Enhanced viral transduction of mammalian cells using material scaffolds

Abstract

To provide compositions and methods for a low cost, tunable, macroporous, alginate scaffold that transduces T cells with vectors under static conditions.SOLUTION: Provided is a method of transducing a cell comprising a) obtaining a cell and a vector encoding an exogenous gene, siRNA, tasiRNA, IncRNA, shRNA, mRNA, gRNA, miRNA, and / or DNA encoding the siRNA, tasiRNA, IncRNA, shRNA, mRNA, gRNA, miRNA, and b) applying the cell and the vector to a dry scaffold.SELECTED DRAWING: Figure 28

Description

[Technical field]

[0001] This invention was made in part with funding from the National Institutes of Health, Grant No. UL1TR002489. This invention was made with Government support under the National Institute of Standards and Technology Act (NISAT). The Government has certain rights in this invention.

[0002] This application is a continuation of U.S. Provisional Patent Application No. 63 / 008,069, filed April 10, 2020. No. 6,399,433, filed on Oct. 23, 2003, which is incorporated by reference in its entirety. [Background technology]

[0003] Adoptive T cell therapy harnesses and redirects a patient's own immune system, but It has emerged as a promising personalized therapeutic modality for treating cancer and various other diseases. The most successful example targets CD19, which is expressed by B cell-derived malignancies. Adoptive transfer of chimeric antigen receptor-redirected T cells (CAR-T cells), which Approved by the FDA in 2017. The compelling success of CD19-specific CAR-T cell therapy As a result, there are currently efforts to expand the use of CAR-T cells to other types of cancer. One of the key steps involved in current CAR-T cell therapy is ex vivo This is a genetic modification of T cells in the tumor, which redirects the specificity of T cells against tumor antigens. The drug confers on T cells the ability to stably express a CAR molecule that targets the target gene.

[0004] Many different approaches to genetically engineer T cells ex vivo are being explored. Among them, lentivirus or retrovirus vector (RV)-based gene transfer is This represents the most successful approach. When using RV to generate CAR-T cells, Because resting T cells have difficulty undergoing RV-mediated recruitment, T cells isolated from patients are the first First, it was stimulated with agonist anti-CD3 and anti-CD28 antibodies, and then with RV. RV particles are incubated for a short distance, so Brownian motion (60 times within one half-life) 0 μm) and activated T cells and RVs can move through the solution. The contact must be effected by centrifugation or spinoculation in the presence of a transfer enhancer. Transduction enhancers such as polybrene and protamine sulfate have been shown to be somewhat successful. The recombinant fibronectin fragment CH296 (Retronectin) is the most commonly used fibronectin for RV. It represents a transduction-enhancing reagent that is widely used clinically. , which binds to RV via its heparin-binding domain and via the CS-1 / RGD domain Retronectin binds to T cells, bringing the cells together with the RV and facilitating gene transfer. For efficient colocalization of V and T cells, polystyrene plates, flasks, or Solid surfaces such as bags must be coated in advance. and subsequent CAR expression remains suboptimal under passive / static conditions. To achieve expression, RV cells were seeded onto Retronectin-coated surfaces. The RetroNectin complement complexes are then centrifuged to concentrate them on the same surface. Assisted spinoculation limits scale-up and entails significant costs. Furthermore, RetroNectin is a recombinant protein and is inherently bioreactive, allowing Therefore, Retronectin or Spinokine may affect the differentiation and proliferation of This allows for one-step ex vivo treatment of T cells under static conditions without any modulation. The development of an efficient culture system will allow RV or other viral vectors to serve as gene delivery systems. This would significantly reduce the manufacturing costs of CAR-T cells when used as a therapeutic agent. Summary of the Invention

[0005] Modified hydrogels or dry scaffolds, and transduction of cells to receive e.g. chimeric antigens. The production of the cells and their use in the treatment of cancer are disclosed.

[0006] In one embodiment, one or more ligands specific for a T cell receptor and / or a NK cell receptor are The antibodies (anti-CD3 antibodies, CD1d, Fc fragments of immunoglobulins, or anti-Fcγ receptors (F Biocompatible hydrogels, including, but not limited to, antibodies against .gamma.RIII. trics and / or dry scaffolds (e.g., dry scaffolds or hydrogels comprising alginate) The disclosed hydrogel matrix and / or dry scaffolds are Cells (e.g., T cells, natural killer (NK) cells, NKT cells, macrophages, tumor Tumor-infiltrating lymphocytes (TIL), tumor-infiltrating NK cells (TINK), and / or bone marrow-infiltrating lymphocytes Immune cells, including but not limited to myeloid leukocytes (MIL), as well as, for example, mesenchymal stem cells (MSCs), SC), hematopoietic stem cells (HSC), dendritic cells, neural stem cells, induced pluripotent stem cells, or pancreatic islet cells. to transduce target cells (e.g., non-immune cells such as thymocytes) to generate chimeric antigen receptor (CAR) cells. In addition, the scaffold can be used for fundamental research purposes. It is possible to transduce cancer cells or any other type of cells with In such a case, the hydrogel matrix and / or dry scaffold can be transfected with exogenous genes, siRNA, t asiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or is the siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, m DNA encoding an iRNA (including but not limited to engineered genes, siRNAs, tas iRNA, lncRNA, shRNA, mRNA, gRNA, and / or miRNA, e.g. A vector (e.g., a chimeric antigen receptor (CAR) gene) encoding and / or encapsulating the , lentivirus, retrovirus, adenovirus, adenovirus-associated virus, virus-like particles, cell mimetic particles, transposons, exosomes, nanoparticles, micelles or liposomes The polymer may include a polymerosome.

[0007] As used herein, one or more antibodies, cytokines (e.g., IL-2, TNF-α, or IFN-γ), and / or T cells, natural killer (NK) cells, NKT cells, macrophages, and Phage, tumor infiltrating lymphocytes (TIL), tumor infiltrating natural killer cells (TINK), or bone marrow infiltration Any of the preceding, further comprising a cytokine costimulatory molecule that activates lymphocytes (MIL). Hydrogel matrices and / or dry scaffolds of embodiments are also disclosed. The scaffold and / or the dried scaffold may also be administered with anti-CD28, B7-1, B7-2, anti-inducible co-stimulatory agents ( ICOS), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, Anti-CD40L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-T IM-2, anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript-1 (LLT -1), anti-CD137, CD48, MICA, anti-2B4, and anti-glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), Ligands that induce signal transduction via costimulatory receptors in cells, NK cells, or NKT cells This also includes antibodies or peptides.

[0008] In one aspect, the present disclosure relates to a hydrochloride composition according to any of the preceding aspects, further comprising a chemotherapeutic agent. A gel matrix and / or a dry scaffold are disclosed.

[0009] Also referred to herein is the term "targeted therapy for a disease or condition (e.g., cancer, metastasis, or graft versus host disease) in a subject. Main disease (GVHD), myocardial infarction, ischemia, bone / cartilage damage, osteoarthritis, epilepsy, hemophilia, Severe osteogenesis imperfecta, Wiskott-Aldrich syndrome, X-linked severe combined immunodeficiency All diseases, β-thalassemia, adrenoleukodystrophy (ALD), metachromatic leukodystrophy ( Treating, inhibiting, reducing, diminishing, ameliorating, and / or preventing the development of any of the following conditions: A method of preventing, comprising administering to a subject a hydrogel and / or dry Disclosed herein is a method comprising administering a scaffold to a subject. Treating, reducing, inhibiting, mitigating, diminishing, and / or preventing cancer and / or metastasis in a subject The method includes the steps of: a) administering cells (e.g., autologous, allogeneic, and / or haploidentical T cells, natural killer (NK) cells, NKT cells, macrophages, etc. obtained from donor sources tumor infiltrating lymphocytes (TIL), tumor infiltrating NK cells (TINK) and / or bone marrow infiltration lymphocytes (MIL), including but not limited to, T cells, NK cells, NKT cells, immune cells such as macrophages, TILs, TINKs, and / or MILs), and exogenous genes A vector (e.g., a lentivirus, a recombinant vector) encoding a gene (e.g., a chimeric antigen receptor) Torovirus, adenovirus, adeno-associated virus, virus-like particles, cytomimetic particles, transposons, exosomes, nanoparticles, micelles, or liposomes); and b. ) The cells and vectors are placed in a scaffold (e.g., a biocompatible material, including but not limited to alginate). (e.g., a biocompatible hydrogel matrix and / or dry scaffold); and (c) applying the Cells were incubated with hydrogel and / or dry scaffolds at 1, 2, 3, 4, 5, 10, 15, 2 0, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 4 8, 54, 60, 66, 72, 84, 96 hours, 5, 6, 7, 8, 9, 10, 11, 12 d) incubating the hydrogel and / or dry scaffold for 1, 2, 3, or 4 days; and administering to the subject the cells with or without incubation in step c). Alternatively, cells incubated with the hydrogel and / or dry scaffolds may be administered to subjects. and administering to the subject a scaffold comprising a T cell receptor, or NK cell receptor ligands or antibodies (anti-CD3 antibody, CD1d, immunoglobulin F c fragment, or anti-Fcγ receptor (FcγRIII) antibody) Thus, in one aspect, the present disclosure provides a method for the treatment of a subject matter of any of the preceding aspects. 2. A method for treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing cancer or metastasis in the foot, The vectors within the field (e.g., hydrogel matrix and / or dry scaffold) transduce the cells. The transduced cells within the scaffold are then administered to the subject at the site of the cancer for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 25, 26, 27, 28, 29, 3 0, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46, 47, 48, 54, 60, 66, 72, 84, 96 hours, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1, 22, 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32, 45 , 58, 59, 60, 61, 62, 90 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 3 Methods are disclosed that can be released over 6 months, 4, 5, 6, 7, 8, 9, or 10 years. In one embodiment, the cells are cultured for about 1 week to about 1 hour after administration of the hydrogel and / or dry scaffold. Release from the hydrogel occurs over a two week period.

[0010] In one aspect, the present specification provides a method for treating cancer or metastasis in a subject of any of the preceding aspects. A method for treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer, comprising administering to a patient a scaffold comprising an anti-cancer agent (e.g., Disclosed are methods further comprising administering to the subject the therapeutic agent of the present invention an immunoblocking inhibitor or a chemotherapeutic agent.

[0011] Also, as used herein, cells (e.g., immune cells (e.g., autologous, allogeneic, and and / or T cells, natural killer (NK) cells from haploidentical donor sources , NKT cells, macrophages, tumor-infiltrating lymphocytes (TIL), tumor-infiltrating NK cells (TI NK) and / or bone marrow infiltrating lymphocytes (MIL), K cells, NKT cells, TIL, macrophages, TINK or MIL, etc.), non-immune cells (e.g., mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), dendritic cells, neural stem cells, induced induced pluripotent stem cells or pancreatic islet cells (immune and non-immune cells include primary cells and cell lines) A method for transducing and producing chimeric antigen receptor immune cells is disclosed, The method includes: a) administering to a subject a cell (e.g., an immune cell (e.g., an autologous, allogeneic, and / or haplotype I cell) T cells, natural killer (NK) cells, NKT cells, and Macrophages, tumor infiltrating lymphocytes (TIL), tumor infiltrating NK cells (TINK) and / or These include, but are not limited to, bone marrow infiltrating lymphocytes (MIL), T cells, NK cells, NKT cells, TILs, macrophages, TINKs or MILs, etc.), non-immune cells (e.g., mesenchymal Myeloid stem cells (MSC), hematopoietic stem cells (HSC), dendritic cells, neural stem cells, induced pluripotent stem cells or pancreatic islet cells) (immune and non-immune cells include primary cells and cell lines), and exogenous Gene, siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA , miRNA, and / or the siRNA, tasiRNA, lncRNA, shRNA, DNA encoding mRNA, gRNA, miRNA (including but not limited to engineered genes) child, siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, and and / or miRNA, e.g., chimeric antigen receptor (CAR) genes) (b) obtaining a vector that is encapsulated in the cell; and (c) providing the cell and vector in a scaffold (e.g., but not limited to, a scaffold). However, biocompatible hydrogel matrices, such as hydrogels or scaffolds containing alginate, and / or dry scaffolds) and incubate the scaffolds, cells and vectors. In some embodiments, the scaffold comprises a T cell receptor or a NK cell receptor (antibody). CD3 antibody, CD1d, Fc fragment of immunoglobulin, or anti-Fcγ receptor (FcγRII I) Ligands or antibodies, including but not limited to antibodies. Scaffolds, cells , and vector incubation was performed at 1, 2, 3, 4, 5, 10, 15, 20, 25, 3 0, 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 54, 6 0, 66, 72, 84, 96 hours, 5, 6, 7, 8, 9, 10, 11, 12, 13, or This can be done for 14 days, or alternatively, the scaffold and cells can be implanted without incubation. Post a comment.

[0012] In one aspect, the present disclosure relates to a cell (e.g., an immune cell (e.g., For example, T cells obtained from autologous, allogeneic, and / or haploidentical donor sources; Natural killer (NK) cells, NKT cells, macrophages, tumor-infiltrating lymphocytes (TI L), tumor infiltrating NK cells (TINK) and / or bone marrow infiltrating lymphocytes (MIL). Not limited to these, T cells, NK cells, NKT cells, TIL, macrophages, TINK or MIL), non-immune cells (e.g., mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), ), dendritic cells, neural stem cells, induced pluripotent stem cells or pancreatic islet cells), primary cells, or cell lines) and a method for producing the chimeric antigen receptor immune cell of any of the preceding aspects. The method of the present invention comprises the steps of: (a) administering to the patient a therapeutically effective amount of anti-CD28, B7-1, B7- 2. Anti-inducible costimulatory agent (ICOS), ICOS ligand, anti-CD27, CD70, 4-1 BBL, anti-41-BB, anti-CD40L, CD40, anti-DAP10, anti-CD30, CD30 L, anti-TIM-1, anti-TIM-2, anti-TIM-3, anti-CD44, anti-NK1.1, lectin -like transcript-1 (LLT-1), anti-CD137, CD48, MICA, anti-2B4, and anti It contains the glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), Signaling through a costimulatory receptor on T cells, NK cells, or NKT cells, including but not limited to these. The method further comprises administering to the subject a ligand or antibody that induces null signaling.

[0013] Also provided herein is a method for producing a cell of any of the preceding embodiments (e.g., For example, immune cells (e.g., from autologous, allogeneic, and / or haploidentical donor sources) T cells, natural killer (NK) cells, NKT cells, macrophages, tumor Tumor-infiltrating lymphocytes (TIL), tumor-infiltrating natural killer cells (TINK) and / or bone marrow infiltrating lymphocytes (MIL), including but not limited to T cells, NK cells, NKT cells, TIL, phages, TINK or MIL, etc.), non-immune cells (e.g., mesenchymal stem cells (MSCs) , hematopoietic stem cells (HSCs), dendritic cells, neural stem cells, induced pluripotent stem cells, or pancreatic islet cells) (immune Methods for transducing immune and non-immune cells (including primary cells and cell lines) and methods for transducing immune and non-immune cells (including primary cells and cell lines) In any one of the embodiments, the method for producing a chimeric antigen receptor immune cell comprises the steps of: The above cytokines (e.g., IL-2, TNF-α, and / or IFN-γ, etc.) A method is disclosed, including: [Brief description of the drawings]

