Ex vivo generation of immune effector cells from monosampled material intermediates

Abstract

Provided herein are methods and compositions 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. Also provided herein are methods of making and using the same.

Description

[0001] This application claims priority benefit of the following U.S. provisional application numbers: 63 / 512,032, filed July 5, 2023; 63 / 515,528, filed July 25, 2023; and 63 / 605,326, filed December 1, 2023. Each of the above applications is incorporated by reference in its entirety. TECHNICAL FIELD

[0002] The field of the invention is immunotherapy technology. BACKGROUND

[0003] The background description includes information that can be useful in understanding the present disclosure. It is not an admission that any information provided herein is prior art or relevant to the presently claimed application, or that 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 if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or otherwise different from the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the incorporated reference does not apply.

[0005] Autologous or allogeneic T cells and natural killer cells provide meaningful benefits for other refractory malignancies. Apheresis generates the starting material for T cell or NK cell manufacturing. Because NK cells comprise only a small fraction of lymphocytes (about 1-20%), methods have been developed to enrich them from large volumes of peripheral blood (such as apheresis products) or to expand NK cell populations from smaller volumes of blood or stem cells. As the clinical indications for T cells and NK cells continue to expand, researchers are attempting to develop new and improved methods for obtaining these cells from peripheral blood mononuclear cells (PBMCs).

[0006] Accordingly, there remains a need in the art for improved methods of isolating and expanding T cells and NK cells for the treatment of cancer or infectious disease in a patient in need thereof. SUMMARY

[0007] The present subject matter provides compositions and methods of 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 disease.

[0008] In embodiments, the inventive subject matter includes a tumor-targeted lymphocyte for use in the treatment of a cancer or an infectious disease. The tumor-targeted lymphocyte is produced by a method comprising: performing therapeutic apheresis on a subject having a cancer or an infectious disease; purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; purifying CD14+ monocytes from the CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; differentiating the CD14+ monocytes into dendritic cells (DCs), and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein the DCs MHC-I or MHC-II present 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.

[0009] In embodiments, the inventive subject matter includes a tumor-targeted natural killer (NK) cell for use in the treatment of a cancer or an infectious disease. The NK cell is produced by a method comprising: performing therapeutic apheresis on a subject having a cancer or an infectious disease; purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; purifying CD14+ monocytes from the apheresised CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; expanding NK cells from the apheresised CD3- CD14- fraction; differentiating the CD14+ monocytes into dendritic cells (DCs), and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein the DCs MHC-I or MHC-II present 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; purifying the expanded T cells; isolating at least one nucleic acid encoding an alpha chain and a beta chain of a T cell receptor (TCR) from the expanded T cells, and fusing the nucleic acid to the 5' end of a second nucleic acid encoding a transmembrane domain and an intracellular signaling domain of a chimeric antigen receptor (CAR), wherein the fused nucleic acid encodes a TCR CAR; and transfecting the enriched and expanded NK cells with the nucleic acid encoding the TCR CAR.

[0010] In embodiments, the inventive subject matter includes a method of expanding tumor infiltrating lymphocytes for use in the treatment of a cancer or infectious disease. The method includes performing therapeutic apheresis on a subject having a cancer or infectious disease; purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; purifying CD14+ monocytes from the apheresised CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; expanding NK cells from the apheresised CD3- / CD14- fraction; differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein the DCs MHC-I or MHC-II present the peptide sequence or portions thereof, thereby activating the DCs; exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cells; purifying the expanded T cells; purifying CD3+ TILs from a solid tumor and exposing the TILs to the activated DCs, thereby expanding the TILs; and purifying the expanded TILs.

[0011] In embodiments, the inventive subject matter includes 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 the patient, wherein the CD3+ T cells are exposed to the activated DCs, thereby activating and expanding the T cells. The pharmaceutical composition is contemplated for use in the treatment of a cancer or infectious disease.

[0012] In embodiments, the inventive subject matter includes a pharmaceutical composition for use in the treatment of a cancer or infectious disease, the pharmaceutical composition comprising dendritic cells (DCs) derived from apheresis of a patient, GM-CSF, and an adenovirus (Ad) encoding an antigenic peptide.

[0013] In embodiments, the inventive subject matter includes a pharmaceutical composition for use in the treatment of a cancer or infectious disease, the pharmaceutical composition comprising 1) natural killer (NK) cells and / or natural killer T (NKT) cells derived from apheresis of a patient, 2) GM-CSF, and 3) an adenovirus (Ad) encoding an antigenic peptide.

[0014] Further disclosed herein is a pharmaceutical composition for use in the treatment of a cancer or infectious disease, the pharmaceutical composition comprising 1) T cells, B cells, and / or monocytes derived from apheresis of a patient, 2) GM-CSF, and 3) an adenovirus (Ad) encoding an antigenic peptide.

[0015] Also disclosed herein is a pharmaceutical composition comprising 1) a dendritic cell (DC), 2) an irradiated biopsy sample, and 3) a T cell, wherein the DC and T cell are derived from apheresis of a patient and the biopsy sample is from a tumor of the same patient, and wherein the pharmaceutical composition is for use in the treatment of cancer.

[0016] In embodiments, the inventive subject matter includes a pharmaceutical composition comprising a dendritic cell (DC) and a T cell, wherein the DC and T cell are isolated from apheresis of a patient, wherein the DC and / or T cell is exposed to a biopsy sample from a tumor of the same patient, and wherein the pharmaceutical composition is formulated for administration to the patient.

[0017] In embodiments, the inventive subject matter comprises a pharmaceutical composition comprising a dendritic cell (DC) and a T cell, wherein an apheresis sample from a patient is exposed to a biopsy sample from a tumor of the same patient, wherein the DC and T cell are then isolated from the apheresis sample, and wherein the pharmaceutical composition is formulated for administration to the patient.

[0018] Further disclosed herein is a method of generating tumor-targeted lymphocytes for use in the treatment of cancer or an infectious disease. The method comprises performing therapeutic apheresis on a subject having cancer or an infectious disease; purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; purifying CD14+ monocytes from the CD3- fraction of the apheresis, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; expanding NK cells from the CD3- / CD14- fraction of the apheresis; differentiating the 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 DCs MHC-I or MHC-II present at least one re-expressed peptide sequence or portion thereof, thereby activating 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 have disclosed a method of generating tumor-targeted natural killer (NK) cells for use in the treatment of cancer or an infectious disease. The method comprises therapeutic apheresis of a subject having cancer or an infectious disease; purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; purifying CD14+ monocytes from the apheresis CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; expanding NK cells from the apheresis CD3- / CD14- fraction; differentiating the 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 DCs MHC-I or MHC-II present at least one re-expressed peptide sequence or portion thereof, thereby activating the DCs; exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cells; isolating at least one nucleic acid encoding an a chain and a b chain of a T cell receptor (TCR) from the expanded T cells, and fusing the nucleic acid to the 5' end of a second nucleic acid encoding a transmembrane domain and an intracellular signaling domain of a chimeric antigen receptor (CAR), wherein the fused nucleic acid encodes a TCR CAR; transfecting the enriched and expanded NK cells with the nucleic acid encoding the TCR CAR.

[0020] In embodiments, the inventive subject matter includes a method of expanding tumor infiltrating lymphocytes for use in the treatment of cancer or an infectious disease. The method comprises therapeutic apheresis of a subject having cancer or an infectious disease; purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; purifying CD14+ monocytes from the apheresis CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; expanding NK cells from the apheresis CD3- / CD14- fraction; differentiating the 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 DCs MHC-I or MHC-II present at least one re-expressed peptide sequence or portion thereof, thereby activating the DCs; purifying CD3 + TIL from a solid tumor and exposing the CD3+ TIL to the activated DCs, thereby expanding the TILs; and purifying the expanded TILs.

[0021] Also disclosed herein are pharmaceutical compositions comprising the tumor-targeted CD3 +A pharmaceutical composition of T lymphocytes, wherein the pharmaceutical composition is for use in the treatment of a cancer or an infectious disease. 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, whereby the DCs MHC-I or MHC-II present at least one re-expressed peptide sequence or portion thereof, thereby activating the DCs; and 3) CD3 + T cells; wherein the T cells are exposed to the activated DCs, thereby activating and expanding the T cells.

[0022] Further disclosed herein is a method of expanding tumor infiltrating lymphocytes for use in the treatment of a cancer or an infectious disease. The method comprises subjecting a subject having a cancer or an infectious disease to therapeutic apheresis, wherein the subject has been treated with at least one therapeutic agent selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor; purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; purifying CD14+ monocytes from the apheresised CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; differentiating the CD14+ monocytes into dendritic cells (DCs), and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein the DCs MHC-I or MHC-II present the peptide sequence or portion thereof, thereby activating the DCs; purifying CD3 + TILs from a solid tumor, and exposing the TILs to the activated DCs, thereby expanding the TILs; purifying the expanded TILs.

[0023] In embodiments, the inventive subject matter includes a method of generating tumor-targeting lymphocytes for use in the treatment of cancer or infectious disease. The method includes subjecting a subject having cancer or infectious disease to therapeutic apheresis, wherein the subject has been treated with at least one therapeutic agent selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor; purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; purifying CD14+ monocytes from the apheresized CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; differentiating the CD14+ monocytes into dendritic cells (DCs), and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein the DCs MHC-I or MHC-II present the peptide sequence or portions thereof, thereby activating the DCs; exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cells; purifying the expanded T cells.

[0024] The various objects, features, aspects and advantages of the present inventive subject matter will become more apparent from the following detailed description of certain embodiments, along with the accompanying drawings in which like numerals represent like components. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 IncuCyte images of donor 001 at day 5 post transduction of 12 well plates are shown.

[0026] Figure 2 Results for donor 001 are shown and demonstrate that different cytokine cocktails (N-803, GM-CSF and IL-4) induce different cell morphologies.

[0027] Figure 3 Results for donor 001 are shown and demonstrate that different cytokine cocktails (N-803, GM-CSF and IL-4) result in different cell morphologies.

[0028] Figure 4 Results for donor 001 are shown and demonstrate that a higher number of GFP+ cells are observed in the GM-CSF treated group at day 3 and day 5 post transduction.

[0029] Figure 5 IncuCyte images of donor 002 at day 5 post transduction of 12 well plates are shown.

[0030] Figure 6 Results for donor 002 are shown and demonstrate that different cytokine cocktails (N-803, GM-CSF and IL-4) induce different cell morphologies.

[0031] Figure 7 Results for donor 002 are presented, and higher numbers of GFP+ cells were observed in the GM-CSF treatment group on days 3 and 5 post-transduction.

[0032] Figure 8 A gating strategy for flow cytometry was demonstrated.

[0033] Figure 9 A chart showing the treatment of locally advanced neoadjuvant pancreatic cancer is presented. Detailed Implementation

[0034] This invention provides compositions and methods for pharmacologically manipulating dendritic cells from patients (cancer patients or patients with viral infections) by exposing them to epigenetic modulators. Specifically, protein arginine methyltransferase 5 (PRMT5), DNA methyltransferase (DNMT), and histone deacetylase (HDAC) mediate epigenetic events. These proteins have been shown to regulate the activity of genes involved in both tumor cell proliferation and tumor suppression in vitro and in vivo (preclinical and clinical). Therefore, epigenetic regulation has become a clinically validated target for reducing tumor cell proliferation and inducing tumor suppression. Just as genes involved in tumor suppression can be epigenetically silenced, the inventors have conceived and proposed, herein, the epigenetic silencing of immunogenic peptides, thereby silencing mutations routinely monitored by the cell-mediated immune system. Epigenetic inhibitor treatment can promote the reexpression of immunogenic peptides derived from mutated genes or cells infected by infectious pathogens.

