Ex vivo generation of immunoeffector cells from apheresis material intermediates

Abstract

This specification provides compositions and methods for generating tumor-targeting lymphocytes and / or tumor-infiltrating lymphocytes and / or tumor-targeting natural killer (NK) cells for use in the treatment of cancer or infectious diseases. Methods for their preparation and use are also provided.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 512,032 filed July 5, 2023, No. 63 / 515,528 filed July 25, 2023, and No. 63 / 605,326 filed December 1, 2023. Each of the above applications is incorporated in whole by reference.

[0002] The field of this invention is immunotherapy technology. [Background technology]

[0003] The background information contains information that may be useful in understanding this disclosure. Nothing provided herein constitutes prior art, or relates to the claimed invention, or any publication specifically or implicitly referenced is prior art.

[0004] All publications and patent applications herein are incorporated by reference to the same extent as each individual publication or patent application is specifically and individually indicated and incorporated by reference. If a definition or use of a term in an incorporated reference conflicts with or is contrary to a definition of that term provided herein, the definition provided herein shall prevail, and the definition in the reference shall not prevail.

[0005] Autologous or allogeneic T cells and natural killer cells have significant effects on refractory malignancies. Apheresis generates starting materials for T cell or NK cell production. Because NK cells make up only a small fraction (about 1-20%) of lymphocytes, methods have been developed to enrich them from large amounts of peripheral blood, such as apheresis products, or to expand NK cell populations from smaller amounts of blood or stem cells. As the clinical applications of T cells and NK cells expand, researchers are seeking to develop new and improved methods to obtain these cells from peripheral blood mononuclear cells (PBMCs). [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Thus, in this field, there is still a need for improved methods for isolating and expanding T cells and NK cells for the treatment of patients requiring treatment for cancer or infectious diseases. [Means for solving the problem]

[0007] The subject of this invention is to provide compositions and methods for generating tumor-targeting lymphocytes and / or tumor-infiltrating lymphocytes and / or tumor-targeting natural killer (NK) cells for use in the treatment of cancer or infectious diseases.

[0008] In one embodiment, the subject matter of the present invention includes tumor-targeted lymphocytes for use in the treatment of cancer or infectious diseases. Tumor-targeted lymphocytes are produced by a method comprising the steps of: performing therapeutic apheresis on a subject with cancer or infectious disease; purifying a CD3+ T cell fraction from the apheresis product (where the remaining apheresis product includes a CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction (where the remaining apheresis product includes a CD3- / CD14- fraction); differentiating CD14+ monocytes into dendritic cells (DCs); exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence (where the MHC-I or MHC-II of the DCs presents the peptide sequence or a portion thereof, thereby activating the DCs); exposing purified CD3+ T cells to activated DCs, thereby expanding the T cells; and purifying the expanded T cells.

[0009] In one embodiment, the subject of the present invention includes tumor-targeted natural killer (NK) cells for use in the treatment of cancer or infectious diseases. The NK cells are obtained by: performing therapeutic apheresis on a subject with cancer or infectious disease; purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product includes the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product includes the CD3- / CD14- fraction); expanding the NK cells from the CD3-CD14- fraction of the apheresis product; differentiating the CD14+ monocytes into dendritic cells (DCs); and infecting the DCs with an antigenic peptide or adenovirus encoding an antigenic peptide sequence. The NK cells are produced by a method comprising the steps of: exposure (where the MHC-I or MHC-II of the DC presents a peptide sequence or a portion thereof, thereby activating the DC); exposure of purified CD3+ T cells to activated DCs, thereby expanding the T cells; purification of the expanded T cells; isolation of at least one nucleic acid encoding the α and β chains of the T cell receptor (TCR) from the expanded T cells and fusion of the nucleic acid to the 5' end of a second nucleic acid encoding the transmembrane domain and intracellular signaling domain of the chimeric antigen receptor (CAR) (where the fused nucleic acid encodes the TCR CAR); and transfecting enriched and expanded NK cells with the nucleic acid encoding the TCR CAR.

[0010] In one embodiment, the subject matter of the present invention includes a method for expanding tumor-infiltrating lymphocytes for use in the treatment of cancer or infectious diseases. The method includes: performing therapeutic apheresis on a subject with cancer or infection; purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); expanding NK cells from the CD3- / CD14- fraction of the apheresis product; differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an adenovirus encoding an antigenic peptide or antigenic peptide sequence (where the MHC-I or MHC-II of the DCs presents the peptide sequence or a portion thereof, thereby activating the DCs); exposing the purified CD3+ T cells to activated DCs to expand the T cells; purifying the expanded T cells; purifying CD3+ TILs from solid tumors and exposing the TILs to activated DCs to expand the TILs; and purifying the expanded TILs.

[0011] In one embodiment, the subject of the present invention is a pharmaceutical composition comprising: 1) activated dendritic cells (DCs) wherein the DCs are differentiated from CD14+ monocytes derived from apheresis of a patient; 2) an adenovirus encoding an antigenic peptide sequence wherein the DCs are activated upon exposure to the adenovirus; and 3) CD3+ T cells derived from apheresis of a patient, wherein the CD3+ T cells are activated and expanded upon exposure to activated DCs. The pharmaceutical composition is intended for use in the treatment of cancer or infectious diseases.

[0012] In one embodiment, the subject of the present invention comprises a pharmaceutical composition for use in the treatment of cancer or infection, comprising patient-derived dendritic cells (DCs), GM-CSF, and an adenovirus (Ad) encoding an antigenic peptide, wherein the DCs are derived from the patient's apheresis.

[0013] In one embodiment, the subject of the present invention comprises a pharmaceutical composition for use in the treatment of cancer or infection, comprising 1) patient-derived natural killer (NK) cells and / or natural killer T (NKT) cells, 2) GM-CSF, and 3) an adenovirus (Ad) encoding an antigenic peptide, wherein the NK cells or NKT cells are derived from the patient's apheresis.

[0014] This specification further discloses a pharmaceutical composition for use in the treatment of cancer or infection, comprising 1) patient-derived T cells, B cells, and / or monocytes, 2) GM-CSF, and 3) an adenovirus (Ad) encoding an antigenic peptide, wherein the T cells, B cells, and / or monocytes are derived from the patient's apheresis.

[0015] Furthermore, this specification also discloses a pharmaceutical composition comprising 1) dendritic cells (DCs), 2) an irradiated biopsy sample, and 3) T cells, wherein the DCs and T cells are derived from the patient's apheresis, the biopsy sample is derived from a tumor of the same patient, and the pharmaceutical composition is for use in the treatment of cancer.

[0016] In one embodiment, the subject of the present invention comprises a pharmaceutical composition comprising dendritic cells (DCs) and T cells, wherein the DCs and T cells are isolated from apheresis products of a patient, the DCs and / or T cells are exposed to a biopsy sample from a tumor of the same patient, and the pharmaceutical composition is formulated for administration to the patient.

[0017] In one embodiment, the subject of the present invention comprises a pharmaceutical composition comprising dendritic cells (DCs) and T cells, wherein an apheresis sample from a patient is exposed to a biopsy sample from a tumor of the same patient, and the DCs and T cells are then isolated from the apheresis sample, and the pharmaceutical composition is formulated for administration to the patient.

[0018] This specification further discloses a method for generating tumor-targeted lymphocytes for use in the treatment of cancer and infectious diseases. The method includes performing therapeutic apheresis on a subject with cancer or an infectious disease; purifying a CD3+ T cell fraction from the apheresis product (where the remaining apheresis product includes a CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product includes a CD3- / CD14- fraction); expanding NK cells from the CD3- / CD14- fraction of the apheresis product; differentiating CD14+ monocytes into dendritic cells (DCs), exposing the DCs to at least one compound selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor, thereby activating the DCs by presenting at least one re-expressed peptide sequence or a part thereof by MHC-I or MHC-II of the DCs; and exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cells.

[0019] In yet another embodiment, the inventors disclose a method for generating tumor-targeted natural killer (NK) cells for use in the treatment of cancer or infection. The method involves: performing therapeutic apheresis on a subject with cancer or infection; purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); expanding NK cells from the CD3- / CD14- fraction of the apheresis product; differentiating the CD14+ monocytes into dendritic cells (DCs); and treating the DCs with a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and histogenic therapy. The method comprises: exposing DCs to at least one compound selected from the group consisting of HDAC inhibitors, thereby causing the MHC-I or MHC-II of the DCs to present at least one re-expressed peptide sequence or a portion thereof, thereby activating the DCs; exposing purified CD3+ T cells to activated DCs, thereby expanding the T cells; isolating at least one nucleic acid encoding the α and β chains of the T cell receptor (TCR) from the expanded T cells and fusing the nucleic acid to the 5' end of a second nucleic acid encoding the transmembrane domain and intracellular signaling domain of the chimeric antigen receptor (CAR) (wherein the fused nucleic acid encodes TCR CAR); and transfecting enriched and expanded NK cells with the nucleic acid encoding TCR CAR.

[0020] In one embodiment, the subject matter of the present invention includes a method for expanding tumor infiltrating lymphocytes for use in the treatment of cancer or an infectious disease. The method includes performing therapeutic apheresis on a subject with cancer or an infectious disease; purifying a CD3+ T cell fraction from the apheresis product (where the remaining apheresis product includes a CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product includes a CD3- / CD14- fraction); expanding NK cells from the CD3- / CD14- fraction of the apheresis product; differentiating CD14+ monocytes into dendritic cells (DCs), and exposing the DCs to at least one compound selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor, whereby the MHC-I or MHC-II of the DCs presents at least one re-expressed peptide sequence or a part thereof, thereby activating the DCs; purifying CD3 TILs from a solid tumor, and exposing the CD3 + TILs to the activated DCs, thereby expanding the TILs; and purifying the expanded TILs.

[0021] Also disclosed herein is a pharmaceutical composition comprising tumor-targeting CD3 + T lymphocytes, wherein the pharmaceutical composition is for use in the treatment of cancer or an infectious disease. The composition comprises 1) dendritic cells (DCs) that are differentiated from apheresis-purified CD14+ monocytes from a patient, 2) at least one compound selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor, whereby the MHC-I or MHC-II of the DCs presents at least one re-expressed peptide sequence or a part thereof, thereby activating the DCs, and 3) CD3 + T cells purified from a patient's apheresis, wherein the T cells are exposed to the activated DCs, thereby activating and expanding the T cells.+ Includes T cells.

[0022] This specification further discloses a method for expanding tumor-infiltrating lymphocytes for use in the treatment of cancer and infectious diseases. The method involves: performing therapeutic apheresis on a subject with cancer or infectious disease (where the subject is treated with at least one therapeutic agent selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors); purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an adenovirus encoding an antigenic peptide or antigenic peptide sequence (where the MHC-I or MHC-II of the DCs presents the peptide sequence or a portion thereof, thereby activating the DCs); and purifying CD3+ T cells from solid tumors. + The process includes purifying the TIL, exposing the TIL to an activated DC, thereby expanding the TIL; and purifying the expanded TIL.

[0023] In one embodiment, the subject matter of the present invention includes a method for generating tumor-targeted lymphocytes for use in the treatment of cancer or infectious diseases. The method includes: performing therapeutic apheresis on a subject with cancer or infection (where the subject is treated with at least one therapeutic agent selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors); purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an adenovirus encoding an antigenic peptide or antigenic peptide sequence (where the MHC-I or MHC-II of the DCs presents the peptide sequence or a portion thereof, thereby activating the DCs); exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cells; and purifying the expanded T cells.

[0024] Various objects, features, aspects, and advantages of the subject matter of the present invention will become apparent from the following detailed description of preferred embodiments in conjunction with the accompanying drawings. In the figures, the same numbers represent the same components. [Brief explanation of the drawing]

[0025] [Figure 1] The IncuCyte image of donor 001 in a 12-well plate 5 days after transduction is shown. [Figure 2] The results for donor 001 are illustrated, showing that different cytokine cocktails (N-803, GM-CSF, IL-4) induce different cell morphologies. [Figure 3] The results for donor 001 are illustrated, showing that different cytokine cocktails (N-803, GM-CSF, IL-4) result in different cell morphologies. [Figure 4-1]The results for donor 001 are shown, illustrating that more GFP+ cells were observed in the GM-CSF treatment group on days 3 and 5 after transduction. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5] This image shows the IncuCyte image of donor 002 in a 12-well plate 5 days after transduction. [Figure 6] The results for donor 002 are illustrated, showing that different cytokine cocktails (N-803, GM-CSF, IL-4) induce different cell morphologies. [Figure 7-1] The results for donor 002 are shown, illustrating that more GFP+ cells were observed in the GM-CSF treatment group on days 3 and 5 after transduction. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 8] The gating strategy for flow cytometry is illustrated. [Figure 9-1] This chart shows the treatment plan for locally advanced neoadjuvant pancreatic cancer. [Figure 9-2] This is a continuation of Figure 9-1. [Figure 9-3] This is a continuation of Figure 9-2. [Modes for carrying out the invention]

[0026] The subject of this invention is to provide compositions and methods for exposing dendritic cells from patients (cancer patients or patients with viral infections) to pharmacological manipulation of epigenetic modifiers. In particular, the protein arginine methyltransferase 5 (PRMT5), DNA methyltransferase (DNMT), and histone deacetylase (HDAC) mediate epigenetic events. These proteins have been shown to modulate the activity of genes involved in both tumor cell proliferation and tumor suppression in vitro and in vivo (preclinical and clinical). Thus, epigenetic regulation has become a clinically effective target for reducing tumor cell proliferation and inducing tumor suppression. Similar to how genes for tumor suppression can be epigenetically silenced, the inventors have devised and present herein epigenetic silencing of immunogenic peptides. This silences mutations that would normally be monitored by the cellular immune system. Treatment with epigenetic inhibitors promotes the re-expression of immunogenic peptides derived from mutated genes or cells infected with infectious agents.