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, Together with the description, the disclosed compositions and methods are illustrated. [Figure 1] Retroviral transduction of human activated T cells mediated by dry macroporous scaffolds. FIG. 1A shows a schematic diagram illustrating the preparation of macroporous alginate scaffolds. FIG. 1B and FIG. 1C show SEM images of dry macroporous scaffolds. FIG. 1D shows GFP expression in retrovirally transduced activated T cells seeded on dry macroporous scaffolds (red) and on retronectin-coated plates by spinoculation (blue). Non-transduced control cells are shown in black. FIG. 1E shows SEM images of dry nanoporous scaffolds. FIG. 1F shows GFP expression in retrovirally transduced T cells seeded on dry macroporous scaffolds (red), hydrated macroporous scaffolds (purple), and dry nanoporous scaffolds (green). Control non-transduced cells are in black. FIG. 1G shows quantification of GFP+ cells (***p<0.0001 vs. dry macroporous scaffolds, Student's t-test). [Diagram 2]We show that scaffold-generated CD19.CAR-T cells display similar functional activity in vitro as retronectin / spinoculation-derived CAR-T cells. Figure 2A shows CD19.CAR expression in transduced T cells on retronectin-coated plates or macroporous scaffolds compared to non-transduced control T cells (NT cells). Figure 2B shows ex vivo proliferation of transduced T cells on retronectin-coated plates or macroporous scaffolds or NT cells. Figures 2C and 2D show the immunophenotypic composition of CAR-T cells obtained via transduction via scaffolds or retronectin / spinoculation and NT cells at day 12 of culture. Gating analysis was performed on CAR-expressing T cells, excluding NT cells. Figure 2E shows the remaining percentage of CD19+ Daudi cells (tumor cells) when co-cultured with scaffold-generated, retronectin / spinoculation-generated, CAR-T cells, or control NT cells. Tumor cells and T cells were plated at an effector to target ratio of 1:5. T cells and tumor cells were quantified by flow cytometry on day 5 of coculture. P<0.0001 for scaffold CAR-T cells or RetroNectin / Spinoculation CAR-T cells compared to control NT cells by unpaired Student's t-test. Figures 2F and 2G show IFN-γ (2F) and IL-2 (2G) release into coculture supernatants by scaffold-generated, RetroNectin / Spinoculation-generated CAR-T cells, and control NT cells after 24 h of coculture with tumor cells, assessed by ELISA. P<0.0001 for scaffold CAR-T cells or RetroNectin / Spinoculation CAR-T cells compared to control NT cells by two-way ANOVA with Tukey correction. Data are presented as mean ± SD of triplicate experiments. [Figure 3A]FACS analysis of T cells transduced with a retroviral vector encoding GFP at different MOIs (1, 2, 4) on day 3 post-transduction. T cells were transduced with a retrovirus encoding GFP at various MOIs on retronectin-coated plates or scaffolds and analyzed for GFP expression by flow cytometry. [Figure 3B] FACS analysis of T cells on days 5 and 9 post-transduction is shown. T cells from three different donors were transduced with a retrovirus encoding CD19.CAR on RetroNectin coated plates or scaffolds and analyzed for CAR expression by flow cytometry. [Figure 4] Shown are CD4 / CD8, CD45RA / CCR7, CD62L / CCR7 expression in CAR-T cells from three different donors generated by scaffold- or retronectin-mediated transduction. Gating analysis was performed on CAR-expressing cells, excluding NT cells. [Diagram 5] Shown is PD-1 / LAG3 expression (exhaustion markers) in CAR-T cells from three different donors generated by scaffold or retronectin mediated transduction. Gating analysis was performed on CAR expressing cells, excluding NT cells. [Figure 6] Figure 1 shows that CAR-T cells generated by scaffold-mediated transduction exhibit toxicity against CD19+ tumor cells.CD19+ tumor cells were co-cultured with control non-transduced cells (NT cells) or with CD19.CAR-T cells generated by scaffold-mediated transduction or retronectin-assisted spinoculation at an E:T ratio of 1:5 (left panel) and 1:2 (right panel) and analyzed by FACS on day 5 of co-culture. [Figure 7]We show that CAR-T cells generated by scaffold-mediated transduction retain antigen specificity and are not functional against CD19- U937 tumor cells.CD19- tumor cells were co-cultured with control non-transduced cells (NT cells) or with CD19.CAR-T cells generated by scaffold-mediated transduction or retronectin-assisted spinoculation at an E:T ratio of 1:5 and analyzed by FACS on day 5 of co-culture. [Figure 8] Figure 8A shows tumor eradication by scaffold-generated CD19.CAR-T cells in a lymphoma xenograft model in mice. Figure 8A shows an experimental timeline of a lymphoma xenograft model in NSG mice using FfLuc-labeled CD19+ human Daudi tumor cells. Figure 8B shows representative tumor bioluminescence images (BLI) of NSG mice inoculated with Daudi cells and treated with control NT cells or CD.19.CAR-T cells generated by scaffold-mediated transduction or retronectin / spinoculation-assisted transduction. Figure 8C shows the kinetics of tumor growth measured by quantification of BLI. **p<0.01, one-way ANOVA, when comparing scaffold or CAR-T cells to control NT cells. Figures 8D and 8E show the weight change (8D) and survival rate (8E) of tumor-bearing mice treated with control NT cells or with CD19.CAR-T cells generated by scaffold-mediated or retronectin / spinoculation-assisted transduction. *p<0.05, log-rank test. [Figure 9] IVIS images of NSG mice inoculated with luciferin-expressing Daudi cells on day 0 and then treated with non-transduced cells (NT cells) or CD.19 CAR-T cells generated by scaffold-mediated transduction or retronectin-assisted spinoculation on day 4 after tumor inoculation are shown. [Figure 10]Figure 10A shows a schematic diagram of DBCO-PEG4-NHS conjugated anti-CD3 or anti-CD28 antibodies and the absorbance of the conjugated antibodies compared to unmodified antibodies. Figures 10B and 10C show the UV absorption spectrum of DBCO-conjugated antibodies, showing a clear absorption peak of DBCO at 309 nm. Figure 10D shows the synthesis scheme of azide-modified alginates. Figure 10E shows the quantification of the number of azides per alginate chain. [Figure 11] We show that MASTER (also referred to herein as CCI-Alg) promotes activation and retroviral-mediated transduction of primary human T cells. Figure 11A shows a schematic of the synthesis of MASTER. Figure 11B shows an SEM image of MASTER showing a homogenous macroporous structure throughout the scaffold. Figure 11C shows a 3D confocal fluorescence micrograph of GFP+ T cells in AF647-labeled MASTER showing cell distribution along the pores. Figure 11D shows quantification of T cells expressing CD69, an early T cell activation marker. MASTER with increasing amounts of anti-CD3 and anti-CD28 antibodies were seeded with PBMCs and analyzed for CD69+ cells by flow cytometry after 24 hours. Figure 11E shows an overview of the MASTER-mediated T cell transduction process. PBMCs and gamma retroviral particles are seeded on MASTER and incubated at 37°C. After 72 hours, cells are harvested from the scaffold and analyzed by flow cytometry. Figure 11F shows representative flow cytometry plots showing GFP expression in T cells transduced on MASTER, on blank scaffolds without anti-CD3 and anti-CD28 antibodies, or by conventional spinoculation. Non-transduced cells were used as controls. Figure 11G shows FACS quantification of GFP+ cells (***p<0.0001, one-way ANOVA with Tukey correction). [Figure 12] 12A and 12B show 3D confocal micrographs depicting the distribution of GFP+ T cells in AF647-labeled MASTERs at 10x (12A) and 40x (12B) magnification. [Figure 13]MASTER-mediated gene transfer generates highly functional CAR-T cells. Figure 13A shows flow cytometry plots showing CD19.CAR expression in T cells transduced on MASTER. Figures 13B and 13C show the immunophenotypic composition of CAR-T cells obtained by MASTER-mediated transduction of PBMC or by conventional spinoculation of activated T cells on day 12 of culture. Non-transduced (NT) cells were used as controls. Gating analysis was performed on CAR-expressing T cells, excluding NT cells. Figure 13D shows FACS quantification of CAR+ cells expressing PD-1 and / or LAG-3. Figure 13E shows ex vivo expansion of non-transduced T cells (NT cells) or T cells transduced on MASTER or by spinoculation. Figure 13F shows the percentage of viable CD19+ Daudi cells when NT cells are co-cultured with MASTER or CAR T cells obtained by spinoculation. Tumor cells and CAR T cells were plated at an effector to target ratio of 1:5. T cells and tumor cells were quantified by flow cytometry on day 5 of coculture. Figures 13G and 13H show IFN-γ and IL-2 released into coculture supernatants by MASTER-produced, spinoculated, CAR-T cells, and control NT cells after 24 hours as assessed by ELISA. ***P<0.0001, one-way ANOVA with Tukey correction. Data are presented as mean ± SD of triplicate experiments, each from a different PBMC donor. [Figure 14] FACS analysis of T cells 3 and 10 days after transduction is shown. T cells from three different donors were transduced on MASTER with a gamma retrovirus encoding CD19.CAR and analyzed for CAR expression by flow cytometry. [Figure 15]Figure 1 shows expression of CD4 / CD8, CD45RA / CCR7, CD62L / CCR7, and PD-1 / LAG3 in CAR-T cells from three different donors generated by MASTER or spinoculation. Analysis was performed on CAR-expressing cells, excluding NT cells. [Figure 16] 16A shows that CAR-T cells generated by MASTER-mediated transduction exhibit toxicity against CD19+ tumor cells (16A) but not against CD19- U937 cells (16B).CD19+ tumor cells or CD19- U937 cells were co-cultured with control non-transduced cells (NT cells) or with CD19.CAR-T cells generated by MASTER-mediated transduction or spinoculation at an E:T ratio of 1:5 and analyzed by FACS on day 5 of co-culture. [Figure 17] This shows that MASTER functions as an efficient T cell release system. Figure 17A shows a schematic of the in vitro release study. Figure 17B shows the percentage of cells released from the scaffold. [Figure 18]Figure 18 shows that subcutaneously implanted MASTER generates and releases fully functional CAR-T cells in a lymphoma xenograft model. Figure 18A shows the experimental timeline of a lymphoma xenograft model in NSG mice engrafted with FFLuc-labeled CD19+ human Daudi tumor cells. Figure 18B shows in vivo tumor bioluminescence imaging (BLI) of NSG mice treated with MASTER, conventional CAR-T cells, or control non-transduced (NT) cells. Figure 18C shows the kinetics of tumor growth measured by quantification of BLI. Figure 18D shows the % change in body weight (BW) of treated mice. Figure 18E shows mouse survival shown as Kaplan-Meier curves. Six mice per treatment group are shown. **p<0.01, log-rank (Mantel-Cox) test, Gehan-Breslow-Wilcoxon test. Figure 18F shows flow cytometry quantification of CD3+CAR+ cells in peripheral blood of mice described in (A). **p<0.01, two-way ANOVA with Sidak's multiple comparison test. Figures 18G and 18H show quantification of CD3+CAR+ cells in bone marrow and spleen of mice euthanized on day 32 in the model described in (A), as determined by flow cytometry **p<0.01, one-way ANOVA with Tukey correction. Figure 18I shows analysis of exhaustion markers PD-1 and LAG3 expression in CAR+ cells isolated from bone marrow of mice treated as described in (A). **p<0.001, two-way ANOVA with Tukey correction. [Figure 19] Individual tumor growth curves for mice injected with NT cells (19A), MASTER (19B), or CAR T cells (19C) are shown. Each line represents one individual animal. Data are shown for 9 mice per treatment condition. [Figure 20] The gating strategy for detection of CAR+ cells in blood, BM or spleen is shown. [Figure 21]Figure 21A shows representative flow cytometry plots showing human CD45+CD3+CAR+ cells isolated from blood, bone marrow, and spleen of mice (n=3) 32 days after tumor cell inoculation. Figure 21B shows the percentage of human CD3+CAR+ relative to total cells in blood, bone marrow, and spleen of mice euthanized 32 days after tumor cell inoculation, as determined by flow cytometry. [Figure 22] Schematic diagram showing conventional CAR-T cell therapy (left, approximately 4 week process) compared to rapid MASTER-mediated CAR T cell generation and therapy (right, 1 day process). [Diagram 23] FIG. 23A shows a scanned volume of the MASTER (left) with a colored surface showing the cross section shown on the right. In the cross section, light values ​​indicate high density (scaffold) and dark values ​​indicate low density (air voids). FIG. 23B shows the CT volume of the MASTER (left) and also isolated and color-labeled pores (right). Color-labeling of the pores is done so that pores with similar volumes have similar colors. When the pores were isolated and analyzed individually, the calculated porosity was 75.8%. FIG. 23C shows the relative frequency of pores of different dimensions. FIG. 23D shows the aspect ratio of the pores, indicating that most pores are elliptical. An aspect ratio of 1 corresponds to a sphere, and an aspect ratio close to 0 corresponds to a plane or rod. FIG. 23E shows the surface area as a function of the plotted volume. The total surface area inside the MASTER is about 810 mm. FIG. 23F shows the connectivity between the pores within the scaffold. The centers or nodes of the pores are shown as spheres, with lines drawn from the spheres to other pores to connect them. The greater the connectivity, the larger the sphere, and the spheres are color coded to indicate the number of pores they are connected to. Connectivity when the sample is partially cut (left) and the sample is fully connected (right). Figure 23G shows the connectivity of the sample, showing that most pores are connected to 0-3 other pores, with very few (~6%) connected to more than 9 pores. [Figure 24]Figure 24 shows that MASTER shows enhanced activity in controlling tumors after tumor rechallenge compared to intravenous infusion of CAR T cells. Figure 24A shows the experimental timeline of lymphoma xenograft model in NSG mice engrafted and rechallenged with FFLuc-labeled CD19+ human Daudi tumor cells. Figure 24B shows in vivo tumor bioluminescence imaging (BLI) of NSG mice treated with MASTER, conventional CAR-T cells or control non-transduced (NT) cells. Figures 24C, 24D, and 24E show individual tumor growth curves of mice treated with NT cells (24C), MASTER (24D), or iv injected with CAR T cells (24E). Each line represents one individual animal. Figure 24F shows mouse survival shown as Kaplan-Meier curves. Six mice per treatment group are shown. **p<0.01, log-rank (Mantel-Cox) test, Gehan-Breslow-Wilcoxon test. [Diagram 25] 1 shows the biocompatibility of MASTER and its components. Representative images of H&E stained sections of five major organs and implantation sites 4 weeks after subcutaneous implantation of MASTER, MASTER+mouse PBMC+GFP-encoding gammaretrovirus in C57Bl6 / J immune-competent mice, as well as untreated controls. [Figure 26] Biocompatibility of MASTER and its components is shown, as revealed by biochemical analysis of mouse blood. Gamma retrovirus encoding MASTER, MASTER+mouse PBMC+GFP was implanted into the subcutaneous space of C57Bl6 / J immune-competent mice (n=3). [Figure 27] PBMC and retrovirus-loaded MASTER do not transduce host cells. Figure 27A shows an in vitro transwell model that mimics an in vivo system. Figure 27B shows GFP expression in fibroblasts seeded on the bottom of a transwell plate, indicating that the virus does not leak out of the scaffold when MASTER is co-seeded with PBMC and virus (MOI 2). [Figure 28]Dry scaffolds were seeded with 1 million HEK293T cells and lentivirus encoding GFP (MOI 2). After 72 hours, cells were isolated from the scaffolds and GFP expression was measured. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, They are not intended to be used in any particular synthetic method or in any particular recombinant biotechnology method unless otherwise specified. The methods are not limited to specific reagents (which may, of course, vary) unless otherwise stated. It should be understood that the terminology used herein is not intended to describe specific embodiments. It should also be understood that these are for illustrative purposes only and are not intended to be limiting.

[0016] A.Definition In this specification and the appended claims, certain terms are defined to have the following meanings: Please refer to the terminology.

[0017] As used in this specification and the appended claims, the singular forms "a," "an," and " and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like. .

[0018] As used herein, ranges may be expressed as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment may include any Including from a particular value and / or to another particular value. Similarly, values ​​may be expressed as approximations. When expressed, the use of the antecedent "about" indicates that the particular value forms another embodiment. It will be understood that each endpoint of a range shall be expressly construed in relation to the other endpoint, and in relation to the other endpoint. It will be understood that both are important independently. Each value is herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. As will be appreciated by one of ordinary skill in the art, a value is disclosed as being "less than or equal to" a given value. It is also understood that when a value is given, "greater than or equal to that value" and the possible range between those values ​​is also disclosed. For example, if the value "10" is disclosed the "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. Also, throughout this application, data is provided in a number of different formats, and this data It is also understood that the ranges represent the starting and end points, as well as any combination of the data points. For example, specific data point "10" and specific data point 15 are disclosed. In addition to between 10 and 15, the numbers are greater than 10, 10 or more, less than 10, 10 or less, and Equal to 10, greater than 15, greater than or equal to 15, less than 15, less than or equal to 15, and equal to 15 It is understood that each unit between two particular units is considered to be disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13 and 14 are also disclosed.

[0019] "Administration to a subject" includes any route of introducing or delivering an agent to a subject. , oral, topical, intravenous, subcutaneous, transcutaneous, tran sdermal), intramuscular, intraarticular, parenteral, intraarterial, intradermal, intraventricular, intracranial, intraperitoneal, Intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, Intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and cranial This can be done by any suitable route, including intravenous injection or infusion techniques. As used herein, "concurrent administration" means n, administration in combination, simultan administrative or administered simul taneously means that the compounds are administered at the same time in time or essentially interchangeably. In the latter case, the two compounds are administered immediately after the , close enough in time that the results are indistinguishable from those obtained when administered at the same time in time. "Systemic administration" refers to administration to a widespread area of ​​a subject's body (e.g., greater than 50% of the body). ) via a route for introducing or delivering a drug to the subject, such as via an entry into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of a drug to the area of ​​the point of administration or and introducing or delivering the agent to an area immediately adjacent to the target tissue, thereby introducing the agent systemically in a therapeutically significant amount. This refers to the introduction or delivery of a drug to a subject via a route that is not directly related to the body. For example, topical administration The drug is readily detectable in the local vicinity of the point of administration, but is not detectable in distal parts of the subject's body. Administration includes self-administration and administration by others. This includes administration of

[0020] "Biocompatible" generally refers to a compound that is generally non-toxic to cells in culture or to the recipient organism. refers to a material and any metabolites or decomposition products thereof that do not cause significant adverse effects in a subject. vinegar.

[0021] "Comprising" means that the composition, method, etc. includes the recited elements, but does not exclude other elements. When used to define compositions and methods, it means "essentially consisting of "In one embodiment" means to include the recited elements, but not to include any other elements of essential importance to the combination. Therefore, as defined herein, The composition, consisting essentially of the components, is free of trace contaminants from the isolation and purification process, as well as phosphate buffer. "Consists of" would not exclude pharma- ceutically acceptable carriers such as saline, preservatives, etc. The present invention provides a method for administering compositions of the present invention in a manner that is consistent with the principles of the present invention. The terms defined by each of these transitional terms are intended to exclude Various embodiments are within the scope of the present invention.

[0022] A "control" is a substitute object or sample used in an experiment for comparison purposes. " or "negative."

[0023] "Controlled release" or "sustained release" achieves a desired pharmacokinetic profile in vivo "Controlled release" refers to the release of a drug from a given dosage form in a controlled manner in order to achieve a desired outcome. One aspect of the present invention is the ability to manipulate the formulation and / or dosage form to establish desired kinetics of drug release. It is power.

[0024] An "effective amount" of a drug refers to an amount of the drug sufficient to provide a desired effect. The amount of a given drug depends on many factors, such as the age and general condition of the subject, and the specific drug(s). Therefore, it is not possible to specify a quantified "effective amount." However, it is not always possible to determine an appropriate "effective amount" in any given subject case. may be determined by one of ordinary skill in the art using routine experimentation. In this context, unless otherwise specified, an "effective amount" of a drug refers to a therapeutically effective amount and a prophylactically effective amount. It can also refer to the amount of a drug that covers both the effective amount and the amount of the drug required to achieve a therapeutic effect. The "amount" may vary according to factors such as the age, sex, and weight of the subject. The dosage may be adjusted to provide a therapeutic response as desired. For example, the dosage may be divided into several doses per day. may be administered in divided doses or proportionately as indicated by the exigencies of the therapeutic situation. It may be reduced.

[0025] "Reduction" refers to a decrease in gene expression, protein expression, degree of symptoms, disease, composition, It can refer to any change that results in a state or activity of a substance. The genetic output of a gene product that contains the substance is reduced compared to the output of a gene product that does not contain the substance. It is understood that a reduction in the genetic output of a gene occurs when, for example, the reduction is insufficient to prevent symptoms. The decrease may be a change in the symptoms of the disorder such that the symptoms are less than previously observed. The present invention may be a method for treating a patient suffering from atopic dermatitis, and ... Therefore, a decrease can be calculated by 1, 2, 3, 4, 5, 6, etc., as long as the decrease is statistically significant. , 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, It may be a 65, 70, 75, 80, 85, 90, 95, or 100% reduction.

[0026] The disclosed hydrogels and dry scaffolds can be used to deliver exogenous genes and other nucleic acids (e.g., siRNA, A, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, etc. As used herein, "exogenous" refers to a vector encoding a specific polypeptide. The term refers to engineered genes, such as siRNA, tasiRNA, lncRNA, sh RNA, mRNA, gRNA, miRNA, and / or the siRNA, tasiRNA, Includes DNA encoding lncRNA, shRNA, mRNA, gRNA, and miRNA "Nucleic acid" refers to nucleic acid from an external source, including but not limited to.

[0027] "Inhibit," "inhibiting," and "inhibition" refer to any action that inhibits or reduces an activity, response, condition, disease, or other This means to decrease a biological parameter of a substance, including an activity, response, state, or This may include, but is not limited to, complete elimination of a disease. This may include, for example, natural or A 10% reduction in activity, response, condition, or disease compared to a control level may also be included. Thus, the reduction can be as low as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 100, 150, 200, 300, 400, 500, 600, 1000, 15 ...500, 1500, It may be a reduction of 70, 80, 90, 100%, or any amount in between.