[0035] As further described in this disclosure, the ablation product is processed to purify CD3+ T cells and CD14+ monocytes, and CD3-CD14- cells are used to enrich and expand NK cells. CD14+ cells are used to differentiate into dendritic cells, which are then exposed to at least one epigenetic regulator 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 regulator induces the expression of novel epitopes presented on the cell surface by MHC-I and MHC-II. Subsequent exposure to T cells leads to the activation and expansion of T cells specific to the newly expressed epitopes. The same logic applies to the expression of epitopes of infectious pathogens.

[0036] Treatment of patients 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 isolated into dendritic cells and T lymphocytes. Dendritic cells re-express the repressive epitopes or immunogenic peptides to stimulate the isolated T cells. The epigenetic modulator thereby reveals genes encoding tumor-promoting peptides (cell cycle inducers) and / or tumor-specific peptides (new epitopes), both of which stimulate the activation and proliferation of tumor-educated lymphocytes via MHC presentation.

[0037] In another embodiment, NK cells are isolated from the apheresis product, activated and expanded in vitro, and then re-infused into the patient along with tumor or virus-acclimated T cells.

[0038] In another embodiment, purified dendritic cells are treated with IL-15 or an agonist derivative thereof (such as N-803). The dendritic cells may be derived from patients already treated with epigenetic modifiers, or the dendritic cells may be treated ex vivo with epigenetic modifiers. The dendritic cells may also be derived from patients already treated with epigenetic modifiers and / or IL-15 agonist derivatives, particularly stable derivatives thereof.

[0039] In another embodiment, the patient may receive IL-15 or a stable agonist derivative thereof concurrently with T-cell and / or NK-cell therapy. Tumor or virus-induced lymphocyte administration may be supplemented with chemotherapy agents, tumor-targeting antibodies, checkpoint inhibitor antibodies, vaccines (adenovirus-based or yeast-based), or radiation.

[0040] In one aspect, this disclosure provides a method for generating tumor-targeting lymphocytes for use in the treatment of cancer or infectious diseases. The method includes therapeutic apheresis of a subject suffering from cancer or an infectious disease. In this regard, it should be understood that apheresis is performed by obtaining circulating blood from a person, separating the blood through a device that separates red blood cells and white blood cells (apheresis product) from plasma, and returning the plasma to the patient's circulation. The apheresis product is then separated into two fractions: a purified CD3+ T cell fraction and a residual apheresis product fraction containing CD3- cells. The CD3- fraction is then further separated into two fractions: a purified CD14+ monocyte fraction and a residual apheresis product fraction containing CD3- CD14- cells. The purified CD14+ monocyte fraction is differentiated into dendritic cells (DCs). The DCs are then exposed to one or more antigenic peptides associated with the patient's cancer or infectious disease, or transfected with one or more antigenic peptides associated with the patient's cancer or infectious disease, or transfected with an expression vector (preferably a viral vector) containing a nucleic acid encoding the one or more antigenic peptides. Most preferably, and as discussed further in more detail below, tumor-associated neoepitaphs include or are neoepitaphs specific to the patient's tumor, while virus-associated neoepitaphs are specific to the virus and the patient. Therefore, dendritic cells thus exposed or transfected will present tumor epitopes via the MHC-I / MHC-II system, thereby activating the dendritic cells (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 infectious diseases.

[0041] Optionally, once CD14+ cells differentiate into dendritic cells, they are exposed to at least one epigenetic regulator 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 regulator 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 leads to the activation and expansion of T cells specific to the newly expressed tumor- and patient-specific or virus-specific neoepitopes.

[0042] Optionally, prior to therapeutic apheresis, the subject 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.

[0043] The methods disclosed above can further 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 a single-collected CD3-CD14 fraction. Then, nucleic acids encoding the α and β chains of the T cell receptor (TCR) are isolated from the expanded T cells. This nucleic acid is then fused to the 5' end of a second nucleic acid encoding the transmembrane domain and intracellular signal transduction domain of a chimeric antigen receptor (CAR). In this way, a fusion nucleic acid encoding a TCR CAR is obtained. Enriched and / or expanded NK cells are then transfected with the fusion nucleic acid encoding the TCR CAR, thereby generating tumor-targeted natural killer (NK) cells for use in the treatment of cancer or infectious diseases.

[0044] In some embodiments, the methods disclosed above can be used to expand tumor-infiltrating lymphocytes (TILs) for use in the treatment of cancer or infectious diseases. In this case, CD3 is obtained from solid tumors. + TILs were amplified by exposing CD3+ TILs to activated dendritic cells (DCs). The amplified TILs were then purified and used for the treatment of cancer or infectious diseases.

[0045] In another aspect, this disclosure provides pharmaceutical compositions comprising one or more of the following components: 1) activated dendritic cells (DCs) differentiated from patient-acquired CD14+ monocytes; 2) an adenovirus encoding an antigenic peptide sequence; 3) patient-acquired 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); 7) patient-derived natural killer (NK) cells, natural killer T (NKT) cells, T cells, B cells, and / or monocytes. In preferred embodiments, the pharmaceutical composition comprises at least two, at least three, at least four, or at least five of the above components.

[0046] 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) differentiated from patient-collected CD14+ monocytes; 2) an adenovirus encoding an antigenic peptide sequence, wherein the DCs are activated upon exposure to the adenovirus; and 3) patient-collected CD3+ T cells, wherein the CD3+ T cells are exposed to activated DCs, thereby activating and expanding the T cells. Alternatively or additionally, the pharmaceutical composition for use in the treatment of cancer or infectious diseases may comprise patient-derived dendritic cells (DCs), GM-CSF, and an adenovirus (Ad) encoding an antigenic peptide, wherein the DCs are derived from patient collection.

[0047] In yet another embodiment, the pharmaceutical composition 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, for use in the treatment of cancer or infectious diseases, wherein the NK or NKT cells are derived from apheresis of the patient. Alternatively or additionally, the pharmaceutical composition may comprise 1) patient-derived T cells, B cells, and / or monocytes, 2) GM-CSF, and 3) an adenovirus (Ad) encoding an antigenic peptide, for use in the treatment of cancer or infectious diseases, wherein the T cells, B cells, and / or monocytes are derived from apheresis of the patient.

[0048] Further, the pharmaceutical composition may comprise 1) dendritic cells (DCs), 2) an irradiated biopsy sample, and 3) T cells, wherein the DCs and T cells are derived from a single biopsy of a patient, and the biopsy sample is from a tumor of the same patient, and wherein the pharmaceutical composition is intended for use in the treatment of cancer. Alternatively or additionally, the pharmaceutical composition may comprise dendritic cells (DCs) and T cells, wherein the DCs and T cells are isolated from a single biopsy of a patient, wherein the DCs and / or T cells are exposed to a biopsy sample from a tumor of the same patient, and wherein the pharmaceutical composition is formulated for administration to a patient.

[0049] It is also anticipated that the pharmaceutical composition may comprise dendritic cells (DCs) and T cells, wherein a single sample from a patient is exposed to a biopsy sample from a tumor from the same patient, wherein DCs and T cells are then isolated from the single sample, and wherein the pharmaceutical composition is formulated for administration to the patient. Alternatively or additionally, the pharmaceutical composition may comprise a tumor-targeting CD3 group. +T lymphocytes for use in the treatment of cancer or infectious diseases, the composition comprising: 1) dendritic cells (DCs) derived from patient-derived, purified CD14+ monocytes; 2) 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, wherein the DCs present at least one re-expressed peptide sequence or a portion thereof from MHC-I or MHC-II, thereby activating the DCs; and 3) CD3+ purified from patient collection. + T cells; in which T cells are exposed to activated DCs, thereby activating and expanding T cells.

[0050] In this disclosure, CD3+ T cells are obtained (and purified) from a patient's tumor or blood sample and expanded in vitro. These expanded T cells can then be reintroduced into the patient as autologous cells, or administered as donor or allogeneic cells to different subjects.

[0051] T cells can be expanded by exposure to a mixture of cytokines containing one or more of IL-2, IL-15, and IL-7, or agonist derivatives thereof. 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 directs IL-2 to endoplasmic reticulum IL-2 (“erIL-2”). Similarly, IL-15 can be expressed with a signaling sequence that directs IL-15 to endoplasmic reticulum IL-15 (“erIL-15”). This allows IL-2 and / or IL-15 to be expressed at levels sufficient for autocrine activation, but without the extracellular release of IL-2. See Konstantinidis et al., “Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92 cells”, Exp Hematol, Feb. 2005; 33(2):159-64.

[0052] Furthermore, in some embodiments, T cells are genetically modified to express a chimeric antigen receptor (CAR), wherein the CAR targets a tumor antigen or a checkpoint inhibitor. The intracellular signaling domain of the CAR may also include an FcεRIγ moiety. U.S. Patent Application #17 / 341098, disclosing such a method, is incorporated herein by reference. Preferably, T cells are engineered to express a TCR that recognizes a peptide presented by MHC-I. 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. After transfection, CAR T cells can be expanded in a bioreactor until a clinically effective number of cells is obtained.

[0053] The expression vector can be a viral vector, and preferably an adenovirus, such as Ad5 adenovirus. Furthermore, it is generally further preferred that the virus be a replication-defective and non-immunogenic virus, typically achieved by targeting the deletion of selected viral proteins (e.g., E1, E3 proteins). Such desired characteristics can be further enhanced by deleting the function of the E2b gene, and high-titer recombinant viruses can be obtained using genetically modified human 293 cells, as recently reported (e.g., JVirol. [Journal of Virology] 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. Such a method is disclosed in patent application PCT / US2017 / 045093.

[0054] Furthermore, dendritic cells can be further exposed to one or more of the peptide pools of modified RNA, lentiviruses, and / or novel epitopes.

[0055] The term "epigenetics" describes heritable changes in cellular phenotype without alteration of genotype. Epigenetic modifications generally refer to alterations in gene expression without changing the DNA sequence. These modifications include DNA and RNA methylation, histone modifications, chromatin remodeling, and non-coding RNA.

[0056] 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 to both DNA and histones. These modifications occur in a tightly regulated and cooperative manner via chromatin-modifying enzymes, leading to changes in chromatin structure. Regulators responsible for these epigenetic modifications on DNA and histones have been categorized into four main classes: “writers,” “erasers,” “readers,” or “mover.” Writers introduce epigenetic markers into DNA or histones, including DNA methyltransferases (DNMTs), histone methyltransferases (HMTs), and histone acetyltransferases (HATs). Erasers, on the other hand, remove epigenetic markers through the action of histone lysine demethylases (KDMs) and histone deacetylases (HDACs). Readers recognize or are recruited to specific epigenetic markers, such as chromogenic domains and brominated domains (BRDs) that recognize methylated or acetylated residues, respectively. Movers are chromatin remodeling proteins that alter the dynamic spatiotemporal positioning of nucleosomes to allow gene transcription.

[0057] As discussed by the inventors of this paper, dysregulation of epigenetic modifications can lead to activation of oncogenes or silencing of tumor suppressor genes, as well as disruption of multiple signal transduction pathways.

