune responses.

[0313] In an optional further embodiment, transduction of CD40L+ and CXCL13+ alloDCs with CD93 also promotes cross-talk between allogeneic DCs and host DCs, thereby generating stable and sustained host anti-tumor immunogenicity.

[0314] In patients whose tumors can be biopsied or resected, the protein expression profiles of excised tumor samples and biopsy samples are screened. If the expression profile indicates at least 3 fragments shared with GMP-MCV, recombinant imDCs are loaded with GMP-MCV (allogeneic or "generic" tumor lysate) prior to DC in vitro maturation.

[0315] For patients whose samples lack any tumor fragments shared with allogeneic / generic lysates, autologous tumor lysates are generated and presented to immature recombinant alloDCs for processing of the autologous tumor lysates and presentation by the recombinant immature alloDCs.

[0316] For any patients who cannot be resected and biopsied, the recombinant CD40L+CXCL13+alloDC (or optionally further including CD93+) cell composition will be administered to the patient as an immune adjuvant without antigen loading. Using this CD40L+CXCL13+(optional)CD93 AlloDC approach, patients with advanced cancer can increase new anti-tumor cellular and humoral immune responses to tumor neoantigens and enhance existing immune responses through rapid, effective and low-cost treatment.

[0317] An overview of these options follows:

[0318] Option 1: Screen the protein expression profiles of resected tumor samples and biopsy samples to cross-match with the MelCancerVac (MCV) tumor lysate expression profile. If the expression profile indicates at least 3 identical fragments to GMP-MCV tumor lysate, the patient can be given the generic product, MCV tumor lysate pulsed enhanced / activated recombinant allogeneic DC vaccine (alloDC / MCV tumor lysate).

[0319] Option 2: If the protein expression profile does not match the MCV lysate profile, present autologous tumor lysate to immature recombinant alloDCs to allow them to mature after in vitro pulse.

[0320] Option 3: For unresectable and non-biopsyable patients, CD40L+CXCL13 (and optionally CD93) recombinant alloDCs can be administered as immune adjuvants without antigen pulse or activation.

[0321] An option for making recombinant DC cells is to transduce CD40L and CXCL13 into purified human CD14+ monocytes using recombinant adenoviral vectors / CRISPR Cas9 instead of lentiviral or retroviral vectors. The transduction efficiency and function of the transduced monocytes, including migration, differentiation, and cytokine secretion, will be assessed to ensure that the recombinant / transgenic monocytes can differentiate into functional and immunogenic DCs.

[0322] For many reasons, the use of adenovirus (AdV) is beneficial, including high transduction efficiency for many cell types (including hematopoietic origin cells that are unrelated to their mitotic state). Other benefits are that replication-deficient AdV has proven its safety clinically. In addition, AdV can provide high levels of transgenic expression, and AdV-transduced DCs can effectively present antigenic proteins. Recombinant adenoviral vectors can successfully transfect immature DCs with an efficiency of 95%. (See: Lei Zhong, et al. Eur. J. Immunol. 1999. 29: 964-972.) However, it is expected that other viral vector systems, including lentiviral vectors, can be utilized. (See, commercial sources such as Vectalys (Toulouse France) and related methods in US Patent No. 10,272,111).

[0323] Chemokine (CXC motif) ligand 13 (CXCL13), also known as B lymphocyte chemotactic factor (BLC) or B cell attracting chemokine 1 (BCA-1), is a protein ligand in humans encoded by the CXCL13 gene. CXCR5 is the receptor for CXCL13. Chemokine expression initiates a forward loop of lymphocyte recruitment and stimulation. Overexpressed CXCL13 in intestinal epithelial cells promotes a significant increase in the number of B cells in the lamina propria and an increase in the size and number of lymphoid follicles in the small intestine. (See, F. Marchesi et al. Mucosal Immunology. 2009. 2(6): 486-494.)

[0324] These results suggest that overexpression of CXCL13 in the intestine during inflammatory conditions favors the mobilization of B cells as well as LTi and NK cells with immunomodulatory and repair functions.

[0325] CD40 ligand (CD40L), also known as CD154, is a protein that is a member of the TNF superfamily of molecules. It binds to CD40 on antigen presenting cells (APCs) and, depending on the target cell type, will result in a number of effects. CD40L has a total of three binding partners: CD40, α5β1 integrin, and αIIbβ3. CD154 acts as a co-stimulatory molecule and is particularly important on a subset of T cells called T follicular helper cells (TFH cells). On TFH cells, CD40L promotes B cell maturation and function by engaging CD40 on the surface of B cells, and thus promotes communication between cells. Stable expression of CD40L allows DC to produce IL-12 to overcome immunosuppression and trigger memory T cell differentiation.

[0326] CD93 is an O-sialoglycoprotein of approximately 120 kDa that is selectively expressed on cells of the myeloid lineage within the hematopoietic system. Its primary structure and function were unknown until recently. Retroviral expression cloning was used to isolate CD93 cDNA. Sequence analysis showed that CD93 is identical to a protein called the C1q receptor (C1qRp) on human phagocytes. C1qRp was previously shown to mediate enhanced monocyte phagocytosis and is considered a receptor for C1q and two other structurally related molecules. When CD93 transducers and control cells were studied, it was found that cells expressing CD93 had an enhanced ability to bind to C1q. In addition, immature dendritic cells (DCs) were shown to express CD93 / C1qRp, while mature DCs, which are known to have reduced antigen uptake capacity and have lost the ability to phagocytose, showed weak to negative CD93 / C1qRp expression.

[0327] Cells used as a cell source for transduction of heterologous proteins provided herein can be any donor. In some embodiments, donors are screened to establish that cells isolated from the donor will be considered to be allogeneic relative to the subject to which it is administered. In some embodiments, donor screening criteria can include donor demographics, including: donor age, donor gender, donor race, donor ABO / Rh; donor BMI (weight and height), donor HLA high-resolution typing.