[0028] The terms "prevent", "preventing", "prevention" and grammatical variations thereof are used herein. As used herein, refers to the development or progression of a disease and / or one or more of its associated symptoms. or partially or completely delay or eliminate recurrence or prevent the subject from acquiring or regaining the disease. or prevents the subject from obtaining a medical treatment that is associated with a disease or one or more of its associated symptoms. This refers to a method of reducing the risk of acquiring or losing

[0029] A "pharmaceutical acceptable" component is a component that is not biologically or otherwise undesirable. i.e., without causing or containing significant undesirable biological effects. The pharmaceutical formulations of the present invention are provided without interacting in a deleterious manner with any of the other components of the formulation. It can refer to a component that can be incorporated and administered to a subject as described herein. When used in relation to human administration, the term generally applies to products that have undergone toxicity testing and manufacturing It meets the necessary standards for manufacturing testing or is certified by the U.S. Food and Drug Administration. Inactive Ingredients Guide This means that

[0030] A "Pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") is generally considered to be safe and means a carrier or excipient useful in the preparation of a pharmaceutical or therapeutic composition for veterinary and and / or a carrier that is acceptable for human pharmaceutical or therapeutic use. The term "acceptable carrier" includes phosphate buffered saline, water, emulsions (oil / water or These may include, but are not limited to, water / oil emulsions, and / or various types of wetting agents. As used herein, the term "carrier" refers to any excipient, diluent, , bulking agents, salts, buffers, stabilizers, solubilizers, lipids, stabilizers, or for use in pharmaceutical formulations These include materials well known in the art and those further described herein, but are not limited to: Not limited to these.

[0031] "Pharmacological activity" (or simply "activity") refers to a "pharmacologically active" derivative or analogue. Derivatives or analogues (e.g., analogs) that have the same type and degree of pharmacological activity as the parent compound. For example, salts, esters, amides, complexes, metabolites, isomers, fragments, etc.

[0032] "Therapeutic agent" refers to any composition that has a beneficial biological effect. Examples include therapeutic effects, such as treatment of disorders or other undesirable physiological conditions, and prevention. Effects, e.g., prevention of disorders or other undesirable physiological conditions (e.g., non-immunogenic cancers) These terms also include those beneficial agents specifically mentioned herein. pharmacologically acceptable active derivatives of the compound of the present invention include, but are not limited to, salts, esters, amides, etc. , prodrugs, active metabolites, isomers, fragments, analogs, etc. When used, then, or when a particular agent is specifically identified, the term refers to the agent. per se, as well as pharma- ceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, complexes, It should be understood that this includes conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0033] What is meant by a "therapeutically effective amount" or a "therapeutically effective dose" of a composition (e.g., a composition containing a drug)? refers to an amount effective to achieve a desired therapeutic result. In some embodiments, the desired therapeutic result is , control of type I diabetes. In some embodiments, the desired therapeutic outcome is control of obesity. The therapeutically effective amount of a given therapeutic agent typically depends on the type and severity of the disorder or disease being treated. The term may vary with respect to factors such as the intensity of the dose, and the age, sex, and weight of the subject. Also, the amount of therapeutic agent, or the concentration of therapeutic agent, effective to promote a desired therapeutic effect, such as pain relief, may be used. It can also refer to the rate of delivery (e.g., amount over time). The exact desired therapeutic effect can be determined by the duration of the treatment. the condition being treated, the tolerance of the subject, the drug and / or drug formulation being administered (e.g., efficacy of the therapeutic agent), , concentration of drug in the formulation, etc.), as well as a variety of other factors that will be appreciated by those of skill in the art. In some cases, the desired biological or medical response may occur over a period of days, weeks, or even years. This is achieved after multiple administrations of the composition over a period of time, or years.

[0034] In this specification and the appended claims, certain terms are defined to have the following meanings: Please refer to the terminology.

[0035] "Optional" or "optionally" means that the following event or circumstance does not occur. This statement includes instances in which the event or circumstance may or may not occur. Includes:

[0036] Throughout this application, various publications are referenced. The disclosures of these publications are hereby incorporated by reference in their entireties. No. 6,393,636, filed on Dec. 13, 2003, in order to more fully describe the state of the art to which this application pertains. The references disclosed are also discussed in the text by which they are referenced, and the material contained therein is incorporated herein by reference. The materials discussed herein are individually and specifically incorporated by reference.

[0037] B. Composition Components used to prepare the disclosed compositions, as well as the methods disclosed herein The compositions themselves are disclosed for use in the compositions of matter disclosed herein. These and other materials are disclosed herein. When any combination, subset, interaction, group, etc. of these materials is disclosed, Specific reference to each of the various individual and collective combinations and permutations of the compounds of the present invention is expressly intended to be included herein. Although not disclosed herein, it is understood that each is specifically contemplated and described herein. For example, specific hydrogel matrices and / or dry scaffolds are disclosed and This can be done for several molecules including hydrogel matrices and / or dry scaffolds. Where some modifications are discussed, unless specifically stated otherwise, the hydrogel matrix Each and every combination and permutation of dry and / or dry scaffolds and Modifications are specifically contemplated. Thus, in addition to the classes of molecules A, B, and C, Disclosed are classes of molecules D, E, and F, as well as AD as an example of a combination molecule. In each case, even if not individually listed, each is contemplated individually and collectively, and all That is, AE, AF, BD, BE, BF, CD, CE, and CF are disclosed. Likewise, any subset or combination of these is also considered disclosed. Thus, for example, the subgroups AE, BF, and CE are considered to be disclosed. This concept will be apparent from steps in the methods of making and using the disclosed compositions. This applies to all aspects of this application, including but not limited to the above. If there are various additional steps that may be included, each of these additional steps is disclosed. It is understood that the method may be practiced in any specific embodiment or combination of embodiments. can be.

[0038] We present an efficient, single-step immunocytochemical method for engineering T cells with RV vectors. We developed a three-dimensional (3D) dry macroporous scaffold as a target platform. reported that a three-dimensional scaffold with macroporosity and high water absorption capacity was fabricated using RetroNectin. We hypothesized that this would allow for colocalization of RV particles and T cells, as achieved by We prepared dry macroporous scaffolds from calcium cross-linked alginate. However, it is important to note that it is biocompatible, has low toxicity, low cost, and is mildly soluble due to divalent cations. For gelation, we use GMP-compliant, FDA-approved biocompatible materials that are widely used in many biomedical applications. The inventors have demonstrated that dry, hygroscopic, and desiccated macroporous alginate scaffolds are R Facilitating the interaction of V vectors with cells (including immune cells such as T cells and non-immune cells); without spinoculation and without affecting the function and viability of recipient cells, Efficient delivery of nucleic acids (genes, siRNAs, tasiRNAs, lncRNAs) in a single step A, shRNA, mRNA, gRNA, and / or miRNA) Therefore, these scaffolds can be used to contain T cells for adoptive cell therapy. A simple, cost-effective method for generating highly functional engineered cells, including but not limited to It represents a highly flexible and scalable platform technology.

[0039] Thus, in one embodiment, a cellular receptor or protein (including but not limited to a T cell receptor) is and / or NK cell receptors) CD3 antibody, CD1d, Fc fragment of immunoglobulin, or anti-Fcγ receptor (FcγRII I) a biocompatible hydrogel matrix and / or a dry scaffold (e.g., Disclosed herein are hydrogels and / or dry scaffolds that include alginate. The resulting hydrogel matrix and / or dry scaffold can be used to infuse cells (e.g., T cells, natural NK cells, NKT cells, macrophages, tumor infiltrating lymphocytes (TIL), tumor These include, but are not limited to, tumor-infiltrating NK cells (TINK), and / or bone marrow infiltrating lymphocytes (MIL). Transduction of non-immune and immune cells, as well as chimeric antigen receptor (CAR) (i) a scaffold used for the production of human cells. The hydrogel matrix and / or dry scaffold can be loaded with exogenous genes, siRNA, tasiRNP, A, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or siR Encoding NAs, tasiRNAs, lncRNAs, shRNAs, gRNAs, and miRNAs DNA (including but not limited to engineered genes, siRNA, tasiRNA, lncRNA A. shRNA, mRNA, gRNA, and / or miRNA, e.g., chimeric antigen receptors A vector (e.g., a lentil vector) encoding and / or carrying a CAR gene (e.g., a CAR gene) viruses, retroviruses, adenoviruses, adeno-associated viruses, virus-like particles, The carrier may include a carrier, such as a sposon or liposome.

[0040] The structure of the device, e.g., hydrogel, may be composed of peptide polymers, polysaccharides, synthetic polymers, hydrogel materials, ceramics (e.g., calcium phosphate or hydroxyapatite), Several proteins, glycoproteins, proteoglycans, metals, and metal alloys Different rigid, semi-rigid, flexible, gel-like, self-organizing, liquid crystalline, or fluid compositions The compositions are constructed from materials that are known in the art, such as by injection molding, preformed The resulting structures can be freeze-dried, printed, self-assembled, phase-inverted, solvent-cast, melt-processed, and galvanized. The scaffolds are constructed using foaming, fibrosis / processing, particle leaching, or a combination thereof. do.

[0041] The hydrogels and / or dry scaffolds disclosed herein may be made of any suitable biodegradable polymer. A "polymer" is a relatively high molecular weight, natural or synthetic Refers to organic compounds whose structure can be represented by small repeating units called monomers Non-limiting examples of polymers include polyethylene, rubber, and cellulose. Synthetic polymers are , typically formed by addition or condensation polymerization of monomers. The term "copolymer" refers to refers to a polymer formed from two or more different repeating units (monomer residues). Examples are Copolymers include, but are not limited to, alternating copolymers, random copolymers, block copolymers, and the like. In a particular embodiment, the copolymer may be a block copolymer or a graft copolymer. It is also contemplated that the various block segments of the mer may themselves comprise copolymers. The term "polymer" includes natural polymers, synthetic polymers, homopolymers and heteropolymers. or any form of polymer, including but not limited to copolymers, addition polymers, etc. It includes attitudes.

[0042] Exemplary materials, e.g., hydrogels, that may be used to form the devices include polystyrene. Polylactic acid, polyglycolic acid, polylactide-co-glycolide (PLG), alginate and alginate lignin derivatives, gelatin, collagen, fibrin, fibronectin, methacryl Amides, acrylamides, decellularized tissues, hyaluronic acid, laminin-rich gels, agarose , natural and synthetic polysaccharides, polyamino acids, polypeptides, polyesters, polyanhydrides, polyhops Sulfadin, poly(vinyl alcohol), poly(alkylene oxide), poly(allyla Poly(amine) (PAM), poly(acrylate), modified styrene polymer, pullol, polyol xamers, poly(uronic acids), poly(vinylpyrrolidone), and copolymers of any of the above copolymers or graft copolymers.

[0043] One preferred hydrogel is RGD-modified alginate. The hydrogel may be a cross-linked polymer, such as cross-linked alginate, gelatin, or the like. Derivatives of these include, for example, methacrylated ones.

[0044] In one embodiment, the hydrogel and / or dry scaffold is made of a biocompatible polymer (e.g., alginate). Such polymers can also be used to treat CAR T cells, CAR NK cells, and the like. The present invention may serve to gradually release cells, TILs, and / or MILs into tissues. As used herein, biocompatible polymers include, but are not limited to, polysaccharides; hydrophilic polypeptides; Poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-asparagine poly(amino acids), such as poly(amino acids), poly-L-serine, or poly-L-lysine; polyethylene; glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) polyalkylene glycols and polyalkylene oxides such as poly( Oxyethylated polyols; Poly(olefin alcohols); Polyvinylpyrrolidone) Poly(hydroxyalkyl methacrylamide); Poly(hydroxyalkyl methacrylamide) Poly(saccharides); Poly(hydroxy acids); Poly(vinyl alcohol), Poly(lactic acid) ), polyhydroxyacids such as poly(glycolic acid), and poly(lactic-co-glycolic acid) Acids; polyhydrogens such as poly 3-hydroxybutyrate or poly 4-hydroxybutyrate Poly(orthoesters);Polyanhydrides;Poly(caprolactones);Poly(orthoesters);Poly(anhydrides); ... (Phosphazene);Poly(lactide-co-caprolactone);Tyrosine polycarbonate Polycarbonates such as polyamides (including synthetic and natural polyamides), polypeptides and poly(amino acids); polyesteramides; polyesters; poly(dioxanone); Poly(alkylene alkylates);Hydrophobic polyethers;Polyurethanes;Polyether ethers steel;polyacetal;polycyanoacrylate;polyacrylate;polymethylmeth Acrylates; Polysiloxanes; Poly(oxyethylene) / Poly(oxypropylene) copolymers Polyketals;Polyphosphates;Polyhydroxyvalerates;Polyalkyleneoxa poly(maleic acid), as well as their copolymers Examples of biocompatible polymers include, but are not limited to, polyamides, Polycarbonate, polyalkylene, polyalkylene glycol, polyalkyleneoxy Polyalkylene terepthalate te), polyvinyl alcohol (PVA), methacrylate PVA (m-PVA), poly Vinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidones , polyglycolide, polysiloxane, polyurethane and their copolymers, alkyl cerium cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, Nitrocellulose, polymers of acrylic and methacrylic esters, methylcellulose, ethylene ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate cellulose acetate butyrate, cellulose acetate phthalate, carboxylate ethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt , poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate) Poly(isobutyl methacrylate), Poly(hexyl methacrylate Poly(isodecyl methacrylate), Poly(lauryl methacrylate) poly(phenyl methacrylate), poly(methyl acrylate), Poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecamethyl Poly(ethylene glycol), polypropylene, poly(ethylene glycol), Poly(ethylene oxide), Poly(ethylene terephthalate), Poly(vinyl alcohol) , poly(vinyl acetate, polyvinyl chloride polystyrene and polyvinyl pyrrolidone ( polyvinylpryrrolidone), their derivatives, their linear and Included are branched and block copolymers, as well as mixtures thereof. Examples of biodegradable polymers include polyesters, poly(orthoesters), and poly(ethylene amine), poly(caprolactone), poly(hydroxybutyrate), poly(hydroxy sivalerate), polyanhydrides, poly(acrylic acid), polyglycolide, poly(urethane) , polycarbonate, polyphosphate ester, polyphosprayene, derivatives thereof, Linear and branched copolymers and block copolymers thereof, as well as mixtures thereof, are included. Can be obtained.

[0045] In some embodiments, the particles are selected from poly(lactic acid), poly(glycolic acid), and poly( Biocompatible and / or biodegradable polyesters or polyamides such as lactic acid-co-glycolic acid The particles may include one or more of the following polyesters: Homopolymers containing glycolic acid units, referred to as "PGA," are collectively referred to herein as "PLA." Poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, Homologs containing lactic acid units such as poly(D-lactide), poly(D,L-lactide), and poly(D,L-lactide)5 Polymers and poly(e-caprolactone), collectively referred to herein as “PCL” ), as well as homopolymers containing caprolactone units, collectively referred to herein as "PLGA." Poly(lactic acid-co-glycolic acid), commonly referred to as Lactic acid and glycolic acid, such as various forms of poly(lactide-co-glycolide) and poly(lactide-co-glycolide), are also known. Copolymers and polyacrylates containing carboxylic acid units, and derivatives thereof. The polymers are also referred to herein collectively as "PEGylated polymers" such as PLGA-PEG or Polyethylene glycol (PEG) and its derivatives, such as various forms of PLA-PEG copolymers, are and copolymers of the aforementioned polyesters. In certain embodiments, the PEG region is a copolymer of PEGylated polymers are covalently associated with the PEG-mer via a cleavable linker to obtain a "PEGylated polymer" In one embodiment, the polymer can be at least 60, 65, 70, 75, 80, 8 5, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent The compound contains an acetal pendant group.

[0046] The triblock copolymers disclosed herein may be, for example, polyethylene glycol ( PEG), polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone (PVP), Polyethylene oxide (PEO), poly(vinylpyrrolidone-co-vinyl acetate), Acrylate, polyoxyethylene alkyl ether, polyoxyethylene castor oil , polycaprolactam, polylactic acid, polyglycolic acid, poly(lactic-glycolic) acid, poly Poly(lactic-co-glycolic) acid (PLGA), hydroxymethylcellulose, hydroxypropyl The core polymer may be a cellulose derivative such as pyrocellulose.

[0047] In some cases, the components of the device, e.g., the hydrogel-containing components, are Geometric shapes (e.g., disks, beads, pellets), niches, planar layers (e.g., thin plates ) are arranged in a diameter of about 0.1 mm to about 50 cm, e.g. For example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 7 0, 80, 90mm, 10, 15, 20, 25, 30, 35, 40, 45, 50 Centimeter diameter disks are implanted subcutaneously. Disks range from 0.1 to 10 mm in diameter. The disk may have a thickness of, for example, 1, 2, or 5 millimeters. Exemplary disks for subcutaneous administration have the following dimensions: It has a diameter of 8 mm and a thickness of 1 mm. It contains, for example, hydrogel, Multicomponent devices can optionally be made up of different physical qualities (polymer %, crosslinked % of polymer, hydrophilicity, etc.). Chemical composition, pore size, porosity, and pore architecture of the rogel, stiffness, toughness, ductility The present invention is constructed of concentric layers characterized by different properties (e.g., viscoelastic, and / or pharmaceutical compositions).

[0048] The scaffolds disclosed herein can be configured in 384-well, 96-well, 48-well, 24-well, Manufactured in the wells of a multi-well plate, such as a 12-well or 6-well plate or may be inserted into the wells of a multi-well plate. 5, T175, or T225 flasks, or The scaffolding is available in sizes 35mm, 60mm, 100mm and 150mm. It may also be produced in a culture dish such as a dish, or inserted into a culture dish. Scaffolds are available in 3mL, 5mL, 7mL, 8mL, 12mL, 14mL, and 15mL sizes. Cell culture tubes, such as 100 mL, 16 mL, 19 mL, 21 mL, or 50 mL tubes. The scaffold can be fabricated in a tube or inserted into a cell culture tube. They can be of any shape and size, with sizes ranging from 1mm3 to 0.1m3. The scaffold can be manufactured in any mold or inserted into a mold. , 100mL, 200mL, 300mL, 500mL, 1000mL, 2000mL, 5 It can also be manufactured in cell culture bags of 10,000mL, 10,000mL, or in cell culture bags. It can also be inserted.

[0049] The scaffolds disclosed herein can be placed in the wells of a multi-well plate or in a culture dish. Since it can be manufactured in a disk shape with a diameter of 1 mm to 50 cm and a thickness of 1 mm to 50 cm, The scaffolding is a square or square structure with a side length of 1 mm to 50 cm and a thickness of 1 mm to 50 cm. In addition, the scaffolds can be regularly shaped or They can be produced in irregularly shaped moulds and can also be made in triangular, pentagonal, hexagonal, star, The shape may be regular, such as a diamond, or irregular. The molds have a surface area of ​​1mm^2~2500cm^2 and a thickness of 1mm~50cm. It is possible.

[0050] The scaffold can consist of an aggregate of individual particles. These particles can be spray-dried, electrochemically Through air spinning, extrusion, emulsification / gelation, milling, spin drying, or other known particle creation techniques. Examples of particles that make up the scaffold include particles with diameters of 50 μm, 100 μm, 20 The particle size may be 0 μm, 500 μm, 1000 μm, 2000 μm or 5000 μm. Scaffolding can also consist of sections cut from a manufactured block. The size of the original block can be from 1 square meter to 1,000 square meters.

[0051] The scaffold structure may contain pores in a microporous or macroporous state. The pore size may be , 10μm, 20μm, 50μm, 100μm, 200μm, 500μm, 1000μm The pore pattern can optionally be homogenous, heterogenous, ordered, repeating, or is random.