[0058] DNMTs are writing enzymes that function in DNA methylation. Writing enzymes (such as DNMT1, DNMT3a, and DNMT3b) add methyl groups to cytosine residues in DNA. Despite having similar structures (a regulatory domain at the N-terminus and a catalytic domain at the C-terminus), these enzymes differ in function and expression patterns. As a maintenance methyltransferase, DNMT1 not only maintains the stability of already methylated DNA sequences, ensuring their preservation during DNA replication and cell division, but also repairs DNA methylation. In contrast, DNMT3a and DNMT3b are known as de novo methyltransferases, which can add new methyl groups to previously unmethylated DNA sequences, thus creating new methylation patterns. DNMT3a / b target specific DNA sequences via TFs (such as CTCF, Sp1, YY1, NRSF / REST, FOXA1, and SALL4.11). Two other DNMTs (DNMT2 and DNMT3L) do not possess cytosine methyltransferase activity. DNMT3L can enhance the activity of DNMT3a and DNMT3b by increasing their binding affinity to the methyl donor S-adenosyl-1-methionine (SAM). DNMT2 primarily functions by introducing the methyl chain into ncRNAs such as transfer RNA, ribosomal RNA, and nuclear RNA.

[0059] PRMT is a family of histone methyltransferases. PRMT is involved in a wide range of disease models, including neurological disorders, inflammatory diseases, cardiovascular diseases, and cancer. In particular, aberrant expression of PRMT has been extensively studied in cancers such as lung cancer, breast cancer, CRC, and leukemia. PRMT is frequently overexpressed in various tumor types, including breast and prostate cancer, and has been shown to promote tumor growth and metastasis. PRMT dysregulation is considered to play a key role in cancer onset and progression, highlighting its potential as a therapeutic target for cancer treatment.

[0060] HDACs possess the ability to remove acetyl groups from lysine residues on 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. Besides changes in expression or genetics, HDACs can drive leukemia by aberrantly recruiting to specific gene promoters through oncogenic fusion proteins. Furthermore, high expression of HDACs is associated with drug resistance in various cancers. For example, HDACs increase temozolomide resistance in glioblastoma and cisplatin and sorafenib resistance in NSCLC. Similarly, significantly upregulated HDACs in glioblastoma contribute to resistance to temozolomide chemotherapy. In conclusion, aberrant expression of HDACs contributes to the development of tumor resistance, and inhibiting these enzymes can prevent the emergence of drug resistance.

[0061] Currently, FDA-approved epigenetic drugs for cancer include azacitidine (5-azacytosine), decitabine (5-aza-2'-deoxycytosine), vorinostat (salicylic acid (SAHA)), romidisin (depeptidase), belistat (Beleodaq, PXD101), prabistat (LBH589), tucidinostat, tazestat (EPZ-6438), entsidipin (AG-221), and evanixib (AG-120), as discussed in Tao L, Zhou Y, Luo Y et al., "Epigenetic regulation in cancer therapy: from mechanisms to clinical advances." MedComm–Oncology [Drug Communications-Oncology] 2024; 3:e59. The contents disclosed in doi:10.1002 / mog2.59 are incorporated herein by reference in their entirety.

[0062] The term "apheresis" generally refers to the removal of whole blood from a patient or donor and the separation of the blood into two or more components. During apheresis, blood is drawn from the subject through a needle inserted into a vein. The needle is connected to one end of a plastic tube that provides a flow path for the blood. The other end of the tube terminates in a container for collecting the blood. The collected blood is then separated into individual components in a separator (such as a centrifuge). Desired blood components can be collected according to the procedure; these components can be red blood cells, platelets, plasma, white blood cells, or stem cells. These components are further processed to purify blood fractions, such as CD3+ fractions, CD3- fractions, CD3-CD14- fractions, CD14+ fractions, etc. One or more of these different fractions are further modified / processed as discussed throughout this application. One or more of these fractions can be infused back into the patient who requires the component / fraction.

[0063] Regarding NK cells, it should be noted that all NK cells are considered applicable herein, and therefore include primary NK cells (preserved, expanded, and / or fresh cells), immortalized subcultured NK cells, autologous or allogeneic NK cells (banked, preserved, fresh, etc.), and modified NK cells, as described in more detail below. In some embodiments, preferably, the NK cells are NK-92 cells. The NK-92 cell line is a distinctive cell line found to proliferate in the presence of interleukin-2 (IL-2) (see, for example, Gong et al., Leukemia 8:652-658 (1994)). NK-92 cells are cancerous NK cells that, upon expansion in suitable culture media, exhibit broad-spectrum antitumor cytotoxicity and predictable yield. Advantageously, NK-92 cells possess high cytolytic activity against a variety of cancers.

[0064] The original NK-92 cell line expressed the surface markers CD56bright, CD2, CD7, CD11a, CD28, CD45, and CD54, but did not exhibit the markers CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34. Growth of these NK-92 cells in culture was dependent on the presence of interleukin-2 (e.g., rIL-2), with doses as low as 1 IU / mL sufficient to maintain proliferation. IL-7 and IL-12 did not support long-term growth, nor did several 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 exhibited higher cytotoxicity even at relatively low effector cell:target (E:T) ratios (e.g., 1:1). The representative NK-92 cells are deposited at the American Center for Type Culture Collection (ATCC) and named CRL-2407. U.S. Patent Nos. 7,618,817, 8,034,332, 8,313,943, 9,150,636, 9,181,322, 10,138,462, and 10,258,649, as well as all other external references, are incorporated herein by reference in their entirety.

[0065] In another aspect of the subject matter of this invention, genetically engineered NK cells can 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, for example, Blood [Blood] 2009 113:3716-3725), and all methods of generation and expression are considered applicable herein. It is believed that expression of such a receptor allows for the specific targeting of tumor cells with antibodies specific to the patient's tumor cells (e.g., novel epitopes), specific tumor types (e.g., her2neu, PSA, PSMA, etc.), or cancer-associated antibodies (e.g., CEA-CAM). Advantageously, such antibodies are commercially available and can be used in conjunction with cells (e.g., binding to the Fcγ receptor). Alternatively, such cells can also be commercially available as haNK cells from NantKwest. These cells can then be further genetically modified to form CARs, as 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

[0066] This paper envisions that gene modification of NK cells can be performed in multiple ways, and all known methods are considered suitable for this purpose. Furthermore, it should be recognized that NK cells can be transfected with either DNA or RNA, and the specific choice of transfection will depend at least in part on the type of recombinant cells expected and the transfection efficiency. For example, in cases where stable transfection of NK cells is expected, linearized DNA can be introduced into the cells for integration into the genome. On the other hand, in cases where transient transfection is expected, circular DNA or linear RNA (e.g., mRNA with a poly-A+ tail) can be used.

[0067] For example, in the case where the NK cells are autologous NK cells or NK-92 cells, it is envisioned that the recombinant nucleic acid would include a fragment encoding a CAR, which includes an FcεRIγ signaling domain, and preferably also includes fragments encoding cytokines to provide 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 fragments encoding CD16 or high-affinity CD16158V. As will be readily understood, including cytokines to provide autocrine growth stimulation would make the modified recombinant independent of the addition of exogenous cytokines, which would make large-scale production of such cells economically feasible. Similarly, in the case where the modified recombinant also expresses CD16 or high-affinity CD16158V, such cells would have further enhanced ADCC properties and further improved targeted cytotoxicity.

[0068] It should be understood that recombinant nucleic acids encoding cytokines and / or CD16 or high-affinity CD16158V can be integrated into the genome of NK cells or can be provided as extrachromosomal units (which can be virally delivered or chemically, mechanically, or electrochemically transfected linear or circular DNA or linear RNA). For example, recombinant NK-92 cells expressing IL-2ER and CD16158V are called haNK cells (Oncotarget [Tumor Targets] Dec 27, 2016; 7(52): 86359-86373) and can be transfected with recombinant nucleic acids that include fragments encoding CARs, which include the FcεRIγ signaling domain. Again, such recombinant nucleic acids can contain additional fragments that can encode additional immunotherapeutic proteins, such as N-803, TxM-type compounds, IL-8 capture proteins, TGF-β capture proteins, etc. Similarly, NK-92 cells may have already been transfected with cDNA encoding IL-2 (e.g., NK-92MI, ATCC CRL-2408). These cells can then be further transfected with recombinant nucleic acids that include fragments encoding CARs containing the FcεRIγ signaling domain and fragments encoding CD16 or high-affinity CD16158V.

[0069] On the other hand, as further disclosed in PCT / US2019 / 033407 (which is incorporated herein by reference in its entirety), NK cells or NK-92 cells (e.g., autologous, fresh, cultured, or previously frozen) can also be transfected with recombinant nucleic acids comprising a fragment encoding a CAR having an FcεRIγ signaling domain, a fragment encoding a cytokine to provide 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 a fragment encoding CD16 (SEQ ID NO:34) or high-affinity CD16158V (SEQ ID NO:35, encoded by SEQ ID NO:36). Most typically, such recombinant nucleic acids will be configured as tricistronic constructs. As previously described, such constructs can be extrachromosomal circular plasmids, linear DNA (which can be integrated into the genome of NK cells), or linear RNA. Such nucleic acids are typically transfected into cells using methods well known in the art (e.g., electroporation, lipid transfection, ballistic gene transfer, etc.). Similarly, nucleic acids can be delivered to cells via recombinant viruses. Therefore, NK cells applicable herein include NK-92 cells (which can be transfected with a tricistronic construct encoding CAR, CD16 or a variant thereof, or a cytokine or a variant thereof), genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof or a cytokine or a variant thereof (which can be transfected with nucleic acids encoding CAR and CD16 or a variant thereof or a cytokine or a variant thereof), and genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof and a cytokine or a variant thereof (which can be transfected with nucleic acids encoding CAR). U.S. Patent Application No. 17 / 056,385, U.S. Patent No. 11,077,143, U.S. Patent No. 10,738,279, and U.S. Patent Application No. 16 / 969,152 are incorporated herein by reference.

[0070] Therefore, in preferred embodiments, it should be noted that genetically modified NK cells (especially those expressing CAR and CD16 or variants thereof) will exhibit three distinct cell-killing modes: general cytotoxicity mediated by activating receptors (e.g., NKG2D receptor), ADCC mediated by antibodies binding to target cells, and CAR-mediated cytotoxicity.

[0071] Therefore, it should be understood that the transfection method will depend at least in part on the type of nucleic acid used. Thus, viral transfection, chemical transfection, and mechanical transfection methods are all considered applicable herein. For example, in one embodiment, the vector described herein is a transient expression vector. Exogenous transgenes introduced using such vectors do not integrate into the nuclear genome of the cell; therefore, in the absence of vector replication, the exogenous transgenes will degrade or dilute over time.

[0072] In another embodiment, the vector described herein allows for stable transfection of cells. In one embodiment, the vector allows for the incorporation of one or more transgenes into the cell's genome. Preferably, such a vector has a positive selection marker, and suitable positive selection markers include any gene that allows cells to grow under conditions that would kill cells that do not express that gene. Non-limiting examples include antibiotic resistance, such as genimycin (from the Neo gene of Tn5). Alternatively, or additionally, the vector is a plasmid vector. In one embodiment, the vector is a viral vector, and preferably an adenovirus vector. As those skilled in the art will understand, any suitable vector can be used, and suitable vectors are well known in the art.