[0328] Additionally, in some embodiments, donor samples can be tested for full transfusion (FDA) testing on leukapheresis material (or material from any source), including: CMV testing; Syphilis serology and antibody screening; and an infectious disease panel including one or more of the following tests: Hepatitis B core antibody (Anti-HBs EIA); Hepatitis B surface antigen (HBsAg EIA); Hepatitis C virus antibody (Anti-HCV EIA); Human immunodeficiency virus antibody (HIV1 / 2 plus O); Human T-lymphotropic virus antibody (HTLV-I / II); HIV-1 / HGV / HBV nucleic acid test; WNV nucleic acid test; Trypanosoma cruzi antibody; and Zika. Thus, in some embodiments, the donor cells or cells administered to the patient are derived from a donor who is free of CMV, syphilis, hepatitis A, hepatitis B, hepatitis C, HIV, HTLV-I / II, West Nile virus (WNV), Trypanosoma cruzi, and / or Zika.

[0329] The sample can be assessed for the percentage of monocytes and counted by hematology analyzer and flow cytometry.

[0330] Samples can be collected by apheresis or by whole blood collection and standard tests performed. In some cases, collection will be performed by Optia leukapheresis collection. Samples will be analyzed for viability and CD14+%, and CD14+ monocytes will be counted.

[0331] Negative isolation of monocytes using CliniMACS according to standard procedures is another option.

[0332] Purity and cell count after isolation

[0333] In some embodiments, purified monocytes are transfected with an adenoviral vector comprising CD40L and CXCL13 (and for certain embodiments CD40L, CXCL13, and CD93).

[0334] In some embodiments, purified monocytes are transfected with a lentiviral vector comprising CD40L and CXCL13 (and for certain embodiments CD40L, CXCL13, and CD93).

[0335] After exposure to the viral vector, positively transduced monocytes can be selected. For example, cells can be selected using one or more of the following positive or negative markers: negative lineage (CD3-, CD56-, CD19-, CD66b-), CD45+, CD14+, CD40L+, CXCL13+, CD1c+, CD11b+, CD11c+, HLA-DR+, CD86+, CD80low, CD83-, CD16low, CD33+, CD163-, CD206+, or CD209.

[0336] In some cases, the transduction efficiency of the cells will be assessed prior to positive sorting so that the yield can be calculated. The transduction efficiency / transduction efficiency is required to determine the expected yield of CD40L+CXCL13+ or CD40L+CXCL13+CD93+ monocytes.

[0337] As provided herein, cell can be monocyte, it can be in vitro differentiated into immature dendritic cell.In some embodiments, cell (transduced or non-transduced) is centrifuged to separate cells.Cell can be for example centrifuged 10 minutes under 400x g low crushing at room temperature (RT).Centrifugal cell can be separated from its supernatant.X-VIVO15, recombinant human GM-CSF can be used, such as (1000 units / ml) and IL-4 (1000 units / ml)) centrifugal cell is resuspended in cell culture bottle, and cell can be allowed to differentiate monocyte in incubator at least or about 1,2,3,4,5,6,7,8,9 or 10 days.In some embodiments, allow cell differentiation about 4 to about 8 days.

[0338] In some embodiments, the differentiated cells can then be collected from the cell culture medium and centrifuged. For example, in some embodiments, the cell culture medium is collected from the culture bottle into a centrifuge tube. PBS can be placed in the cell culture bottle to cover the surface of the bottle. The cell culture bottle can be incubated with PBS at 37°C, 5% CO2 in an incubator for about 30 minutes. In some embodiments, the contents of the flask are collected into a centrifuge tube by tapping the flask after incubation. The flask can be rinsed and collected into a centrifuge tube. In some embodiments, the collected cells can be centrifuged for 10 minutes at 400 × g under low fragmentation conditions, for example, at RT. After centrifugation, the cells are separated from their supernatant and the cells are separated. The cells can be resuspended and analyzed for viability.

[0339] In some embodiments, samples are analyzed for counting and flow cytometry identification of relevant biomarkers. They can be analyzed or selected by one or more of the following positive or negative markers: negative lineage (CD3-, CD56-, CD19-, CD66b-), CD45+, CD14+, CD40L+, CXCL13+, CD1c+, CD11b+, CD11c+, HLA-DR+, CD86+, CD80 low, CD83-, CD16 low, CD33+, CD163-, CD206+ or CD209.

[0340] As provided herein, monocytes can be transduced with viral vectors such as lentivirus or adenovirus. Any protocol for viral transduction can be used. For example, cells can be cultured in a culture medium mixture. In some embodiments, the culture medium includes 100ng / mL of mFlt3L / mTPO / mSCF and 30ng / mL of mIL-3. For example, cells can be cultured in this culture medium and then incubated at 37°C and 5% CO2 for 24 hours or until activated. After activation, cells can be pretreated with PGE2. Cells can then be transduced with appropriate vectors. In some embodiments, viruses are added with an MOI (multiplicity of infection) of 10, 100 or 100. After transduction, cells can be separated using beads or purified products, for example, bound to one or more heterologous proteins encoded by the vector. For example, cells can be collected using CD40L microbeads (Miltenyi) after the transduction process to purify cells expressing CD40L.

[0341] In some embodiments, to differentiate transduced monocytes, such as CD34+ lentivirus+ transduced cells into CD14+CD16+ monocytes, purified CD34+Lv+ cells can be cultured in the following expansion medium: 5 CD34 +Cells can be expanded for 3-10 days at 10 cells / ml: G-Rex 10M (X-VIVO 10 medium; human AB serum 10%; rhSCF 50ng / ml (R&D systems); TPO 15ng / ml (R&D systems); IL-3 30ng / ml (R&D systems); Flt-3L 30ng / ml (R&D systems). After cell expansion (1-10 days), the cells can be moved to differentiation medium in G-Rex 100M for 14 days. A non-limiting example of differentiation medium includes, but is not limited to, IMDM with 20% human AB serum; SCF 25ng / ml (R&D systems); M-CSF 30ng / ml (R&D systems); IL-3 30ng / ml (R&D systems); and Flt-3L 30ng / ml (R&D Systems). The cells can then be incubated in the differentiation mixture for about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. A non-limiting example of a differentiation mixture is RPMI-1640; 3% human AB serum; GM-CSF 900IU / ml; IL-4 1000IU / ml; TNFα 400IU / ml; and TGFβ1 0.2ng / ml. The differentiation mixture can enhance the differentiation of monocytes into immature dendritic cells.