[0027] As further described in this disclosure, apheresis products are processed to purify CD3+ T cells and CD14+ monocytes, and NK cells are enriched and expanded using CD3-CD14- cells. CD14+ cells are used to differentiate into dendritic cells and are then exposed to at least one epigenetic modifier, the drug selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors. The presence of the drug induces the expression of neoepitopes presented by MHC-I and MHC-II on the cell surface. Subsequent exposure to T cells activates and expands T cells specific to the newly expressed neoepitopes. The same logic applies to the expression of infectious factor epitopes.

[0028] Furthermore, it is intended that the patient is treated with at least one epigenetic modulator selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors. The apheresis product is derived from the treated patient and is isolated into dendritic cells and T lymphocytes. The dendritic cells reexpress suppressed neoepitopes or immunogenic peptides to stimulate the isolated T cells. The epigenetic modulators expose genes encoding tumor-promoting peptides (cell cycle inducers) and / or tumor-specific peptides (neoepitopes), both of which stimulate the activation and proliferation of tumor-educating lymphocytes via MHC presentation.

[0029] In another embodiment, NK cells are isolated from apheresis products, activated and expanded ex vivo, and reinjected into a patient along with tumor or virus-educated T cells.

[0030] In another embodiment, purified dendritic cells are treated with IL-15 or its agonist derivative (such as N-803). The dendritic cells may be from patients treated with epigenetic modifiers, or they may be ex vivo treated with epigenetic modifiers. The dendritic cells may be derived from patients treated with epigenetic modifiers and / or IL-15 agonist derivatives, particularly their stabilized derivatives.

[0031] In another embodiment, the patient may be administered IL-15 or its stabilized agonist derivatives in conjunction with T cell and / or NK cell therapy. Administration of tumor or virus-educated lymphocytes may be complemented by chemotherapeutic agents, tumor-targeting antibodies, checkpoint inhibitor antibodies, vaccines (adenovirus or yeast-based), or radiation.

[0032] In one embodiment, the present disclosure provides a method for generating tumor-targeted lymphocytes for use in the treatment of cancer or infection. The method comprises performing therapeutic apheresis on a subject with cancer or infection. In this regard, it should be recognized that apheresis is performed by taking circulating blood from a person, passing the blood through a device that separates red blood cells and white blood cells (apheresis products) from the plasma, and returning the plasma to the patient's circulation. The apheresis products are separated into a purified CD3+ T cell fraction and a remaining apheresis product fraction containing CD3- cells. The CD3- fraction is then further separated into two fractions: a purified CD14+ monocyte cell fraction and a remaining apheresis product fraction containing CD3-CD14- cells. The purified CD14+ monocyte cell fraction is differentiated into dendritic cells (DCs). The DCs are then exposed to or transfected with an expression vector (preferably a viral vector) containing one or more antigenic peptides related to the patient's cancer or infection, or nucleic acids encoding one or more antigenic peptides. Most preferably, as will be explained in more detail below, tumor-associated neoepitopes include or are specific to the patient's tumor, while virus-associated neoepitopes are specific to the virus and the patient. As a result, dendritic cells thus exposed or transfected present tumor epitopes via the MHC-I / MHC-II system, thereby activating DCs. The previously purified CD3+ T cell fraction is then exposed to the activated DCs, thereby expanding the T cells. Finally, the expanded T cells are purified for use in the treatment of cancer or infection.

[0033] Selectively, when CD14+ cells differentiate into dendritic cells, they are exposed to at least one epigenetic modifier, selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors. The presence of at least one epigenetic modifier induces the expression of tumor and patient-specific or virus-specific neoepitopes presented by MHC-I and MHC-II on the cell surface. Subsequent exposure to T cells activates and expands T cells specific to the newly expressed tumor and patient-specific or virus-specific neoepitopes.

[0034] Optionally, prior to initiating therapeutic apheresis, subjects may be treated with at least one therapeutic agent selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors.

[0035] The method disclosed above may also be used to generate tumor-targeted natural killer (NK) cells for use in the treatment of cancer or infectious diseases. In this embodiment, NK cells are expanded from the CD3-CD14 fraction of the apheresis product. Next, nucleic acids encoding the α and β chains of the T cell receptor (TCR) are isolated from the expanded T cells. These nucleic acids are then fused to the 5' end of a second nucleic acid encoding the transmembrane domain and intracellular signaling domain of a chimeric antigen receptor (CAR). Thus, a fusion nucleic acid encoding TCR-CAR is obtained. Next, tumor or infectious disease-targeted natural killer (NK) cells for use in the treatment of cancer or infectious diseases are generated by transfecting enriched and / or expanded NK cells with the fusion nucleic acid encoding TCR-CAR.

[0036] In some embodiments, the methods disclosed above may be used to enlarge tumor-infiltrating lymphocytes (TILs) for use in the treatment of cancer or infection. In this case, CD3 + TILs are obtained from solid tumors, and CD3+ TILs are exposed to activated DCs, which enlarges the TILs. The enlarged TILs are then purified and used for the treatment of cancer or infection.

[0037] In another embodiment, the present disclosure provides a pharmaceutical composition comprising the following components: 1) activated dendritic cells (DCs) wherein the DCs are differentiated from patient apheresis-derived CD14+ monocytes; 2) an adenovirus encoding an antigenic peptide sequence; 3) patient apheresis-derived CD3+ T cells; 4) an irradiated biopsy sample; 5) at least one compound selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors; 6) granulocyte-macrophage colony-stimulating factor (GM-CSF); and 7) one or more patient-derived natural killer (NK) cells, natural killer T (NKT) cells, T cells, B cells, and / or monocytes. In a preferred embodiment, the pharmaceutical composition comprises at least two, or at least three, or at least four, or at least five of the above components.

[0038] In a preferred embodiment, the pharmaceutical composition comprises tumor or viral disease-targeting lymphocytes for use in the treatment of cancer or infectious diseases. The composition comprises 1) activated dendritic cells (DCs) wherein the DCs are differentiated from patient apheresis-derived CD14+ monocytes; 2) an adenovirus encoding an antigenic peptide sequence wherein the DCs are activated upon exposure to the adenovirus; and 3) patient apheresis-derived CD3+ T cells wherein the CD3+ T cells are activated and expanded upon exposure to activated DCs. Alternatively, or in addition, the pharmaceutical composition may comprise patient-derived dendritic cells (DCs), GM-CSF, and an adenovirus encoding an antigenic peptide (Ad) for use in the treatment of cancer or infectious diseases, wherein the DCs are derived from patient apheresis.

[0039] In yet another embodiment, the pharmaceutical composition for use in the treatment of cancer or infection comprises 1) patient-derived natural killer (NK) cells and / or natural killer T (NKT) cells, 2) GM-CSF, and 3) an adenovirus (Ad) encoding an antigenic peptide, wherein the NK cells or NKT cells are derived from the patient's apheresis. Alternatively, or in addition, the pharmaceutical composition for use in the treatment of cancer or infection may comprise 1) patient-derived T cells, B cells, and / or monocytes, 2) GM-CSF, and 3) an adenovirus (Ad) encoding an antigenic peptide, wherein the T cells, B cells, and / or monocytes are derived from the patient's apheresis.

[0040] It is further contemplated that the pharmaceutical composition may comprise 1) dendritic cells (DCs), 2) irradiated biopsy samples, and 3) T cells, where the DCs and T cells are derived from the patient's apheresis, the biopsy sample is derived from the tumor of the same patient, and the pharmaceutical composition is for use in the treatment of cancer. Alternatively, or in addition thereto, the pharmaceutical composition may comprise dendritic cells (DCs) and T cells, where the DCs and T cells are isolated from the patient's apheresis product, the DCs and / or T cells are exposed to a biopsy sample from the tumor of the same patient, and the pharmaceutical composition is formulated for administration to the patient.

[0041] It is also contemplated that the pharmaceutical composition may comprise dendritic cells (DCs) and T cells, where an apheresis sample from the patient is exposed to a biopsy sample from the tumor of the same patient, then the DCs and T cells are isolated from the apheresis sample, and the pharmaceutical composition is formulated for administration to the patient. Alternatively, or in addition thereto, the pharmaceutical composition may be for use in the treatment of cancer or an infectious disease and comprise tumor-targeted CD3 + T lymphocytes, the composition comprising 1) dendritic cells (DCs) that are differentiated from the patient-derived apheresis-purified CD14+ monocytes, 2) at least one compound selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor, whereby the MHC-I or MHC-II of the DCs presents at least one re-expressed peptide sequence or a part thereof, thereby activating the DCs, and 3) CD3 + T cells that are CD3 T cells exposed to the activated DCs, whereby the T cells are activated and expanded. + T cells.

[0042] In this disclosure, CD3+ T cells are obtained (and purified) from a tumor or patient blood sample and expanded ex vivo. These expanded T cells can be reintroduced into the patient as autologous cells or administered to a different subject as donor cells or allogeneic cells.

[0043] T cells can be enlarged by exposure to a cytokine cocktail containing one or more of IL-2, IL-15, and IL-7, or their agonist derivatives. Alternatively, T cells can be genetically modified to express endoplasmic reticulum-localized IL-15 (erIL-15). In this case, the genetic modification of T cells involves introducing nucleic acids encoding cytokines such as IL-2 or IL-15 into the T cells. IL-2 can be expressed with a signaling sequence that induces IL-2 to become endoplasmic reticulum IL-2 ("erIL-2"). Similarly, IL-15 can be expressed with a signaling sequence that induces IL-15 to become endoplasmic reticulum IL-15 ("erIL-15"). This allows for the expression of IL-2 and / or IL-15 at levels sufficient for autocrine activation, but without the release of IL-2 into the extracellular space. See Konstantinidis et al., “Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92 cells,” Exp Hematol. 2005 Feb;33(2):159-64.

[0044] Furthermore, in some embodiments, T cells are genetically modified to express a chimeric antigen receptor (CAR), where the CAR targets a tumor antigen or checkpoint inhibitor. The intracellular signaling domain of the CAR may also include an FcεRIγ moiety. U.S. Patent Application No. 17 / 341098 discloses such a method and is incorporated herein by reference. T cells are preferably engineered to express a TCR that recognizes an MHC-I-presenting peptide. Preferably, T cells are genetically modified by transfecting them with one or more nucleic acids encoding one or more CARs. Transfection techniques include, but are not limited to, viral transduction, mRNA transfection, and the Sleeping Beauty transposon system. Following transfection, CAR T cells may be expanded in a bioreactor until a clinically effective cell number is obtained.

[0045] The expression vector may be a viral vector, preferably an adenovirus such as Ad5 adenovirus. Furthermore, it is even more generally preferred that the virus be a replication-deficient nonimmunogenic virus, which is typically achieved by targeted deletion of selected viral proteins (e.g., E1, E3 proteins). Such desirable properties may be further enhanced by deleting the function of the E2b gene, and high titers of recombinant viruses can be achieved using recombinant human 293 cells, as recently reported (e.g., J Virol. 1998 Feb;72(2):926-933). Most typically, the desired nucleic acid sequence (for expression from virus-infected cells) is under the control of suitable regulatory elements well known in the art. Patent application PCT / US2017 / 045093 discloses such a method.

[0046] Furthermore, dendritic cells may be further exposed to one or more modified RNAs, lentiviruses, and / or neoepitope peptide pools.

[0047] The term "epigenetics" refers to genetic changes in cellular phenotype that do not involve changes in genotype. Epigenetic modifications generally refer to changes in gene expression that do not involve alterations in DNA sequence. These modifications include DNA and RNA methylation, histone modifications, chromatin remodeling, and non-coding RNA.

[0048] Epigenetic regulation is a dynamic and reversible process characterized by the addition and removal of modifications to DNA and histones. In most cases, these modifications are covalent modifications of DNA and histones. These modifications are strictly controlled and coordinated by chromatin-modifying enzymes, resulting in changes to the structure of chromatin. The regulators responsible for these epigenetic modifications of DNA and histones are classified into four main categories: "writers," "erasers," "readers," or "movers." Writers introduce epigenetic marks into DNA or histones through the action of enzymes such as DNA methyltransferase (DNMT), histone methyltransferase (HMT), and histone acetyltransferase (HAT). Erasers, on the other hand, remove epigenetic marks through the action of histone lysine demethylase (KDM) and histone deacetylase (HDAC). Leaders recognize or are recruited to specific epigenetic marks, such as chromodomain and bromodomain (BRD) proteins that recognize methylated or acetylated residues, respectively. Movers are chromatin remodeling proteins that alter the dynamic spatiotemporal arrangement of nucleosomes to enable gene transcription.