[0073] In other embodiments, cells are transfected with mRNA encoding a target protein (e.g., CAR). mRNA transfection results in transient protein expression. In one embodiment, mRNA is transfected into NK-92 cells immediately before cell administration. In one embodiment, "immediately before cell administration" means approximately 15 minutes to approximately 48 hours prior to administration. Preferably, mRNA transfection is performed approximately 5 hours to approximately 24 hours prior to administration. In at least some embodiments described in more detail below, NK cell transfection with mRNA results in unexpectedly consistent and high expression of CAR in a high proportion of transfected cells. Moreover, such transfected cells exhibit high specific cytotoxicity even at relatively low effector cell to target cell ratios.

[0074] Regarding the envisioned CAR, it is noted that NK or NK-92 cells will be genetically modified to express the CAR as a membrane-bound protein, thereby exposing a portion of the CAR on the cell surface while retaining its signal transduction domain in the intracellular space. Most typically, the CAR will include at least the following elements (in order): an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signal transduction domain.

[0075] In a preferred embodiment, the cytoplasmic domain of the CAR includes or is composed of the FcεRIγ signaling domain. It is noteworthy, and as described in more detail below, that the FcεRIγ signaling domain provides a significant increase in CAR expression levels and a significant extension of cytotoxicity over time. In some embodiments, the FcεRIγ cytoplasmic domain is the only signaling domain. However, it should be understood that additional elements, such as 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 an alternative embodiment, the cytoplasmic domain of the CAR may also include a CD3ζ (CD3 zeta) signaling domain. In one embodiment, the cytoplasmic domain of the CAR is composed of the CD3ζ signaling domain.

[0076] Therefore, the envisioned CAR would include a general structure comprising a desired antigen-binding domain coupled to a hinge domain coupled to a transmembrane domain coupled to a signal transduction domain. Alternatively, the envisioned CAR could have a desired binding domain, which would then be coupled to a hybrid protein containing, consisting of, or substantially consisting of a hinge domain coupled to a transmembrane domain coupled to a signal transduction domain.

[0077] Most typically, but not necessarily, the extracellular binding domain of a CAR will be an scFv or other natural or synthetic binding motif that specifically binds to the target antigen. Particularly suitable binding motifs include small antibody fragments with single, dual, or multiple target specificities, β-barrel domain conjugates, phage-displayed fusion proteins, etc. Among other suitable extracellular binding domains, preferred domains will specifically bind to tumor-specific antigens, tumor-associated antigens, or patient-specific antigens 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, CTAGIB, and CD33. Table 1 lists other non-restrictive tumor-associated antigens and the malignancies associated with them. By way of non-restrictive examples, additional tumor-specific antigens are described in US 2013 / 0189268; WO 1999024566 A1; US ​​Patent No. 7,098,008; and WO2000020460, each of which is incorporated herein by reference in its entirety. Similarly, other preferred domains will specifically bind to (pathogenic) virus-specific antigens, such as antigens of HIV (e.g., gp120), HPV, RSV, influenza, Ebola, or HCV.

[0078] Therefore, the envisioned CAR would target antigens associated with specific cancer types. For example, targeted cancers include leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloid, 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, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, and lymphangioma. Endothelial sarcoma, synovial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Examples of infectious diseases that are expected to be treated using the method of the present invention include AIDS, H1N1, Ebola, BSE, Zika virus, SARS, coronavirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E.

[0079] In embodiments, the subject matter of the invention includes a method for treating cancer, the method comprising determining the MHC-I expression level in a tumor. Upon determining that the MHC-I level is low relative to control non-cancerous tissue, a variety of NK cells are administered to the patient, wherein these 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 intratumorally. After a period of time, a variety of T cells are administered to the patient. The T cells may be autologous or allogeneic. The T cells may be further genetically engineered to express a targeting agent, wherein the agent comprises a CAR or a TCR.

[0080] Appropriate methods for determining MHC-I levels in tumor and normal tissue samples involve measuring MHC-I expression on the cell surface. Enzymatic and / or mechanical dissociation of tissue (thereby isolating and purifying live whole cells from primary tissue) is essential for determining MHC-I surface expression using Ab staining and flow cytometry. Alternative methods may include transcriptomic analysis, proteomics, Western blotting, and surface plasmon resonance (SPR).

[0081] As used in this article, t-haNK cells are NK cells expressing genetically engineered CARs. Without being bound by any particular theory, it should be understood that the targeting portion of the CAR serves a dual purpose. First, the targeting portion guides NK cells to the expression sites of antigens in the patient, thereby facilitating NK cell delivery to tumor tissue. Second, the targeting portion can be immunogenic, thereby facilitating the delivery of antibodies and T cells to the tumor.

[0082] Primary NK cells can also be enriched and expanded from whole blood or cord blood mononuclear cells using standard methods, including exposing primary NK cells to CD16 antibodies, dexamethasone, and / or IL-15. Stabilized IL-15 can be used, including IL-15 hyperagonists such as nogapendenkin alpha imbakicept (Alt-803, N-803, Vesanktiva) and stabilized IL-15 / IL-15Ra fusion proteins. U.S. Patent Application Nos. 16 / 985,728, 16 / 505,528, 11,351,196, 63 / 156,269, 8,163,879, 8,507,222, and 10,537,615 are incorporated herein by reference.

[0083] The cytokine-enhanced NK (ceNK) cells disclosed herein refer to NK cells in which cytotoxic activity is enhanced by cytokine stimulation. ceNK cells are prepared by inducing NK cells with a composition of corticosteroids and optionally cytokines comprising IL-15, IL-15:IL-15Rα, or an agonist derivative thereof, such as N-803. The cytokine composition may comprise a fusion protein containing IL-15 or an agonist derivative thereof. Preferably, the fusion protein comprises IL-15, wherein the fusion protein has increased stability relative to IL-15. While not limiting the scope of the invention, it is generally preferred that the corticosteroid be hydrocortisone and the optional cytokine be N-803.

[0084] The memory-like cytokine-enhanced NK cells (m-ceNK) disclosed in this article comprise enriched and expanded NK cells obtained from donor peripheral blood using apheresis techniques to generate NK cells with a memory-like phenotype. m-ceNK cells exhibit high cytotoxicity and increased interferon-γ production. These m-ceNK cells can be generated from individual donors for autologous cell therapy or as an allogeneic product derived from umbilical cord blood. In addition to enhanced efficacy, m-ceNK cells can be readily infused in an outpatient setting.

[0085] As a non-limiting example, m-ceNK cells can be generated by the following steps: obtaining a plurality of monocytes and contacting the plurality of monocytes with a corticosteroid and optionally a cytokine. In another step, the plurality of monocytes are incubated in the presence of a corticosteroid and optionally a cytokine to enrich monocytes in NK cells, and then the enriched NK cells are induced with a cytokine composition comprising IL-15, IL-12, and IL-18 or agonist derivatives thereof. The composition may comprise one or more fusion proteins, wherein these fusion proteins comprise at least one of the IL-15, IL-12, and IL-18 cytokines or agonist derivatives thereof. The cytokine composition may comprise a TxM fusion protein to generate m-ceNK cells, wherein 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.

[0086] Further description of the preparation of m-ceNK cells and their advantageous properties is given in PCT / US2022 / 018290, which is incorporated herein by reference in its entirety. U.S. Patent Application 17 / 375,985 and U.S. Patent 11,453,862 provide additional alternative methods for inducing NK cell enrichment and expansion. Each of the above references is incorporated herein by reference in its entirety.

[0087] The "T-cell receptor," or "TCR," refers to a dimeric polypeptide typically found on the surface of T cells. Each peptide chain of the TCR generally contains an extracellular domain, a transmembrane domain, and an intracellular domain, comprising a variable region and a constant region. The variable region is the portion of the TCR that interacts with antigens presented by the MHC. The constant region is the region within each of the two peptide chains, covalently linked by disulfide bonds. The intracellular domain generally contains CD3z, which contains one or more immune receptor tyrosine activation motifs (ITAMs). ITAMs mediate the binding of the variable region to appropriate intracellular signaling pathways.

[0088] The intracellular signal transduction domain of CARs may also include the FcεRIγ moiety. U.S. Patent Application No. 17 / 341098 is incorporated herein by reference.

[0089] T cells may optionally contain a modified TCR, which relates to 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 linker peptide), a transmembrane domain, and an intracellular domain. In a particular embodiment, the variable region and the constant region are attached via a linker. In another particular embodiment, the linker peptide is located between the constant region and the transmembrane domain. In yet another particular embodiment, the two peptide chains are linked to each other by disulfide bonds between the linker peptides of each peptide chain. The modified TCR does not interact with the endogenous TCR produced by the T cell. The contents of U.S. Patent Application No. 63 / 227,195 are incorporated herein by reference.

[0090] Once a CAR-based therapeutic agent (e.g., an antigen-binding domain conjugated to a CAR scaffold) binds to an antigen expressed by cancer cells, cytotoxic cells can induce cancer cell destruction. While all cytotoxic cells are generally expected to be suitable for this purpose, particularly preferred cytotoxic cells include NK cells, activated NK cells, high-affinity NK cells, CD8+ T cells, and CD4+ T cells that have been modified to recombinantly express the CAR-based therapeutic agent, any of which may be of different origin. Cytotoxic cells are engineered to express TCRs that recognize MHC-I-presented peptides.

[0091] Therapeutic T cells, as used herein, can be patient-derived (autologous) or donor-derived (allogeneic). T cells are typically obtained via leukapheresis and further isolated based on the expression of surface markers (CD4, CD8). Purified T cells can be activated by exposure to CD3 and / or CD28 Abs, or by exposure to antigen-presenting cells (APCs). Cells can be expanded by exposure to a mixture of cytokines containing one or more of IL-2, IL-15, and IL-7. In a preferred embodiment, T cells or primary NK cells are expanded on an automated platform and can be transfected via microfluidic electroporation as described in U.S. Patent 11,377,652, the contents of which are incorporated herein by reference.

[0092] T cells can be purified from tumors and are therefore tumor-infiltrating lymphocytes (TILs). TILs can be purified from tumor tissue and expanded in vitro. TILs can be reintroduced into patients as autologous cells or administered as donor cells to different subjects. Stimulation of MHC-I expression by NK cells is expected to enhance the cytotoxic efficacy of TILs.

[0093] In embodiments of the invention, tumor-associated endothelial cells (TILs) are isolated from a patient's tumor using standard techniques. The TILs are then exposed to the patient's tumor tissue, in which the patient has been treated with NK cells, thereby inducing MHC-I expression. NK exposure can be performed via IV injection or intratumoral injection. NK exposure to tumor tissue can also be performed ex vivo. This activates and expands the TILs exposed ex vivo to tumor tissue. The expanded TILs, containing CD4 and CD8 cytotoxic T cells, are then administered to the patient.

[0094] T cells can be transfected to express one or more CARs. Transfection techniques include, but are not limited to, viral transduction, mRNA transfection, and the Sleeping Beauty transposon system. After transfection, CAR T cells can be expanded in a bioreactor until a clinically effective number of cells is obtained.

[0095] However, it should be understood that, in other respects, cytotoxic cells can also be macrophages, monocytes, neutrophils, basophils, or eosinophils. Therefore, and from different perspectives, the cells considered in this article can exert cytotoxic effects through phagocytosis, pore formation, induction of antibody-dependent cell-mediated cytotoxicity (ADCC), or by initiating TNF or fas-mediated killing pathways.