[0342] In some embodiments, in order to further enhance the maturation of immature dendritic cells, cells can be incubated in a maturation mixture. In some embodiments, the maturation mixture includes antigens, tumor lysates or live tumor cells. These examples are provided herein. The maturation mixture can include, for example, GM-CSF 500IU / ml; IL-15 400ng / nl; IFNγ100ng / ml; TNFα2ng / ml; and PgE2 2mcg / ml. This amount is only exemplary and is not limited to such an amount. Therefore, in some embodiments, the maturation composition (mixture) includes GM-CSF, IL-15, IFNγ, TNFα and / or PgE2. Cells can be incubated in a maturation composition for about 12 to about 48 hours, about 20 to about 40 hours, about 30 to about 38 hours, about or at least 12, 16, 18, 20, 24, 28, 32, 36, 40, 44 or 48 hours. The maturation cocktail may also include TNF-α, IL-1β, IFN-α, IFN-γ, and / or pIC.

[0343] Experimental results using one or more of these embodiments are Figure 2A -F shows representative phenotyping and purity assessment of CD14+ monocytes isolated using CliniMACS.

[0344] It showed that immature recombinant DCs exhibited phagocytic ability. Figure 4A -B. Figure 4A -B shows the results, the phagocytosis of fluorescently labeled Escherichia coli (E. coli) particles by immature DC products was observed by flow cytometry. Figure 4A is a negative control; Figure 4B It shows that 86.13% of the product has engulfed the fluorescently labeled E. coli particles.The assay used can be any engulfment assay.

[0345] in addition, Figure 5A -D shows that recombinant DC stimulated allogeneic T cell proliferation as indicated by dilution of carboxyfluorescein succinimidyl ester (CFSE). Figure 5A -D shows the results, allogeneic T cells were labeled with CFSE and co-cultured with recombinant alloDC. The proliferation of T cells can be observed by diluting CFSE. Figure 5A and Figure 5C ) shows proliferation at day 4, and the right column ( Figure 5B and Figure 5D ) shows proliferation on day 7. These results indicate that recombinant alloDC stimulated allogeneic T cell proliferation.

[0346] Useful aspects of the compositions and methods of the invention include the ability to bank recombinant DCs to provide a generic vaccine for use in local hospitals, thereby making it easier to treat patients. Such recombinant DCs can be processed in three different ways: 1) they can be loaded with autologous tumor lysate, or (2) in other cases, they can be loaded with MCV tumor lysate (Dandrit GMP, if ≥3 epitopes in the patient's tumor are present in the MCV antigen library) or other commercially available allogeneic tumor lysate; or (3) in the case of unresectable tumors, there may be no antigen loading.

[0347] In the next step of recombinant DC vaccine preparation, recombinant alloDCs are matured with a cocktail of recombinant human cytokines, such as those described above.

[0348] Then, the cells are phenotyped and functionally evaluated (as shown in Figures 4-5), as well as to evaluate the expression of relevant transgenes (e.g., CD40L, CXCL13, and CD93). Finally, the recombinant DCs are frozen or, more specifically, cryopreserved, usually at a controlled rate, and then used in a clinical setting after thawing. Such cells are used as the basis for recombinant DC biovaccines / immune adjuvants for the treatment of cancers, tumors, and malignancies. Fig. 9 Schematic diagram of the treatment, cell preparation and vaccination process is shown.

[0349] In some embodiments, cells are frozen, which can be referred to as cryopreservation. For example, cells can be frozen using CryoStorCS5 (freezing medium). However, this is a non-limiting example of freezing medium, and other freezing mediums can be used. Initially, cells can be refrigerated to 4°C. Cells can then be further cooled to about -20°C. Cells can be centrifuged or resuspended in additional freezing medium, and then cooled to -90°C in a stepwise manner. Cells can then be stored in liquid nitrogen. In some embodiments, cells are frozen in a freezing bag.

[0350] The cells can be thawed by thawing in a 37°C water bath. In some embodiments, the cells are thawed without moving the cells in the water bath, without a figure-of-eight motion or flicking. The cells can then be contacted with plasma and a warm thawing medium. The viability of the cells can then be analyzed prior to administration to a subject. Examples of markers are provided herein and above.

[0351] In some embodiments, dendritic cells are matured and / or DC is loaded with autologous tumor lysate / MCV with a cytokine mixture. For example, live immature DC can be cultured with tumor lysate or MCV and DC cells. This can be carried out in the presence of a mature mixture as described above or a composition such as comprising TNF-α, IL-1β, IFN-α, IFN-γ and pIC. Cells can be incubated with this composition for about 12 to about 36 hours, such as or about or at least 20, 22, 24, 26, 28 or 30 hours. Mature cells can then be collected and analyzed. Cryogenic media and progressive freezing methods can also be used to freeze mature cells, such as described herein.

[0352] As described herein, cells can be incubated or loaded with tumor lysates. Tumor lysates can be prepared in any way. For example, in some embodiments, tumor material is provided (isolated, obtained, excised, etc.). The sample can be quickly frozen in liquid nitrogen. In some embodiments, the sample does not contain non-malignant tissue. This may mean that the sample is removed without any non-malignant tissue, or the sample has been further processed to remove such non-malignant tissue. The sample can then be thawed and quickly frozen 1-5 times to help lyse the cells. The lysate can be centrifuged and filtered to prepare a tumor lysate. The lysate can be stored frozen, for example in a -80°C freezing device.