[0049] As discussed herein, dysregulation of epigenetic modifications can lead to activation of oncogenes or silencing of tumor suppressor genes, and disruption of multiple signaling pathways.

[0050] DNMTs are a type of writing enzyme that plays a role in DNA methylation. Writing enzymes such as DNMT1, DNMT3a, and DNMT3b add methyl groups to cytosine residues in DNA. Although these enzymes have similar structures with a regulatory domain at the N-terminus and a catalytic domain at the C-terminus, they differ in function and expression patterns. As a maintenance methyltransferase, DNMT1 not only maintains the stability of already methylated DNA sequences and ensures their preservation during DNA replication and cell division, but can also repair DNA methylation. In contrast, DNMT3a and DNMT3b are known as de novo methyltransferases and can add new methyl groups to previously unmethylated DNA sequences, thereby forming new methylation patterns. DNMT3a / b are targeted to specific DNA sequences by TFs such as CTCF, Sp1, YY1, NRSF / REST, FOXA1, and SALL4.11. The other two DNMTs, DNMT2 and DNMT3L, do not possess cytosine methyltransferase activity. DNMT3L can enhance the activity of DNMT3a and DNMT3b by increasing their ability to bind to the methyl donor S-adenosyl-l-methionine (SAM). DNMT2 primarily functions by introducing methyl chains into ncRNAs such as transfer RNA, ribosomal RNA, and nuclear RNA.

[0051] PRMT is a type of histone methyltransferase. PRMT is involved in a wide range of disease models, including neurological disorders, inflammatory diseases, cardiovascular diseases, and cancer. In particular, PRMT abnormal expression has been extensively studied in cancers such as lung cancer, breast cancer, CRC, and leukemia. PRMT is frequently overexpressed in various types of tumors, including breast cancer and prostate cancer, and has been shown to promote tumor growth and metastasis. Dysregulation of PRMT is thought to play a crucial role in cancer development and progression, highlighting its potential as a therapeutic target for cancer treatment.

[0052] HDACs have the ability to remove acetyl groups from lysine residues in both histone and non-histone proteins, resulting in a more compact chromatin structure and reduced transcriptional activity. Overexpression of HDACs is frequently observed in many cancer patients. In addition to expression or genetic alterations, HDACs can be abnormally recruited to specific gene promoters by oncogenic fusion proteins, driving leukemic development. Furthermore, high HDAC expression is associated with drug resistance in various cancers. For example, HDACs enhance temozolomide resistance in glioblastoma and cisplatin and sorafenib resistance in NSCLC. Similarly, significantly upregulated HDACs in glioblastoma contribute to resistance to temozolomide chemotherapy. In summary, abnormal expression of HDACs contributes to the development of tumor resistance, and inhibiting these enzymes may prevent the emergence of drug resistance.

[0053] Currently approved epigenetic therapies by the FDA include azacitidine (5-azacitidine), decitabine (5-aza-2'-deoxycytidine), vorinostat (suberoylanilide hydroxamic acid (SAHA)), romidepsin (depsipeptide), belinostat (Beleodaq, PXD101), panobinostat (LBH589), tidamide (tucidinostat), tazemetostat (EPZ-6438), enasidenib (AG-221), and ivosidenib (AG-120). (Tao L, Zhou Y, Luo Y, et al. Epigenetic regulation in cancer therapy: from mechanisms to clinical) This information is disclosed in advances.MedComm-Oncology.2024;3:e59.doi:10.1002 / mog2.59, and its entirety is incorporated herein by reference.

[0054] The term "apheresis" generally refers to the process of collecting whole blood from a patient or donor and separating it into two or more components. In an apheresis procedure, blood is collected from the subject through a needle inserted into a vein. The needle is attached to one end of a plastic tube that provides a pathway for the blood. The other end of the tube is connected to a container for collecting the blood. The collected blood is separated into its components using a separator, such as a centrifuge. Depending on the procedure, the desired blood components may be collected, which may be red blood cells, platelets, plasma, white blood cells, or stem cells. These components are further processed to purify blood fractions such as the CD3+ fraction, CD3- fraction, CD3-CD14- fraction, and CD14+ fraction. One or more of these different fractions are further modified / processed as discussed throughout this application. One or more of these fractions can be injected back into a patient who needs that component / fraction.

[0055] With respect to NK cells, it should be noted that all NK cells are considered suitable for use as described herein, and therefore include primary NK cells (stored, expanded, and / or fresh cells), immortalized secondary NK cells, autologous or xenogeneic NK cells (banked, stored, fresh, etc.), and modified NK cells as described in more detail below. In some embodiments, the NK cells are preferably NK-92 cells. The NK-92 cell line is a unique cell line that has been found to proliferate in the presence of interleukin-2 (IL-2) (see, for example, Gong et al., Leukemia 8:652-658 (1994)). After expansion in a suitable culture medium, NK-92 cells are cancerous NK cells with broad antitumor cytotoxicity and predictable yields. Advantageously, NK-92 cells have high cytolytic activity against a variety of cancers.

[0056] The original NK-92 cell line expressed the CD56bright, CD2, CD7, CD11a, CD28, CD45, and CD54 surface markers, but did not display the CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers. Growth of such NK-92 cells in culture depends on the presence of sufficient interleukin-2 (e.g., rIL-2) to maintain proliferation even at low doses of around 1 IU / mL. IL-7 and IL-12 do not support long-term growth, nor do various other cytokines tested, including IL-1a, IL-6, tumor necrosis factor α, interferon α, and interferon γ. Compared to primary NK cells, NK-92 typically exhibits high cytotoxicity even at relatively low effector:target (E:T) ratios (e.g., 1:1). Representative NK-92 cells are deposited in the American Type Culture Collection (ATCC) under the name CRL-2407. U.S. Patents 7,618,817, 8,034,332, 8,313,943, 9,150,636, 9,181,322, 10,138,462, and 10,258,649 are incorporated herein by reference in their entirety, as are all other external references.

[0057] In another aspect of the subject matter of the present invention, genetically engineered NK cells may also be NK-92 derivatives modified to express a high-affinity Fcγ receptor (CD16). Sequences of high-affinity variants of the Fcγ receptor are well known in the art (see, e.g., Blood 2009 113:3716-3725), and all methods of generation and expression are considered suitable for the uses described herein. Expression of such receptors is thought to enable specific targeting of tumor cells using antibodies specific to patient tumor cells (e.g., neoepitope), specific tumor types (e.g., her2neu, PSA, PSMA, etc.), or cancer-related ones (e.g., CEA-CAM). Advantageously, such antibodies are commercially available and can be used in combination with cells (e.g., conjugated to the Fcγ receptor). Alternatively, such cells may also be commercially available from NantKwest as haNK cells. Such cells may then be further genetically modified to become CARs, as will be described in more detail below. U.S. Patent Nos. 10,738,279, 10,456,420, 10,736,921, 11,000,550, 10,801,013, and 10,774,310.

[0058] The genetic modification of NK cells as envisioned herein can be carried out in many ways, and all known methods are considered suitable for use herein. Furthermore, it should be recognized that NK cells can be transfected with DNA or RNA, and that the specific selection of transfection will depend, at least in part, on the desired recombinant cell type and transfection efficiency. For example, if stable transfection of NK cells is desired, linearized DNA can be introduced into the cells for integration into the genome. On the other hand, if transient transfection is desired, circular DNA or linear RNA (e.g., mRNA with a polyA+ tail) can be used.

[0059] For example, if the NK cells are autologous NK cells or NK-92 cells, the recombinant nucleic acid includes a segment encoding a CAR containing an FcεRIγ signaling domain, and preferably a segment encoding a cytokine that provides autocrine growth stimulation (e.g., IL-2, IL-2 modified with an ER retention sequence, IL-15, or IL-15 modified with an ER retention sequence) and / or CD16 or high-affinity CD16. 158V It is intended to include a segment encoding and . As will be readily apparent, the incorporation of cytokines that provide autocrine growth stimulation will give modified recombinants that are independent of the addition of exogenous cytokines, thus making the large-scale production of such cells economically feasible. Similarly, the modified recombinants will include CD16 or high-affinity CD16 158V When this gene is expressed, such cells will have further enhanced ADCC properties and, consequently, improved targeted cytotoxicity.

[0060] Cytokines and / or CD16 or high affinity CD16 158V It should be recognized that recombinant nucleic acids encoding IL-2 can be integrated into the genome of NK cells or supplied as extrachromosomal units (which may be linear or circular DNA or linear RNA delivered via viral delivery or chemical, mechanical, or electrical transfection). For example, recombinant NK-92 cells expressing IL-2ER and CD16158V are known as haNK cells (Oncotarget 2016 Dec 27;7(52):86359-86373) and can be transfected with recombinant nucleic acids containing a segment encoding a CAR with an FcεRIγ signaling domain. Again, such recombinant nucleic acids may contain further segments that can encode additional immunotherapy proteins, such as N-803, TxM-type compounds, IL-8 traps, TGF-β traps, etc. Similarly, NK-92 cells may already be transfected with cDNA encoding IL-2 (e.g., NK-92MI, ATCC CRL-2408). Next, such cells are treated with CD16 or high-affinity CD16158V It can be further transfected with recombinant nucleic acids that include a segment encoding a CAR containing the FcεRIγ signaling domain, along with the segment encoding the CAR.

[0061] On the other hand, (e.g., autologous, fresh, cultured, or pre-frozen) NK cells or NK-92 cells also contain a segment encoding a CAR having an FcεRIγ signaling domain, a segment encoding a cytokine that provides autocrine growth stimulation (e.g., IL-2, IL-2 modified with an ER retention sequence, IL-15, or IL-15 modified with an ER retention sequence), and CD16 (SEQ ID NO: 34) or high-affinity CD16 158VThe recombinant nucleic acid may be transfected with a segment encoding SEQ ID NO: 35 (encoded by SEQ ID NO: 36), which is further disclosed in PCT / US2019 / 033407, the entirety of which is incorporated herein by reference. Most typically, such recombinant nucleic acid would be configured as a tricistronic construct. As described above, such construct may be an extrachromosomal circular plasmid, linear DNA (which can be integrated into the genome of NK cells), or linear RNA. Such nucleic acid would typically be transfected into cells by methods well known in the art (e.g., electroporation, lipofection, ballistic gene transfer, etc.). Similarly, nucleic acid may be delivered into cells via recombinant virus. Accordingly, suitable NK cells for use as described herein include NK-92 cells (which may be transfected with a tricistronic construct encoding CAR, CD16 or its variant, and a cytokine or its variant), genetically modified NK cells or NK-92 cells expressing CD16 or its variant or a cytokine or its variant (which may be transfected with nucleic acids encoding CAR, CD16 or its variant, or a cytokine or its variant), and genetically modified NK cells or NK-92 cells expressing CD16 or its variant and a cytokine or its variant (which may be transfected with nucleic acids encoding CAR). U.S. Patents 17 / 056,385, 11,077,143, 10738279, and 16 / 969,152 are incorporated herein by reference.

[0062] Therefore, it should be noted that in preferred embodiments, genetically modified NK cells (particularly when the cells express CAR and CD16 or their variants) will exhibit three distinguishable modes of cell death: general cytotoxicity mediated by receptor activation (e.g., NKG2D receptor), ADCC mediated by antibodies bound to target cells, and CAR-mediated cytotoxicity.

[0063] Therefore, it should be recognized that the transfection method will depend, at least in part, on the type of nucleic acid used. Accordingly, viral transfection, chemical transfection, and mechanical transfection methods are all considered suitable for use as described herein. For example, in one embodiment, the vector described herein is a transient expression vector. Since the exogenous transgene introduced using such a vector is not integrated into the nuclear genome of the cell, in the absence of vector replication, the exogenous transgene will degrade or be diluted over time.

[0064] In another embodiment, the vector described herein enables stable transfection of cells. In one embodiment, the vector enables the uptake of a transgene into the cell's genome. Preferably, such a vector has a positive selection marker, and a preferred positive selection marker includes any gene that enables cell growth under conditions that can kill cells that do not express the gene. Examples, but not limited to, include antibiotic resistance, such as Geneticin (the Neo gene derived from Tn5). Alternatively or additionally, the vector is a plasmid vector. In one embodiment, the vector is a viral vector, preferably an adenovirus vector. As will be understood by those skilled in the art, any preferred vector can be used, and preferred vectors are well known in the art.

[0065] In yet another embodiment, cells are transfected with mRNA encoding a target protein (e.g., CAR). mRNA transfection results in transient expression of the protein. In one embodiment, mRNA transfection of NK-92 cells is performed immediately before administration of the cells. In one embodiment, "immediately before" administration of cells means approximately 15 minutes to approximately 48 hours before administration. Preferably, mRNA transfection is performed approximately 5 hours to approximately 24 hours before administration. In at least some embodiments, as described in more detail below, mRNA transfection of NK cells resulted in unexpectedly consistent and strong expression of CAR in high-percentage transfected cells. Furthermore, such transfected cells also exhibited high specific cytotoxicity at relatively low effector cell-to-target cell ratios.

[0066] Regarding the intended CAR, it should be noted that NK or NK-92 cells will be genetically modified to express the CAR as a membrane-bound protein by exposing a portion of the CAR on the cell surface while maintaining the signaling domain within the intracellular space. Most typically, the CAR will contain at least the following elements: an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signaling domain (in order).