[0096] As part of the cytotoxic antitumor process, cytotoxic cells can release various types of cytotoxic particles (e.g., granulosin, perforin, granzymes). A variety of assays can be used to monitor cell-mediated cytotoxicity, including flow cytometry assays (e.g., based on the presence of lysed particles such as perforin, granzymes, or the production of TNF family members such as TNF-α, FasL, or TRAIL) (Zaritskaya 2010, Clay, T. et al., Clin. Cancer Res. [Clinical Cancer Research] (2001) &:1127-1135).

[0097] In one embodiment, a body fluid is obtained after NK cell therapy, wherein the body fluid contains cellular components, such as tumorigenic cells or cancer cells displaying antigens that bind to cytotoxic cells expressing CARs as described herein, and cytotoxic cells expressing antigen-binding moieties are in contact with these cells. Assays are then performed to detect an immune response, such as an indication that an ADCC or ADCP response has been triggered by the patient's own immune cells.

[0098] Assays for detecting immune responses are known in the art and are described herein. For example, assays for detecting such responses may detect the release of cytotoxic particles (e.g., granulosin, perforin, granzyme), or phagocytosis, or receptor-ligand-mediated cell lysis (e.g., mediated by the Fas / APO pathway). A variety of flow cytometry assays may be used to monitor cell-mediated cytotoxicity, such as based on the presence of lysing particles like perforin, granzyme, or the production of TNF family members such as TNF-α, FasL, or TRAIL (Zaritskaya 2010, Clay, T. et al., Clin. Cancer Res. [Clinical Cancer Research] (2001) &:1127-1135).

[0099] 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 conjugated to a CAR scaffold) to promote or elicit an immune response. Cytokines include, but are not limited to, IL2, IL4, IL7, IL11, IL15, IL21, TNF-α, IFN-γ, etc. 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.

[0100] Other techniques for treating cancer include surgery, radiation therapy, chemotherapy, immunosuppressants (such as azathioprine, cyclosporine, methotrexate, mycophenolate mofetil, 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.

[0101] It should be understood that embodiments of the present invention may be administered to patients using appropriate formulations, indications, and dosing regimens that are suitable for government regulatory agencies such as the U.S. Food and Drug Administration (FDA).

[0102] In some embodiments, cytotoxic cells expressing a TCR, a modified TCR, or a CAR-based therapeutic agent (e.g., an antigen-binding domain coupled to a CAR scaffold) are administered as a pharmaceutical composition to a patient. In another embodiment, a method of treating cancer by administering cytotoxic cells to a subject is contemplated. In yet another embodiment, a method of inhibiting or reducing the proliferation of cells expressing a corresponding antigen (with the antigen-binding domain binding thereto) on their cell surface is contemplated by administering cytotoxic cells to a subject.

[0103] In one embodiment, the patient may be lymphocyte depleted, thereby reducing the number of endogenous lymphocytes, which in turn increases the availability of essential endogenous cytokines and promotes the survival of infused T cells.

[0104] In some embodiments, relative to a negative control, cytotoxic cells expressing TCR, modified TCR, or CAR-based therapeutic agents (e.g., antigen-binding domains conjugated to a CAR scaffold) reduce the amount (e.g., cell number, tumor size, etc.) of 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%.

[0105] Examples of cancers for which cytotoxic cell therapy may be considered in this article include any cancer that expresses or overexpresses cancer-associated antigens on its cell surface. Examples of cancers for which cytotoxic cell therapy may be used with TCRs, modified TCRs, or CAR-based therapeutic agents (e.g., antigen-binding domains conjugated 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.

[0106] Dendritic cells (DCs) refer to multiple populations of morphologically similar cell types found in various lymphoid and non-lymphoid tissues, as detailed in Steinman (1991) Ann. Rev. Immunol. [Annual Review of Immunology] 9:271-296. Dendritic cells constitute the most efficient and preferred antigen-presenting cells (APCs) in organisms. Dendritic cells can differentiate from monocytes and have a different phenotype. Mature DCs can provide all the signals required for T cell activation and proliferation. Furthermore, mature dendritic cells are not phagocytic, while monocytes and immature dendritic cells are strong phagocytic cells. Immature DCs are capable of capturing antigens through endocytosis, phagocytosis, macrocytosis, or adsorbent endocytosis and receptor-mediated antigen uptake, and have high intracellular concentrations of MHC class II molecules.

[0107] For example, dendritic cells can be obtained from immune-competent cells of a patient diagnosed with cancer (immunocompetent cells are typically obtained from a single patient apheresis). Alternatively, dendritic cells can also be derived from progenitor cells that respond to specific growth factors (e.g., GM-CSF). Regardless of the type of isolation, it is then envisioned that the dendritic cells be transfected with one or more tumor-associated epitopes of the patient's tumor or with an expression vector (preferably a viral vector) containing nucleic acids encoding one or more tumor-associated epitopes of the patient's tumor. Most preferably, the tumor-associated epitopes include or are novel epitopes specific to the patient's tumor. Thus, the dendritic cells so transfected will present tumor epitopes via the MHC-I and / or MHC-II systems.

[0108] The antigen can be exposed to naked dendritic cells (DCs) or in a viral vector (such as an adenovirus vector). Adenovirus vectors are particularly preferred. Furthermore, it is generally even more preferred that the virus is a replication-deficient and non-immunogenic virus, typically by targeting the deletion of selected viral proteins (e.g., E1, E3 proteins). Such desired properties can be further enhanced by deleting the function of the E2b gene, and high titers of recombinant viruses can be obtained using genetically modified human 293 cells, as recently reported (e.g., J Virol. [Journal of Virology] 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 will provide several benefits for triggering a strong and durable immune response against cancer-associated sequences. First, and after dendritic cells are infected with a recombinant virus, expression and presentation of cancer-associated sequences using the MHC-I and / or MHC-II presentation pathways will increase the production of appropriately activated CD4. + and CD8 + The likelihood of cells, which in turn is believed to increase the likelihood of appropriate antibody production and proper T-cell and B-cell memory.

[0109] Therefore, dendritic cells obtained from a single collection from a patient with a tumor are exposed in vitro to one or more tumor-associated epitopes of the patient's tumor, or to nucleic acids encoding one or more tumor-associated or tumor-specific epitopes of the patient's tumor. In this way, the immune response can be specifically targeted at a particular tumor (and even a tumor subpopulation), and the patient's immune-competent cells will not be rejected.

[0110] The pharmaceutical composition may comprise cytotoxic cells containing antigen-binding domains conjugated or linked to the CAR scaffold as described herein, and one or more pharmaceutically or physiologically acceptable loads, diluents, or excipients. Additionally, the pharmaceutical composition may comprise one or more adjuvants (e.g., aluminum hydroxide), antioxidants, antibacterial agents, buffers, carbohydrates, chelating agents such as EDTA or glutathione; colorants, flavorings and / or aromatic substances, emulsifiers, excipients, lubricants, pH buffers, preservatives, salts for influencing osmotic pressure, peptides (e.g., glycine), proteins, solubilizers, stabilizers, wetting agents, etc., which do not harmfully react with or otherwise interfere with the activity of the active compound (e.g., antigen-binding domains conjugated to the CAR scaffold). Buffers include, but are not limited to, neutral buffered saline, phosphate buffered saline, etc. Carbohydrates include, but are not limited to, dextran, glucose, mannose, mannitol, sucrose, etc.

[0111] Pharmaceutical compositions can be formulated for specific modes of administration. Modes of administration may include, but are not limited to: intra-articular, intradermal, intranasal, intraperitoneal, intrathecal, intratumoral, intravenous, intraventricular, subcutaneous, transdermal, transmucosal, or local routes.

[0112] In a preferred embodiment, the cytotoxic cells are administered via intravenous infusion. Such formulations can be prepared according to standard techniques known to those skilled in the art. For example, the composition to be administered intravenously may contain one or more components (e.g., diluents, suspension buffers, saline or glucose / water, other components such as cytokines, etc.) before being infused into the patient.

[0113] Many such techniques for the formulation and administration of pharmaceutical compositions are known in the art, for example, U.S. Patent Application Publication No. 2014 / 0242025, and all such references are incorporated herein by reference in their entirety.

[0114] In some embodiments, the cytotoxic cells proliferate in vivo, thereby persisting in the patient for months or even years after administration to provide a sustained mechanism for inhibiting tumor growth or recurrence. In some aspects, these cytotoxic cells persist for at least three months, six months, nine months, twelve months, fifteen months, eighteen months, two years, three years, four years, or five years after administration to the patient.

[0115] Cytotoxic cells can be obtained from a variety of sources (e.g., isolated from humans, isolated from commercially available cytotoxic cells, isolated from cell banks, etc.). Procedures for in vitro expansion of 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 limit us to any particular method for in vitro expansion of cytotoxic cells.

[0116] The pharmaceutical composition containing cytotoxic cells described herein can 10 4 Up to 10 9 Cells / kg body weight, 10 5 Up to 10 6 Administer doses of cells / kg body weight or any integer value within these ranges. Cytotoxic cell compositions may be administered at these doses once or continuously (over several days, weeks, or months). Infusion techniques for 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).

[0117] In other embodiments, the pharmaceutical composition is administered in a therapeutically effective amount, which is the amount that is effective in treating a particular indication. Administration may be as a single dose or based on intervals. As used herein, "interval" indicates periodic administration of a therapeutically effective amount (as opposed to a single dose). The administration interval for an individual does not need to be fixed but may vary over time. The terms "in combination with" or "co-administered" indicate that the composition may be administered shortly before, simultaneously with, or approximately simultaneously with, or shortly after another composition. Example

[0118] Example 1 In one example disclosed herein, tumor-targeting lymphocytes for use in the treatment of cancer or infectious diseases are generated. The method includes the following steps: therapeutic apheresis of a subject with cancer or infectious disease; purification of CD3+ T cell fractions from the apheresis product, wherein the remaining apheresis product contains CD3- fractions; purification of CD14+ monocytes from the CD3- fraction, wherein the remaining apheresis product contains CD3- / CD14- fractions; differentiation of CD14+ monocytes into dendritic cells (DCs) and exposure of DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein DCs present the peptide sequence or a portion thereof using MHC-I or MHC-II, thereby activating the DCs; exposure of purified CD3+ T cells to activated DCs, thereby expanding T cells; and purification of the expanded T cells.

[0119] T cells can expand in the presence of IL-15 or its agonist derivatives. Furthermore, T cells can be genetically modified to express endoplasmic reticulum-localized IL-15 (erIL-15) and / or chimeric antigen receptors (CARs), where the CARs target tumor antigens or checkpoint inhibitors. The adenovirus disclosed herein may be Ad5 adenovirus. Additionally, dendritic cells can be further exposed to modified RNA and / or lentiviruses and / or peptide pools of novel epitopes.

[0120] Example 2In another instance disclosed in this article, tumor-targeting natural killer (NK) cells are generated for use in the treatment of cancer or infectious diseases. The method includes the following steps: performing therapeutic apheresis on a subject with cancer or an infectious disease; purifying CD3+ T cell fractions from the apheresis product, wherein the remaining apheresis product contains CD3- fractions; purifying CD14+ monocytes from the apheresis CD3- fractions, wherein the remaining apheresis product contains CD3- / CD14- fractions; expanding NK cells from the apheresis CD3- / CD14- fractions; differentiating CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein the DCs present the peptide sequence or a portion thereof with MHC-I or MHC-II, thereby activating the DCs; exposing the purified CD3+ T cells to the 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 a 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 signal transduction domain of a chimeric antigen receptor (CAR), wherein the fused nucleic acid encodes the TCR. CAR; and NK cells enriched and expanded by transfection with nucleic acids encoding TCR CAR.