[0353] Embodiments are now described with reference to the following examples. These embodiments are provided only for illustrative purposes, and these embodiments should not be construed as being limited to these embodiments, but should be construed as covering any and all variations that become apparent due to the teachings provided herein. Those skilled in the art will readily recognize that various non-critical parameters that can be changed or modified to produce substantially similar results.

[0354] Example 1

[0355] Patients with unresectable metastatic colorectal cancer

[0356] A male patient was diagnosed with colorectal cancer at the age of 29, and a 6cm primary colorectal tumor was removed immediately after diagnosis. The patient chose to withdraw from adjuvant chemotherapy. The patient presented with recurrence of primary tumor lesions 18 months after the initial surgery, accompanied by multiple mesenteric and liver metastases. Because the primary tumor recurrence (16cm) blocked the colorectal passage, the patient underwent emergency colostomy surgery. During the colostomy surgery, a core biopsy was performed from the patient's primary tumor. Three weeks after surgery, the patient began to receive allogeneic DCs that were impacted with autologous tumor cells presenting CD40L and matured with a maturation mixture, as described above. The patient did not receive other treatments. The vaccine dosage schedule is 1x10 subcutaneously injected every 15 days. 6 Allo-DC CD40L+ loaded cells. Fig. 6A -B is a CAT / PET scan (CT) image showing a significant reduction in tumor mass 6 months after Allo-DC CD40L+ cell treatment (12 injections). After 6 months of treatment with alloDC CD40L+ vaccine loaded with autologous tumor cells, the primary tumor shrank from 12 cm to 17.3 mm x 34.5 mm ( Figure 6B ). These cells are transduced to express CD40L, and alloDC cells further transduced with CXCL13 and optionally CD93 are expected to further augment the anti-tumor effect (loaded with autologous tumors) together with the alloDC CD40L+ cell line.

[0357] Example 2: 69-year-old male with unresectable metastatic colorectal cancer

[0358] A male patient was diagnosed with colorectal cancer at the age of 68 years. Five centimeters (5 cm) of primary colorectal cancer were removed immediately after diagnosis. The patient received a 6-month FOLFOX regimen. In a follow-up evaluation after FOLFOX treatment, the patient presented with a recurrence of the primary tumor, accompanied by liver and lung metastases. After follow-up, the patient began to receive the FOLFIRINOX regimen. Six months after FOLFIRINOX treatment, the patient underwent emergency colostomy surgery due to the recurrence of the primary tumor blocking the colorectal passage. The patient began to receive an alloDC vaccine with an autologous tumor pulse presenting CD40L matured with a maturation mixture, as described above.

[0359] Patients received capecitabine only during vaccine treatment. The vaccine dosing schedule was 2x10 6cells. Fig. 7A -D shows that the CT / PET images in the alloDC CD40L+ vaccine loaded with autologous tumor cells for treatment six months (12 injections) showed a significant reduction in liver and lung masses. These cells were transduced to express CD40L, and alloDC cells further transduced with CXCL13 and optionally CD93 are expected to further increase the anti-tumor effect (loaded with autologous tumor) together with the alloDC CD40L+ cell line.

[0360] Example 3: 45-year-old woman with metastatic invasive ductal breast cancer

[0361] A female patient was diagnosed with HER-2 positive, ER and PR negative breast cancer and had a mastectomy in 2008 (T1cN2M0). The patient was treated with TAC for 6 months and achieved remission. In 2010, PET showed evidence of metastasis. The patient received gemcitabine, carboplatin, trastuzumab but the disease progressed. The patient then received bortezomib and lapatinib but the disease continued to progress. Third line treatment with ado-ente trastuzumab was started in 2013 until 2015. The disease progression in the lung continued.

[0362] The patient then received fifth-line treatment with trastuzumab and vinorelbine starting in August 2015 and interrupted in April 2016 due to progression. Sixth-line treatment with trastuzumab, bortezomib, and eribulin started in April 2016 and interrupted in July 2016 due to progression. Treatment with ado-entetrastuzumab and pablociclib started in July 2017 and interrupted in January 2018 due to progression. Eighth-line treatment with fulvestrant, trastuzumab, and pablociclib was given from January to August 2017 and interrupted due to progression.

[0363] The patient was diagnosed with brain metastases in October 2017 and underwent a right craniotomy and all five brain lesions were treated with stereotactic radiosurgery. The patient did not receive any other brain treatment at that time. The patient started treatment with trastuzumab, ixabepilone, and capecitabine in February 2018 and discontinued treatment in April 2018 due to progression. The patient received whole-brain radiation in August 2018 to become medically fit for immunotherapy. The patient received adoptive cellular therapy as initial treatment before a bronchoscopic tumor biopsy in September 2018.

[0364] Patients began receiving autologous tumor cell pulsed alloDC expressing CD40L and CXCL13 in September 2018, which were matured with a maturation cocktail as described herein. The vaccination dose was subcutaneous injection (sub-Q) of 10 6 The vaccine schedule was as follows: injections #1-#4 every 7 days, injections #5-#8 every 10 days, and injections #9-#12 every 15 days (a total of 12 sub-Q injections over 130 days). Fig. 8A -D CT and PET / CT images show a significant reduction in lung lesions one month after treatment with alloDCCD40L+CXCL13+ cells pulsed with autologous tumor cells.

[0365] See below Figure 10-12 Based on the synergistic in vitro results shown in , these cells were transduced to express CD40L and CXCL13, and it is expected that alloDC cells further transduced with CD93 will further amplify the anti-tumor effect (loaded with autologous tumors) together with the alloDC CD40L+CXCL13+ cell line.

[0366] Example 4: In vitro testing

[0367] T cell proliferation ( Fig.10 )

[0368] Tumor lysates obtained from breast cancer patients were used to generate mature DCs genetically modified with lentiviral vectors expressing CD40L+CXCL13+CD93 or CD40L+CXCL13 or CD40L alone. Mature allo-DCs (2x10 4 ) were co-cultured in triplicate in TexMACS (Miltenyi) medium supplemented with 500 IU / ml IL-2 (R&D Systems). 6 ). 48 hours after seeding, the persistence of allo-DCs was measured by flow cytometry (CD86 and CD1a). In each condition, no viable allo-DCs were detected after 48 h of co-culture.