[0067] In preferred embodiments, the cytoplasmic domain of CAR includes or consists of the signaling domain of FcεRIγ. Notably, as will be described in more detail below, the FcεRIγ signaling domain provides substantially increased expression levels of CAR, along with significantly long-term cytotoxicity over time. In some embodiments, the FcεRIγ cytoplasmic domain is the sole signaling domain. However, it should be recognized that additional elements, e.g., other signaling domains (e.g., CD28 signaling domain, CD3ζ signaling domain, 4-1BB signaling domain, etc.), may also be included. These additional signaling domains may be located downstream and / or upstream of the FcεRIγ cytoplasmic domain. In alternative embodiments, the cytoplasmic domain of CAR may also include the signaling domain of CD3 zeta (CD3ζ). In one embodiment, the cytoplasmic domain of CAR consists of the signaling domain of CD3 zeta.

[0068] Therefore, the intended CAR would have a general structure consisting of a signaling domain, a transmembrane domain, a hinge domain, and a desired antigen-binding domain. From another perspective, the intended CAR may have a desired binding domain, which then binds to a hybrid protein that contains, consists of, or is essentially composed of, a signaling domain, a transmembrane domain, and a hinge domain.

[0069] While not always the case, most typically the extracellular binding domain of a CAR will be an scFv or other native or synthetic binding site that specifically binds to the target antigen. Particularly preferred binding sites include small antibody fragments with single, dual, or multi-target specificity, β-barrel main binders, and phage display fusion proteins. Among the preferred extracellular binding domains, particularly preferred domains will specifically bind to tumor-specific antigens, tumor-associated antigens, or patient- and tumor-specific antigens. Tumor-specific antigens include, but are not limited to, NKG2D ligand, CS1, GD2, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, AFP, CEA, CTAG1B, and CD33. Additional tumor-associated antigens and associated malignancies may be found in Table 1, though not limited to those described herein. Other further tumor-specific antigens are described, for example, in U.S. Patent Application Publication 2013 / 0189268, International Publication 1999024566A1, U.S. Patent No. 7,098,008, and International Publication 2000020460 (each incorporated herein by reference in its entirety). Similarly, other preferred domains would specifically bind to (pathogenic) virus-specific antigens, such as those of HIV virus (e.g., gp120), HPV virus, RSV virus, influenza virus, Ebola virus, or HCV virus.

[0070] Therefore, the intended CARs would target antigens associated with specific oncological types. For example, target cancers include leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, including but not limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, and endothelial tumors). Examples include sarcomas, lymphangiosarcomas, intralymphatic sarcomas, synoviomas, mesotheliomas, Ewing's tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancers, pancreatic cancers, breast cancers, ovarian cancers, prostate cancers, squamous cell carcinomas, basal cell carcinomas, adenocarcinomas, sweat gland carcinomas, sebaceous gland carcinomas, papillary carcinomas, papillary adenocarcinomas, cystadenocarcinomas, medullary carcinomas, bronchogenic carcinomas, renal cell carcinomas, hepatomas, cholangiocarcinomas, choriocarcinomas, seminomas, embryonic carcinomas, Wilms' tumors, cervical cancers, testicular cancers, lung cancers, small cell lung cancers, bladder cancers, epithelial carcinomas, gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pineal glandomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, and retinoblastomas. Examples of infectious diseases for which treatment by the method of the present invention is intended include AIDS, H1N1, Ebola hemorrhagic fever, BSE, Zika fever, SARS, coronavirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E.

[0071] In one embodiment, the subject matter of the present invention includes a method for treating cancer, the method including determining the level of MHC-I expression in a tumor. If it is determined that the MHC-I level is lower than that of a control non-cancerous tissue, the patient is administered multiple NK cells, in which case the NK cells include at least one of aNK cells, haNK cells, t-haNK cells, or primary ceNK or memory-like ceNK (m-ceNK) cells. The NK cells may be autologous or allogeneic. The NK cells may be administered intravenously or intratumor. After some time, the patient is administered multiple T cells. The T cells may be autologous or allogeneic. The T cells may further be genetically modified to express a targeting agent, in which case the agent includes a CAR or TCR.

[0072] A suitable method for determining MHC-I levels in tumor and normal tissue samples involves measuring MHC-I cell surface expression. Enzymatic or mechanical dissociation of tissues, from which all viable cells are purified and isolated from primary tissue, is required for Ab staining and flow cytometric determination of MHC-I surface expression. Alternative methods may include transcriptomics, proteomics, Western blotting, and surface plasmon resonance (SPR).

[0073] As used herein, t-haNK cells are NK cells expressing a genetically modified CAR. While not bound by any particular theory, the targeting portion on the CAR is understood to have a dual purpose. Firstly, the targeting portion facilitates the delivery of NK cells to tumor tissue by directly targeting antigen expression sites in the patient. Secondly, the targeting portion may be immunogenic, thereby facilitating the delivery of antibodies and T cells to the tumor.

[0074] Primary NK cells can also be enriched and expanded from whole cells or umbilical cord blood mononuclear cells via standard methods including exposure of primary NK cells to CD16 antibody, dexamethasone, and / or IL-15. Stabilized IL-15 may be used, in which case it includes IL-15 superagonists, such as nogapendekin alpha-invacicept (Alt-803, N-803, Vesanktiva), and even stabilized IL-15 / IL15-Ra fusion proteins. U.S. Patent Application No. 16 / 985,728, U.S. Patent Application No. 16 / 505,528, U.S. Patent No. 11,351,196, U.S. Patent Application No. 63 / 156,269, U.S. Patent No. 8,163,879, U.S. Patent No. 8,507,222, and U.S. Patent No. 10,537,615 are incorporated herein by reference.

[0075] Cytokine-enhanced NK (ceNK) cells disclosed herein mean NK cells whose cytotoxic activity is enhanced by cytokine stimulation. ceNK cells are prepared by inducing NK cells with a corticosteroid and optionally with a cytokine composition containing IL-15, IL-15:IL-15Rα, or their agonist derivatives, such as N-803. The cytokine composition may include a fusion protein, in which case the fusion protein contains IL-15 or its agonist derivative. A fusion protein containing IL-15, which is a fusion protein with increased stability compared to IL-15, is preferred. While not limiting the subject matter of the present invention, it is generally preferred that the corticosteroid is hydroxycortisone and the optionally selected cytokine is N-803.

[0076] The memory-like cytokine-enhanced NK cells (m-ceNK) disclosed herein include enriched and enlarged NK cells obtained from donor peripheral blood using apheresis techniques to generate NK cells with a memory-like phenotype. m-ceNK cells exhibit both high cytotoxicity and increased interferon-gamma production. Such m-ceNK cells can be generated from individual donors for autologous cell therapy or from umbilical cord blood as an allogene. In addition to efficacy enhancement, m-ceNK cells are readily injectable in an outpatient setting.

[0077] For example, m-ceNK cells may be generated by obtaining multiple mononuclear cells and exposing them to corticosteroids and optionally cytokines. In another step, multiple mononuclear cells are incubated in the presence of corticosteroids and optionally cytokines to enrich the mononuclear NK cells, and then the enriched NK cells are induced with a cytokine composition comprising IL-15, IL-12, and IL-18 or their agonist derivatives. The composition may comprise one or more fusion proteins, in which case the fusion protein comprises at least one of the IL-15, IL-12, and IL-18 cytokines or their agonist derivatives. The cytokine composition may comprise a TxM fusion protein for generating m-ceNK cells, in which case the TxM fusion protein comprises a protein moiety having IL-12 activity, a protein moiety having IL-15 activity, and a protein moiety having IL-18 activity.

[0078] Further descriptions of how to create m-ceNK cells and their advantageous properties are found in PCT / US2022 / 018290 (which is incorporated in its entirety by reference). U.S. Patent Application No. 17 / 375,985 and U.S. Patent No. 11,453,862 provide further alternative methods for inducing NK cell enrichment and expansion. Each of the above references is incorporated in its entirety by reference.

[0079] "T cell receptor" or "TCR" typically refers to a dimeric polypeptide found on the surface of T cells. Each peptide chain of a TCR generally contains an extracellular domain including a variable region and a constant region, a transmembrane domain, and an intracellular domain. The variable region is the part of the TCR that interacts with antigens presented by the MHC. The constant region is the region of each of the two peptides, in which case the two peptide chains are covalently linked by a disulfide bond. The intracellular domain generally contains CD3z, which contains one or more immunoreceptor tyrosine-based activation motifs (ITAMs). The ITAMs mediate the binding of the variable region to the appropriate intracellular signaling pathway.

[0080] The intracellular signaling domain of CAR may also include an FcεRIγ moiety. U.S. Patent Application No. 17 / 341098 is incorporated herein by reference.

[0081] T cells may optionally contain a modified TCR, which is associated with a dimeric polypeptide based on the TCR structure. Specifically, the modified TCR comprises two peptide chains, each containing an extracellular domain (including a variable region, a constant region, and a linking peptide), a transmembrane domain, and an intracellular domain. In specific embodiments, the variable region and the constant region are attached via a linker. In another specific embodiment, the linking peptide is located between the constant region and the transmembrane domain. In yet another specific embodiment, the two peptide chains are linked to each other by disulfide bonds between the linking peptides of each peptide chain. The modified TCR does not interact with the endogenous TCR produced by T cells. The contents of U.S. Patent Application No. 63 / 227,195 are incorporated herein by reference.

[0082] Once a CAR-based therapeutic agent (e.g., an antigen-binding domain bound to a CAR scaffold) binds to an antigen expressed by cancer cells, cytotoxic cells can trigger the destruction of the cancer cells. All cytotoxic cells are generally intended to be suitable for use as described herein, and particularly preferred cytotoxic cells include NK cells, activated NK cells, high-affinity NK cells, CD8+ T cells, and CD4+ T cells modified to recombinantly express the CAR-based therapeutic agent (all of which may be of different origins). The cytotoxic cells are engineered to express a TCR that recognizes an MHC-I-presenting peptide.

[0083] The therapeutic T cells used herein may be patient-derived (autologous) or donor-derived (allogeneic). T cells are typically obtained via leucorpheresis and further isolated according to surface marker (CD4, CD8) expression. Purified T cells may be activated by exposure to CD3 and / or CD28 Ab or by exposure to antigen-presenting cells (APCs). Cells may be enlarged by exposure to a cytokine cocktail containing one or more IL-2, IL-15, and IL-7. In preferred embodiments, T cells or primary NK cells may be enlarged on an automated platform and transfected by microflow-through electroporation as described in U.S. Patent No. 11,377,652 (the contents of which are incorporated herein by reference).

[0084] T cells, which can be purified from tumors, are tumor-infiltrating lymphocytes (TILs). TILs can be purified from tumor tissue and expanded ex vivo. TILs can be reintroduced into patients as autologous cells or administered to different subjects as donor cells. Stimulation of MHC-I expression by NK cells is expected to enhance the cytotoxic efficacy of TILs.

[0085] In one embodiment of the present invention, TILs are isolated from a patient tumor using standard techniques. The TILs are then exposed to the patient's tumor tissue, in which case the patient is treated with NK cells, thereby inducing MHC-I expression. NK exposure may be by IV infusion or intratumoral injection. NK exposure to tumor tissue may be performed ex vivo. TILs exposed to tumor tissue ex vivo are thereby activated and expanded. Expanded TILs containing CD4 and CD8 cytotoxic T cells are administered to the patient.

[0086] T cells can be transfected to express one or more CARs. Transfection techniques, though not limited to them, include viral transduction, mRNA transfection, and the Sleeping Beauty transposon system. Following transfection, CAR T cells can be expanded in a bioreactor until a clinically effective number of cells is obtained.

[0087] However, it should be recognized that in other embodiments, cytotoxic cells can also be macrophages, monocytes, neutrophils, basophils, or eosinophils. Thus, from a different perspective, the cells envisioned herein can exert cytotoxic effects by triggering TNF or fas-mediated cell death pathways through phagocytosis, pore formation, or induction of antibody-dependent cell-mediated cytotoxicity (ADCC).

[0088] Cytotoxic cells may release various types of cytotoxic granules (e.g., granulysin, perforin, granzymes) as part of the cytotoxic antitumor process. To monitor cell-mediated cytotoxicity, various assays are available, including flow cytometric assays, based on the presence of soluble granules such as perforin and granzymes, or the production of TNF family members such as TNF-α, FasL, and TRAIL (Zaritskaya 2010, Clay, T. et al., Clin. Cancer Res. (2001) 1127-1135).

[0089] In one embodiment, the body fluid is obtained after treatment with NK cells, in which case the body fluid includes cellular components, for example, tumorigenic or cancer cells that present antigens for binding to CAR-expressing cytotoxic cells as described herein and for contact with cytotoxic cells expressing antigen-binding moieties. An assay is then performed to detect an immune response suggesting, for example, that an ADCC response or ADCP response was triggered by the patient's own immune cells.

[0090] Immune response detection assays are known in the art and are described herein. For example, such response detection assays can detect the release of cytotoxic granules (e.g., granulicin, perforin, granzyme), phagocytosis, or receptor-ligand-mediated cell lysis (e.g., mediated by the Fas / APO pathway). To monitor cell-mediated cytotoxicity, various flow cytometric assays are available, for example, based on the presence of soluble granules such as perforin, granzyme, or the production of TNF family members such as TNF-α, FasL, and TRAIL (Zaritskaya 2010, Clay, T. et al., Clin. Cancer Res. (2001) 1127-1135).