[0121] The NK cells disclosed in this article may include NK-92 cells or memory cytokine-enriched NK cells (M-CENK). Furthermore, M-CENK cells may be genetically modified to express CD16 and / or CARs targeting tumor antigens or checkpoint inhibitors. Additionally, NK-92 cells may contain CD16 and / or they may be further genetically modified to express a second CAR.

[0122] Example 3 In yet another example, the inventors have disclosed 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 an infectious disease; purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product contains a CD3- fraction; purifying CD14+ monocytes from the apheresis CD3- fraction, wherein the remaining apheresis product contains a CD3- / CD14- fraction; expanding NK cells from the apheresis CD3- / CD14- fraction; differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein the DCs present the peptide sequence or a portion thereof under MHC-I or MHC-II, thereby activating the DCs; exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cells; purifying the expanded T cells; purifying CD3+ TILs from a solid tumor and exposing the TILs to the activated DCs, thereby expanding the TILs; and purifying the expanded TILs.

[0123] Example 4 In another example, a pharmaceutical composition is disclosed. This pharmaceutical composition comprises 1) activated dendritic cells (DCs), wherein the DCs are differentiated from patient-donated CD14+ monocytes; 2) an adenovirus encoding an antigenic peptide sequence, wherein the DCs are activated upon exposure to the adenovirus; and 3) patient-donated CD3+ T cells, wherein the CD3+ T cells are exposed to the activated DCs, thereby activating and expanding the T cells. The pharmaceutical composition is intended for use in the treatment of cancer or infectious diseases.

[0124] In the pharmaceutical compositions disclosed above, DCs present an antigenic peptide sequence or a portion thereof using MHC-I or MHC-II, thereby activating the DCs. Activated CD3+ T cells may contain T cell receptors (TCRs) that are specific to the MHC-presented antigenic peptide sequence on the DCs.

[0125] The pharmaceutical composition may further comprise natural killer (NK) cells. In some cases, NK cells are expanded from patient apheresis. NK cells may also comprise cytokine-enriched (CENK) cells or memory-like cytokine-enriched (M-CENK) cells. NK cells are expanded in a culture medium containing IL-15, IL-12, and / or IL-18. In some embodiments, NK cells are NK-92 cells, and more preferably NK-92 cells comprising high-affinity Fc receptor and / or endoplasmic reticulum-targeting IL-2 (erIL-2) or erIL-15. NK cells may also comprise a CAR. The CAR is intended to comprise a targeting domain, wherein the targeting domain comprises the α and β chains of a TCR. Preferably, the α and β chains are derived from activated T cells of the patient. In some embodiments, the CAR comprises a targeting domain, wherein the targeting domain comprises an antibody-binding domain. Preferably, the antibody-binding domain is specific to tumor-associated antigens, tumor-specific antigens, or novel epitopes. Alternatively or additionally, the antibody-binding domain is specific to checkpoint inhibitors.

[0126] It is further anticipated that the pharmaceutical composition comprises IL-15 or an agonist derivative thereof, wherein T cells are exposed to IL-15 or an agonist derivative thereof prior to and / or during exposure to activated DCs. Dendritic cells of the pharmaceutical composition disclosed herein are preferably activated in a medium containing GM-CSF (granulocyte-macrophage colony-stimulating factor). Furthermore, dendritic cells may be activated in a medium further containing IL-4.

[0127] Example 5In another example, the inventors disclosed a pharmaceutical composition for use in the treatment of cancer or infectious diseases, comprising patient-derived dendritic cells (DCs), GM-CSF, and an adenovirus (Ad) encoding an antigenic peptide, wherein the DCs are derived from apheresis of the patient. The pharmaceutical composition may further comprise IL-4 and / or IL-15. The MOI (multiple of infection) of the pharmaceutical composition is expected to be in the range of 20-20,000.

[0128] Example 6 In yet another example, the inventors disclose a pharmaceutical composition for use in the treatment of cancer or infectious diseases, 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 apheresis of a patient. The pharmaceutical composition may further comprise IL-4 and / or IL-15. The MOI (multiple of infection) of the pharmaceutical composition is expected to be in the range of 20-20,000.

[0129] Example 7 A pharmaceutical composition for use in the treatment of cancer or infectious diseases, the pharmaceutical composition 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 apheresis of the patient. The pharmaceutical composition may further comprise IL-4 and / or IL-15. The MOI (multiple of infection) of the pharmaceutical composition is expected to be in the range of 20-20,000.

[0130] 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 a single apheresis of a patient, and the biopsy sample is derived from a tumor of the same patient, and wherein the pharmaceutical composition is intended for use in the treatment of cancer.

[0131] The pharmaceutical composition may further comprise IL-4 and / or IL-15. The MOI (multiple of infection) of the pharmaceutical composition is expected to be in the range of 20-20,000. The pharmaceutical composition may further comprise adenovirus (Ad), wherein the Ad comprises nucleic acid encoding an antigenic peptide sequence.

[0132] The pharmaceutical composition may further comprise natural killer (NK) cells. In some cases, NK cells are expanded from patient apheresis or patient PBMCs. NK cells may also comprise cytokine-enriched (CENK) cells or memory-like cytokine-enriched (M-CENK) cells. NK cells are expanded in a culture medium containing IL-15, IL-12, and / or IL-18. In some embodiments, NK cells are NK-92 cells, and more preferably NK-92 cells comprising high-affinity Fc receptor and / or endoplasmic reticulum-targeting IL-2 (erIL-2) or erIL-15. NK cells may also comprise a CAR. The CAR is intended to comprise a targeting domain, wherein the targeting domain comprises the α and β chains of a TCR. Preferably, the α and β chains are derived from activated T cells of the patient. In some embodiments, the CAR comprises a targeting domain, wherein the targeting domain comprises an antibody-binding domain. Preferably, the antibody-binding domain is specific to tumor-associated antigens, tumor-specific antigens, or novel epitopes. Alternatively or additionally, the antibody-binding domain is specific to checkpoint inhibitors.

[0133] In some cases, biopsy samples and T cells are added to the dendritic cells (DCs) sequentially. For example, the biopsy sample may be combined with the DCs first, and then T cells, which are expanded T cells, may be added.

[0134] Example 9 In another instance, the inventors disclosed a pharmaceutical composition comprising dendritic cells (DCs) and T cells, wherein the DCs and T cells are isolated from a patient’s apheresis, wherein the DCs and / or T cells are exposed to a biopsy sample of a tumor from the same patient, and wherein the pharmaceutical composition is formulated for administration to the patient.

[0135] Preferably, the DCs are further exposed to adenovirus (Ad), wherein the Ad contains nucleic acid encoding an antigenic peptide sequence. The DCs and / or T cells may also be further exposed to IL-15 or an agonist derivative thereof. Ideally, the biopsy sample is from a tumor of a patient who has been treated with a DAMP inducer, wherein the DAMP inducer contains radiation and / or a histone deacetylase (HDAC) inhibitor. The DCs are exposed to the biopsy in the presence of an activating medium containing granulocyte colony-stimulating factor (GMCSF) and IL-4, wherein the DCs are activated and mature. The DCs and biopsy are exposed to the activating medium for 24–48 hours, wherein the DCs are then isolated and combined with T cells. The pharmaceutical composition can be formulated for intravenous, subcutaneous, intratumoral, or intravenous administration.

[0136] Example 10In another example, the inventors disclosed a pharmaceutical composition comprising dendritic cells (DCs) and T cells, wherein a single sample from a patient is exposed to a biopsy sample from a tumor from the same patient, wherein DCs and T cells are then isolated from the single sample, and wherein the pharmaceutical composition is formulated for administration to the patient.

[0137] Preferably, the DCs are further exposed to adenovirus (Ad), wherein Ad contains nucleic acid encoding an antigenic peptide sequence. The DCs and / or T cells may also be further exposed to IL-15 or an agonist derivative thereof. The isolated DCs may be further exposed to adenovirus (Ad), wherein Ad contains nucleic acid encoding an antigenic peptide sequence. Ideally, the biopsy sample is from a tumor of a patient who has been treated with a DAMP inducer, wherein the DAMP inducer contains radiation and / or a histone deacetylase (HDAC) inhibitor. The DCs and / or CD14+ monocytes are exposed to the biopsy in the presence of an activating medium containing granulocyte colony-stimulating factor (GMCSF) and IL-4, wherein the DCs are activated and mature. The exposure of the DCs and / or CD14+ monocytes and the biopsy to the activating medium can be sustained for 24-48 hours, and wherein the differentiated DCs are then isolated and combined with purified CD3+ T cells. The pharmaceutical composition can be formulated for intravenous, subcutaneous, intratumoral, or intravenous administration.

[0138] Example 11 In another example, the inventors have disclosed a method for generating tumor-targeting lymphocytes for use in the treatment of cancer or 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, wherein the remaining apheresis product contains a CD3- fraction; purifying CD14+ monocytes from the apheresis CD3- fraction, wherein the remaining apheresis product contains a CD3- / CD14- fraction; expanding NK cells from the apheresis CD3- / CD14- fraction; differentiating the CD14+ monocytes into dendritic cells (DCs) and 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, whereby the DCs MHC-I or MHC-II 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 cells.

[0139] Example 12In yet another example, the inventors have disclosed a method for generating tumor-targeted natural killer (NK) cells for use in the treatment of cancer or infectious diseases. The method includes: therapeutically apheresis of a subject with cancer or an infectious disease; purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product contains a CD3- fraction; purifying CD14+ monocytes from the apheresis CD3- fraction, wherein the remaining apheresis product contains a CD3- / CD14- fraction; expanding NK cells from the apheresis CD3- / CD14- fraction; differentiating the CD14+ monocytes into dendritic cells (DCs) and 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 activating the DCs by presenting at least one re-expressed peptide sequence or a portion thereof under MHC-I or MHC-II; and converting the purified CD3+ T cell fraction into a CD14+ T cell fraction. T cells are exposed to activated dendritic cells (DCs) to expand T cells; at least one nucleic acid encoding the a-chain and b-chain of a T cell receptor (TCR) is isolated from the expanded T cells and fused to the 5' end of a second nucleic acid encoding the transmembrane domain and intracellular signal transduction domain of a chimeric antigen receptor (CAR), wherein the fused nucleic acid encodes the TCR CAR; enriched and expanded NK cells are transfected with the nucleic acid encoding the TCR CAR.

[0140] Example 13 In this example, the 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 an infectious disease; purifying CD3+ T cell fractions from the apheresis product, wherein the remaining apheresis product contains CD3- fractions; purifying CD14+ monocytes from the apheresis CD3- fractions, wherein the remaining apheresis product contains CD3- / CD14- fractions; expanding NK cells from the apheresis CD3- / CD14- fractions; differentiating CD14+ monocytes into dendritic cells (DCs) and 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 activating the DCs by presenting at least one re-expressed peptide sequence or a portion thereof from MHC-I or MHC-II; purifying CD3+ T cell fractions from solid tumors; purifying CD14+ T cell fractions from solid tumors; differentiating CD14+ T cell fractions from the apheresis product containing CD3- / CD14- fractions; differentiating CD14+ T cell fractions from the apheresis product containing CD3- / CD14- fractions; differentiating CD14+ T cell fractions into dendritic cells (DCs) and 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 activating the DCs by presenting at least one re-expressed peptide sequence or a portion thereof from MHC-I or MHC-II; and purifying CD3+ T cell fractions from solid tumors. + TILs were amplified by exposing CD3+ TILs to activated DCs; and the amplified TILs were purified.