[0369] NK cell proliferation ( Fig.11 )

[0370] Tumor lysates obtained from breast cancer patients were used to generate mature DCs genetically modified with lentiviral vectors expressing CD40L+CXCL13+CD93 or CD40L+CXCL13 or CD40L alone. Mature allo-DCs (1x10 4 ) Peripheral blood CD16+CD56+ NK cells (5x105 ). 48 hours after seeding, the persistence of allo-DCs was measured by flow cytometry (CD86 and CD1a). In each condition, no viable allo-DCs were detected after 48 h of co-culture.

[0371] B cell activation ( Fig.12 )

[0372] Tumor lysates obtained from breast cancer patients were used to generate mature DCs genetically modified with lentiviral vectors expressing CD40L+CXCL13+CD93 or CD40L+CXCL13 or CD40L alone. Mature allo-DCs (1x10 4 ) Peripheral blood CD19+ B cells (2.5×10 5 The activation level of B cells was detected by flow cytometry to determine the expression level of CD69 in CD19+ cells on day 4.

[0373] From Figure 10-12 The results showed a synergistic effect of co-expressing all three receptors on recombinant DC. Fig.10 In , T cell proliferation was used as a surrogate marker to indicate an increased cellular immune response to recombinant DC. The greatest increase in T cell proliferation was shown for triple recombinant DC (CD40L+CXCL13+ and CD93). Fig.11 In , the proliferation of NK (natural killer) cells co-cultured with recombinant DCs was used as another surrogate marker for increased cellular immune responses in vivo. Fig.11 The largest increase in NK cell proliferation was shown for the triple construct. Fig.12 In the present study, the activation level of B cells in co-culture was used as a surrogate marker for B cell activation in vivo. B cell activation was determined by analyzing CD69 expression in CD19+ cells in co-cultures on day 4. In these co-cultures, triple reconstituted DCs (CD40L+CXCL13+ and CD93) also showed the highest levels of CD69+ expression, similarly indicating activation of the humoral immune system.

[0374] Standard Methods

[0375] Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 19892 nd Edition,2001 3 rded.) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Sambrook and Russell (2001) Molecular Cloning, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley & Sons, New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0376] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley & Sons, New York). Chemical analysis, chemical modifications, post-translational modifications, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley & Sons, New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley & Sons, New York, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) Bio Directory, Piscataway, NJ, pp. 384-391). The production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0377] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent was specifically and individually indicated as incorporated by reference. This statement of incorporation by reference is intended to refer to each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, each of which is incorporated by reference in accordance with 37 CFR §1.57(b)(1), even if such citation is not immediately adjacent to a specific statement of incorporation by reference. The specific statement of incorporation by reference, if any, covers and in no way impairs such general statement of incorporation by reference. Citation of a reference herein is not intended to admit that the reference is relevant prior art, nor does it constitute any admission of the contents or date of such publications or documents.