[0052] In one aspect, the scaffold may be a "dry scaffold." As used herein, "dry scaffold" refers to a "Dry scaffold" refers to any scaffold having water in an amount not exceeding 10% by mass. In other words, a dry scaffold The scaffold has a moisture content of less than 10% by weight. In some instances, the dry scaffold is 9, 8, 7, 6 , 5, 4, 3, 2, or 1% by weight water. For example, the scaffold may have less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, Contains 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10% by weight of water It is possible.

[0053] As noted above, one preferred material for the hydrogel is alginate or modified alginate. Alginate is a material that allows control over molecular weight, degradation rate, and scaffold formation. This results in versatile polysaccharide-based polymers that can be formulated for specific applications. The alginate molecule is made up of (1-4) linked β-D-mannuronic acid (M units) and α It consists of L-guluronic acid (G unit) monomers, which are arranged in a uniform ratio and order along the polymer chain. Alginate polysaccharides can change the distribution of divalent cations (e.g., C a +2 , Mg +2 , B.A. +2 ) and when exposed to these molecules A polyelectrolyte system that forms stable hydrogels. Martinsen A., et al., Biotech. & Bioeng., 33 (1989) 79-89.) Examples For example, cross-linked calcium alginate hydrogels are useful in the methods described herein. For example, the polymer of the hydrogel, e.g., alginate, may be 0-100% crosslinked, e.g., At least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 8 In another embodiment, the polymer of the hydrogel is crosslinked to 0%, 90% or more. In some instances, the polymers of the hydrogel, such as alginate, are not crosslinked. - e.g., alginate is less than 50%, e.g., less than 50%, less than 40%, less than 30% , less than 20%, less than 10%, less than 50%, less than 2%, less than 1% crosslinked.

[0054] Alginates can be chemically modified to obtain new properties. For example, alginates can be , can be oxidized to increase the rate of biodegradation. Alternatively, alginate can be biocompatible. Alginates can also be reduced to change their crosslinking behavior. For example, alginate can be modified with bioorthogonal click groups. In another example, alginate can be modified with acrylic groups to allow for click crosslinking. Modifications can be made to allow radical polymerization crosslinking. Modification with to-guest chemistry allows for host-guest cross-linking.

[0055] Alginate polymers can be formed into various hydrogel types. Alginate scaffolds are The alginate is formed from alginates with molecular weights ranging from 1,000 Da to 500,000 Da. The alginate scaffolds are made from alginates with a G / M ratio of 0.5 to 5. The kinetics of hydrogel degradation can be controlled by different hydrogel formulations. Pharmaceutical compositions from alginate hydrogels, such as small molecules, morphogens, or other The release rate of the bioactive agent is controlled by the hydrogel formulation to spatially distribute the pharmaceutical composition. This controlled release reduces systemic side effects and Eliminates the need for multiple injections.

[0056] Useful polysaccharides other than alginate include agarose and microbial polysaccharides, as follows: These include, but are not limited to: fungal pullulan, scleroglucan, chitin, chitosan , elsinan, bacterial xanthan gum, curdlan, dextran, gelatin, levan, ethan Mulsan, cellulose, hyaluronic acid, etc.

[0057] In one aspect, the hydrogel matrix and / or dry scaffold comprises a receptor (e.g., epidermal growth factor receptor). Receptor (EGFR, Platelet-derived growth factor receptor (PDGFR)), Ligand (but not limited to Although it is not a marker for inflammatory bowel disease, epidermal growth factor (EGF), platelet-derived growth factor, granulocyte-macrophage cofactor Ronnie stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), granulocyte colony stimulation Granulocyte-macrophage colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (GM-CSF), Phage colony stimulating factor (M-CSF), fibroblast growth factor (FGF), insulin including IGF-1, and / or IGF-2, bone morphogenetic transcription factors Proteins (BMP), ephrins (A1, A2, A3, A4, A5, B1, B2, B3), Erythropoietin, Fibrocyte Growth Factor (FGF1, FGF2, FGF3, FGF4, FG F5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12 , FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF 19, FGF20, FGF21, FGF22, FGF23), glial cell line-derived neurotrophin Factors (GDNF), hepatocyte growth factor (HGF), neurotrophins (BDNF, NGF, NT- 3, NT-4), T cell growth factor (TCGF), transforming growth factor (TGF-α, TGF -β), tumor necrosis factor α (TNF-α), Wnt signaling pathway, integrins (VL A-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, FLJ252 20, RLC, PRO827, HsT18964, FLJ39841, HUMINAE, LFA1A, MAC-1, VNRA, MSK8, GPIIb, cadherin (e.g., E-ca adherin), and / or immune activating and / or sustaining antibodies IL-2, IL-6, IL-7, IL-15 , IL-21, TNF-α, or IFN-γ, etc.

[0058] It is understood that to fully activate immune cells, costimulatory signals may be necessary; Contemplated herein. Thus, in one aspect, the present invention relates to a combination of anti-CD28, B7-1 , B7-2, anti-induced costimulatory agent (ICOS), ICOS ligand, anti-CD27, CD70 , 4-1BBL, anti-41-BB, anti-CD40L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TIM-2, anti-TIM-3, anti-CD44, anti-NK1.1, Lectin-like transcript-1 (LLT-1), anti-CD137, CD48, MICA, anti-2B4 , and anti-glucocorticoid-inducible tumor necrosis factor receptor-related protein (GITR) These include, but are not limited to, T cells, natural killer (NK) cells, NKT cells, mammary phages, tumor infiltrating lymphocytes (TIL), tumor infiltrating NK cells (TINK), or bone marrow The hydrogel matrix further comprises one or more costimulatory molecules that activate infiltrating lymphocytes (MIL). Thus, hydrogel matrices and / or dry scaffolds are disclosed. Alternatively, the dry scaffold may also be coated with anti-CD28, B7-1, B7-2, or anti-induced co-stimulatory agent (ICOS). , ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, anti-CD40 L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TIM-2, Anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript-1 (LLT-1), anti CD137, CD48, MICA, anti-2B4, and anti-glucocorticoid-induced tumor necrosis T cell, NK cell, including but not limited to, factor receptor-associated protein (GITR) Ligand or antibody that induces signal transduction via a costimulatory receptor in NKT cells It may also include.

[0059] 1. Delivery of Compositions to Cells Transduction of immune cells can be carried out by any means known in the art. In one aspect, the transduction of immune cells is by the incorporation of a vector encoding a transgene (e.g., a CAR, etc.). Thus, in one embodiment, the transgene (e.g., Vectors (e.g., lentivirus, retrovirus, etc.) encoding a target antigen receptor (CAR) Virus, adenovirus, adeno-associated virus, virus-like particle, liposome, or transfection Any of the hydrogels disclosed herein further comprising a transposon or the like. It is a dry scaffold.

[0060] Several methods can be used to deliver nucleic acids to cells, either in vitro or in vivo. There are several compositions and methods. These methods and compositions involve the use of viral-based delivery systems. and non-viral based delivery systems. For example, nucleic acids can be , electroporation, lipofection, calcium phosphate precipitation, plasmids, virus vector, viral nucleic acid, phage nucleic acid, phage, cosmid, virus-like particle ( VLPs), transposons (e.g., Sleeping Beauty transposase, Frog-Prince, piggyBac, Tol2 and other Tc1 / Mariner type transformants Class II transposable elements containing posases), zinc finger nucleases, meganucleases enzymes, transcription activator-like effectors (e.g., TALENs), triplexes, epigenetic Modification mediators, as well as CRISPR and rAAV technologies), small circular DNA, etc. By a number of direct delivery systems, or by, for example, virus-like particles, cell mimicking particles, trans Cells or intracellular genetic material such as pososomes, exosomes, nanoparticles, micelles or liposomes Delivery can be via the transport of vectors, chemical transfectants, or Transfection methods include physical and mechanical methods such as electroporation and direct DNA diffusion. Suitable means for transfection are described, for example, in Wolff, JA, et al., Sc ience, 247, 1465-1468, (1990), and Wolff, JAN Ature, 352, 815-818, (1991). Methods for the preparation of glycerol-containing amines are well known in the art and may be used with the compositions and methods described herein. In certain cases, the method works specifically with large DNA molecules. Additionally, these methods may be used to exploit the targeting properties of carriers. By this, it is possible to target specific diseases and cell populations.

[0061] (1) Retrovirus vector Retroviruses are animal viruses that belong to the Retroviridae family of viruses and are Retroviral vectors are classified according to type, subfamily, genus, or tropism. rma,IM,Retroviral vectors for gene tra Examples of retroviruses that can be used as vectors are described in Human T-lymphotropic virus (HTLV)-1 (HTLV-1), HTLV-2 HTLV-3, HTLV-4, simian foamy virus, human foamy virus, simian immunodeficiency virus These include the HIV, SIV, and Rous sarcoma virus. Examples include, but are not limited to:

[0062] Retroviruses are essentially packages that contain nucleic acid cargo. The ribosomal protein contains a packaging signal, which allows the replicated daughter molecules to be efficiently packaged within the package coat. In addition to the packaging signal, The required cis site for the replication of the virus and for packaging of the replicated virus. There are several molecules. Typically, the retroviral genome is composed of a protein coat. The gag, pol, and env genes involved in the production of ribosomal RNA are introduced into the target cell. Typically, the gag, pol, and env genes are replaced by foreign DNA. Retroviral vectors are typically packaged in a package envelope for incorporation into the package coat. The gag transcription signal, a sequence that signals the initiation of the gag transcription unit, and the tRNA promoter for reverse transcription An element required for reverse transcription that contains a primer binding site that binds to the ima during DNA synthesis. Terminal repeat sequences that guide the switch of the RNA strand, the promoter for synthesis of the second strand of DNA synthesis The purine-rich sequence on the 5' side of the 3'LTR, which acts as a priming site, and the host genome Features near the ends of the LTR that allow for insertion of DNA in a retroviral state for insertion into the The deletion of the gag, pol, and env genes results in a fragment of approximately 8 kb. The foreign sequence is inserted into the viral genome, reverse transcribed, and then produced into new retroviral particles during replication. This amount of nucleic acid can be packaged in as little as one transcript, depending on the size of each transcript. It is sufficient to deliver multiple genes from the same It is preferred to include either a positive or negative selectable marker.

[0063] Most of the replication machinery and packaging proteins in retroviral vectors are Since the genes for gag, pol, and env have been removed, the vector typically The packaging cell line is generated by placing the The recombinant plasmid was designed to contain the ribosomal protein and the packaging machinery, but lack any packaging signals. A cell line that has been transfected or transformed with a retrovirus. When vectors carrying the .DELTA..DELTA. gene are transfected into these cell lines, they are transformed by helper cells into The machinery provided by IS allows the vector containing the gene of interest to replicate and produce new The genome of the virus lacks the necessary signals. Therefore, it is not packaged.

[0064] Human immunodeficiency virus (HIV) vectors and simian immunodeficiency virus (SIV) vectors Lentiviral vectors (LV), including but not limited to, LV vectors, can be used in cultured cells or in cultured cells. It is a versatile vector for in vivo gene transfer in dividing and non-dividing cells. This allows for flexibility in transducing a variety of lung cancer cells without time-consuming selection for stable expression. Replication-deficient VSV G-pseudotyped lentiviral vectors (G Cells can be transduced using eneCopoeia (manufactured to order by Cut.

[0065] (2) Adenovirus Vector The construction of replication-defective adenoviruses has been described previously (Berkner et al., 2002). t al., J. Virology 61:1213-1220 (1987), Mass ie et al.,Mol.Cell.Biol.6:2872-2883(1986 ), Haj-Ahmad et al., J. Virology 57:267-274 (1986), Davidson et al., J. Virology 61:122 6-1239(1987), Zhang “Generation and ident ification of recombinant adenovirus by l iposome-mediated transfection and PCR an alysis” BioTechniques 15:868-872 (1993)). The advantage of using these viruses as vectors is that they replicate within the initially infected cell. can transmit the virus to other organisms, but cannot form new infectious virus particles. The extent to which recombinant adenovirus can spread to other cell types is limited. The drug has been shown to be directly linked to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and several other tissue sites. It has been shown that highly efficient gene transfer can be achieved following in vivo delivery of .Clin.Invest.92:1580-1586(1993), Kirshenb aum, J. Clin. Invest. 92:381-387 (1993), Roess ler, J. Clin. Invest. 92:1085-1092 (1993), Mou llier, Nature Genetics 4:154-159(1993), La Salle, Science 259:988-990 (1993), Gomez-F. oix, J. Biol. Chem. 267:25129-25134 (1992), Ri ch,Human Gene Therapy 4:461-476(1993), Za bner, Nature Genetics 6:75-83(1994), Guzma n,Circulation Research 73:1201-1207(1993 ), Bout, Human Gene Therapy 5:3-10(1994), Z abner, Cell 75:207-216 (1993), Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993), and Rago t, J. Gen. Virology 74:501-507 (1993). Nonoviruses achieve gene transfer by binding to specific cell surface receptors, The virus then transduces in a receptor-mediated manner in the same manner as wild-type or replication-defective adenoviruses. Internalized by endocytosis (Chardonnet and Dales , Virology 40:462-477 (1970), Brown and Bur. Lingham, J. Virology 12:386-396 (1973), Sven sson and Persson, J. Virology 55:442-449(1 985), Seth, et al., J. Virol. 51:650-655 (1984 ), Seth, et al.,Mol.Cell.Biol.4:1528-1533( 1984), Varga et al., J. Virology 65:6061-60 70 (1991), Wickham et al., Cell 73:309-319( 1993).

[0066] The viral vector should be based on an adenovirus with the E1 gene deleted. These virions can be produced in cell lines such as the human 293 cell line. In a preferred embodiment, both the E1 and E3 genes are removed from the adenoviral genome. can be.

[0067] (3) Adeno-associated virus vector Another type of viral vector is based on adeno-associated viruses (AAVs). Certain parvoviruses are preferred because they can infect many cell types and are nonpathogenic to humans. AAV-type vectors are preferred vectors. They can transport approximately 4 to 5 kb and are highly effective in the wild-type genome. AAV-type chromosomes are known to stably integrate into chromosome 19 (e.g., AAV recombinants). Vectors with this site-specific integration property are preferred. A particularly preferred embodiment of this type of vector is the vector available from Avigen (San Francis Group, The P4.1C vector produced by Sancisco, CA, is a simple vector. Rupesvirus thymidine kinase gene, HSV-tk, and / or marker genes, e.g. For example, it may have a gene encoding the green fluorescent protein, GFP.

[0068] In another type of AAV virus, AAV expresses a cell-specific .alpha.- A pair of fusion proteins flanking at least one cassette having a promoter for directing heterologous expression. In this context, heterologous refers to AAV or B19 It refers to any nucleotide sequence or gene that is not native to a parvovirus.

[0069] Typically, the AAV and B19 coding regions are deleted to produce a safe, non-cytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration capabilities. The promoter induces cell-specific expression. Patent No. 6,261,834 is incorporated herein by reference for material relating to AAV vectors. INCORPORATED INTO THE SPECIFICATION.

[0070] Thus, the disclosed vectors integrate into mammalian chromosomes without substantial toxicity. The present invention provides a DNA molecule capable of

[0071] The inserted gene in viruses and retroviruses usually directs expression of a desired gene product. It contains a promoter and / or enhancer that help control the Generally, a DNA fragment that functions when in a relatively fixed position with respect to the transcription start site. The promoter is a sequence required for the basic interaction of RNA polymerase and transcription factors. It contains the necessary core elements and may contain upstream elements and response elements.

[0072] (4) Large payload viral vectors Molecular genetic experiments using large human herpesviruses have revealed that large heterologous DNA fragments , cloning, propagation, and establishment in cells permissive for infection with herpes viruses. (Sun et al., Nature Genetics, s 8:33-41,1994, Cotter and Robertson,.Cur. r Opin Mol Ther 5:633-644,1999). These large D NA virus (Herpes simplex virus (HSV) and Epstein-Barr virus (EBV) V)) has the ability to deliver fragments of human heterologous DNA of >150 kb to specific cells. EBV recombinants are able to maintain large pieces of DNA within infected B cells as episomal DNA. Individual clones carrying human genomic inserts of up to 330 kb can be genetically Maintenance of these episomes appears to be constitutively dependent on EBV during infection. A specific EBV nuclear protein, EBNA1, which is expressed in the EBV nucleoprotein 1 (EBNA1) is required. The vector can be used to perform transfection, where large amounts of protein can be The herpes virus amplification system can also be used to transiently produce Packages DNA fragments larger than 220 kb and stably stores the DNA as episomes It has also been used to infect cells that can be maintained in a stable manner.

[0073] Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors. Kutar is one example.

[0074] 2. Expression system The nucleic acids delivered to cells typically contain expression control systems. For example, viruses and The inserted gene in a retroviral system usually contains a promoter to control the expression of the desired gene product. The promoter generally contains a promoter and / or enhancer that is useful for transcription. A sequence of DNA that functions when it is in a fixed location relatively to the start of transcription. The promoter contains core elements required for the basic interaction of RNA polymerase and transcription factors. and may contain upstream elements and response elements.

[0075] a) Viral promoters and enhancers Preferred promoters controlling transcription from the vector in mammalian host cells include a variety of Sources, e.g., polyoma, Simian Virus 40 (SV40), adenovirus, Viruses such as hepatitis B virus, hepatitis B virus, and most preferably cytomegalovirus. from the genome of the mammalian plant or from a heterologous mammalian promoter, e.g., the β-actin promoter. The early and late promoters of the SV40 virus can be obtained from the The SV40 restriction fragment also contains the SV40 origin of replication (Fiers et al. ., Nature, 273:113 (1978)). The promoter is conveniently obtained as a HindIII E restriction fragment (Greenway, PJ et al., Gene 18:355-360 (1982)). Promoters from the host cell or related species are also useful herein.

[0076] Enhancers generally function at any convenient distance from the transcription start site and act as transcriptional regulators of transcription units. 5' side (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or the 3' side (Lusky, ML, et al. , Mol. Cell Bio. 3:1108 (1983)). Furthermore, enhancers are sequences located within introns (Banerji, JLett. al., Cell 33:729 (1983)), and within the coding sequence itself (Osbo rne,TF,et al.,Mol.Cell Bio.4:1293(1984 )) which are usually 10-300 bp in length and function in cis. Enhancers function to increase transcription from a nearby promoter. Enhancers also often contain response elements that mediate the regulation of transcription. Enhancers can also contain response elements that mediate the regulation of transcription. Determines the regulation of expression. Many enhancer sequences are now known to exist in mammalian genes (globin, elastase, albumin, -fetoprotein, and insulin), Typically, one uses enhancers from eukaryotic cell viruses for general expression. Preferred examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), Tomegalovirus early promoter enhancer, polyoma enhancer on the late side of the replication origin and adenovirus enhancers.

[0077] Promoters and / or enhancers can be activated by light or specific chemicals to trigger their function. The system can be specifically activated by either tetracycline or dexamethasone. It can be regulated by agents such as methazone. It can also be regulated by exposure to radiation such as gamma irradiation. Exposure to IL-14 or alkylating chemotherapy drugs enhances gene expression of viral vectors. There are also ways to do this.