[0141] Example 14 This article discloses information including tumor-targeting CD3. +A pharmaceutical composition for T lymphocytes, wherein the pharmaceutical composition is intended for use in the treatment of cancer or infectious diseases. The composition comprises 1) dendritic cells (DCs) differentiated from patient-derived, single-aborted, purified CD14+ monocytes; 2) 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, whereby the DCs present at least one re-expressed peptide sequence or a portion thereof under MHC-I or MHC-II, thereby activating the DCs; and 3) CD3+ purified from patient apheresis. + T cells; in which T cells are exposed to activated DCs, thereby activating and expanding T cells.

[0142] Example 15 In another example, the inventors disclose a method for expanding tumor-infiltrating lymphocytes for use in the treatment of cancer or infectious diseases. The method includes therapeutic apheresis of a subject with cancer or an infectious disease, wherein the subject has been 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; purification of CD3+ T cell fractions from the apheresis product, wherein the remaining apheresis product contains CD3- fractions; purification of CD14+ monocytes from the apheresis CD3- fractions, wherein the remaining apheresis product contains CD3- / CD14- fractions; differentiation of CD14+ monocytes into dendritic cells (DCs) and exposure of DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein DCs MHC-I or MHC-II present the peptide sequence or a portion thereof, thereby activating the DCs; and purification of CD3+ T cells from solid tumors. + TILs were amplified by exposing them to activated DCs; the amplified TILs were then purified.

[0143] Example 16The inventors also disclose a method for generating tumor-targeting lymphocytes for use in the treatment of cancer or infectious diseases. The method includes: therapeutic apheresis of a subject suffering from cancer or an infectious disease, wherein the subject has been 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; purification of CD3+ T cell fractions from the apheresis product, wherein the remaining apheresis product contains CD3- fractions; purification of CD14+ monocytes from the apheresis CD3- fractions, wherein the remaining apheresis product contains CD3- / CD14- fractions; differentiation of CD14+ monocytes into dendritic cells (DCs) and exposure of DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein DCs present the peptide sequence or a portion thereof under MHC-I or MHC-II, thereby activating the DCs; exposure of the purified CD3+ T cells to the activated DCs, thereby expanding the T cells; and purification of the expanded T cells.

[0144] Example 17

[0145] In another example, the inventors disclosed a method for transducing fresh PBMCs using an adenovirus control, AD5-[E1-, E2b-]-GFP. Fresh blood from two donors was used for PBMC isolation and Ad5-GFP transduction. Cells were infected with an MOI of 20. After transduction, cells were cultured in AIM-V medium containing either N-803 or GM-CSF, or GM-CSF + IL-4. GFP expression was monitored using IncuCyte and evaluated using flow cytometry. The following conditions were evaluated:

[0146] Table 1

[0147]

[0148] The MOI used in the above experiments was 20. Transduction details are as follows: 12-well plate, for donor 1, using 1 x 102 6 1 cell / well, and for donor 2, use 5 x 10 5 Cells / well. Use the following amounts of N-803, GM-CSF, and IL-4: 74 ng / mL N-803, 100 ng / mL GM-CSF, and 20 ng / mL IL-4. The virus used for transduction was Ad5-[E1-, E2b-]-GFP.

[0149] Transduction was performed in 12-well plates. Isolated PBMCs were transduced in AIM-V medium (250 μL). Ad5 virus (250 μL) was added with MOI 20. The virus-infected culture was incubated at 37°C, 5% CO2 for 1 hour. After 1 hour, 0.5 mL of medium containing the corresponding cytokines was added, and the plate was incubated for another 6 days (Note: longer incubation yields more cells for staining by flow cytometry). GFP expression was monitored using IncuCyte and evaluated by flow cytometry.

[0150] The results for the two donors (donor 001 and donor 002) are shown in Figures 1-8 middle. Figures 1-3 Figures 5-6 show that different mixtures of cytokines (N-803, GM-CSF, and IL-4) induced different cell morphologies in donor 001 and donor 002, respectively. Figure 4 and 7 The results showed that on days 3 and 5 post-transduction, higher numbers of GFP+ cells were observed in donor 001 and donor 2, respectively, in the GM-CSF treatment group.

[0151] flow cytometry gating strategies are shown in Figure 8 The results are presented in the tables below. The following two tables (Tables 2-3) show the percentage of GFP+ cells calculated by flow cytometry on day 6.

[0152] Table 2: Donor 1, Day 6

[0153]

[0154] Table 3: Donor 2, Day 6

[0155]

[0156] The percentage of GFP+ cells on day 23, as measured by flow cytometry, is shown in Table 4-5 below.

[0157] Table 4: Donor 1, Day 23

[0158]

[0159] Table 5: Donor 2, Day 23

[0160]

[0161] Figure 9A treatment diagram for locally advanced neoadjuvant pancreatic cancer is shown. 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 Publications US 20190381156A1 and US 20190318804A1, which are incorporated herein by reference in their entirety. The apheresis product is transfected with an adenovirus encoding at least one immunogenic peptide sequence as shown, wherein the peptide sequence contains a tumor-associated antigen or a novel epitope. The transduced apheresis product is stimulated with an immunostimulant. N-803 is shown in the figure. T cells exposed to the transduced apheresis product are expanded and formulated for administration to patients before or after surgery. Of course, incorporating the therapeutic apheresis product of the present invention into treatment regimens is not limited to this example.

[0162] The foregoing discussion provides many exemplary embodiments of the subject matter of this invention. Although each embodiment represents a single combination of inventive elements, the subject matter of this invention is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, the subject matter of this invention is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0163] It will be apparent to those skilled in the art that further modifications are possible beyond those already described without departing from the inventive concept described herein. Therefore, the subject matter of the invention is not limited except in the spirit of the appended claims. Furthermore, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to an element, component, or step in a non-exclusive manner, indicating that the referenced element, component, or step may be present or utilized, or combined with other elements, components, or steps not explicitly referenced. Where the specification or claims refer to at least one element selected from the group consisting of A, B, C… and N, the text should be interpreted as requiring only one element from that group, rather than A plus N, or B plus N, etc.

[0164] All publications identified herein are incorporated by reference to the same degree as each individual publication or patent application is specifically and individually indicated as incorporated by reference. If a definition or use of a term in an incorporated reference is inconsistent with or contradicts the definition of that term provided herein, the definition provided herein shall apply, and the definition in the references shall not apply.

[0165] In some embodiments, the numerical values ​​used to describe and claim certain embodiments of the subject matter of this invention, representing components, properties (such as concentrations), reaction conditions, etc., should be understood to be modified by the term "about" in some cases. Therefore, in some embodiments, the numerical parameters set forth in the written description and appended claims are approximations that may vary depending on the desired properties sought to be obtained in a particular embodiment. In some embodiments, numerical parameters should be interpreted based on the number of significant figures reported and by applying common rounding techniques. Although the wide range of numerical ranges and parameters set forth in some embodiments of the subject matter of this invention are approximations, the numerical values ​​set forth in particular instances are reported as precisely as possible. The numerical values ​​presented in some embodiments of the subject matter of this invention may contain some error necessarily caused by the standard deviation found in their respective test measurements.

[0166] In some embodiments, the numerical values ​​used to describe and claim certain embodiments of the subject matter of this invention, representing components, properties (such as concentrations), reaction conditions, etc., should be understood to be modified by the term "about" in some cases. Therefore, in some embodiments, the numerical parameters set forth in the written description and appended claims are approximations that may vary depending on the desired properties sought to be obtained in a particular embodiment. In some embodiments, numerical parameters should be interpreted based on the number of significant figures reported and by applying common rounding techniques. Although the wide range of numerical ranges and parameters set forth in some embodiments of the subject matter of this invention are approximations, the numerical values ​​set forth in particular instances are reported as precisely as possible. The numerical values ​​presented in some embodiments of the subject matter of this invention may contain some error necessarily caused by the standard deviation found in their respective test measurements.

[0167] Unless the context indicates otherwise, all scopes described herein should be interpreted as including their endpoints, and open scopes should be interpreted as including only business-useful values. Similarly, all lists of values ​​should be considered to include intermediate values ​​unless the context indicates otherwise.

[0168] As used herein and throughout the claims, unless the context clearly indicates otherwise, the meaning of “a” and “the” includes plural pronouns. Similarly, as used herein, unless the context clearly indicates otherwise, the meaning of “in” includes both “in” and “on”.

[0169] The description of value ranges herein is intended only as a shorthand method of individually referring to each individual value falling within that range. Unless otherwise specified herein, each individual value is incorporated into the specification as if it were described separately herein. All methods described herein may be performed in any suitable order unless otherwise specified herein or otherwise obviously contradictory to the context. The use of any and all instances or exemplary language (e.g., "for example") provided with respect to certain embodiments herein is intended only to better illustrate the subject matter of the invention and does not constitute a limitation on the scope of the additionally claimed subject matter of the invention. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of the subject matter of the invention.

[0170] The grouping of alternative elements or embodiments of the subject matter of this invention disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually, or in any combination with other members of that group or other elements found herein. For convenience and / or patentability reasons, one or more members of a group may be included in or removed from the group. When any such inclusion or removal occurs, this specification is deemed to contain the modified group thereby satisfying the written description of all Markush groups as used in the appended claims.

Claims

1. A method of generating tumor-targeted lymphocytes for use in the treatment of cancer or infectious disease, the method comprising: i. performing therapeutic apheresis on a subject having cancer or infectious disease; ii. purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; iii. purifying CD14+ monocytes from the CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; iv. differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein DC MHC-I or MHC-II presents the peptide sequence or portion thereof, thereby activating the DCs; v. exposing the purified CD3+ T cells to the activated DCs, thereby expanding T cells; vi. purifying the expanded T cells.

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

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

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

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

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

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

8. The method of any one of claims 1-2, wherein the dendritic cells are further exposed to a pool of peptides of neoepitopes.

9. A method of generating tumor-targeted natural killer (NK) cells for use in the treatment of cancer or infectious disease, the method comprising: i. performing therapeutic apheresis on a subject having cancer or infectious disease; ii. purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; iii. purifying CD14+ monocytes from the apheresised CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; iv. expanding NK cells from the apheresised CD3- CD14- fraction; v. differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein DC MHC-I or MHC-II presents the peptide sequence or portion thereof, thereby activating the DCs; vi. exposing the purified CD3+ T cells to the activated DCs, thereby expanding T cells; vii. purifying the expanded T cells; viii. isolating at least one nucleic acid encoding an alpha chain and a beta chain of a T cell receptor (TCR) from the expanded T cells and fusing the nucleic acid to the 5' end of a second nucleic acid encoding a transmembrane domain and an intracellular signaling domain of a chimeric antigen receptor (CAR), wherein the fused nucleic acid encodes a TCR CAR; ix. transfecting the enriched and expanded NK cells with the nucleic acid encoding the TCR CAR.

10. The method of claim 9, wherein the NK cells comprise memory cytokine enriched NK cells (M-CENK).

11. The method of any one of claims 9-10, wherein the M-CENK are genetically modified to express CD 16.

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

13. The method of any one of claims 9-12, wherein the NK cells comprise NK-92 cells, and optionally wherein the NK-92 cells comprise CD 16.