[0378] The scope of the present invention is not limited by the specific embodiments described herein. In fact, in addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art based on the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Sequence Listing <110> RENOVARO BIOPHARMA INC. <120> Methods and compositions for using recombinant dendritic cells for cancer treatment <130> 146616.00302 <140> CN 201980051688.7 <141> 2019-07-15 <150> 62 / 698,254 <151> 2018-07-15 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 783 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 1 atgatcgaaa catacaacca aacttctccc cgatctgcgg ccactggact gcccatcagc 60 atgaaaattt tttgtattt acttactgtt tttcttatca cccagatgat tgggtcagca 120 cttttgctg tgtatcttca tagaaggttg gacaagatag aagatgaaag gaatcttcat 180 gaagattttg tattcatgaa aacgatacag agatgcaaca caggagaaag atccttatcc 240 ttactgaact gtgaggagat taaaagccag tttgaaggct ttgtgaagga tataatgtta 300 aacaaagagg agacgaagaa agaaaacagc tttgaaatgc aaaaaggtga tcagaatcct 360 caaattgcgg cacatgtcat aagtgaggcc agcagtaaaa caacatctgt gttacagtgg 420 gctgaaaaag gatactacac catgagcaac aacttggtaa ccctggaaaa tgggaaacag 480 ctgaccgtta aaagacaagg actctattat 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ccatgggcct gctgctgctg ctgctgctgc tcctgaccca gcccggggcg 60 gggacgggag ctgacacgga ggcggtggtc tgcgtgggga ccgcctgcta cacggcccac 120 tcgggcaagc tgagcgctgc cgaggcccag aaccactgca accagaacgg gggcaacctg 180 gccactgtga agagcaagga ggaggcccag cacgtccagc gagtactggc ccagctcctg 240 aggcgggagg cagccctgac ggcgaggatg agcaagttct ggattgggct ccagcgagag 300 aagggcaagt gcctggaccc tagtctgccg ctgaagggct tcagctgggt ggggcgggggg 360 gaggacgc cttactctaa ctggcacaag gagctccgga actcgtgcat ctccaagcgc 420 tgtgtgtctc tgctgctgga cctgtcccag ccgctccttc ccagccgcct ccccaagtgg 480 tctgagggcc cctgtgggag cccaggctcc cccggagaga aattgaggg cttcgtgtgc 540 aagttcagct tcaaaggcat gtgccggcct ctggccctgg ggggcccagg tcaggtgacc 600 tacaccaccc ccttccagac caccagttcc tccttggagg ctgtgccctt tgcctctgcg 660 gccaatgtag cctgtgggga aggtgacaag gacgagactc agagtcatta tttcctgtgc 720 aaggagaagg cccccgatgt gttcgactgg ggcagctcgg gccccctctg tgtcagcccc 780 aagtatggct gcaacttcaa caatggggc tgccaccagg actgctttga agggggggat 840 ggctccttcc tctgcggctg ccgaccagga ttccggctgc tggatgacct ggtgacctgt 900 gcctctcgaa acccttgcag ctccagccca tgtcgtgggg gggccacgtg cgtcctggga 960 ccccatggga aaaactacac gtgccgctgc ccccaagggt accagctgga ctcgagtcag 1020 ctggactgtg tggacgtgga tgaatgccag gactccccct gtgcccagga gtgtgtcaac 1080 acccctgggg gcttccgctg cgaatgctgg gttggctatg agccgggcgg tcctggagag 1140 ggggcctgtc aggatgtgga tgagtgtgct ctgggtcgct cgccttgcgc ccagggctgc 1200 accaacacag atggctcatt tcactgctcc tgtgaggagg gctacgtcct ggccggggag 1260 gacgggactc agtgccagga cgtggatgag tgtgtgggcc cggggggccc cctctgcgac 1320 agcttgtgct tcaacacaca agggtccttc cactgtggct gcctgccagg ctgggtgctg 1380 gccccaaatg gggtctcttg caccatgggg cctgtgtctc tgggaccacc atctgggccc 1440 cccgatgagg aggacaaagg agagaaagaa gggagcaccg tgccccgtgc tgcaacagcc 1500 agtcccacaa ggggccccga gggcaccccc aaggctacac ccaccacaag tagaccttcg 1560 ctgtcatctg acgcccccat cacatctgcc ccactcaaga tgctggcccc cagtgggtcc 1620. ccaggcgtct ggagggagcc cagcatccat cacgccacag ctgcctctgg cccccaggag 1680. cctgcaggtg gggactcctc cgtggccaca caaaacaacg atggcactga cgggcaaaag ctgcttttat tctacatcct aggcaccgtg gtggccatcc tactcctgct ggccctggct 1800. ctggggctac tggtctatcg caagcggaga gcgaagaggg aggagaga ggagagaga 1860. ccccagaatg cggcagacag ttactcctgg gttccagagc gagctgagag cagggccatg gagaaccagt acagtccgac acctgggaca gactgc <210> 4 <211> 261 <212> PRT <213> The snowstorm <220> <223> Make a snowflake: a snowflake <400> 4 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu Free Mp3 Download 20 25 30 Ile Thr Gln Met Ile Gly Ser Free Mp3 Download 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> 5 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 5 Met Lys Phe Ile Ser Thr Ser Leu Leu Leu Met Leu Leu Val Ser Ser 1 5 10 15 Leu Ser Pro Val Gln Gly Val Leu Glu Val Tyr Tyr Thr Ser Leu Arg 20 25 30 Cys Arg Cys Val Gln Glu Ser Ser Val Phe Ile Pro Arg Arg Phe Ile 35 40 45 Asp Arg Ile Gln Ile Leu Pro Arg Gly Asn Gly Cys Pro Arg Lys Glu 50 55 60 Ile Ile Val Trp Lys Lys Asn Lys Ser Ile Val Cys Val Asp Pro Gln 65 70 75 80 Ala Glu Trp Ile Gln Arg Met Met Glu Val Leu Arg Lys Arg Ser Ser 85 90 95 Ser Thr Leu Pro Val Pro Val Phe Lys Arg Lys Ile Pro 100 105 <210> 6 <211> 652 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 6 Met Ala Thr Ser Met Gly Leu Leu Leu Leu Leu Leu Leu Leu Leu Thr 1 5 10 15 Gln Pro Gly Ala Gly Thr Gly Ala Asp Thr Glu Ala Val Val Cys Val 20 25 30 Gly Thr Ala Cys Tyr Thr Ala His Ser Gly Lys Leu Ser Ala Ala Glu 35 40 45 Ala Gln Asn His Cys Asn Gln Asn Gly Gly Asn Leu Ala Thr Val Lys 50 55 60 Ser Lys Glu Glu Ala Gln His Val Gln Arg Val Leu Ala Gln Leu Leu 65 70 75 80 Arg Arg Glu Ala Ala Leu Thr Ala Arg Met Ser Lys Phe Trp Ile Gly 85 90 95 Leu Gln Arg Glu Lys Gly Lys Cys Leu Asp Pro Ser Leu Pro Leu Lys 100 105 110 Gly Phe Ser Trp Val Gly Gly Gly Glu Asp Thr Pro Tyr Ser Asn Trp 115 120 125 His Lys Glu Leu Arg Asn Ser Cys Ile Ser Lys Arg Cys Val Ser Leu 130 135 140 Leu Leu Asp Leu Ser Gln Pro Leu Leu Pro Ser Arg Leu Pro Lys Trp 145 150 155 160 Ser Glu Gly Pro Cys Gly Ser Pro Gly Ser Pro Gly Ser Asn Ile Glu 165 170 175 Gly Phe Val Cys Lys Phe Ser Phe Lys Gly Met Cys Arg Pro Leu Ala 180 185 190 Leu Gly Gly Pro Gly Gln Val Thr Tyr Thr Thr Pro Phe Gln Thr Thr 195 200 205 Ser Ser Ser Leu Glu Ala Val Pro Phe Ala Ser Ala Ala Asn Val Ala 210 215 220 Cys Gly Glu Gly Asp Lys Asp Glu Thr Gln Ser His Tyr Phe Leu Cys 225 230 235 240 Lys Glu Lys Ala Pro Asp Val Phe Asp Trp Gly Ser Ser Gly Pro Leu 245 250 255 Cys Val Ser Pro Lys Tyr Gly Cys Asn Phe Asn Asn Gly Gly Cys His 260 265 270 Gln Asp Cys Phe Glu Gly Gly Asp Gly Ser Phe Leu Cys Gly Cys Arg 275 280 285 Pro Gly Phe Arg Leu Leu Asp Asp Leu Val Thr Cys Ala Ser Arg Asn 290 295 300 Pro Cys Ser Ser Ser Pro Cys Arg Gly Gly Ala Thr Cys Val Leu Gly 305 310 315 320 Pro His Gly Lys Asn Tyr Thr Cys Arg Cys Pro Gln Gly Tyr Gln Leu 325 330 335 Asp Ser Ser Gln Leu Asp Cys Val Asp Val Asp Glu Cys Gln Asp Ser 340 345 350 Pro Cys Ala Gln Glu Cys Val Asn Thr Pro Gly Gly Phe Arg Cys Glu 355 360 365 Cys Trp Val Gly Tyr Glu Pro Gly Gly Pro Gly Glu Gly Ala Cys Gln 370 375 380 Asp Val Asp Glu Cys Ala Leu Gly Arg Ser Pro Cys Ala Gln Gly Cys 385 390 395 400 Thr Asn Thr Asp Gly Ser Phe His Cys Ser Cys Glu Glu Gly Tyr Val 405 410 415 Leu Ala Gly Glu Asp Gly Thr Gln Cys Gln Asp Val Asp Glu Cys Val 420 425 430 Gly Pro Gly Gly Pro Leu Cys Asp Ser Leu Cys Phe Asn Thr Gln Gly 435 440 445 Ser Phe His Cys Gly Cys Leu Pro Gly Trp Val Leu Ala Pro Asn Gly 450 455 460 Val Ser Cys Thr Met Gly Pro Val Ser Leu Gly Pro Pro Ser Gly Pro 465 470 475 480 Pro Asp Glu Glu Asp Lys Gly Glu Lys Glu Gly Ser Thr Val Pro Arg 485 490 495 Ala Ala Thr Ala Ser Pro Thr Arg Gly Pro Glu Gly Thr Pro Lys Ala 500 505 510 Thr Pro Thr Thr Ser Arg Pro Ser Leu Ser Ser Asp Ala Pro Ile Thr 515 520 525 Ser Ala Pro Leu Lys Met Leu Ala Pro Ser Gly Ser Pro Gly Val Trp 530 535 540 Arg Glu Pro Ser Ile His His Ala Thr Ala Ala Ser Gly Pro Gln Glu 545 550 555 560 Pro Ala Gly Gly Asp Ser Ser Val Ala Thr Gln Asn Asn Asp Gly Thr 565 570 575 Asp Gly Gln Lys Leu Leu Leu Phe Tyr Ile Leu Gly Thr Val Val Ala 580 585 590 Ile Leu Leu Leu Leu Ala Leu Ala Leu Gly Leu Leu Val Tyr Arg Lys 595 600 605 Arg Arg Ala Lys Arg Glu Glu Lys Lys Glu Lys Lys Pro Gln Asn Ala 610 615 620 Ala Asp Ser Tyr Ser Trp Val Pro Glu Arg Ala Glu Ser Arg Ala Met 625 630 635 640 Glu Asn Gln Tyr Ser Pro Thr Pro Gly Thr Asp Cys 645 650