[0091] In other embodiments, immunostimulatory cytokines are administered to a patient in combination with cytotoxic cells expressing CAR-based therapeutic agents (e.g., antigen-binding domains bound to a CAR scaffold) to promote or trigger an immune response. Examples of cytokines include, but are not limited to, IL2, IL4, IL7, IL11, IL15, IL21, TNF-alpha, and IFN-gamma. In some embodiments, cytokines can reactivate exhausted T cells. In other cases, immune-competent cells can be engineered to recombinantly express one or more cytokines.

[0092] Other cancer treatment techniques include surgery, radiation therapy, chemotherapy, immunosuppressant agents (e.g., azathioprine, cyclosporine, methotrexate, mycophenolates, etc.), immunotherapy, targeted therapy, hormone therapy, stem cell transplantation, or other precision methods. Any of these techniques can be combined with embodiments of the present invention to treat cancer.

[0093] Embodiments of the present invention are understood to be administered to patients using appropriate formulations, indications, and administration regimens preferred by government regulatory authorities such as the U.S. Food and Drug Administration (FDA).

[0094] In some embodiments, cytotoxic cells expressing a TCR, a modified TCR, or a CAR-based therapeutic agent (e.g., an antigen-binding domain bound to a CAR scaffold) are administered to a patient as a pharmaceutical composition. In another embodiment, a method for treating cancer by administering cytotoxic cells to a subject is envisioned. In yet another embodiment, a method for inhibiting or reducing the proliferation of cells expressing a corresponding antigen (to which the antigen-binding domain binds) on the cell surface is envisioned by administering cytotoxic cells to a subject.

[0095] In one embodiment, the patient may be lymph-depleted, which reduces the number of endogenous lymphocytes, thereby increasing the utilization of intrinsic endogenous cytokines and promoting the survival of injected T cells.

[0096] In some embodiments, cytotoxic cells expressing a TCR, a modified TCR, or a CAR-based therapeutic agent (e.g., an antigen-binding domain bound to a CAR scaffold) reduce the amount (e.g., cell number, mass size, etc.) in subjects with cancer associated with the expression of the corresponding antigen on the cell surface by at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, or at least 99% compared to negative controls.

[0097] Examples of cancers that can be treated with cytotoxic cells as intended herein include any cancer that expresses or overexpresses cancer-associated antigens on its cell surface. Examples of cancers that can be treated with cytotoxic cells expressing TCRs, modified TCRs, or CAR-based therapeutic agents (e.g., antigen-binding domains bound to a CAR scaffold) include, but are not limited to, breast cancer, colon cancer, leukemia, lung cancer, melanoma, neuroblastoma, pancreatic cancer, pediatric intracranial ependymoma, and prostate cancer.

[0098] Dendritic cells (DCs) refer to a diverse population of morphologically similar cell types found in various lymphoid and non-lymphoid tissues (Steinman (1991) Ann. Rev. Immunol. 9:271-296). Dendritic cells constitute the most potent and desirable antigen-presenting cells (APCs) in the body. Dendritic cells can differentiate from monocytes and have a different phenotype from monocytes. Mature DCs can provide all the signals necessary for T cell activation and proliferation. Mature dendritic cells are not phagocytic, while monocytes and immature dendritic cells are highly phagocytic. Immature DCs can capture antigens through endocytosis, phagocytosis, macropinocytosis or adhesive pinocytosis, and receptor-mediated antigen uptake, and have high intracellular concentrations of MHC class II molecules.

[0099] For example, dendritic cells may be isolated from immune cells of a patient diagnosed with cancer (immune cells are typically obtained from apheresis of the patient). Alternatively, dendritic cells may also be induced from progenitor cells in response to specific growth factors (e.g., GM-CSF). Regardless of the type of isolation, the dendritic cells are intended to be transfected with an expression vector (preferably a viral vector) containing one or more tumor-associated epitopes of the patient's tumor, or nucleic acids encoding one or more tumor-associated epitopes of the patient's tumor. Most preferably, the tumor-associated epitopes include or are neoepitopes specific to the patient's tumor. As a result, the dendritic cells thus transfected present tumor epitopes via the MHC-I and / or MHC-II systems.

[0100] Antigens can be exposed to DCs either in their naked state or in a viral vector (such as an adenovirus vector). Adenovirus vectors are particularly preferred. Furthermore, it is even more generally preferred that the virus be a non-immunogenic virus with replication defects, which is typically achieved by targeted deletion of selected viral proteins (e.g., E1, E3 proteins). Such desirable properties may be further enhanced by deleting the function of the E2b gene, and high titers of recombinant viruses can be achieved using recombinant human 293 cells, as recently reported (e.g., J Virol. 1998 Feb;72(2):926-933). Most typically, the desired nucleic acid sequence (for expression from virus-infected cells) is under the control of appropriate regulatory elements well known in the art. Viral vectors offer several advantages for eliciting a potent and sustained immune response to cancer-associated sequences. First, when recombinant virus infects dendritic cells, cancer-associated sequences are expressed and presented using the MHC-I and / or MHC-II presentation pathway, thereby activating CD4 + and CD8 + This is thought to increase the likelihood of cell production, and consequently, the likelihood of appropriate antibody production and appropriate T-cell and B-cell memory.

[0101] Thus, dendritic cells obtained from apheresis in patients with tumors are ex vivo exposed to nucleic acids encoding one or more tumor-associated epitopes of the patient's tumor, or one or more tumor-associated or tumor-specific epitopes of the patient's tumor. In this way, the immune response can be specifically directed to a particular tumor (or even a subpopulation of tumors), and the patient's immune cells do not experience rejection.

[0102] The pharmaceutical composition may contain cytotoxic cells containing antigen-binding domains bound to or linked to a CAR scaffold as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. In addition, the pharmaceutical composition may contain one or more adjuvants (e.g., aluminum hydroxide), antioxidants, bacteriostatic agents, buffers, carbohydrates, chelating agents (e.g., EDTA or glutathione), colorants, flavoring agents, and / or aromatic agents, emulsifiers, excipients, lubricants, pH buffers, preservatives, salts affecting osmotic pressure, polypeptides (e.g., glycine), proteins, solubilizers, stabilizers, wetting agents, etc., provided that they do not react adversely with the active compound (e.g., antigen-binding domains bound to a CAR scaffold) or otherwise interfere with its activity. Examples of buffers include, but are not limited to, neutral buffered saline and phosphate-buffered saline. Examples of carbohydrates, though not limited to them, include dextran, glucose, mannose, mannitol, and sucrose.

[0103] Pharmaceutical compositions may be formulated using specific administration modes. These modes of administration are not limited to, but may include: intra-articular, intradermal, intranasal, intraperitoneal, intrathecal, intratumoral, intravenous, ventricular / ventricular, subcutaneous, transdermal, transmucosal, or local routes.

[0104] In preferred embodiments, cytotoxic cells are administered by intravenous infusion. Such formulations may be prepared according to standard techniques known to those skilled in the art. For example, a composition administered intravenously may have one or more components (e.g., diluents, suspension buffers, saline or dextrose / water, other components such as cytokines) before infusion to the patient.

[0105] Many such techniques for formulating and administering pharmaceutical compositions are known in the art, for example, in U.S. Patent Application Publication No. 2014 / 0242025, and all such references are incorporated herein by reference in their entirety.

[0106] In some embodiments, cytotoxic cells proliferate in vivo, persisting in the patient for months or even years after administration, providing a sustained mechanism for inhibiting tumor growth or recurrence. In some embodiments, cytotoxic cells persist for at least 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 2 years, 3 years, 4 years, or 5 years after administration of cytotoxic cells to the patient.

[0107] Cytotoxic cells can be obtained from any of the following sources (e.g., from humans, from commercially available cytotoxic cells, or cells isolated from repositories). Ex vivo expansion procedures for NK cells, T cells, or other types of cytotoxic cells are known in the art (e.g., Smith et al., Clinical & Translational Immunology (2015) 4:e31). The examples presented herein are not intended to be limited to any particular method of ex vivo expansion of cytotoxic cells.

[0108] The cytotoxic cell-containing pharmaceutical compositions described herein may be administered in doses of 10⁴–10⁹ cells / kg body weight, 10⁵–10⁶ cells / kg body weight, or any integer value within these ranges. The cytotoxic cell compositions may be administered in these doses in a single dose or sequentially (over several days, weeks, or months). Techniques for injecting cytotoxic cells, such as T cells, are known in the art (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0109] In other embodiments, the pharmaceutical composition is administered in a therapeutically effective dose, which is an amount effective in treating a specific indication. Administration may be as a single dose or based on intervals. As used herein, “interval” suggests that the therapeutically effective dose is administered periodically (to distinguish it from a single dose). The administration interval for a single individual does not need to be a fixed interval and can vary over time. The terms “in combination with” or “co-administered” suggest that the composition may be administered immediately before, simultaneously with, or nearly simultaneously with another composition, or immediately after. [Examples]

[0110] Example 1: In one example disclosed herein, tumor-targeting lymphocytes are generated for use in the treatment of cancer or infectious diseases. The method comprises the steps of: performing therapeutic apheresis on a subject with cancer or infectious disease; purifying a CD3+ T cell fraction from the apheresis product (where the remaining apheresis product includes a CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction (where the remaining apheresis product includes a CD3- / CD14- fraction); differentiating the CD14+ monocytes into dendritic cells (DCs); exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence (where the MHC-I or MHC-II of the DCs presents the peptide sequence or a portion thereof, thereby activating the DCs); exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cells; and purifying the expanded T cells.

[0111] T cells may be enlarged in the presence of IL-15 or its agonist derivatives. Furthermore, T cells may be genetically modified to express endoplasmic reticulum-localized IL-15 (erIL-15) and / or to express chimeric antigen receptors (CARs) that target tumor antigens or checkpoint inhibitors. The adenovirus disclosed herein may be an Ad5 adenovirus. Furthermore, dendritic cells may be further exposed to a peptide pool of modified RNA and / or lentivirus and / or neoepitope.

[0112] Example 2: In another example disclosed herein, tumor-targeted natural killer (NK) cells are generated for use in the treatment of cancer or infection. The method involves the steps of: performing therapeutic apheresis on a subject with cancer or infection; purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); expanding the NK cells from the CD3-CD14- fraction of the apheresis product; differentiating the CD14+ monocytes into dendritic cells (DCs); and activating the DCs with an antigenic peptide or adenovirus encoding an antigenic peptide sequence. The method includes the steps of: exposing (where the MHC-I or MHC-II of the DC presents a peptide sequence or a portion thereof, thereby activating the DC); exposing purified CD3+ T cells to activated DCs, thereby expanding the T cells; purifying the expanded T cells; isolating at least one nucleic acid encoding the α and β chains of the T cell receptor (TCR) from the expanded T cells and fusing the nucleic acid to the 5' end of a second nucleic acid encoding the transmembrane domain and intracellular signaling domain of the chimeric antigen receptor (CAR) (where the fused nucleic acid encodes TCR CAR); and transfecting enriched and expanded NK cells with the nucleic acid encoding TCR CAR.

[0113] The NK cells disclosed herein may include NK-92 cells or memory cytokine-enriched NK cells (M-CENK). Furthermore, M-CENK cells may be genetically modified to express CARs targeting CD16 and / or tumor antigens or checkpoint inhibitors. Furthermore, NK-92 cells may contain CD16 and may be further genetically modified to express a second CAR.

[0114] Example 3: In yet another example, the present inventors disclose a method for expanding tumor-infiltrating lymphocytes for use in the treatment of cancer or infectious diseases. The method includes: performing therapeutic apheresis on a subject with cancer or infection; purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); expanding NK cells from the CD3- / CD14- fraction of the apheresis product; differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an adenovirus encoding an antigenic peptide or antigenic peptide sequence (where the MHC-I or MHC-II of the DCs presents the peptide sequence or a portion thereof, thereby activating the DCs); exposing the purified CD3+ T cells to activated DCs to expand the T cells; purifying the expanded T cells; purifying CD3+ TILs from solid tumors and exposing the TILs to activated DCs to expand the TILs; and purifying the expanded TILs.

[0115] Example 4: In a further example, a pharmaceutical composition is disclosed. The pharmaceutical composition comprises: 1) activated dendritic cells (DCs) wherein the DCs are differentiated from patient apheresis-derived CD14+ monocytes; 2) an adenovirus encoding an antigenic peptide sequence wherein the DCs are activated upon exposure to the adenovirus; and 3) patient apheresis-derived CD3+ T cells wherein the CD3+ T cells are activated and expanded upon exposure to activated DCs. The pharmaceutical composition is intended for use in the treatment of cancer or infection.

[0116] In the pharmaceutical composition disclosed above, the MHC-I or MHC-II of the DC presents an antigen peptide sequence or a part thereof, thereby activating the DC. The activated CD3+ T cell may contain a T cell receptor (TCR) that is specific to the MHC-presented antigen peptide sequence on the DC.