[0078] In certain embodiments, the promoter and / or enhancer region is a transcription unit that is transcribed. It acts as a constitutive promoter and / or enhancer to maximize expression of the target region. In certain constructs, the promoter and / or enhancer region may be All eukaryotic cell types, even if they are only expressed in certain types of cells at certain times A preferred promoter of this type is the CMV promoter (650 Other preferred promoters include the SV40 promoter, the cytomegalovirus the promoter (full length promoter), and the LTR of the retroviral vector.

[0079] All specific regulatory elements have been cloned and expressed in specific cell types, such as melanoma cells. It has been shown that this method can be used to construct selectively expressed expression vectors. The glial fibrillary acetate protein (GFAP) promoter is involved in the selection of genes in cells of glial origin. It has been used to selectively express

[0080] It can also be used in eukaryotic host cells (yeast, fungal, insect, plant, animal, human, or nucleated cells). The expression vector used may contain sequences necessary for the termination of transcription that may affect the expression of mRNA. These regions may be located in the untranslated portion of the mRNA encoding the tissue factor protein. The 3' untranslated region contains a polyadenylated segment that is transcribed at 3 min. Preferably, the transcription unit also contains a polyadenylation region. One advantage of the region is that the transcription unit may be processed and transported like an mRNA. The identification and use of polyadenylation signals in expression constructs It is well established that homologous polyadenylation signals are used in transgene constructs. In certain transcription units, the polyadenylation region is preferably an SV40 early polyadenylation region. It is derived from the re-adenylation signal and consists of about 400 bases. The transcription unit can be either alone or can be combined with other standard sequences to improve expression or stability from the construct. It is also preferred that the compound contains

[0081] b) Marker The vector can include a nucleic acid sequence that encodes a marker product. The method uses the gene to deliver it to a cell and determine whether it is expressed once delivered. A preferred marker gene is the lacZ gene of E. coli (β-galactosidase ), and green fluorescent protein.

[0082] In some embodiments, the marker may be a selectable marker. Mammalian cells Examples of suitable selectable markers include dihydrofolate reductase (DHFR), thymidine dinucleotide Kinase, neomycin, neomycin analog G418, hydromycin, and puromycin Such selectable markers have been successfully transferred to mammalian host cells. The transformed mammalian host cell is then capable of surviving if placed under selective pressure. There are two different categories of selective regimes that are widely used. The category relies on the use of mutant cell lines that lack the ability to grow independently of a supplemented medium, as well as on cellular metabolism. Two examples are CHO DHFR-cells and mouse LTK-cells. These cells have the ability to grow without the addition of nutrients such as thymidine or hypoxanthine. These cells lack certain genes required for a complete nucleotide synthesis pathway. Therefore, they cannot survive unless the missing nucleotides are provided in a supplemented medium. An alternative to supplementing the medium would be to introduce intact DHFR or The first step is to change their growth requirements by introducing the TK gene. Individual cells that were not transformed with the TK gene were able to survive in non-supplemented medium. It would be impossible.

[0083] The second category refers to the selection schemes used in any cell type, including the use of mutant cell lines. These schemes are dominant selection schemes that do not require the use of a gene that stops the growth of the host cell. Those cells that have the new gene transmit drug resistance. In an example of such dominant selection, the drug nephrectomy is used to express the protein and survive the selection. Omycin, (Southern P. and Berg, P., J. Molec. Ap pl. Genet. 1:327(1982)), mycophenolic acid (Mulligan, RCand Berg, P.Science 209:1422(1980)), and was hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5:410-413 (1985). Three examples are shown in which the cellular functions of eukaryotic cells are controlled by The bacterial genes were then used to inject the appropriate drugs, G418 or neomycin (Geneticin), It transmits resistance to XGPT (mycophenolic acid) or hygromycin. These include the omycin analogs G418 and puramycin.

[0084] 3. Pharmaceutical Carriers / Delivery of Pharmaceutical Products As mentioned above, the composition can also be administered in vivo in a pharma- ceutically acceptable carrier. "Pharmaceutically acceptable" means that the compound does not cause any undesirable biological effects. does not interact in any harmful manner with any of the other components of the pharmaceutical composition with which it comes into contact. Materials that are not functional and biologically or otherwise undesirable, i.e., nucleic acids A carrier refers to a material that can be administered to a subject together with a vector. thus minimizing any degradation of the active ingredient and preventing any adverse side effects in the subject. can be necessarily selected to minimize

[0085] The compositions can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneally, transdermally, extracorporeally, It may be administered topically (including topical intranasal administration or by inhalation), or the like. As used herein, "topical intranasal administration" refers to administration of a composition to the nose via one or both nostrils. means to deliver to the tract by a spray or droplet mechanism, or by delivery of nucleic acid or vector Administration of the composition by inhalation can include delivery by aerosolization of the composition. Delivery can be through the nose or mouth via a droplet mechanism. Delivery can be through intubation. Alternatively, the compound can be delivered directly to any area of ​​the respiratory system (e.g., the lungs). The exact amount of ingredient will depend on the species, age, weight, and general condition of the subject, the type of allergic disorder being treated, and the severity of the allergic disorder. Depending on the severity, the particular nucleic acid or vector used, and its method of administration, it may vary from subject to subject. Therefore, it is not possible to specify an exact amount for every composition. However, the appropriate amount can be determined using only routine experimentation given the teachings herein. and can be determined by one of ordinary skill in the art.

[0086] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables are liquids. in liquid form or suspension, solid form suitable for solution of suspension in liquid prior to injection, or as emulsions The recently revised approach to parenteral administration is This involves the use of sustained or extended release such that a constant dosage is maintained. See U.S. Pat. No. 3,610,795, which is incorporated herein by reference.

[0087] The material may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), These may be directed to specific cell types via antibodies, receptors, or receptor ligands. The following references provide literature recommendations for targeting specific proteins to tumor tissue: An example of the use of the technology is shown in Senter, et al., Bioconjugate Chemistry. m.,2:447-451,(1991),Bagshawe,KD,Br.JC ancer,60:275-281,(1989),Bagshawe,et al., Br. J. Cancer, 58:700-703, (1988), Senter, et al. al.,Bioconjugate Chem.,4:3-9,(1993), Batt elli,et al.,Cancer Immunol.Immunother.,3 5:421-425, (1992), Pietersz and McKenzie,I. Reviews, 129:57-80, (1992), and Roff ler,et al.,Biochem.Pharmacol,42:2062-206 5, (1991)). Vehicles such as "stealth" and other antibody-conjugated lipoproteins. , which contains lipid-mediated drugs targeting colon cancer, through cell-specific ligands. Receptor-mediated targeting of DNA, lymphocyte-specific tumor targeting, and in vivo mouse tumor targeting Highly specific therapeutic retroviral targeting of glioma cells. The following references are based on tumor tissue An example of the use of this technology to target specific proteins in vivo is shown in Hughes et al. l., Cancer Research, 49:6214-6220, (1989), and and Litzinger and Huang, Biochimica et Bioph. ysica Acta, 1104:179-187, (1992)). In general, the receptors , which are involved in either constitutive or ligand-induced endocytosis pathways. The receptors cluster within clathrin-coated pits and are transported to the cell via clathrin-coated vesicles. They enter the cell, pass through acidified endosomes where receptors are sorted, and then are recycled to the cell surface. These proteins are either transported through the blood, stored intracellularly, or degraded in the lysosomes. The internalization pathway is involved in the uptake of nutrients, removal of activated proteins, and clearance of macromolecules. opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and regulation of receptor levels Many receptors function differently depending on the cell type, receptor concentration, ligand type, and Depending on the ligand valency and concentration, two or more intracellular pathways are followed. The molecular and cellular mechanisms of endocytosis have been reported (Brown and Gr eene,DNA and Cell Biology 10:6,399-409(1 991).

[0088] a) Pharmaceutically acceptable carrier The compositions comprising the antibodies may be used therapeutically in combination with a pharma- ceutically acceptable carrier. can.

[0089] Suitable carriers and their formulations are described in Remington: The Science and d Practice of Pharmacy(19th ed.)ed.ARG ennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of glycerol is added to the formulation to render the formulation isotonic. Examples of pharma- ceutically acceptable carriers include physiological saline. Solutions include, but are not limited to, water, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, and more preferably about 7 to about 7.5. Suitable carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing the antibody. The matrix may be a shaped article, such as a film, a liposome, or Certain carriers may be used depending on, for example, the route of administration and the concentration of the composition being administered. It will be apparent to one skilled in the art that this may be more preferable depending on the circumstances.

[0090] Pharmaceutical carriers are known to those of skill in the art. They most typically include sterile water at physiological pH, Standard carriers for administration of drugs to humans include solutions such as saline and buffered solutions. These compositions can be administered intramuscularly or subcutaneously. Other compounds are known to those skilled in the art. The drug will be administered according to standard procedures used by

[0091] Pharmaceutical compositions may contain, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surface active agents, The pharmaceutical compositions may also include antimicrobial agents, anti-inflammatory agents, anesthetic agents, etc. The composition may contain one or more active ingredients such as:

[0092] The pharmaceutical composition may be administered in a variety of ways depending on whether local or systemic treatment is desired and the area to be treated. Depending on the application, administration can be in a number of ways. Administration can be topical (including ophthalmic, vaginal, rectal, intranasal), Orally, by inhalation, or parenterally, e.g., by intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. may be administered to

[0093] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, oligosaccharides, and the like. vegetable oils, such as bean oil, and injectable organic esters, such as ethyl oleate. Carriers include water, alcoholic / aqueous solutions, emulsions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, or a suspension. Contains sucrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as Ringer's dextrose), Preservatives and other additives, such as antimicrobial agents, antioxidants, Chelating agents, inert gases, and the like may also be present.

[0094] Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, and sprays. Conventional pharmaceutical carriers, aqueous, powder or oily bases may be used in the form of sprays, liquids and powders. , thickeners, and the like may be necessary or desirable.

[0095] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, Capsules, sachets or tablets. Thickeners, flavourings, diluents, emulsifiers, dispersing aids , or a binder may be desirable.

[0096] Some of the compositions contain hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and Acid, inorganic acids such as phosphoric acid, as well as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, Organic acids such as pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid or by the reaction of sodium hydroxide, ammonium hydroxide, potassium hydroxide, etc. Organic bases, and mono-, di-, trialkyl-, and arylamines, and substituted ethanolamines. As pharma- ceutically acceptable acid or base addition salts formed by reaction with organic bases such as benzene, It may be possible to administer the drug via a single injection.

[0097] b) Therapeutic use Effective dosages and schedules for administering the compositions may be determined empirically; Therefore, making such a determination is within the skill of one of ordinary skill in the art. The dosage ranges are large enough to produce the desired effect which affects the symptoms of the disorder. The dosage may be adjusted to avoid adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, etc. In general, dosage should be adjusted according to the age of the patient. , condition, sex, and extent of disease, route of administration, or whether other drugs are included in the regimen The dosage will vary depending on the dosage and can be determined by one of skill in the art. The dosage may be adjusted by the individual physician in the event of any contraindications. The drug may be administered in one or more doses daily for one or several days. Guidance on appropriate dosages for these medications can be found in the literature. For example: Guidance on selecting an appropriate dose for an antibody can be found in the literature on therapeutic use of antibodies, For example, Handbook of Monoclonal Antibodies,Fe rrone et al., eds., Noges Publications, Par. k Ridge, NJ, (1985) ch.22 and pp.303-357, Smith et al., Antibodies in Human Diagnos is and Therapy, Haber et al., eds., Raven P. Ress, New York (1977) pp. 365-389. A typical daily dosage of antibody used alone might range from about 1 µg to 100 µg, depending on the factors mentioned above. The range may be from 100 mg / kg body weight up to 100 mg / kg body weight or more.

[0098] C. Methods of Using the Compositions 1. Methods for transducing immune cells and generating chimeric antigen receptor cells In one aspect, the present disclosure provides a method for the production of cells (e.g., immune cells (e.g., autologous, allogeneic, , and / or T cells, natural killer (NK) cells obtained from a haploidentical donor source. ) cells, NKT cells, macrophages, tumor-infiltrating lymphocytes (TIL), tumor-infiltrating NK cells (TINK) and / or bone marrow infiltrating lymphocytes (MIL), cells, NK cells, NKT cells, macrophages, TIL, TINK or MIL, etc.), immune cells (e.g., mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), dendritic cells, neural stem cells, etc.) cells, induced pluripotent stem cells, or pancreatic islet cells) (immune and non-immune cells include primary cells and cell lines) Methods for transducing a chimeric antigen receptor (CAR) and generating chimeric antigen receptor immune cells are disclosed. The method includes: a) i) a cell (e.g., an immune cell (e.g., autologous, allogeneic, and / or are T cells, natural killer (NK) cells, and N cells obtained from haploidentical donor sources. KT cells, macrophages, tumor infiltrating lymphocytes (TIL), tumor infiltrating NK cells (TINK ) and / or bone marrow infiltrating lymphocytes (MIL), including but not limited to T cells, NK cells, cells, NKT cells, macrophages, TIL, TINK or MIL, etc.), non-immune cells (e.g. For example, mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), dendritic cells, neural stem cells, induced (e.g., immune and non-immune cells, including primary cells and cell lines); and ii) exogenous genes, siRNAs, tasiRNAs, lncRNAs, shRNAs. , mRNA, gRNA, miRNA, and / or said gene, siRNA, tasiRNA DNA encoding lncRNA, shRNA, mRNA, gRNA, miRNA (limited Not specified, but engineered genes, siRNAs, tasiRNAs, lncRNAs, shRs NA, mRNA, gRNA, and / or miRNA, such as chimeric antigen receptors (CARs) b) obtaining a vector encoding and / or encapsulating a gene or gene product (such as a gene for a cell); and a scaffold (e.g., but not limited to, a hydrogel or dry scaffold, including alginate; Biocompatible hydrogel matrices and / or dry scaffolds, etc., have been applied to In some embodiments, the method includes incubating the scaffold, the cells, and the vector. In the present invention, the scaffold is a ligand or antibody for the T cell receptor or NK cell receptor (anti-CD3 antibody, CD1d, Fc fragments of immunoglobulins, or anti-Fcγ receptor (FcγRIII) antibodies The present invention further includes, but is not limited to,

[0099] The cells to be transduced are incubated with the scaffold prior to administration to allow for transduction. The cells to be transferred can be integrated into the scaffold and reliably transduced with the vector It is understood and contemplated herein that, in one aspect, the immune cells are at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 ,50,55 minutes,1,2,3,4,5,6,7,8,9,10,11,12,13,1 4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 54, 60, 66, 72, 8 Incubate for 4, 96 hours, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days In one aspect, the cells and scaffolds can be administered without incubation.

[0100] In one aspect, the present disclosure relates to cells (e.g., immune cells (e.g., autologous, allogeneic, and / or T cells, natural killer (NK) from haploidentical donor sources cells, NKT cells, macrophages, tumor-infiltrating lymphocytes (TIL), tumor-infiltrating NK cells ( TINK), or bone marrow infiltrating lymphocytes (MIL), etc. cells, NK cells, NKT cells, macrophages, TILs, TINKs, and / or MILs; and any commercially available cell or cell line), as well as methods for transducing the A method for producing a chimeric antigen receptor immune cell comprising the steps of: As mentioned above, the cells to be transduced can be cells from a commercially available cell line. Examples of cells that can be transduced include NCI-H295R, 5637, and HT- 1376, J82, SW 780, T24, T24-Luc-Neo, T24P, BT1 42, D54-Luc, DBTRG (tumor), DBTRG-05MG, Gli36-Ds Red-R-Luc (Rescue), LN-18, LN-229, LN-827 (pMM P-LucNeo), M059K, SF-295, SF-539, SF-767, SNB -19, U-251, U-251-Luc-mCh-Puro, U-87 MG, U-8 7 MG-Luc, Ca Ski, HeLa, KB, C2BBe1, Caco-2, CO LO 205, COLO 205-Luc#2, DLD-1, HCC2998, HCT- 116, HCT-116-Luc, HCT-15, HCT-8, HT-29, HT-29 -Luc, LoVo, LoVo-6-Luc1, LS 174T, LS411N, NCI -H508, SW-480, SW-620, A-431, HEKn, HEL, HEL 9 2.1.7, HEL 92.1.7-Luc-Neo, HEL-Luc-Neo, TF- 1a-Luc-Neo, OE33, A4573, Hs 895.T, NHDF (normal human human skin fibroblasts), TE 353.Sk, TE 354.T, GIST-T1, NCI -N87、NUGC-4、SNU-5、CAL 27、FaDu、L1210、M-NF S-60、HL-60、EOL-1、Kasumi-1、Kasumi-3、Kasum i-3-Luc-mCh-Pure、KG-1-Luc-mCh-Pure、MOLM- 13、MV-4-11、MV-4-11-Luc-mCh-Pure、NOMO-1、T HP-1、NALM6、NALM6-Luc-MCh-Pure、Reh、Reh(pM MP-Luc-Neo)、K-562、K-562-Luc2、ARH-77、CCRF -CEM、DND-41-Luc-mCh-Puro、Jurkat、Jurkat-C lone E6-1、MOLT-4、MOLT-4-Luc-MCh-Pure、Hep 3B2.1-7、Hep G2、LL、LL / 2、LL / 2-Luc-M38、NCI -H596、Calu-6、NCI-H322M、A549、A549-Luc-C8、 Head-1、Head-3、HCC827、HCC827-Luc-mCh-Pure 、NCI-H125、NCI-H125-Luc、NCI-H1299、NCI-H16 50、NCI-H1703、NCI-H1703-Luc-mCh-Pure、NCI- H1975、NCI-H1975-Luc、NCI-H2110、NCI-H2122、 NCI-H23、NCI-H292、NCI-H3122、NCI-H441、NCI- H460、NCI-H460-Luc2、NCI-H522、PC-9、DMS 114 、NCI-H446、NCI-H69、NCI-H82、SHP-77、EBC-1、S K-MES-1、RL、DB、DB / M2、GRANTA-519、Farange、B -JAB、David、David-Luke-mCh-Pure、NAMALWA、Ra ji、Raji-Luc、Branches、Branches-Luc、HuT78、HT、SU- DHL-6、SU-DHL-6-Luc-mCh-Pure、OCI-Ly1 LN、O CI-Ly19-Luc-Neo、OCI-Ly3-Luc-mCh-Pure、OCI -Ly7-Luc-mCh-Pure(レスキュー)、OCI-Ly7-Luc-Neo 、Pfeiffer、SU-DHL-10、SU-DHL-10-LN-High、SU -DHL-16、SU-DHL-4-Luc-mCh-Pure、SU-DHL-8、T MD8、Toledo-Luc-Neo、WSU-DLCL2、WSU-FSCCL、W SU-FSCCL-CMV-Luc-Pure、WSU-FSCCL-MSCV-Luc -Pure-copGFP、NK-92MI、CARPAS 299、BT-20、BT -474、HCC1395、HCC70、Hs 578Bst、Hs 578T、MCF 10A、MCF-7、MCF7-Luc-mCh-Pure、MDA-MB-231、 MDA-MB-231-2LMP、MDA-MB-231-Luc-D3H1、MDA- MB-231-Luc-D3H2、MDA-MB-231-Luc-D3H2LN、MD A-MB-231-Luc-D3H3、MDA-MB-361、MDA-MB-453、 MDA-MB-468、MX-1、MX-1-Luc、SK-BR-3、T47D、UI SO-BCA-1、ZR-75-1、A2058、A375、COLO 829、G-3 61、LOX-IMVI、M14、MDA-MB-435S、OCM-1、OCM-1- Luc-mCh-Pure、PA-NUT、SK-MEL-28、SK-MEL-28- Luc-mCh-Puro, SK-MEL-5, UACC-62, WM-115, WM- 266-4, JJN-3-Luc, MM.1S(pMMP-Luc-Neo), NCI- H929, NCI-H929-Luc-mCh-Puro, OPM-2, RPMI 82 26, U266B1, SK-N-AS, SK-N-FI, SK-N-SH, MKL-1, A2780, A2780-Luc, IGROV1, IGROV1-Luc-Mch-Pu ro, OVCAR-4, OVCAR-5, OVCAR-5-Luc-mCh-Puro, OVCAR-8, OVCAR-8-Luc-mCh-Puro, SK-OV-3 (subcutaneous) , SK-OV-3-Luc-D3 (intraperitoneal), Bx-PC-3, BxPC-3-Luc2 , Capan-1, Capan-2, KP4, MIA PaCa-2, MIA PaCa -2-Luc, PANC-1, PANC-1-Luc, SU-86.86, SW 199 0, 22Rv1, CWR-22-R, DU 145, DU 145-Luc, LnCap , LnCap clone FGC, PC-3, PC-3-Luc, PC-3M-Luc-C6 (intracardiac), PC-3M-Luc-C6 (orthotopic), PC-3M-Luc-C6 (Per itibial), PC-3M-Luc-C6 (SC-Axilla), VCaP, 76 9-P, 786-O, 786-O-Luc-Neo (rescue), A-498, ACH N, Caki-1, TK-10, A-673, HT-1080, MG-63, Saos- 2, SJSA-1, SW 872, MB-1, TT, SK-LMS-1, 293T, HE K293, or HeLa cells, among others, are not limited thereto. The cells to be transfected The cells may be adherent (e.g., HEK cells) or non-adherent (e.g., T cells) or It is also understood and contemplated herein that the cell line may be a cell line.