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

15. A method of expanding tumor infiltrating lymphocytes for use in the treatment of a cancer or an infectious disease, the method comprising: i. performing therapeutic apheresis on a subject having a cancer or an infectious disease; ii. purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; iii. purifying CD14+ monocytes from the apheresised CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; iv. expanding NK cells from the apheresised CD3- / CD14- fraction; v. differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein the DCs MHC-I or MHC-II present the peptide sequence or portions thereof, thereby activating the DCs; vi. exposing the purified CD3+ T cells to the activated DCs, thereby expanding T cells; vii. purifying the expanded T cells; viii. purifying CD3+ TILs from a solid tumor and exposing the TILs to the activated DCs, thereby expanding the TILs; ix. purifying the expanded TILs.

16. A pharmaceutical composition comprising tumor targeting lymphocytes for use in the treatment of a cancer or an infectious disease, the composition comprising 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 exposed to the activated DCs, thereby activating and expanding T cells.

17. The pharmaceutical composition of claim 16, wherein the DC MHC-I or MHC-II presents the antigenic peptide sequence or portion thereof, thereby activating the DC.

18. The pharmaceutical composition of any one of claims 16-17, wherein the activated CD3+ T cell comprises a T cell receptor (TCR) specific for the MHC-presented antigenic peptide sequence on the DC.

19. The pharmaceutical composition of any one of claims 16-18, further comprising natural killer (NK) cells.

20. The pharmaceutical composition of claim 19, wherein the NK cells are expanded from the patient’s leukapheresis.

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

22. The pharmaceutical composition of any one of claims 19-21, wherein the NK cells are expanded in a medium comprising IL-15, IL12, and IL-18.

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

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

25. The pharmaceutical composition of claim 24, wherein the NK-92 cells comprise a high affinity Fc receptor.

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

27. The pharmaceutical composition of any one of claims 19-26, wherein the NK cells comprise a CAR.

28. The pharmaceutical composition of claim 27, wherein the CAR comprises a targeting domain, and wherein the targeting domain comprises an alpha chain and a beta chain of a TCR.

29. The pharmaceutical composition of claim 28, wherein the alpha chain and beta chain are from an activated T cell of the patient.

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

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

32. The pharmaceutical composition of claim 30, wherein the Ab binding domain is specific for a checkpoint inhibitor.

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

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

35. The pharmaceutical composition of any one of claims 19-34, 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 of any one of claims 19-35, wherein the dendritic cells are activated in a culture medium comprising GM-CSF.

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

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

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

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

41. The pharmaceutical composition of any one of claims 38-40, wherein the MOI is 20-20,000.

42. A pharmaceutical composition for use in the treatment of a cancer or an infectious disease, 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 apheresis of the patient.

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

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

45. The pharmaceutical composition of any one of claims 42-44, wherein the MOI is 20-20,000.

46. A pharmaceutical composition for use in the treatment of a cancer or an infectious disease, 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 apheresis of the patient.

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

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

49. The pharmaceutical composition of any one of claims 46-48, wherein the MOI is 20-20,000.

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 apheresis of a patient, and the biopsy sample is from a tumor of the same patient, and wherein the pharmaceutical composition is for use in the treatment of a cancer.

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

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

53. The pharmaceutical composition of any one of claims 50-52, further comprising an adenovirus (Ad), wherein the Ad comprises a nucleic acid encoding an antigenic peptide sequence.

54. The pharmaceutical composition of any one of claims 50-53, further comprising natural killer (NK) cells.

55. The pharmaceutical composition of claim 54, wherein the NK cells are expanded from the patient’s PBMCs.

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

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

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

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

60. The pharmaceutical composition of claim 59, wherein the NK-92 cells comprise a high affinity Fc receptor.

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

62. The pharmaceutical composition of any one of claims 54-61, wherein the NK cells comprise a CAR.

63. The pharmaceutical composition of claim 62, wherein the CAR comprises a targeting domain, and wherein the targeting domain comprises an alpha chain and a beta chain of a TCR.

64. The pharmaceutical composition of claim 63, wherein the alpha chain and beta chain are from an activated T cell of the patient.

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

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

67. The pharmaceutical composition of claim 65, wherein the Ab binding domain is specific for a checkpoint inhibitor.

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

69. The pharmaceutical composition of claim 68, wherein the biopsy sample is combined with the DC first, followed by the addition of the T cells, wherein 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 apheresis of a patient, wherein the DCs and / or T cells are exposed to a biopsy sample from a tumor of the same patient, and wherein the pharmaceutical composition is formulated for administration to the patient.

71. The pharmaceutical composition of claim 70, wherein the DCs are further exposed to an adenovirus (Ad), wherein the Ad comprises a nucleic acid encoding an antigenic peptide sequence.

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

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

74. The pharmaceutical composition of any one of claims 70-73, wherein the DCs are further exposed to an adenovirus (Ad), wherein the Ad comprises a nucleic acid encoding an antigenic peptide sequence.

75. The pharmaceutical composition of any one of claims 70-74, wherein the biopsy sample is from a tumor of a patient that has been treated with a DAMP-inducing agent, wherein the DAMP-inducing agent comprises radiation and / or a histone deacetylase (HDAC) inhibitor.

76. The pharmaceutical composition of any one of claims 70-75, wherein the DCs are exposed to the biopsy in the presence of an activation medium comprising granulocyte colony-stimulating factor (GMCSF) and IL-4, wherein the DCs are activated and matured.

77. The pharmaceutical composition of any one of claims 70-76, wherein the DCs and biopsy are exposed to the activation medium for 24-48 hours, and wherein the DCs are then isolated and combined with the T cells.

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

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, wherein the DCs and T cells are then isolated from the apheresis, and wherein the pharmaceutical composition is formulated for administration to the patient.

80. The pharmaceutical composition of claim 79, wherein the isolated DCs are further exposed to an adenovirus (Ad), wherein the Ad comprises a nucleic acid encoding an antigenic peptide sequence.

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

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

83. The pharmaceutical composition of any one of claims 79-82, wherein the isolated DCs are further exposed to an adenovirus (Ad), wherein the Ad comprises a nucleic acid encoding an antigenic peptide sequence.

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

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

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

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

88. A method of generating tumor-targeting lymphocytes for use in the treatment of a cancer or infectious disease, the method comprising: i. performing therapeutic apheresis on a subject having a cancer or infectious disease; ii. purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; iii. purifying CD14+ monocytes from the apheresised CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; iv. expanding NK cells from the apheresised CD3- / CD14- fraction; v. differentiating the 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 DCs MHC-I or MHC-II present at least one re-expressed peptide sequence or portion thereof, thereby activating the DCs; vi. exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cells.

89. A method of generating tumor-targeting natural killer (NK) cells for use in the treatment of a cancer or infectious disease, the method comprising: i. performing therapeutic apheresis on a subject having a cancer or infectious disease; ii. purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; iii. purifying CD14+ monocytes from the apheresised CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; iv. expanding NK cells from the apheresised CD3- / CD14- fraction; v. differentiating the 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 DCs MHC-I or MHC-II present at least one re-expressed peptide sequence or portion thereof, thereby activating the DCs; vi. exposing the purified CD3+ T cells to the activated DCs, thereby expanding the T cells; vii. isolating from the expanded T cells at least one nucleic acid encoding an alpha chain and a beta chain of a T cell receptor (TCR) and fusing the nucleic acid to a 5' end of a second nucleic acid encoding a transmembrane domain and an intracellular signaling domain of a chimeric antigen receptor (CAR), wherein the fused nucleic acid encodes a TCR CAR; viii. transfecting the enriched and expanded NK cells with the nucleic acid encoding the TCR CAR.

90. A method of expanding tumor infiltrating lymphocytes for use in the treatment of a cancer or an infectious disease, the method comprising: i. performing therapeutic apheresis on a subject having a cancer or an infectious disease; ii. purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; iii. purifying CD14+ monocytes from the apheresised CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; iv. expanding NK cells from the apheresised CD3- / CD14- fraction; v. differentiating the 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 DCs MHC-I or MHC-II present at least one re-expressed peptide sequence or portion thereof, thereby activating the DCs; vi. purifying CD3+ TILs from a solid tumor and exposing the CD3+ TILs to the activated DCs, thereby expanding the TILs; vii. purifying the expanded TILs.

91. A pharmaceutical composition comprising a tumor-targeted CD3 + A pharmaceutical composition of T lymphocytes, the composition comprising 1) dendritic cells (DCs), wherein the DCs are differentiated from patient-derived apheresis purified CD14+ mononuclear cells; 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 DCMHC-I or MHC-II present at least one re-expressed peptide sequence or portion thereof, thereby activating the DC; and 3) CD3 purified from the patient apheresis + T cells; wherein the T cells are exposed to the activated DCs, thereby activating and expanding the T cells.

92. A method of expanding tumor infiltrating lymphocytes for use in the treatment of a cancer or an infectious disease, the method comprising: i. performing therapeutic apheresis on a subject having a cancer or an infectious disease, wherein the subject has been treated with at least one therapeutic agent selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor; ii. purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; iii. purifying CD14+ monocytes from the apheresised CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; iv. expanding NK cells from the apheresised CD3- / CD14- fraction; v. differentiating the 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 DCs MHC-I or MHC-II present at least one re-expressed peptide sequence or portion thereof, thereby activating the DCs; vi. purifying CD3+ TILs from a solid tumor and exposing the CD3+ TILs to the activated DCs, thereby expanding the TILs; vii. purifying the expanded TILs. wherein the T cells are exposed to the activated DCs, thereby activating and expanding the T cells. iii. purifying CD14+ monocytes from the apheresed CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; iv. differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein DC MHC-I or MHC-II presents the peptide sequence or portion thereof, thereby activating the DCs; v. purifying CD3+ TILs from a solid tumor and exposing the TILs to the activated DCs, thereby expanding the TILs; vi. purifying the expanded TILs.

93. A method of generating tumor-targeting lymphocytes for use in the treatment of a cancer or an infectious disease, the method comprising: i. performing therapeutic apheresis on a subject having a cancer or an infectious disease, wherein the subject has been treated with at least one therapeutic agent selected from the group consisting of a protein arginine methyltransferase 5 (PRMT5) inhibitor, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase (HDAC) inhibitor; ii. purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; iii. purifying CD14+ monocytes from the apheresed CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; iv. differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein DC MHC-I or MHC-II presents the peptide sequence or portion thereof, thereby activating the DCs; v. exposing the purified CD3+ T cells to the activated DCs, thereby expanding T cells; vi. purifying the expanded T cells.

94. A method of generating tumor-targeting lymphocytes for use in the treatment of a cancer or an infectious disease, the method comprising: i. performing therapeutic apheresis on a subject having a cancer or an infectious disease; ii. purifying a CD3+ T cell fraction from the apheresis product, wherein the remaining apheresis product comprises a CD3- fraction; iii. purifying CD14+ monocytes from the CD3- fraction, wherein the remaining apheresis product comprises a CD3- / CD14- fraction; iv. differentiating the CD14+ monocytes into dendritic cells (DCs) and exposing the DCs to an antigenic peptide or an adenovirus encoding an antigenic peptide sequence, wherein DC MHC-I or MHC-II presents the peptide sequence or portion thereof, thereby activating the DCs; v. exposing the purified CD3+ T cells to the activated DCs, thereby expanding T cells; vi. purifying the expanded T cells.

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