[0117] The pharmaceutical composition may further contain natural killer (NK) cells. In some examples, the NK cells are expanded from patient apheresis products. The NK cells may also contain cytokine-enriched (CENK) cells or memory-like cytokine-enriched (M-CENK) cells. The NK cells are expanded in a medium containing IL-15, IL-12, and / or IL-18. In some embodiments, the NK cells are NK-92 cells, more preferably NK-92 cells containing a high-affinity Fc receptor and / or endoplasmic reticulum-targeted IL-2 (erIL-2) or erIL-15. The NK cells may also contain CARs. The CAR is intended to contain a targeting domain, and the targeting domain is intended to contain the α and β chains of the TCR. Preferably, the α and β chains are derived from patient-activated T cells. In some embodiments, the CAR contains a targeting domain, and the targeting domain contains an antibody-binding domain. Preferably, the antibody-binding domain is specific to a tumor-associated antigen, tumor-specific antigen, or neoepitope. Alternatively, or in addition, the antibody-binding domain is specific to checkpoint inhibitors.

[0118] The pharmaceutical composition comprises IL-15 or its agonist derivative, and it is further intended that T cells are exposed to IL-15 or its agonist derivative before and / or during exposure to activated DCs. The dendritic cells of the pharmaceutical compositions disclosed herein are preferably activated in a culture medium containing GM-CSF (granulocyte-macrophage colony-stimulating factor). Furthermore, the dendritic cells may be activated in a culture medium further containing IL-4.

[0119] Example 5: In another example, the inventors disclose a pharmaceutical composition for use in the treatment of cancer or infection, comprising patient-derived dendritic cells (DCs), GM-CSF, and an adenovirus (Ad) encoding an antigenic peptide, where the DCs are derived from the patient's apheresis. The pharmaceutical composition may further comprise IL-4 and / or IL-15. The MOI (Multiplicity of Infection) of the pharmaceutical composition is intended to be in the range of 20 to 20,000.

[0120] Example 6: In yet another example, the inventors disclose a pharmaceutical composition for use in the treatment of cancer or infection, comprising 1) patient-derived natural killer (NK) cells and / or natural killer T (NKT) cells, 2) GM-CSF, and 3) an adenovirus (Ad) encoding an antigenic peptide, wherein the NK or NKT cells are derived from the patient's apheresis. The pharmaceutical composition may further comprise IL-4 and / or IL-15. The MOI (Multiplicity of Infection) of the pharmaceutical composition is intended to be in the range of 20 to 20,000.

[0121] Example 7: A pharmaceutical composition for use in the treatment of cancer or infection, comprising 1) patient-derived T cells, B cells, and / or monocytes, 2) GM-CSF, and 3) an adenovirus (Ad) encoding an antigenic peptide, wherein the T cells, B cells, and / or monocytes are derived from the patient's apheresis. The pharmaceutical composition may further contain IL-4 and / or IL-15. The MOI (Multiplicity of Infection) of the pharmaceutical composition is intended to be in the range of 20 to 20,000.

[0122] Example 8: A pharmaceutical composition comprising 1) dendritic cells (DCs), 2) an irradiated biopsy sample, and 3) T cells, wherein the DCs and T cells are derived from the patient's apheresis, the biopsy sample is derived from a tumor of the same patient, and the pharmaceutical composition is for use in the treatment of cancer.

[0123] The pharmaceutical composition may further contain IL-4 and / or IL-15. The MOI (Multiplicity of Infection) of the pharmaceutical composition is intended to be in the range of 20 to 20,000. The pharmaceutical composition may also further contain adenovirus (Ad), where Ad comprises nucleic acid encoding an antigenic peptide sequence.

[0124] The pharmaceutical composition may further contain natural killer (NK) cells. In some examples, NK cells are expanded from patient apheresis products or patient PBMCs. NK cells may also contain cytokine-enriched (CENK) cells or memory-like cytokine-enriched (M-CENK) cells. NK cells are expanded in a medium containing IL-15, IL-12, and / or IL-18. In some embodiments, the NK cells are NK-92 cells, more preferably NK-92 cells containing a high-affinity Fc receptor and / or endoplasmic reticulum-targeted IL-2 (erIL-2) or erIL-15. NK cells may also contain CARs. The CAR is intended to contain a targeting domain, and the targeting domain is intended to contain the α and β chains of the TCR. Preferably, the α and β chains are derived from patient-activated T cells. In some embodiments, the CAR contains a targeting domain, and the targeting domain contains an antibody-binding domain. Preferably, the antibody-binding domain is specific to a tumor-associated antigen, tumor-specific antigen, or neoepitope. Alternatively, or in addition, the antibody-binding domain is specific to checkpoint inhibitors.

[0125] In some cases, the biopsy sample and T cells are added to the DCs sequentially. For example, the biopsy sample may be added to the DCs first, and then the T cells may be added, where the T cells are enlarged T cells.

[0126] Example 9: In another example, the present inventors disclose a pharmaceutical composition comprising dendritic cells (DCs) and T cells, wherein the DCs and T cells are isolated from apheresis products of a patient, the DCs and / or T cells are exposed to biopsy samples from tumors of the same patient, and the pharmaceutical composition is formulated for administration to the patient.

[0127] Preferably, DCs are further exposed to adenovirus (Ad), where Ad comprises a nucleic acid encoding an antigenic peptide sequence. DCs and / or T cells may also be further exposed to IL-15 or its agonist derivatives. Biopsy samples are ideally from tumors of patients treated with DAMP inducers, where DAMP inducers comprise radioactive and / or histone deacetylase (HDAC) inhibitors. DCs are exposed to the biopsy in the presence of an activated medium containing granulocyte colony-stimulating factor (GMCSF) and IL-4, where DCs are activated and matured. Exposure of DCs and biopsies to the activated medium may be 24-48 hours, after which DCs are isolated and combined with T cells. The pharmaceutical composition may be formulated for intravenous, subcutaneous, intratumoral, or intravenous administration.

[0128] Example 10: In another example, the present inventors disclose a pharmaceutical composition comprising dendritic cells (DCs) and T cells, wherein an apheresis sample from a patient is exposed to a biopsy sample from a tumor of the same patient, and the DCs and T cells are then isolated from the apheresis sample, and the pharmaceutical composition is formulated for administration to the patient.

[0129] Preferably, DCs are further exposed to adenovirus (Ad), where Ad contains nucleic acid encoding an antigenic peptide sequence. DCs and / or T cells may also be further exposed to IL-15 or its agonist derivatives. Isolated DCs may be further exposed to adenovirus (Ad), where Ad contains nucleic acid encoding an antigenic peptide sequence. Biopsy samples are ideally from tumors of patients treated with DAMP inducers, where DAMP inducers include radiation and / or histone deacetylase (HDAC) inhibitors. DCs and / or CD14+ cells are exposed to the biopsy in the presence of activated medium containing granulocyte colony-stimulating factor (GMCSF) and IL-4, where DCs are activated and matured. Exposure of DCs and / or CD14+ monocytes and biopsy to activated medium may be 24-48 hours, where differentiated DCs are isolated and combined with purified CD3+ T cells. The pharmaceutical composition may be formulated for administration by intravenous, subcutaneous, intratumoral, or intravenous infusion.

[0130] Example 11: In another example, the present inventors disclose a method for generating tumor-targeted lymphocytes for use in the treatment of cancer or infectious diseases. The method comprises: performing therapeutic apheresis on a subject with cancer or infection; purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product includes the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product includes the CD3- / CD14- fraction); expanding NK cells from the CD3- / CD14- fraction of the apheresis product; differentiating the CD14+ monocytes into dendritic cells (DCs); exposing the DCs to at least one compound selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors, thereby causing the MHC-I or MHC-II of the DCs to present at least one re-expressed peptide sequence or a portion thereof, thereby activating the DCs; and exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cell population.

[0131] Example 12: In yet another example, the inventors disclose a method for generating tumor-targeted natural killer (NK) cells for use in the treatment of cancer or infection. The method involves: performing therapeutic apheresis on a subject with cancer or infection; purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); expanding NK cells from the CD3- / CD14- fraction of the apheresis product; differentiating the CD14+ monocytes into dendritic cells (DCs); and treating the DCs with a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and histogenic therapy. The method comprises: exposing DCs to at least one compound selected from the group consisting of HDAC inhibitors, thereby causing the MHC-I or MHC-II of the DCs to present at least one re-expressed peptide sequence or a portion thereof, thereby activating the DCs; exposing purified CD3+ T cells to activated DCs, thereby expanding the T cells; isolating at least one nucleic acid encoding the α and β chains of the T cell receptor (TCR) from the expanded T cells and fusing the nucleic acid to the 5' end of a second nucleic acid encoding the transmembrane domain and intracellular signaling domain of the chimeric antigen receptor (CAR) (wherein the fused nucleic acid encodes TCR CAR); and transfecting enriched and expanded NK cells with the nucleic acid encoding TCR CAR.

[0132] Example 13: In this example, the present inventors disclose a method for expanding tumor-infiltrating lymphocytes for use in the treatment of cancer or infectious diseases. The method involves performing therapeutic apheresis on subjects with cancer or infection; purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); expanding NK cells from the CD3- / CD14- fraction of the apheresis product; differentiating CD14+ monocytes into dendritic cells (DCs); exposing the DCs to at least one compound selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors, thereby causing the MHC-I or MHC-II of the DCs to present at least one re-expressed peptide sequence or a portion thereof, thereby activating the DCs; and activating CD3 from solid tumors. + TIL is purified and CD3 + This includes exposing the TIL to an activated DC to enlarge the TIL, and purifying the enlarged TIL.

[0133] Example 14: Tumor target CD3 + A pharmaceutical composition comprising T lymphocytes, wherein the pharmaceutical composition is for use in the treatment of cancer or infection. The composition comprises: 1) dendritic cells (DCs) wherein the DCs are differentiated from patient-derived apheresis-purified CD14+ monocytes; 2) at least one compound selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor, wherein the MHC-I or MHC-II of the DCs presents at least one re-expressed peptide sequence or a portion thereof, thereby activating the DCs; and 3) CD3 purified from patient apheresis. + T cells, in which T cells are exposed to activated DCs, thereby becoming activated and expanding, CD3+ Includes T cells.

[0134] Example 15: In another example, the present inventors disclose a method for expanding tumor-infiltrating lymphocytes for use in the treatment of cancer or infectious diseases. The method involves performing therapeutic apheresis on subjects with cancer or infection (where the subjects are treated with at least one therapeutic agent selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors); purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an adenovirus encoding an antigenic peptide or antigenic peptide sequence (where the MHC-I or MHC-II of the DCs presents the peptide sequence or a portion thereof, thereby activating the DCs); and purifying CD3+ T cells from solid tumors. + The process includes purifying the TIL, exposing the TIL to an activated DC, thereby expanding the TIL; and purifying the expanded TIL.

[0135] Example 16: The present inventors also disclose a method for generating tumor-targeted lymphocytes for use in the treatment of cancer or infectious diseases. The method includes: performing therapeutic apheresis on a subject with cancer or infection (where the subject is treated with at least one therapeutic agent selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors); purifying the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); purifying CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an adenovirus encoding an antigenic peptide or antigenic peptide sequence (where the MHC-I or MHC-II of the DCs presents the peptide sequence or a portion thereof, thereby activating the DCs); exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cells; and purifying the expanded T cells.

[0136] Example 17: In another example, the inventors disclose a method for transduction of fresh PBMCs with adenovirus-controlled AD5-[E1-,E2b-]-GFP. Fresh blood from two donors was used for the isolation of PBMCs and transduction of Ad5-GFP. Cells were infected at an MOI of 20. After transduction, cells were cultured in AIM-V medium containing either N-803, GM-CSF, or GM-CSF+IL-4. GFP expression was monitored using IncuCyte and evaluated by flow cytometry. The following conditions were evaluated:

[0137] [Table 1]

[0138] The MOI used in the above experiment was 20. Details of the transduction are as follows: 12-well plate, donor 1 contains 1 × 10⁶ 6 Cells / well, donor 2 has 5 x 10 5 Cells / well were used. The following amounts of N-803, GM-CSF, and IL-4 were used: 74 ng / mL of N-803, 100 ng / mL of GM-CSF, and 20 ng / mL of IL-4. The virus used for transduction was Ad5-[E1-,E2b-]-GFP.

[0139] Transduction was performed in 12-well plates. Isolated PBMCs were used for transduction in AIM-V medium (250 μL). Ad5 virus was added at an MOI of 20 (250 μL). The virus-infected cultures were placed in a 37°C, 5% CO2 incubator for 1 hour. After 1 hour, 0.5 mL of medium containing the corresponding cytokine was added, and the plate was incubated for a further 6 days (Note: The plate was left longer to obtain more cells for staining by flow cytometry). GFP expression was monitored using IncuCyte and evaluated by flow cytometry.

[0140] The results for two donors (Donor 001 and Donor 002) are shown in Figures 1-8. Figures 1-3 and 5-6 show that different cytokine cocktails (N-803, GM-CSF, and IL-4) induce different cell morphologies in Donor 001 and Donor 002, respectively. Figures 4 and 7 show that more GFP+ cells were observed in the GM-CSF treatment group on days 3 and 5 post-transduction for Donor 001 and Donor 2, respectively.

[0141] The gating strategy for flow cytometry is shown in Figure 8, and the results are shown below in table format. The following two tables (Tables 2-3) show the percentage of GFP+ cells on day 6 determined by flow cytometry.