[0101] Other examples of cells that can be transduced in this way include primary cells of human or animal origin. Examples of such primary cells include hematopoietic cells, mononuclear cells (peripheral blood mononuclear cells, umbilical cord blood Mononuclear cells, macrophages, etc.), germ cells, primordial germ cells, oocytes, eggs, spermatocytes, sperm Erythroid progenitor cells, lymphocyte progenitor cells, mature blood cells, lymphocytes (B cells, CD4 T cells) These include, but are not limited to, CD8 T cells, γδ T cells, NK cells, and NKT cells. a), fibroblasts, neuroblasts, nerve cells, endothelial cells, vascular endothelial cells, hepatocytes, epithelial cells , keratinocytes, pancreatic islet cells, myoblasts, scaffold muscle cells, smooth muscle cells, cancer cells, myeloma cells, and Examples of cells that can be cultured include, but are not limited to, leukemia cells and leukemia cells.

[0102] It is understood that costimulatory signals may be required to fully activate immune cells; Contemplated herein. Thus, in one aspect, the present invention provides , cells (e.g., T cells, natural killer (NK) cells, NKT cells, macrophages, Phage, tumor infiltrating lymphocytes (TIL), tumor infiltrating natural killer cells (TINK), or bone marrow infiltration Immune cells such as lymphocytes (MIL) (including but not limited to autologous, allogeneic, and / or T cells, NK cells, NKT cells, and macrophages from haploidentical donor sources The present invention also relates to a method for transducing any of the following cells: The method for producing any of the chimeric antigen receptor immune cells disclosed herein, wherein the scaffold comprises a T Ligands that induce signal transduction via costimulatory receptors on cells, NK cells, or NKT cells or antibodies, including but not limited to anti-CD28, B7-1, B7-2, anti-inducible costimulatory agent (I COS), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, anti CD40L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TI M-2, anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript-1 (LLT- 1) Anti-CD137, CD48, MICA, anti-2B4, and anti-glucocorticoid-induced Methods are disclosed further comprising tumor necrosis factor receptor-related protein (GITR).

[0103] Also disclosed herein are cells (e.g., immune cells (e.g., autologous, allogeneic, and / or T cells, natural killer (NK) from haploidentical donor sources cells, NKT cells, macrophages, tumor-infiltrating lymphocytes (TIL), tumor-infiltrating NK cells ( T cells, NK cells, NKT cells, such as T cells, NK cells, NKT ... cells, macrophages, TILs, TINKs, and / or MILs, but are not limited to these. Methods for transduction of the chimeric antigen receptor (not shown) and the generation of chimeric antigen receptor immune cells disclosed herein In any of the methods, the scaffold may further comprise one or more cytokines (e.g., IL-2, TNF Also disclosed herein are methods that further comprise administering to the patient IFN-α, IFN-γ, and / or IFN-α.

[0104] 2. How to treat cancer The disclosed hydrogels and / or dry scaffolds can be used to treat, inhibit, reduce, ameliorate, or prevent cancer or metastasis. It is understood and contemplated herein that the present invention can be used for the prevention and / or prophylaxis of Disclosed herein are methods for treating, inhibiting, reducing, diminishing, or inhibiting cancer and / or metastasis in a subject. The method includes administering to a subject a hydrogel matrix as disclosed herein. and / or dry scaffold (ligand or antibody that activates immune cell receptors and immune cells is applied, including a vector encoding a chimeric antigen receptor incorporated into a scaffold, The present invention relates to a method for administering a scaffold to a subject, comprising administering to the subject a scaffold comprising a human subject, the human subject being administered a human subject, or the human subject a human subject. In such a manner, the present invention relates to a method for treating, reducing, inhibiting, ameliorating, or reducing cancer and / or metastasis in a subject. A method for reducing and / or preventing the proliferation and / or proliferation of a subject comprising the steps of: a) administering to a subject a cell (e.g., autologous, allogeneic, and T cells, natural killer (NK) cells obtained from a haploidentical donor source and / or Cells, NKT cells, macrophages, tumor-infiltrating lymphocytes (TIL), tumor-infiltrating NK cells (T T cells, including but not limited to, bone marrow infiltrating lymphocytes (INK), or bone marrow infiltrating lymphocytes (MIL), including NK cells, NKT cells, macrophages, TILs, TINKs, and / or MILs , immune cells, etc., including but not limited to, and ii) exogenous genes, siRNA, t asiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or indicates the gene, siRNA, tasiRNA, lncRNA, shRNA, mrNA, gR NA, DNA encoding miRNA (manipulated gene, siRNA, tasiRNA , lncRNA, shRNA, mRNA, gRNA, and / or miRNA, (e.g., but not limited to, chimeric antigen receptor (CAR) genes) and / or or the vector to be packaged (e.g., lentivirus, retrovirus, adenovirus, Adeno-associated virus, virus-like particle, cell mimicking particle, transposon, exosome, and b) obtaining a scaffold (e.g., nanoparticle, micelle, or liposome) containing the cells and vectors. For example, biocompatible scaffolds include, but are not limited to, alginate-containing scaffolds and hydrogels. and applying the resulting mixture to a biocompatible dry scaffold or a synthetic hydrogel matrix. c) incubating the immune cells with the hydrogel and / or the dry scaffold. d) administering the hydrogel and / or dry scaffold together with immune cells to a subject, or administering to a subject administering to a subject immune cells incubated with the hydrogel. In some aspects, the hydrogel and / or dry scaffold with immune cells is provided by: The scaffold may be administered without incubation prior to administration. In some embodiments, the scaffold is (T cell receptor, or NK cell receptor (anti-CD3 antibody, CD1d, immunoglobulin Fc or anti-Fcγ receptor (FcγRIII) antibodies, Or it may further comprise a ligand or antibody for any protein of interest.

[0105] The cells to be transduced are placed on a scaffold (any dry scaffold or hybrid scaffold disclosed herein) prior to administration. By incubating the cells with the scaffold containing the soluble collagen, the cells were able to incorporate into the scaffold and It is understood that the vector can be reliably transduced by the vector, and the vectors contemplated herein can be used in combination with the vectors contemplated herein. In one embodiment, the immune cells are administered in combination with the scaffold at least 1, 2, 3, 4, 5, 1 0, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 0, 21, 22, 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 54, 60, 66, 72, 84, 96 hours, 5, 6, 7, 8, 9, 1 In one embodiment, the cells and scaffold are incubated for 0, 11, 12, 13 or 14 days. , may be administered without incubation.

[0106] It is understood that to fully activate immune cells, costimulatory signals may be necessary; Contemplated herein. Thus, in one aspect, the present invention provides Methods of Treating, Inhibiting, Reducing, Decreasing, Ameliorating, and / or Preventing Cancer or Metastasis in a Subject Any of the above, wherein the scaffold is selected from the group consisting of anti-CD28, B7-1, B7-2, and anti-inducible co-stimulatory agent. (ICOS), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB , anti-CD40L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti- TIM-2, anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript-1 (LL T-1), anti-CD137, CD48, MICA, anti-2B4, and anti-glucocorticoid-induced Inducible tumor necrosis factor receptor-related protein (GITR), Ligands that induce signal transduction via costimulatory receptors on T cells, NK cells, or NKT cells Disclosed are methods further comprising the step of:

[0107] One advantage of the scaffold is that it can be used to deliver exogenous genes, siRNAs, tasiRNAs, lncRNAs, s hRNA, mRNA, gRNA, miRNA, and / or the siRNA, tasiRNA DNA encoding lncRNA, shRNA, mRNA, gRNA, miRNA (limited Not specified, but engineered genes, siRNAs, tasiRNAs, lncRNAs, shRs NA, mRNA, gRNA, and / or miRNA) and Transduced cells activated with a ligand or antibody against a target (e.g., immune cell receptor, etc.) It is understood and contemplated herein that the sustained release of cells (i.e., immune cells or non-immune cells) is Thus, in one aspect, the present disclosure relates to a method for treating or inhibiting cancer or metastasis in a subject. The present invention relates to a method for preventing, reducing, decreasing, ameliorating, and / or preventing the harm of a scaffold (hydrogel matrix and and / or dried scaffolds) to transduce immune cells transduced within the scaffold. The immune cells are administered to the subject at least 1, 2, 3, 4, or 5 days after administration of the scaffold at the site of the cancer. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 3 2, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 54, 60, 66, 72, 84, 96 hours, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2 3, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32, 45, 58, 59 , 60, 61, 62, 90 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 months, 4, Methods are disclosed that can be released over 5, 6, 7, 8, 9, 10 years. In this study, immune cells were detected in the grafts approximately 1 week to approximately 12 weeks after administration of the hydrogel and / or dry scaffold. The drug is released from the hydrogel and / or dry scaffold over time.

[0108] The disclosed hydrogels and / or dry scaffolds can be used to treat cancer in recipient subjects. Additional agents for stimulating an endogenous immune response may also be included. Thus, in one embodiment As used herein, the term "compounds" refers to compounds that are capable of treating, inhibiting, reducing, diminishing, ameliorating, and / or treating cancer or metastasis in a subject. or a method for preventing, comprising administering to the scaffold an anti-cancer agent (e.g., an immune-blocking inhibitor or a chemotherapeutic agent, The method further includes the steps of:

[0109] The present invention is used in the disclosed methods of treating, preventing, inhibiting, and / or reducing cancer or metastasis. The hydrogel matrix and / or dry scaffolds disclosed herein may comprise one or more immunoblocking inhibitors and / or It is understood and contemplated herein that the disclosed hydrochloride compositions may further comprise a chemotherapeutic agent. Chemotherapeutic agents that can be used in the gel matrix and / or dry scaffold include those known in the art. Any chemotherapeutic agent that has been shown to be effective in treating rheumatoid arthritis, including, but not limited to, abemaciclib, abiraterone, Acetate, Avitrexate (Methotrexate), Abraxane (Paclitaxel albumin-stabilized nanoparticle formulation, ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, ado-trastuzumab emtansine , Adriamycin (doxorubicin hydrochloride), Afatinib dimaleate, Afinito (everolimus), Aquinzeo (netupitant and palonosetron hydrochloride), Aldara ( Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alem Tuzumab, Alimta (pemetrexed disodium), Alicopa (copanlisib hydrochloride) , Alkeran for injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chloram bucil), ambochlorin (chlorambucil), amifostine, aminolevulinic acid, ana Strozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanon (nelarabine) , arsenic trioxide, Arzera (ofatumumab), asparaginase Erwinia chrysante Mi, Atezolizumab, Avastin (bevacizumab), Avelumab, Axitinib, Azacitinib Chizin, Babencio (avelumab), BEACOPP, Besenam (carmustine), Bere Odak (belinostat), bendamustine hydrochloride, BEP, Bespo Instax (inotuzumab ozogamicin), bevacizumab, bexarotene, Bexar (tocitabine) tositumomab and iodine I 131, bicalutamide, BiCNU (carmustine) ), bleomycin, blinatumomab, blinatumomab, bortezomib , Bosurif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, Mel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometiq Cabozantinib-S-malate, CAF, Campagnolo (Alemtuzumab), Camptosar, (Irinotecan hydrochloride), Capecitabine, CAP OX, Carac (fluorouracil - topical), Carboplatin, CARBOPLATIN -TAXOL, Carfilzomib, Carmbris (Carmustine), Carmustine, Cal Mustine implant, Casodex (bicalutamide), CEM, Ceritinib, Cerbidi Daunorubicin hydrochloride, Cervarix (recombinant HPV bivalent vaccine), Cetuximab , CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin , Cladribine, Clafen (cyclophosphamide), Clofarabine, Clofarex clofarabine, chloramphenicol, CMF, cobimetinib, cometri cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, COP P, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib) , crizotinib, CVP, cyclophosphamide, Cyphos (ifosfamide), Cyra Muza (ramucirumab), cytarabine, cytarabine liposome, Cytosar-U (cytarabine ), Cytoxan (cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (De Citabine), dactinomycin, daratumumab, Darzalex (daratumumab), Dasa Daunorubicin hydrochloride, Daunorubicin hydrochloride and cytarabine liposome, dextranib Tabine, defibrotide sodium, Defitelio (defibrotide sodium), Dega Relix, denileukin diffitoxib, denosumab, Depocyto (cytarabine liposomal ), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, Xyl (Doxorubicin Hydrochloride Liposomal), Doxorubicin Hydrochloride, Doxorubicin Salt Doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dom e (dacarbazine), durvalumab, efdex (fluorouracil - topical), elimination Tec (rasburicase), Elence (epirubicin hydrochloride), elotuzumab, Eloxa Tin (oxaliplatin), Eltrombopag olamine, Emend (aprepitant), Empliciti (elotuzumab), enasidenib mesylate, enzalutamide, Epirubici eribulin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, eribulin Di(vismodegib), erlotinib hydrochloride, erwinase(asparaginase erwini) Acrisanthemi), Ethyl (Amifostine), Etopofos (Etoposide phosphate), Etoposide Toposide, Etoposide phosphate, Everset (doxorubicin hydrochloride liposome), Eve Rolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), Xemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil topical ), Fareston (toremifene), Faridak (panobinostat), Faslodec Fux (fulvestrant), FEC, Femera (letrozole), Filgrastim, Rudara (Fludarabine phosphate), Fluoplex (Fluorophosphate) Fluorouracil-topical), Fluorouracil injection, Fluorouracil-topical, Flutamide, Fo Rex (methotrexate), Forex PFS (methotrexate), Forfi Forfiri-bevacizumab, Forfiri-cetuximab, Forfirinox , Forfox, Forotyn (pralatrexate), FU-LV, Fulvestran Gardasil (recombinant HPV 4-valent vaccine), Gardasil 9 (recombinant HPV 9-valent vaccine) Gemcitabine hydrochloride, Gemtuzumab o Zogamicin, Gemzar (gemcitabine hydrochloride), Giletrif (afatinib dimaleate) (imatinib mesylate), Gleevec (imatinib mesylate), Gliadel (carmustine implant) (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, glycerol acetate Cerelin, Haraben (Eribulin mesylate), Hemangeol (Propranolol hydrochloride) , Herceptin (Trastuzumab), HPV bivalent vaccine, recombinant, HPV 9-valent vaccine HPV vaccine, recombinant, HPV quadrivalent vaccine, recombinant, Hicamucine (topotecan hydrochloride), Rhea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Pa Rubociclib, ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (Po Natinib hydrochloride), Idamycin (idarubicin hydrochloride), Idarubicin hydrochloride, Idera Lisib, Idifa (enasidenib mesylate), Ifex (ifosfamide), Ifo Sulfamide, Ifosfamidam (Ifosfamide), IL-2 (Aldesleukin), Me Imatinib silate, Imbruvica (ibrutinib), Imfinzi (durvalumab), Imiquimod, Imligic (talimogene laherparepvec), Inrita (axitinib ), inotuzumab ozogamicin, interferon α-2b, recombinant, interleukin Interferon-2 (aldesleukin), Intron A (recombinant interferon α-2b), I131 Tositumomab and Tositumomab, Ipilimumab, Iressa (gefitinib), Irinotecan hydrochloride, Irinotecan hydrochloride liposomal, Istodax (romidepsin) , ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jacaf (ruxolitinib phosphate), JEB, Jevtana (cabazitaxel), Kadcyla ( Adantrastinemabuem), Keoxifene (raloxifene hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (tisagenlecleucel), Cyprolis (carfilzomib), and lanreotide acetate doxorubicin, lapatinib ditosylate, raltruvo (ora) latumab), lenalidomide, mesylate Lenvatinib, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcitonin um, Leukeran (chlorambucil), leuprolide acetate, leustatin (cladribine ), Levran (aminolevulinic acid), Lympholidine (chlorambucil), Lipodox (doxorubicin hydrochloride liposome), lomustine, Lonsurf (trifluridine and thiamine Pyracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate) ), Lupron Depo-Ped (leuprolide acetate), Lynparza (olaparib), Vincristine sulfate liposome, Matulan (procarbazine hydrochloride), Meclo hydrochloride Retamine, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride Phalan, mercaptopurine, mesna, mesnex (mesna), methazolaston (temo Zolomide), methotrexate, methotrexate LPF (methotrexate), methotrexate bromide Tilnaltrexone, Mexate (Methotrexate), Mexate-AQ (Methotrexate) Cert), midostaurin, mitomycin C, mitoxantrone hydrochloride, mitozytre (mitomycin C), MOPP, Mozobil (plelixafor), Mustagen ( Mechlorethamine hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfam Mylotarg (gemtuzumab ozogamicin), Mylosal (azacytidine), Mylotarg (gemtuzumab ozogamicin), Nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide ), neratinib maleate, Nerlinx (neratinib maleate), netupitant and Palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim) Chim), Nexavar (sorafenib tosylate), Nilandrone (nilutamide), Nilo Tinib, Nilutamide, Ninlaro (ixazomib citrate), Niraparib tosylate monohydrate hydrate, nivolumab, nolvadex (tamoxifen citrate), N-plate (Romipro stim), obinutuzumab, odomuzo (sonidegib), OEPA, ofatumumab, OF F, olaparib, olaratumab, omacetaxine mepecate, oncaspar (pegaspar Ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposome), Ntac (denileukin diftitox), Opdivo (nivolumab), OPPA, Osimertinib, oxaliplatin, paclitaxel, albumin-stabilized paclitaxel Nanoparticle formulation, PAD, Palbociclib, Palifermin, Palonoset Hydrochloride Ron, Hydrochloride Palonosetron and netupitant, pamidronate disodium, panitumumab, panobinostat Stat, Paraplatin (Carboplatin), Paraplatin (Carboplatin), Paclitaxel Zopanib, PCV, PEB, pegaspargase, pegfilgrastim, peginterf PEG-Interferon α-2b, PEG-Intron (peginterferon α-2b), Pembroliz Mab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Pla Cisplatin, Platinol-AQ (cisplatin), Plerixafor, Po Maridomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portolazza (Ne Situmumab), pralatrexate, prednisone, procarbazine hydrochloride, proleukine Aldesleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopagulola) amine), propranolol hydrochloride, Provenge (sipuleucel-T), Prinetol (melamine) Captopurine), Pulixan (mercaptopurine), radium 223 dichloride, Raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant Human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) PV) 9-valent vaccine, recombinant human papillomavirus (HPV) 4-valent V-accin, recombinant Interferon alpha-2b, regorafenib, liristor (methylnaltrexone bromide) , R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate ), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hysera (rituximab) Rituximab and hyaluronidase human), Rituximab, Rituximab, hyaluronidase Zehit, Rolapitant hydrochloride, Romidepsin, Romiplostim, Rubidomycin (hydrochloride Unorubicin), Rubraca (rucaparib camsylate), rucaparib camsylate, rucaparib phosphate Xolitinib, Redapt (midostaurin), Sclerosol intrapleural aerosol (talc) , siltuximab, sipuleucel-T, somatulin depot (lanreotide acetate), sonidegi b, sorafenib tosylate, Sprycel (dasatinib), Stanford V, sterile talc Powder (talc), Steritalc (talc), Stivarga (regorafenib), Sodium Malate Nitinib, Sutent (sunitinib malate), Silatron (peginterferon α- 2b), Silvant (siltuximab), Synribo (omacetaxine mepecinate), Broyd (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (ophthalmopathy) Simertinib), talc, talimogene laherparepvec, tamoxifen citrate, ta Rabin PFS (cyclochloride), Targretin (bexarotene), Tasigna (nilotinib) (paclitaxel), Taxotere (docetaxel), Tecentriq, (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, Lidomide, thalomid (thalidomide), thioguanine, thiotepa, tisagenlecleuce Tolac (fluorouracil - topical), topotecan hydrochloride, toremifene, trisodium Temsirolimus, Tositumomab and Iodine I and Tipiracil Hydrochloride, Trisenoc (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (Dinutuximab) ), uridine triacetate, VAC, vandetanib, VAMP, Barbi (rolapitant hydrochloride) , Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Ve Lucade (bortezomib), Versal (vinblastine sulfate), vemurafenib, Crexita (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacytidine), vinblastine sulfate, Vincristine sulfate, Vincristine sulfate, Vincristine sulfate Vinorelbine tartrate, VIP, Vismodegib, Vistogard (Uridinetriamine) acetate), boraxase (glucarpidase), vorinostat, votrient (pazopa Nib hydrochloride), Vyxeos (daunorubicin hydrochloride and cytarabine liposome), Well Covorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine) Zerili, Xerox, Zugeba (denosumab), Zofigo (radium 223 dichloride) , Zutanzi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabectate) Zin), Zaltrap (Ziv-aflibercept), Zalxio (filgrastim) , Zejula (niraparibut tosylate monohydrate), Zelboraf (vemurafenib), Zevali ibritumomab tiuxetan, Zinecard (dexrazoxane hydrochloride), Ziv- Aflibercept, Zofran (ondansetron hydrochloride), Zoladex (gossereli acetate) Zolin (zoledronic acid), Zolinza (vorinostat), Zometa (zoledronic acid), Zyderi (idelalisib), Zykadia (ceritinib), and / or Zytiga (abiraterone acetate). Checkpoint inhibitors include PD-1 (e.g., nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), P D-L1 (e.g., MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (e.g., rHIgM12B7), CTLA -4 (e.g., ipilimumab (MDX-010), tremelimumab (CP-675, 20 6), IDO, B7-H3 (e.g., MGA271, MGD009, ombaltamab) , B7-H4, B7-H3, Ig and ITIM domain-containing T cell immunoreceptor (TI GIT) (e.g., BMS-986207, OMP-313M32, MK-7684, A B-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96 , and T-lymphocyte attenuator (BTLA), a V-domain Ig suppressor of T cell activation. VISTA (e.g. JNJ-61610588, CA-170, etc.), TI M3 (e.g., TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661) , LAG-3 (e.g., BMS-986016, LAG525, MK-4280, REG N3767, TSR-033, BI754111, Sym022, FS118, MGD0 13, and Immutep), as well as antibodies that block PD-1, CTLA-4, L AG-3, TIGIT, CD96, BTLA, B7-H3, VISTA, and TIM-3 Antibodies that block ligands that stimulate PD-L1, fibronectin, and the like (e.g., but not limited to, PD-L1, fibronectin, and the like) FGL1, CD112, CD155, herpes virus endonucleases of hepatic vascular mediator (HVEM), and Ceacam-1, and their respective receptors Antibodies that block binding to the body.