Claims

1. A dendritic cell comprising one or more heterologous nucleic acid molecules encoding CD40L, CXCL13 and CD93.

2. A dendritic cell comprising a heterologous CD40L protein, a heterologous CXCL13 protein and a heterologous CD93 protein.

3. An antigen-activated dendritic cell, wherein: The dendritic cells include one or more heterologous nucleic acid molecules encoding CD40L, CXCL13, and CD93.

4. The antigen-activated dendritic cell according to claim 3, wherein The cells are activated by exposure to one or more antigens.

5. The antigen-activated dendritic cell according to claim 4, wherein The antigen is a tumor antigen.

6. The antigen-activated dendritic cell according to claim 4, wherein The antigen is a viral antigen.

7. The antigen-activated dendritic cell according to any one of claims 4 to 6, wherein: The antigen is a cell lysate.

8. The antigen-activated dendritic cell according to claim 7, wherein The cell lysate is either allogeneic or autologous with respect to the antigen-activated dendritic cells.

9. The antigen-activated dendritic cell according to claim 7, wherein The cell lysate is a lysate comprising one or a combination of allogeneic melanoma cell lysates.

10. The antigen-activated dendritic cell according to claim 9, wherein The allogeneic melanoma cell lysate is DDM-1.7 cell lysate, DDM-1.13 cell lysate or a combination thereof.

11. The antigen-activated dendritic cell according to claim 7, wherein The cell lysate is a whole cell lysate.

12. The antigen-activated dendritic cell according to claim 7, wherein The cell lysate is a tumor cell lysate.

13. A composition comprising the cells of any one of claims 1-12, wherein The composition does not include a heterologous antigen.

14. A pharmaceutical composition comprising the cell of any one of claims 1-12.

15. Use of the dendritic cell of any one of claims 1 to 12 or the composition of claim 13 or 14 in the preparation of a medicament for treating breast cancer, colorectal cancer or pancreatic cancer in a subject.

16. The use according to claim 15, wherein the medicament is prepared for use in a method comprising: Prior to administration, screening the protein expression profile of a resected tumor or biopsy sample from the subject to cross-match the protein expression profile of an allogeneic tumor lysate; and If at least three segments of the protein expression profile of the resected tumor or biopsy sample cross-match the protein expression profile of the allogeneic tumor lysate, then the allogeneic tumor lysate-activated dendritic cells or activated dendritic cell composition are administered.

17. The use according to claim 16, wherein The allogeneic tumor lysate is derived from an allogeneic melanoma cell line selected from the group consisting of DDM-1.7, DDM-1.13, or a combination thereof.

18. The use according to any one of claims 15 to 17, wherein The dendritic cells do not have the same HLA type as the subject.

19. The use according to claim 15, wherein The drug is administered via intratumoral, peritumoral, intradermal, subcutaneous, intramuscular or intraperitoneal injection.

20. The use according to claim 15, wherein The drug is administered via subcutaneous, intratumoral or intradermal injection.

21. The use according to claim 15, wherein The dendritic cells or compositions are cryopreserved and thawed prior to administration.