[0142] [Table 2]

[0143] [Table 3]

[0144] Tables 4-5 below show the percentage of GFP+ cells (%) on day 23, as determined by flow cytometry.

[0145] [Table 4]

[0146] [Table 5]

[0147] Figure 9 shows a treatment chart for locally advanced neoadjuvant pancreatic cancer. In this example, therapeutic apheresis is performed prior to treatment with the Nant cancer vaccine. The Nant cancer vaccine is discussed in more detail in U.S. Patent Publication Nos. 20190381156A1 and 20190318804A1, which are incorporated by reference. The apheresis product is transfected with an adenovirus encoding at least one immunogenic peptide sequence, as shown, where the peptide sequence includes a tumor-associated antigen or neoepitope. The transductioned apheresis product is stimulated with an immunostimulant. N-803 is shown in the figure. T cells exposed to the transductioned apheresis formulation are enlarged and formulated for administration to patients before or after surgery. Of course, the incorporation of the therapeutic apheresis product of the present invention into a treatment plan is not limited to this example.

[0148] The above considerations provide numerous embodiments of the subject matter of the present invention. While each embodiment represents a single combination of the elements of the present invention, the subject matter of the present invention is considered to encompass all possible combinations of the disclosed elements. Therefore, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, the subject matter of the present invention is also considered to encompass other remaining combinations of A, B, C, or D, even if not expressly disclosed herein.

[0149] It will be apparent to those skilled in the art that many more modifications beyond those already described are possible without departing from the concept of the invention as described herein. Therefore, the subject matter of the invention should not be limited beyond the spirit of the appended claims. Furthermore, in interpreting both this specification and the claims, all terms should be interpreted in the broadest possible sense that is consistent with the context. Specifically, the terms “comprises” and “comprising” should be interpreted as non-exclusively referring to elements, components, or processes, suggesting that the referenced elements, components, or processes may exist, be utilized, or be combined with other elements, components, or processes not explicitly referenced. Where this specification or the claims refer to at least one selected from the group consisting of A, B, C, ..., and N, the wording should be interpreted as requiring only one element from that group, and not A+N, B+N, etc.

[0150] All publications specified herein are incorporated by reference to the same extent as each individual publication or patent application is specifically and individually indicated by reference. If a definition or use of a term in an incorporated reference conflicts with a definition of that term provided herein, the definition provided herein shall prevail, and the definition in the reference shall not.

[0151] In some embodiments, numbers representing component amounts, properties, such as concentrations and reaction conditions, used to describe and claim certain embodiments of the subject matter of the present invention should be understood to be modified in some cases by the term "approximately." Therefore, in some embodiments, numerical parameters specified in the written description and appended claims are approximations that may vary depending on the desired properties to be obtained by the particular embodiment. In some embodiments, numerical parameters should be interpreted in light of the reported number of significant figures and by applying common rounding techniques. Although the numerical ranges and parameters representing the broad scope of some embodiments of the subject matter of the present invention are approximations, the numerical values ​​shown in the specific examples are reported as accurately as possible. The numerical values ​​presented in some embodiments of the subject matter of the present invention may contain certain errors that inevitably arise from the standard deviation found in their respective test measurements.

[0152] In some embodiments, numbers representing component amounts, properties, such as concentrations and reaction conditions, used to describe and claim certain embodiments of the subject matter of the present invention should be understood to be modified in some cases by the term "approximately." Therefore, in some embodiments, numerical parameters specified in the written description and appended claims are approximations that may vary depending on the desired properties to be obtained by the particular embodiment. In some embodiments, numerical parameters should be interpreted in light of the reported number of significant figures and by applying common rounding techniques. Although the numerical ranges and parameters representing the broad scope of some embodiments of the subject matter of the present invention are approximations, the numerical values ​​shown in the specific examples are reported as accurately as possible. The numerical values ​​presented in some embodiments of the subject matter of the present invention may contain certain errors that inevitably arise from the standard deviation found in their respective test measurements.

[0153] Unless otherwise specified in the context, all ranges expressed herein should be interpreted as including their endpoints, and open-ended ranges should be interpreted as including only commercially useful values. Similarly, all lists of values ​​should be considered to include intermediate values ​​unless otherwise indicated in the context.

[0154] As used herein and throughout the following claims, the meanings of “a,” “an,” and “the” include plural references unless otherwise explicitly defined by the context. Furthermore, as used herein, the meaning of “in” includes “in” and “on” unless otherwise explicitly defined by the context.

[0155] The enumeration of value ranges in this specification is merely intended to function as a simplified notation for individually referring to each separate value that falls within that range. Unless otherwise specified herein, each individual value is incorporated herein as if it were individually enumerated herein. All methods described herein can be implemented in any preferred order unless otherwise specified herein or unless there is a clear contextual inconsistency. The use of any examples or exemplary expressions provided herein for any particular embodiment (e.g., "such as") is merely intended to improve the understanding of the subject matter of the invention and does not impose any limitation on the scope of the subject matter of the invention as otherwise claimed. The expressions herein should not be construed as suggesting that any non-claim element is essential to the implementation of the subject matter of the invention.

[0156] The grouping of alternative elements or alternative embodiments of the subject matter of the Invention disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in combination with other members of that group or other elements found herein. One or more members of a group may be included or excluded for convenience and / or patentability reasons. In any such inclusion or exclusion, the designation shall be deemed herein to include the group as modified, and thus satisfy the description of all Markush groups used in the appended claims.

Claims

1. A method for generating tumor-targeted lymphocytes for use in the treatment of cancer or infectious diseases, wherein the method is i. Performing therapeutic apheresis on subjects with cancer or infectious diseases; ii. Purify the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); iii. Purify CD14+ monocyte cells from the CD3- fraction (where the remaining apheresis product includes the CD3- / CD14- fraction); iv. Differentiating the CD14+ monocytes into dendritic cells (DCs), and exposing the DCs to an adenovirus encoding an antigenic peptide or an antigenic peptide sequence (wherein the MHC-I or MHC-II cells of the DCs present the peptide sequence or a portion thereof, thereby activating the DCs); v. Exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cell population; vi. A method comprising purifying the enlarged T cells.

2. The method according to claim 1, wherein the T cells are enlarged in the presence of IL-15 or an agonist derivative thereof.

3. The method according to any one of claims 1 to 2, wherein the T cells are genetically modified to express endoplasmic reticulum-localized IL-15 (erIL-15).

4. The method according to any one of claims 1 to 3, wherein the T cells are genetically modified to express a chimeric antigen receptor (CAR), and the CAR targets a tumor antigen or a checkpoint inhibitor.

5. The method according to any one of claims 1 to 4, wherein the adenovirus comprises Ad5 adenovirus.

6. The method according to any one of claims 1 to 5, wherein the dendritic cells are further exposed to modified RNA.

7. The method according to any one of claims 1 to 6, wherein the dendritic cells are further exposed to a lentivirus.

8. The method according to any one of claims 1 to 2, wherein the dendritic cells are further exposed to the peptide pool of NeoEptiOpen.

9. A method for generating tumor-targeted natural killer (NK) cells for use in the treatment of cancer or infectious diseases, wherein the method is i. Performing therapeutic apheresis on subjects with cancer or infectious diseases; ii. Purify the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); iii. Purify CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); iv. Expanding NK cells from the CD3-CD14-fraction of the apheresis product; v. Differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an adenovirus encoding an antigenic peptide or an antigenic peptide sequence (wherein the MHC-I or MHC-II cells of the DCs present the peptide sequence or a portion thereof, thereby activating the DCs); vi. Exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cell population; vii. Purify the enlarged T cells; viiii. Isolate at least one nucleic acid encoding the α and β chains of the T cell receptor (TCR) from the enlarged T cell, and fuse this nucleic acid to the 5' end of a second nucleic acid encoding the transmembrane domain and intracellular signaling domain of the chimeric antigen receptor (CAR) (wherein the fused nucleic acid encodes TCR-CAR); ix. A method comprising transfecting enriched and enlarged NK cells with the nucleic acid encoding the TCR CAR.

10. The method according to claim 9, wherein the NK cells include memory cytokine-enriched NK cells (M-CENK).

11. The method according to any one of claims 9 to 10, wherein the M-CENK is genetically modified to express CD16.

12. The method according to any one of claims 10 to 11, wherein the M-CENK cells are further genetically modified to express CAR, and the CAR targets a tumor antigen or a checkpoint inhibitor.

13. The method according to any one of claims 9 to 12, wherein the NK cells include NK-92 cells, and optionally the NK-92 cells include CD16.

14. The method according to claim 13, wherein the NK-92 cells are further genetically modified to express a second CAR.

15. A method for expanding tumor-infiltrating lymphocytes for use in the treatment of cancer or infection, wherein the method is i. Performing therapeutic apheresis on subjects with cancer or infectious diseases; ii. Purify the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); iii. Purify CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); iv. Expanding NK cells from the CD3- / CD14- fraction of the apheresis product; v. Differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an adenovirus encoding an antigenic peptide or an antigenic peptide sequence (wherein the MHC-I or MHC-II cells of the DCs present the peptide sequence or a portion thereof, thereby activating the DCs); vi. Exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cell population; vii. Purify the enlarged T cells; viiii. Purifying CD3+ TILs from solid tumors, exposing the TILs to activated DCs, and thereby enlarging the TILs; ix. A method comprising purifying the expanded TIL.

16. A pharmaceutical composition comprising tumor-targeting lymphocytes for use in the treatment of cancer or infectious diseases, wherein the composition comprises: 1) activated dendritic cells (DCs) wherein the DCs are differentiated from patient apheresis-derived CD14+ monocytes; 2) an adenovirus encoding an antigenic peptide sequence wherein the DCs are activated upon exposure to the adenovirus; and 3) patient apheresis-derived CD3+ T cells wherein the CD3+ T cells are activated and expanded upon exposure to the activated DCs.

17. The pharmaceutical composition according to claim 16, wherein the MHC-I or MHC-II of the DC presents the antigen peptide sequence or a part thereof, thereby activating the DC.

18. The pharmaceutical composition according to any one of claims 16 to 17, wherein the activated CD3+ T cells include a T cell receptor (TCR) that is specific to the MHC-presenting antigen peptide sequence on the DC.

19. A pharmaceutical composition according to any one of claims 16 to 18, further comprising natural killer (NK) cells.

20. The pharmaceutical composition according to claim 19, wherein the NK cells are expanded from the patient apheresis product.

21. The pharmaceutical composition according to any one of claims 19 to 20, wherein the NK cells include cytokine-enriched (CENK) cells or memory-like cytokine-enriched (M-CENK) cells.

22. The pharmaceutical composition according to any one of claims 19 to 21, wherein the NK cells are expanded in a culture medium containing IL-15, IL-12, and IL-18.

23. The pharmaceutical composition according to any one of claims 19 to 22, wherein the NK cells are expanded in a culture medium containing IL-15.

24. The pharmaceutical composition according to any one of claims 19 to 23, wherein the NK cells are NK-92 cells.

25. The pharmaceutical composition according to claim 24, wherein the NK-92 cells contain a high-affinity Fc receptor.

26. The pharmaceutical composition according to claim 24, wherein the NK-92 cells contain endoplasmic reticulum-targeted IL-2 (erIL-2) or erIL-15.

27. The pharmaceutical composition according to any one of claims 19 to 26, wherein the NK cells include CAR.

28. The pharmaceutical composition according to claim 27, wherein the CAR comprises a targeting domain, and the targeting domain comprises the α-chain and β-chain of the TCR.

29. The pharmaceutical composition according to claim 28, wherein the α chain and β chain are derived from the activated T cells of the patient.

30. The pharmaceutical composition according to any one of claims 27 to 29, wherein the CAR comprises a targeting domain, and the targeting domain comprises an antibody (Ab) binding domain.

31. The pharmaceutical composition according to claim 30, wherein the Ab-binding domain is specific to a tumor-associated antigen, a tumor-specific antigen, or a neoepitope.

32. The pharmaceutical composition according to claim 30, wherein the Ab-binding domain is specific to a checkpoint inhibitor.

33. The pharmaceutical composition according to any one of claims 19 to 32, wherein the nucleic acid comprises an adenovirus Ad5 vector.

34. The pharmaceutical composition according to any one of claims 19 to 33, further comprising IL-15 or an agonist derivative thereof, wherein the T cells are exposed to IL-15 or an agonist derivative thereof before exposure to activated DCs.

35. The pharmaceutical composition according to any one of claims 19 to 34, further comprising IL-15 or an agonist derivative thereof, wherein the T cells are exposed to IL-15 or an agonist derivative thereof during exposure to activated DCs.

36. The pharmaceutical composition according to any one of claims 19 to 35, wherein the dendritic cells are activated in a culture medium containing GM-CSF.

37. The pharmaceutical composition according to any one of claims 19 to 36, wherein the dendritic cells are activated in a culture medium further containing IL-4.

38. A pharmaceutical composition for use in the treatment of cancer or infection, comprising patient-derived dendritic cells (DCs), GM-CSF, and an adenovirus (Ad) encoding an antigenic peptide, wherein the DCs are derived from the patient's apheresis.

39. The pharmaceutical composition according to claim 38, further comprising IL-4.

40. A pharmaceutical composition according to any one of claims 38 to 39, further comprising IL-15 or an agonist derivative thereof.