[0110] The compositions of the present disclosure can be used to treat any disease in which unregulated cell proliferation occurs, such as cancer. Representative, but non-limiting, examples of conditions that can be treated using the disclosed compositions include: The list of typical cancers is as follows: Lymphoma, B-cell lymphoma, T-cell lymphoma. , mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer , squamous cell carcinoma of the head and neck, lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma Breast cancer / glioma, ovarian cancer, skin cancer, liver cancer, melanoma, oral, pharyngeal, and laryngeal cancer, and Squamous cell carcinoma of the lung, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, kidney cancer, urinary Reproductive cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon cancer, Rectal, prostate, or pancreatic cancer. EXAMPLES

[0111] The following examples will enable one of ordinary skill in the art to easily understand the compounds, compositions, articles, and devices claimed herein. The present disclosure provides a complete disclosure and description of the methods and / or methods of making and evaluating the These are intended to be purely illustrative and not limiting of the present disclosure. Efforts have been made to ensure accuracy with respect to values ​​(e.g. amounts, temperatures, etc.). However, some errors and deviations should be accounted for. Parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is atmospheric or below. It is near.

[0112] 1. Example 1: Scaffold-Mediated Static Engineering of T Cells for CAR-T Cell Therapy G The present inventors have developed a method for engineering cells with vectors (e.g., T cells with RV vectors). Three-dimensional (3D) macroporous scaffolds as efficient single-step static platforms The inventors have developed a three-dimensional scaffold with macroporosity and high water absorption capacity that can Similar to that achieved by using nectins and spinoculation, The present inventors have discovered that the colocalization of the viral vector and T cells is possible by using a calcitonin-containing IgG antibody. A macroporous scaffold was prepared from sodium cross-linked alginate, which has been shown to be biocompatible, Due to their low toxicity, low cost, and gentle gelation by divalent cations, they are widely used in many biomedical applications. It is a GMP-compliant, FDA-approved biomaterial that is widely used for various applications. The dry, hygroscopic and macroporous alginate scaffold contains vectors and cells (e.g., RV vectors). and T cells) and without spinoculation Single-stage T cell proliferation without affecting the function and viability of cells (e.g., engineered T cells) We observed that these scaffolds enabled efficient gene transfer in a single scaffold. A simple, cost-effective, and affordable method to generate highly functional T cells for adoptive cell therapy Represents a scalable platform technology.

[0113] Macroporous alginate scaffolds were prepared by gentle cryogelation (Figure 1A). SEM imaging revealed well-connected 100–20 dendritic cells throughout the scaffold. RV particles were used to transduce human T cells. We tested alginate scaffolds for the introduction of erythrocytes into the scaffolds. The T cells were then activated with agonistic anti-CD3 and anti-CD28 antibodies. RV-RetroNectin-coated T cells were cultured using conventional spinoculated RV-RetroNectin-coated RV particles were seeded on either plates, or alginate scaffolds. Transduction at multiplicities of infection (MOI) of 1, 2, and 4 with the retrovirus encoding Preliminary screening of transfection efficiency demonstrated efficient transduction (Figure 3A), and an MOI of 2 was further increased. As shown in Figure 1D, 72% of the cells were incubated with RV particles. After 2 h, GFP-expressing T cells on the scaffold were retronecrotized by spinoculation. The results were comparable to those in the GFP-coated plates (85±3% vs. 90±5% GFP). + Thin These results are indicative of a significant improvement in the efficacy of IFN-γ in both groups compared with viral transduction in preliminary experiments. showed improved transduction in the 3G-T cells (Figure 3A, 49% vs. 73.5%, respectively). There was little variation across the three different PBMC donors (Figure 1). 1D).

[0114] To evaluate whether the macroporosity of the scaffold affects the interaction of RV particles with T cells, To achieve this, the inventors demonstrated that RV particles and T cells can be adsorbed, but the T cells cannot attach to the scaffold. We created a nanoporous scaffold that lacks macroporosity to allow RV particles to enter (Figure 1E). Test whether the sponge-like effect was also necessary to create proximity between the child and T cells To achieve this, we have developed a membrane that has large pores but does not allow active flow of fluid into the inner pores. We tested hydrated macroporous scaffolds without any hygroscopic properties. We also tested dry nanoporous scaffolds. Both the hydrated macroporous gel and the dry macroporous scaffold showed efficient gene transfer. We found that transduction was not possible (Figures 1F and 1G).

[0115] Next, CAR-T cells generated using the dried macroporous scaffolds were tested in vitro. These experiments evaluated whether the CD19 antigen (CD19.CAR) could function as a target for the CD19 antigen. We used RV particles encoding heterologous CARs. Activated CARs obtained from three healthy donors were T cells were cultured on RV particle-loaded alginate scaffolds or retronectin-coated plates. Upon removal from the scaffold or RetroNectin, T cells were maintained in culture and CAR expression was analyzed on days 3 and 9 after transduction. As shown in Figure 2A, the scaffold Both retronectin and retronectin promoted equivalent transduction efficacy, and CAR expression was significantly increased during proliferation at day 9. The results remained unchanged until 2013 (Fig. 2A, Fig. 3B). The immune composition of CAR-T cells was also analyzed. There were no differences in expression of markers of fatigue (PD-1, Lag3) or inflammatory bowel disease (CRD1, CCR7, 45RA, CCR7) or fatigue (PD-1, Lag3) No significant difference was observed (Figures 4 and 5). Next, to evaluate the antitumor effect in vitro, Generated by either scaffold-mediated or retronectin-mediated transduction Control non-transduced T cells (NT cells) and CD19.CAR-T cells were The cells were co-cultured with CD19 positive target cells (Daudi) and CD19 negative target cells (U937). While the cells did not eliminate any of the tumor cells, the cells CD19.CAR-T cells transduced with + Eliminate Daudi cells (Fig. 2E and Fig. 6), but CD19 - U937 cells did not exclude (Figure 7). From the culture experiment, cytokines were measured in the supernatants collected after 24 hours of incubation. In response to Daudi cells, CD19.CAR-T cells secreted IL-2 and interferon. Finally, both the scaffold and retronectin methods released IFN-γ. CD19.CAR-T cells generated using this method responded equally well to Daudi cells. The alginate scaffolds showed high proliferation capacity (Figure 2B). We show that it generates functional CAR-T cells.

[0116] The antitumor activity of CAR-T cells observed in vitro was confirmed in an in vivo tumor model. Daudi cells labeled with firefly luciferase (FfLuc) were used in non-obese mice. Intravenously (i.v.) injected into diabetic severe combined immunodeficiency / γc- / - (NSG) mice for 4 h. Mice were treated with control non-transduced (NT) cells or scaffold or retronectin transduced cells for 14 days. The CD19.CAR-T cells generated by CAR-T cell transduction were intravenously injected into the The bioluminescence intensity of the luminescence generated by either method was evaluated. CD19.CAR-T cells controlled tumor cell proliferation (Figures 8B, 8C, and 9). Tumor control correlated with improved overall survival (Figure 8E), as assessed by changes in body weight. There was no significant toxicity (Figure 8D). Therefore, the scaffold-generated CAR-T cells CAR-T cells generated by a conventional retronectin / spinoculation approach The cells functioned similarly in vivo.

[0117] In conclusion, we have demonstrated that biocompatible macroporous alginate scaffolds can Due to its porosity and hygroscopicity, it is difficult to obtain a retrovirally engineered fibronectin, similar to the commercially available RetroNectin. This demonstrates that the dry cell therapy developed by the present inventors is effective in generating T cells that have been Dried scaffolds were plated coated with Retronectin and seeded with retroviral particles. This eliminates the need for spinocutting, thereby simplifying the manufacturing process. CAR-T cells generated through static transduction on chloroporous alginate gels These data suggest that highly functional T cells could be used for adoptive cell therapy. To enable the generation of This simple platform has also been shown to be effective in other intractable cell types. This would enable addressing the need for a convenient and efficient transduction method.

[0118] a) Materials and methods (1) Preparation of macroporous alginate scaffolds Macroporous alginate scaffolds were prepared using 2% ultrapure alginate (Pronov a, MVG) aqueous solution was vigorously stirred with 4% calcium gluconate for 15 minutes. Then, The resulting mixture (final calcium concentration 0.01 M) was plated in a 24-well plate (1 mL / well). The scaffolds were cast in PBS, frozen at -20 °C overnight, and lyophilized. The scaffolds were either used immediately or or stored at 4°C prior to use in in vitro or in vivo experiments.

[0119] (2) Preparation of dry nanoporous alginate scaffolds Cross-linked calcium alginate gels were cast into 24-well plates and allowed to gel overnight. The gels were then washed with increasing concentrations of ethanol (10, 30, 50, 70, 90, and The extract was subjected to multiple solvent exchanges with 100% v / v water and subsequently dried with supercritical CO2.

[0120] (3) Scanning electron microscope observation: Cut the dried macroporous scaffold with a sharp razor and apply 70 nm of AuPd (Au:6 0%, Pd:40%) for 10 minutes at 7 nm / min. The surface morphology of the nanoporous scaffolds was analyzed by field emission scanning electron microscopy. The results were analyzed using a FESEM (Verios FE1).

[0121] (4) Cell line and retronectin-mediated generation of CAR-T cells. Daudi cells expressing firefly luciferase were cultured in 10% fetal bovine serum (Gibco ), 2 mmol / L GlutaMax (Gibco), penicillin (100 units / mL ), and streptomycin (100 mg / mL; Gibco). All cells were maintained in 5% CO2 at 37°C. The T cells expressing CAR-T cells will be used to manufacture CAR-T cells for clinical use at UNC-CH. Peripheral blood mononuclear cells were generated according to standard operating procedures currently used for the detection of lymphoma. Hoprep medium (Accurate Chemical and Scientific Buffy Coat (Gulf Coast Reg Corporation) The mice were isolated from the Milton Blood Center and treated with 1mg / mL CD3 (Milt enyi Biotec) and CD28 (BD Biosciences) monoclonal antibodies The cells were activated on plates coated with either GFP or CD19.CA antibodies. Retroviruses encoding R were prepared according to a previously reported method. was measured by standard flow cytometry assay. Serially diluted virus stocks was added to HEK293T cells. After 72 hours, GFP expression was analyzed by flow cytometry. The population with 5-20% GFP+ cells was used to analyze the GFP expression using the following formula: The viral titer (transducing units / mL) was calculated: Titer (TU / mL) = (initial cell number x % of GFP+ / (virus volume × dilution factor). MOI is the amount of transducing virus used The number of activated T cells was calculated as the ratio of the number of particles to the actual number of cells. GF on a 24-well plate (Takara Bio Inc.) coated with The mice were transduced with retroviral supernatants encoding P or CD19 CAR. Two days after transduction, the transduced T cells were cultured in 10% HyClone fetal bovine serum (GE HeLa)-treated 10-well plate. althcare), 2 mmol / L GlutaMax (Gibco), penicillin ( 100 units / mL), and streptomycin (100 mg / mL, Gibco), and supplemented with 10 ng / mL IL7 and 5 ng / mL IL15 (PeproTech). 50% Click medium (Irvine Scientific) and 50% RPMI- After proliferation in 1640 for 10-14 days, they were used for functional assays.

[0122] (5) Scaffold-mediated generation of CAR-T cells. Containing a gamma retrovirus encoding GFP or encoding human CD19.CAR The retroviral supernatant was centrifuged at 4°C, 2500g, for 15-20 minutes using an Amicon centrifuge (M The dried alginate foot was concentrated 20-fold using WCO100Kda (Milipore). The field was transferred to a non-tissue culture coated 24-well plate (Falcon). Concentrated retrovirus in μl of medium (2 mL of viral supernatant concentrated to 200 μL) (those obtained by multiplying the 6 Human PBMCs (isolated from buffy coats) were 1 x 10 cells suspended in cell culture medium were placed on the scaffold with a pipette. 6 Control leg with PBMCs The seeded scaffolds were incubated in a 5% CO2 incubator at 37°C without any additional medium. After incubation in 0.5% CO for 1 hour, 1 ml of complete medium was added. After 3 days of culture, Isolate cells from scaffolds by digestion with 125M EDTA and analyze by flow cytometry. Cells were analyzed for GFP expression or CD19.CAR expression by immunofluorescence.

[0123] (6) Flow cytometry and antibodies Human CD3 (APC-Cy7, 557832), CD4 (APC-Cy7, 56183 9), CD8(PerCP-Cy5.5,565310), CD20, CD45RA(P E,555489), CD62L(BV421,563861), LAG3(PE,56 5617), PD-1 (FITC, 561035), and TIM3 (BV421, 565 Monoclonal antibodies specific for 563) were purchased from BD Biosciences. The one for CCR7 (FITC, FAB197F-100) was from R&D System The anti-idiotypic scFv monoclonal antibody was purchased from Sigma-Aldrich Co., Ltd., and used to identify CD19. CAR expression was detected. All samples were analyzed using a BD LSRII, with 1000 bp per sample. We obtained at least 10,000 events from each event. The analysis was performed using the Sigma-Jo LLC.

[0124] (7)Cytokine production by CAR-T cells. CAR-T cells were co-cultured with Daudi tumor cells in a 1:5 effector to target [E:T The cells were co-cultured at a ratio of 1:1 for 24 hours, and the culture supernatant was collected. IL-2 and IFN-γ were added according to the manufacturer's instructions. Quantification was by ELISA using a protocol (R&D Systems).

[0125] (8) In vitro cytotoxicity. Tumor cells (Daudi) were cultured at 1 × 10 per well. 5 cells in a 24-well plate CAR-T cells, normalized for transduction efficiency, were added at an E:T ratio of 1:5. On the fifth day of co-culture, cells were harvested and the T cells and residual tumor ( The frequency of CD20+ cells was measured.