22. The use according to claim 15, wherein The dendritic cells were not pulsed with antigen prior to administration.

23. A kit comprising the dendritic cell of any one of claims 1 to 12 or any one of the compositions of claims 13 or 14, wherein: The dendritic cells or composition are frozen or cryopreserved in a container and optionally include an autologous tumor lysate or an allogeneic tumor lysate frozen or cryopreserved in a separate container.

24. The kit of claim 23, further comprising a separate container comprising one or more buffers and, optionally, one or more activators.

25. The kit according to claim 24, further comprising instructions for incubating and / or processing the dendritic cells or composition.

26. The dendritic cell of any one of claims 1 to 12; or (a) a composition comprising a dendritic cell according to any one of claims 1 to 7 and 11 or any one of the compositions according to claim 13 pulsed with autologous tumor lysate, or (b) The cell according to claim 1 or 2 or the composition according to claim 13 which has not been subjected to antigen pulse Use in the preparation of a medicament for treating breast cancer, colorectal cancer or pancreatic cancer in a subject, wherein the medicament is prepared for use in a method comprising: Prior to administration, screening a protein expression profile of a resected tumor or biopsy sample from the subject to cross-match with a protein expression profile of an allogeneic tumor lysate; If at least three segments of the protein expression profile of the resected tumor or biopsy sample match the protein expression profile of the allogeneic tumor lysate, administering any one of the dendritic cells of any one of claims 1-12 to the subject; or If the at least three segments of the protein expression profile of the resected tumor or biopsy sample do not match the allogeneic tumor lysate protein expression profile, then (a) administering to the subject a composition comprising the dendritic cells of any one of claims 1 to 7 and 11 or any one of the compositions of claim 13 pulsed with autologous tumor lysate, or (b) administering to the subject any one of the cells of claim 1 or 2 or the composition of claim 13 that has not been pulsed with an antigen.

27. Use of the dendritic cells of claim 1 or 2 or the composition of claim 13 in the preparation of a medicament for treating breast cancer, colorectal cancer or pancreatic cancer in a subject, wherein (i) the dendritic cells or the composition have not been pulsed with a tumor antigen; or (ii) the dendritic cells have been pulsed with a tumor antigen or a lysate.

28. A method for producing immature dendritic cells, comprising: CD14+ and / or CD1a+ monocytes heterologously expressing CD40L, CXCL13 and CD93 were cultured into immature dendritic cells in vitro.

29. The method of claim 28, further comprising contacting the immature dendritic cells with a composition comprising one or more of: TNF-α, IL-1β, IFN-α, IFN-γ, and pIC.

30. The method of claim 28, further comprising generating CD14+ monocytes heterologously expressing CD40L, CXCL13, and CD93.

31. The method according to claim 30, wherein: The generating comprises contacting CD14+ monocytes with one or more vectors encoding CD40L, CXCL13 and CD93.

32. The method according to claim 31, wherein: The vector is a recombinant adenoviral vector, a recombinant retroviral vector or a recombinant lentiviral vector or a combination thereof.

33. The method according to claim 31, wherein: The generating of CD14+ monocytes heterologously expressing CD40L, CXCL13 and CD93 comprises contacting the monocytes with one or more CRISPR constructs to generate CD14+ monocytes heterologously expressing CD40L, CXCL13 and CD93.

34. The method of claim 33, wherein: The CRISPR construct is CAS9.

35. The method according to any one of claims 31 to 34, wherein: The contacting includes transfection, transduction, electroporation, infection, or any combination thereof.

36. The method of claim 28, further comprising freezing the immature dendritic cells.

37. The method of claim 28, further comprising contacting the immature dendritic cells with autologous tumor lysate or allogeneic tumor lysate prior to maturation.

38. The method of claim 37, further comprising freezing the dendritic cells after maturation.

39. The method of claim 37, wherein: The allogeneic tumor lysate is derived from an allogeneic melanoma cell line selected from the group consisting of DDM-1.7, DDM-1.13, or a combination thereof.

40. The method of claim 37, wherein: The lysate is a whole cell lysate.

41. The use according to claim 15 or 27, wherein A graft-versus-tumor (GVT) response is increased in the subject.

42. The use according to claim 15 or 27, wherein the subject is further administered one or more additional agents for treating cancer.

43. The use according to claim 42, wherein Within a time range of 4-14 days after administration of the dendritic cells or the composition, the subject is further administered an immune checkpoint inhibitor, which includes an inhibitor of PD-1 or PD-L1, an antagonist of CTLA-4, or a combination thereof.

44. The use according to claim 43, wherein The PD-1 or PD-L1 inhibitor is selected from the group consisting of nivolumab, MK-3475, pidilizumab, AMP-224, MPDL3280A, MDX-1105, MEDI-4736 and MSB-0010718 C.

45. The use according to claim 43, wherein The CTLA-4 antagonist is selected from the group consisting of ipilimumab, tremelimumab, decilimumab, AMGP-224, trastuzumab, a tumor-specific antibody for breast cancer, and rituximab and cetuximab for lymphoma.

46. ​​The use according to claim 42, wherein The additional agent is selected from radiation, chemotherapy, an antibody drug conjugate, and an immunomodulatory antibody.

47. The use according to claim 46, wherein The chemotherapy is cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, vinblastine, irinotecan, etoposide or pemetrexed or a combination thereof, or a pharmaceutically acceptable salt thereof.

48. The use according to claim 46, wherein The immunomodulatory antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD40 antibody, an anti-CTLA-4 antibody or an anti-OX40 antibody, or any combination thereof.

49. The use according to claim 46, wherein The antibody drug conjugate targets c-Met kinase, LRRC15, EGFR or CS1 or any combination thereof.

50. The use according to claim 15 or 27, wherein As long as the subject shows improvement or stable / non-progressive disease, the treatment or increase the immune response as a maintenance therapy is repeated regularly within the following time frame: from once every 5 days, once every 10 days, once every 14 days, once every 21 days, once a month to once every two months, to once every 3 months, to once every 4 months, to once every 5 months, to once every 6 months, or once every 7 months, or once every 8 months, or once every 9 months, or once every 10 months, or once every 11 months, or once a year.

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