41. A pharmaceutical composition according to any one of claims 38 to 40, wherein the MOI is 20 to 20,000.

42. A pharmaceutical composition for use in the treatment of cancer or infectious diseases, comprising 1) natural killer (NK) cells and / or natural killer T (NKT) cells derived from a patient, 2) GM-CSF, and 3) an adenovirus (Ad) encoding an antigenic peptide, wherein the NK cells or NKT cells are derived from the apheresis of the patient.

43. The pharmaceutical composition according to claim 42, further comprising IL-4.

44. A pharmaceutical composition according to any one of claims 42 to 43, further comprising IL-15.

45. A pharmaceutical composition according to any one of claims 42 to 44, wherein the MOI is 20 to 20,000.

46. A pharmaceutical composition for use in the treatment of cancer or infection, comprising 1) patient-derived T cells, B cells, and / or monocytes, 2) GM-CSF, and 3) an adenovirus (Ad) encoding an antigenic peptide, wherein the T cells, B cells, and / or monocytes are derived from the patient's apheresis.

47. The pharmaceutical composition according to claim 46, further comprising IL-4.

48. A pharmaceutical composition according to any one of claims 46 to 47, further comprising IL-15 or an agonist derivative thereof.

49. A pharmaceutical composition according to any one of claims 46 to 48, wherein the MOI is 20 to 20,000.

50. A pharmaceutical composition comprising 1) dendritic cells (DCs), 2) an irradiated biopsy sample, and 3) T cells, wherein the DCs and T cells are derived from the patient's apheresis, the biopsy sample is derived from a tumor of the same patient, and the pharmaceutical composition is intended for use in the treatment of cancer.

51. The pharmaceutical composition according to claim 50, further comprising IL-4.

52. A pharmaceutical composition according to any one of claims 50 to 51, further comprising IL-15 or an agonist derivative thereof.

53. A pharmaceutical composition according to any one of claims 50 to 52, further comprising adenovirus (Ad), wherein the Ad comprises a nucleic acid encoding an antigen peptide sequence.

54. A pharmaceutical composition according to any one of claims 50 to 53, further comprising natural killer (NK) cells.

55. The pharmaceutical composition according to claim 54, wherein the NK cells are expanded from the patient's PBMC.

56. The pharmaceutical composition according to any one of claims 54 to 55, wherein the NK cells include cytokine-enriched (CENK) cells or memory-like cytokine-enriched (M-CENK) cells.

57. The pharmaceutical composition according to any one of claims 54 to 56, wherein the NK cells are expanded in a culture medium containing IL-15, IL-12, and IL-18.

58. The pharmaceutical composition according to any one of claims 54 to 56, wherein the NK cells are expanded in a culture medium containing IL-15.

59. The pharmaceutical composition according to any one of claims 54 to 58, wherein the NK cells are NK-92 cells.

60. The pharmaceutical composition according to claim 59, wherein the NK-92 cells contain a high-affinity Fc receptor.

61. The pharmaceutical composition according to any one of claims 59 to 60, wherein the NK-92 cells contain endoplasmic reticulum-targeted IL-2 (erIL-2) or erIL-15.

62. The pharmaceutical composition according to any one of claims 54 to 61, wherein the NK cells include CAR.

63. The pharmaceutical composition according to claim 62, wherein the CAR comprises a targeting domain, and the targeting domain comprises the α-chain and β-chain of the TCR.

64. The pharmaceutical composition according to claim 63, wherein the α chain and β chain are derived from the activated T cells of the patient.

65. The pharmaceutical composition according to any one of claims 62 to 64, wherein the CAR comprises a targeting domain, and the targeting domain comprises an antibody (Ab) binding domain.

66. The pharmaceutical composition according to claim 65, wherein the Ab-binding domain is specific to a tumor-associated antigen, a tumor-specific antigen, or a neoepitope.

67. The pharmaceutical composition according to claim 65, wherein the Ab-binding domain is specific to a checkpoint inhibitor.

68. The pharmaceutical composition according to any one of claims 50 to 67, wherein the biopsy sample and the T cells are sequentially added to the DC.

69. The pharmaceutical composition according to claim 68, wherein the biopsy sample is first combined with the DC, then the T cells are added, and the T cells are expanded.

70. A pharmaceutical composition comprising dendritic cells (DCs) and T cells, wherein the DCs and T cells are isolated from an apheresis product of a patient, the DCs and / or T cells are exposed to a biopsy sample from a tumor of the same patient, and the pharmaceutical composition is formulated for administration to the patient.

71. The pharmaceutical composition according to claim 70, wherein the DC is further exposed to adenovirus (Ad), and the Ad comprises a nucleic acid encoding an antigen peptide sequence.

72. The pharmaceutical composition according to any one of claims 70 to 71, wherein the DC and / or T cells are further exposed to IL-15 or its agonist derivative.

73. The pharmaceutical composition according to any one of claims 70 to 72, wherein the DC and / or T cells are further exposed to IL-15 or its agonist derivative.

74. The pharmaceutical composition according to any one of claims 70 to 73, wherein the DC is further exposed to adenovirus (Ad), and the Ad comprises a nucleic acid encoding an antigen peptide sequence.

75. The pharmaceutical composition according to any one of claims 70 to 74, wherein the biopsy sample is from a tumor of the patient who has been treated with a DAMP inducer, and the DAMP inducer comprises a radioactive and / or histone deacetylase (HDAC) inhibitor.

76. The pharmaceutical composition according to any one of claims 70 to 75, wherein the DC is exposed to the biopsy in the presence of an activating medium containing granulocyte colony-stimulating factor (GMCSF) and IL-4, and the DC is activated and matured.

77. The pharmaceutical composition according to any one of claims 70 to 76, comprising exposing the DCs and biopsy samples to an activated culture medium for 24 to 48 hours, then isolating the DCs and combining them with the T cells.

78. The pharmaceutical composition according to any one of claims 70 to 77, wherein the composition is formulated for administration by intravenous, subcutaneous, intratumoral, or intravenous infusion.

79. A pharmaceutical composition comprising dendritic cells (DCs) and T cells, wherein an apheresis sample from a patient is exposed to a biopsy sample from a tumor of the same patient, the DCs and T cells are then isolated from the apheresis sample, and the pharmaceutical composition is formulated for administration to the patient.

80. The pharmaceutical composition according to claim 79, wherein the isolated DC is further exposed to adenovirus (Ad), and the Ad comprises a nucleic acid encoding an antigen peptide sequence.

81. The pharmaceutical composition according to any one of claims 79 to 80, wherein the isolated DC and / or T cells are further exposed to IL-15 or its agonist derivative.

82. The pharmaceutical composition according to any one of claims 79 to 81, wherein the isolated DC and / or T cells are further exposed to IL-15 or its agonist derivative.

83. The pharmaceutical composition according to any one of claims 79 to 82, wherein the isolated DC is further exposed to adenovirus (Ad), and the Ad comprises a nucleic acid encoding an antigen peptide sequence.

84. The pharmaceutical composition according to any one of claims 79 to 83, wherein the biopsy sample is from a tumor of the patient who has been treated with a DAMP inducer, and the DAMP inducer comprises a radioactive and / or histone deacetylase (HDAC) inhibitor.

85. The pharmaceutical composition according to any one of claims 79 to 84, wherein purified CD14+ monocytes are exposed to the biopsy sample in the presence of an activated medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4.

86. The pharmaceutical composition according to any one of claims 79 to 85, wherein the CD14+ monocytes are exposed to an activation medium for 24 to 48 hours, and then differentiated DCs are isolated and combined with purified CD3+ T cells.

87. The pharmaceutical composition according to any one of claims 79 to 86, wherein the composition is formulated for administration by intravenous, subcutaneous, intratumoral, or intravenous infusion.

88. A method for generating tumor-targeted lymphocytes for use in the treatment of cancer or infectious diseases, wherein the method is i. Performing therapeutic apheresis on subjects with cancer or infectious diseases; ii. Purify the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); iii. Purify CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); iv. Expanding NK cells from the CD3- / CD14- fraction of the apheresis product; v. Differentiating the CD14+ monocytes into dendritic cells (DCs), exposing the DCs to at least one compound selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor, thereby causing the MHC-I or MHC-II of the DCs to present at least one re-expressed peptide sequence or a portion thereof, thereby activating the DCs; vi. A method comprising exposing the purified CD3+ T cells to the activated DC, thereby expanding the T cells.

89. A method for generating tumor-targeted natural killer (NK) cells for use in the treatment of cancer or infectious diseases, wherein the method is i. Performing therapeutic apheresis on subjects with cancer or infectious diseases; ii. Purify the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); iii. Purify CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); iv. Expanding NK cells from the CD3- / CD14- fraction of the apheresis product. v. Differentiating the CD14+ monocytes into dendritic cells (DCs), exposing the DCs to at least one compound selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor, thereby causing the MHC-I or MHC-II of the DCs to present at least one re-expressed peptide sequence or a portion thereof, thereby activating the DCs; vi. Exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cell population; vii. Isolate at least one nucleic acid encoding the α and β chains of the T cell receptor (TCR) from the enlarged T cell, and fuse this nucleic acid to the 5' end of a second nucleic acid encoding the transmembrane domain and intracellular signaling domain of the chimeric antigen receptor (CAR) (wherein the fused nucleic acid encodes TCR-CAR); viiii. A method comprising transfecting enriched and enlarged NK cells with the nucleic acid encoding the TCR CAR.

90. A method for expanding tumor-infiltrating lymphocytes for use in the treatment of cancer or infection, wherein the method is i. Performing therapeutic apheresis on subjects with cancer or infectious diseases; ii. Purify the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); iii. Purify CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); iv. Expanding NK cells from the CD3- / CD14- fraction of the apheresis product; v. Differentiating the CD14+ monocytes into dendritic cells (DCs), exposing the DCs to at least one compound selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor, thereby causing the MHC-I or MHC-II of the DCs to present at least one re-expressed peptide sequence or a portion thereof, thereby activating the DCs; vi. Purifying CD3+ TILs from solid tumors, exposing the CD3+ TILs to the activated DCs, thereby enlarging the TILs; and vii. A method comprising purifying the expanded TIL.

91. Tumor-targeted CD3 for use in the treatment of cancer or infection ＋ A pharmaceutical composition comprising T lymphocytes, wherein the composition comprises: 1) dendritic cells (DCs) wherein the DCs are differentiated from apheresis-purified CD14+ monocytes derived from a patient; 2) at least one compound selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor, wherein the MHC-I or MHC-II of the DCs presents at least one re-expressed peptide sequence or a portion thereof, thereby activating the DCs; and 3) CD3 purified from the patient's apheresis. ＋ CD3, a T cell, wherein the T cell is exposed to an activated DC, thereby activating and expanding the T cell. ＋ A pharmaceutical composition containing T cells.

92. A method for expanding tumor-infiltrating lymphocytes for use in the treatment of cancer or infection, wherein the method is i. Performing therapeutic apheresis on subjects with cancer or infection (where the subjects are treated with at least one therapeutic agent selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors); ii. Purify the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); iii. Purify CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); iv. Differentiating the CD14+ monocytes into dendritic cells (DCs), and exposing the DCs to an adenovirus encoding an antigenic peptide or an antigenic peptide sequence (wherein the MHC-I or MHC-II cells of the DCs present the peptide sequence or a portion thereof, thereby activating the DCs); v. Purifying CD3+ TILs from solid tumors, exposing the TILs to activated DCs, and thereby enlarging the TILs; vi. A method comprising purifying the expanded TIL.

93. A method for generating tumor-targeted lymphocytes for use in the treatment of cancer or infectious diseases, wherein the method is i. Performing therapeutic apheresis on subjects with cancer or infection (where the subjects are treated with at least one therapeutic agent selected from the group consisting of protein arginine methyltransferase 5 (PRMT5) inhibitors, DNA methyltransferase (DNMT) inhibitors, and histone deacetylase (HDAC) inhibitors); ii. Purify the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); iii. Purify CD14+ monocyte cells from the CD3- fraction of the apheresis product (where the remaining apheresis product contains the CD3- / CD14- fraction); iv. Differentiating the CD14+ monocytes into dendritic cells (DCs), and exposing the DCs to an adenovirus encoding an antigenic peptide or an antigenic peptide sequence (wherein the MHC-I or MHC-II cells of the DCs present the peptide sequence or a portion thereof, thereby activating the DCs); v. Exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cell population; vi. A method comprising purifying the enlarged T cells.

94. A method for generating tumor-targeted lymphocytes for use in the treatment of cancer or infectious diseases, wherein the method is i. Performing therapeutic apheresis on subjects with cancer or infectious diseases; ii. Purify the CD3+ T cell fraction from the apheresis product (where the remaining apheresis product contains the CD3- fraction); iii. Purify CD14+ monocyte cells from the CD3- fraction (where the remaining apheresis product includes the CD3- / CD14- fraction); iv. Differentiating the CD14+ monocytes into dendritic cells (DCs), and exposing the DCs to an adenovirus encoding an antigenic peptide or an antigenic peptide sequence (wherein the MHC-I or MHC-II cells of the DCs present the peptide sequence or a portion thereof, thereby activating the DCs); v. Exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cell population; vi. A method comprising purifying the enlarged T cells.

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