[0126] (9) In vivo antitumor activity. 8-10 week old female non-obese, diabetic, severe combined immunodeficiency / γc- / - (NSG) mice Mouse, 1 x 10 6 ffLuc-expressing Daudi cells were injected intravenously. Days later, each mouse was inoculated with 4 × 10 6 CD19.CAR-T cells or non-transduced (NT ) cells were injected intravenously. Tumor burden was assessed using Xenogen-IVIS imaging. The mice were monitored for signs of discomfort and early signs of Animals were euthanized when they lost more than 15% of their body weight or developed hind limb weakness. The procedures are described in detail in the Institutional Animal Care and Use Center at the University of Tokyo. This was carried out in accordance with the Committee.

[0127] (10) Statistical analysis. All statistical analyses were performed using Tukey post-hoc analysis using GraphPad Prism. The results were analyzed using two-way Student's t-test, one-way analysis of variance, or two-way analysis of variance. The results are shown in the figures as *=p<0.05, **=p<0.01, and ***p<0.001.

[0128] 2. Example 2: Promoting CAR-T cell therapy with multifunctional biomaterial scaffolds CAR T cell therapy has shown unprecedented success against CD19-expressing B cell malignancies It has led to two FDA approvals and stimulated hundreds of ongoing clinical trials to treat human malignancies. Despite the groundbreaking potential of CAR-T cell therapy to treat rheumatoid arthritis, The complex procedures and costs required to produce RT cells have limited their widespread clinical use. The manufacturing of therapeutic CAR-T cells involves a number of steps, including: Steps need to be taken: (i) collection of T cells from patients via leukopheresis, and production (ii) shipping the vectors to a center to activate, expand, and manipulate T cells; (iii) labor-intensive procedures under Good Manufacturing Practice (GMP) conditions for the production and (iv) shipping the final CAR-T cell product to hospitals and patients. The full manufacturing process can cost up to $500,000 and take several weeks to complete. This delay may give the aggressiveness of many cancers enough time to complete their production. Moreover, extensive ex vivo culture of T cells is problematic because it may not be possible to This leads to cytoplasmic differentiation, which impairs CAR- This impairs the efficacy of T cells.

[0129] More rapid methods for generating CAR-T cells with high therapeutic potential are desperately needed. To reduce the effort required to produce CAR-T cells ex vivo, , Closed automated manufacturing facilities are in operation, but this approach still requires some Allogeneic, off-the-shelf CAR-T cells require ex vivo cell engineering for individual patient-specific C This could help meet the need for AR-T cell manufacturing, but often comes at the cost of life-threatening graft versus placebo. They may cause host disease or may be rapidly eliminated by the host's immune system. Systemic delivery of CAR constructs to T cells in vivo using nanoparticles or lentivirus However, this approach is limited by the short plasma half-life of the virus injected into the system. The drug is limited by its shelf life and non-specific targeting of nanoparticles. In situ generation of CAR-T cells by ex vivo engineering remains highly attractive Such an approach would largely eliminate the need for ex vivo culture and would allow for the rapid development of Prevents terminal differentiation of CAR-T cells and promotes autologous rather than allogeneic CAR-T cell products Promotes the use of T cells.

[0130] Provided herein is in vivo CAR-T cell generation with minimal ex vivo manipulation. All-in-one macroporous alginate scaffold (MAS) for T cell engineering and delivery TER, also referred to herein as CCI-Alg). Patient-derived T cells and viral particles encoding CARs were directly loaded and transplanted on the same day. CAR-T cells can be generated in vivo (Figure 22). MASTER is as follows: The antibodies are designed to: (i) retain T cells and viral particles; (ii) activate T cells. (iii) stimulate T cell transduction and proliferation; and (iv) promote full functioning T cell proliferation. The aim of the study was to develop a method to treat tumors by continuously releasing functional CAR-T cells into the circulation to control tumor growth.

[0131] Alginate is a material that is biocompatible, biodegradable, has gentle gelling requirements, and is suitable for cell culture. These multifunctional scaffolds (MASTER, herein) have been widely applied as 3D structures for The scaffold was chosen as the basis for T cell activation (also called CCI-Alg in the literature). To promote cell proliferation, cyclooctyne-conjugated anti-CD3 and anti-CD28 antibodies were administered in a gentle manner. Immobilized on azide-modified alginate via a simple and efficient copper-free click chemistry reaction (Figure 10). The cytokine IL2 was administered to the scaffold to activate and expand T cells within the scaffold. Finally, gentle cryogelation was used to mechanically encapsulate the alginate scaffold. The construction of large pores throughout the scaffold was the first step in the construction of the scaffold. In addition, it provides an interface for efficient contact between cells and the retrovirus, which then propagates. These are important for providing an interface for the mass transfer of nutrients to the CAR-T cells. Figure 1 As shown in 1B, the SEM image of MASTER shows well-connected structures throughout the scaffold. The distribution of cells in the scaffold was examined using AF-647-labeled pores. GFP-expressing T cells were seeded onto the MASTER. 3D confocal imaging Microscopic observation revealed that T cells (approximately 12 μm in diameter) were uniformly distributed throughout the pores of the scaffold. It was shown (FIG. 11C, FIG. 12).

[0132] Efficient T cell activation and transduction are essential for the production of CAR-T cells. STER-loaded T cells are required for proper activation of retroviral-mediated gene transfer To ensure receipt of the cue, an agonist anti-CD20 conjugated to the MASTER 3 antibody and anti-CD28 antibody (1:1 w / w per mg alginate at 0, 0.5, 1 Increasing doses of 100 μg each (1, 2, or 4 μg) were tested. Human peripheral blood mononuclear cells from three different donors were used. PBMCs were seeded into MASTER and 18 hours later, the early T cell activation marker CD6 As shown in Figure 11D, cells seeded on MASTER expressed the antibody 9. Approximately 10-fold increase in CD69 expression compared to cells seeded on alginate scaffolds without lysosomes Maximal CD69 expression was achieved at an antibody concentration of 1 μg / mg alginate. This density was used for further studies. To test activation and transduction, we used naive PBMCs and GFP-encoding retroviruses. The antibody was added to the MASTER (Figure 11E) and to a scaffold control lacking antibody, resulting in The CAR-T cell population was then cultured using conventional spinoculation (RetroNectin-coated The results were compared to those made using a 72-hour inkjet printer (centrifugation onto a plate with ink on it). After incubation, GFP expression was measured by incubating non-activated PBMCs and the virus on blank scaffolds. It was barely detectable when incubated or spinoculated (0 In contrast, incubation of non-activated PBMCs on MASTER resulted in 20% transduction of T cells (Figures 11F and 11G), and MASTER A platform for both T cell activation and static transduction via retroviral gene delivery This shows that it functions as a form.

[0133] Next, the functionality of CAR-T cells generated in MASTER was confirmed by using CD19-specific CAR The study was performed using a retrovirus encoding (CD19.CAR). D19.CAR gamma retroviral particles were loaded into MASTER and incubated for 3 or 10 days. As shown in FIG. 13A, CAR expression was increased in 22%±1% of T cells at day 3. The T cell phenotype was detected in CA1 cells and remained stable at day 10 (Figure 14). As a key determinant for successful engraftment and persistence of RT cells, MASTE The generated CAR-T cells were analyzed for phenotypic composition and compared with conventional clinical procedures (activated T cells) Spinoculum of cells and retroviruses on retronectin-coated plates The results were compared with CAR-T cells generated using a 3D fusion technique. The cells expressed similar amounts of CD4 + and CD8+ T cells (Figure 13B). MASTER-generated CAR-T cells have a higher CCR7 activity than conventionally generated CAR-T cells. + CD 45RA + Central memory T cells (14.8% vs. 2.57%), CCR7 + CD45 RA + Stem cell-like T cells (21.5% vs. 9.8%) and have lymphocyte homing capacity CCR7 + CD62L + The proportion of T cells (13.1% vs. 2.98%) was higher (Figure 13C , Figure 15). Characterization of the expression of fatigue markers PD-1 and LAG3 on day 12 was performed. However, less than 1% of CAR-T cells generated by either method expressed PD-1 + LAG3 + In addition, we showed that the expression of β-lactamase in three cell types (untransduced cells, MA STER cells and conventionally produced CAR-T cells) all express CD19 + Tumor cells The cells showed comparable proliferation capabilities in response to IFN-α / α-glucose (Figure 13E).

[0134] To evaluate the in vitro antitumor effect, control non-transduced T cells (NT cells) were used. ) and CD19 generated by either MASTER or spinoculation. CAR-T cells were + Target cells (Daudi) and CD19 - Target cells (U93 7) at an effector-to-target ratio (E:T) of 1:5. NT cells were cultured with tumor cells While it did not eliminate any of the inflammatory cytokines produced by MASTER cells or spinoculation, The CD19.CAR-T cells produced + Cells were eliminated, but CD19 - In these co-culture experiments, CD19 cells were not excluded (Fig. 13F, Fig. 16). .CAR-T cells are CD19 + Cells respond to IL-2 and interferon IFN- γ was released (Fig. 13G and 13H). Highly functional CAR-T with improved differentiation phenotype compared to conventionally produced CAR-T cells It is shown that the cells are produced.

[0135] Next, we evaluated whether MASTER releases resident T cells. Then, PBMCs (1 × 10 6 ) onto transwell inserts ( At a given time point, each scaffold was transferred to a new well and placed in the bottom chamber. The MASTER efficiently released cells over a 5-day period. We have demonstrated that the specific migratory properties of activated T cells and their ability to bind to the scaffold are well known (Figure 17). This is likely due to the macroporous structure.

[0136] All-in-one platform for T cell activation, transduction, expansion, and release Motivated by the above results demonstrating the function of MASTER as a marker for MASTER-produced CA The antitumor activity of RT cells was tested in an in vivo xenograft tumor model. Retroviral particles encoding MC and CD19.CAR were seeded and incubated for 1 hour. The mice were then transplanted subcutaneously into NSG mice engrafted with Ffluc-labeled Daudi cells. Tumor-bearing mice treated with conventionally generated CAR-T cells injected intravenously served as positive controls. The mice were subcutaneously implanted with MASTER seeded with PBMCs alone (Figure 18A). Mice injected intravenously with transduced or non-transduced cells served as negative controls. Tumors grew rapidly in mice treated with untransduced cells, whereas MASTER The generated CAR-T cells and conventional intravenously infused CAR-T cells showed a significant difference in body weight. The tumor progression was controlled equally up to day 45 without any changes (Figures 18B-18D, Figure 19). At day 100, intravenous infusion of 4 million conventionally produced CAR-T cells This resulted in a tumor-free survival rate of 16.6%. In contrast, MA with 2 million PBMCs Implantation of STER increased tumor-free survival by 50% (p=0.15, FIG. 18E). The in vivo persistence of CAR-T cells was also examined. On day 32, when the control mice reached the humane endpoint, each of the CAR-T treatment groups Three mice were euthanized and blood, bone marrow (BM), and spleen were collected. CD45 + CD3 + CAR + Surprisingly, MA cells were analyzed (Figure 20). STER-generated CAR-T cells showed nearly 30-fold higher absolute concentration in peripheral blood at day 22. (FIG. 18F) and in the bone marrow and spleen at day 32, the conventional The increase was markedly greater than that of CAR-T cells (Figures 18G and H, Figure 21). In this study, bone marrow-isolated CAR was used because bone marrow is the primary site of lymphoma. + Cells, The expression of fatigue markers PD-1 and LAG3 was further analyzed. The released CAR-T cells were then expanded ex vivo and administered intravenously to the CAR-T cells. The expression of PD-1 and LAG-3 was significantly lower in these tumors compared with the tumors. The results showed that CAR-T cells generated using MASTER were effective in controlling tumor growth. They function similarly to conventional CAR-T cells, but have better spreading and persistence, and are easier to manufacture. This demonstrates a significant reduction in manufacturing time, complexity, and cost.

[0137] In summary, we have addressed one key aspect of CAR-T cell production and delivery: They generated a multifunctional scaffold that was integrated into a single platform, shortening the entire process to one day. The field contains anchor anti-CD3 and anti-CD28 antibodies and mAbs that ensure proper T cell activation and proliferation. The macroporosity promotes uniform distribution of T cells and the transport of viral particles. Fully functionally reprogrammed CAs form an interface for interaction between T cells The MASTER system supports the in vivo release of exogenously seeded RT cells. Instead, MASTER is not limited to local release of CAR-T cells, but instead is highly functional. To provide in situ reprogramming and release of effective CAR-T cells, This is a major improvement over current biomaterials. Furthermore, MASTER has the potential to reprogram other immune cells. or deliver immune modulators to synergistically support cellular function. It is a modular platform technology that can be deployed in a variety of ways.

[0138] a) Experimental section (1) DBCO modification of antibodies 10-fold molar excess of NHS-PEG4-DBCO (A134-10, a click chemistry tool) (Clinical Cell, Inc., USA) was added to anti-CD3 or anti-CD28 antibody (Figure 11) and incubated at room temperature for 1 hour. The mixture was then incubated at 4°C until the flow-through was free of NHS-PEG4-DBCO. , 10000g, 10 min (DBCO fraction at 309 nm using Nanodrop) The solution was centrifuged using an Amicon centrifuge (MWCO 10 kD The purified conjugate was purified by the absorbance scan (2 35–400 nm) to the extent of DBCO incorporation (i.e., number of DBCOs per antibody). ) was decided. (molar concentration of DBCO) / (molar concentration of antibody)=(A_309 DBCOxε_280 Ab) / (ε_309 DBCOxA_280c Ab) A309 DBCO = absorbance at 309 nm of DBCO-Ab conjugate ε280 Ab=210,000M-1cm-1 A280C Ab = corrected absorbance of the conjugate at 280 nm = A280-(A 309×CF DBCO) ε309 DBCO=12000M-1cm-1 A280Ab = absorbance at 280 nm of DBCO-Ab conjugate CF DBCO = DBCO correction factor at 280 nm = 1.089

[0139] (2) Preparation of CCI-Alg (MASTER) Azide-modified alginate was prepared. A 2% (w / v) solution of azidoalginate in grade water was used to study the DBCO-modified CD3 and CD The cells were incubated with antibody 28 (1 μg / mg alginate) at 4° C. overnight. Recombinant human IL2 was added at a concentration of 0.2ug / mg alginate and the solution was stirred for 15 minutes. Finally, the resulting solution was vigorously mixed with an equal volume of 4% calcium gluconate for 15 min. Mix thoroughly, cast into 24-well plates (1 mL / well), and freeze overnight at -20°C. The MASTER was used immediately or stored at 4°C for in vitro or used within one week for in vivo experiments.

[0140] (3) Scanning electron microscope observation (SEM): The dried macroporous scaffolds were cut with a sharp razor blade and coated with 70 nm of AuPd (Au : 60%, Pd: 40%) was measured for 10 minutes at 7 nm / min. Analyzed with a 0N variable pressure SEM.

[0141] (4) Confocal microscopy: AF647 DBCO (click chemistry tool) modified with anti-CD3 and anti-CD28 antibodies The excess of unbound dye was removed by permeabilization with water for 1 day. The AF647-labeled scaffolds were prepared as described above. 6 GFP+ T cells were seeded onto these scaffolds and incubated overnight at 37°C and then incubated for 1 h at 4°C for 1 h. ssImaged using an LSM880 confocal microscope.

[0142] (5) Generation of cell lines and CAR-T cells Daudi cells expressing firefly luciferase were cultured in 10% FBS (Gibco), 2 mmol / L GlutaMax (Gibco), and penicillin (100 units / mL) , and RPMI 164 supplemented with streptomycin (100 mg / mL, Gibco). 0 (Gibco). All cells were maintained at 37° C. and 5% CO2.

[0143] (6) Generation of conventional CAR-T cells Peripheral blood mononuclear cells were cultured in Lymhoprep medium (Accurate Chemical Buffy coat (Guid Scientific Corporation) was used lf Coast Regional Blood Center) and 1 mg / mL CD3 (Miltenyi Biotec) and CD28 (BD Biosci) The antibodies were activated on plates coated with monoclonal antibodies. Two days after activation, the isolated T cells were plated onto RetroNectin-coated 24-well plates. The cells were transduced with the retroviral supernatant on a 30-well plate (Takara Bio Inc.). Transduced T cells were incubated with 10 ng / mL IL7 and 5 ng / mL IL15 (Pepro Tech) for 10–14 days, and then incubated with 10% HyClone FBS (GE Healthcare re), 2 mmol / L GlutaMax (Gibco) and penicillin (100 units 50% clones supplemented with streptomycin (100 mg / mL, Gibco) and streptomycin (100 mg / mL, Gibco). in Lick's medium (Irvine Scientific) and 50% RPMI-1640 After spreading, they were used in functional assays.

[0144] (7) Scaffold-mediated generation of CAR-T cells Gamma retroviruses encoding GFP or encoding human CD19.CAR (RV ) was centrifuged at 4°C, 2500g, 15-20 min, and centrifuged at 10°C for 10 min at 37°C. (MWCO 100Kda, Millipore) was used to concentrate 10 times. Drying: Freeze-drying MASTER scaffolds were cultured in non-tissue culture coated 24-well plates (Falcon). Transfer 200 µL of concentrated RV and 1 x 10 isolated from the buffy coat. 6 PBMCs (Total volume of virus and PBMC = 300 μl) was pipetted onto each scaffold. 1 x 10 cells suspended in nutrient medium 6 Control scaffolds were seeded with 10 PBMCs. Only in these cases, the seeded scaffolds were incubated at 37 °C in a 5% CO2 incubator without additional medium. The cells were incubated for 1 hour and then 1 ml of complete medium was added. Isolated from scaffolds after 96 hours by digestion with DTA and washing twice with excess PBS. , analyzed for GFP expression or CD19.CAR expression by flow cytometry. For in vivo studies, encode PBMCs and CD19.CAR in MASTER. The retrovirus was seeded and incubated at 37°C for approximately 1 hour until the medium was completely absorbed into the scaffold. The mice were then immunized with IgG and implanted into the subcutaneous space of NSG mice on the same day.

[0145] (8) Flow cytometry and antibodies Human CD3 (APC-Cy7, 557832), CD4 (APC-Cy7, 56183 9), CD8 (PerCP-Cy5.5,565310), CD20 (FITC,555 622), CD45RA(PE,555489), CD62L(BV421,56386 1), LAG3 (PE, 565617), PD-1 (FITC, 561035), and T A monoclonal antibody specific for IM3 (BV421,565563) was purchased from BD Bios. The antibody against CCR7 (FITC, FAB197F-100) was purchased from Science. It was purchased from R&D Systems. Anti-idiotypic scFv monoclonal Antibodies were used to detect expression of CD19.CAR. All samples were analyzed using the BD LSRII. Samples were run using FlowJo9 ( The analysis was performed on FlowJo LLC.

[0146] (9) Cytokine production by CAR-T cells CAR-T cells were co-cultured with Daudi tumor cells in a 1:5 effector to target [E:T The cells were co-cultured at a ratio of 1:1 for 24 hours, and the culture supernatant was collected. IL-2 and IFN-γ were added according to the manufacturer's instructions. Quantification was performed by ELISA according to the protocol (R&D Systems).

[0147] (10) In vitro cytotoxicity Tumor cells (Daudi) were added at 1 × 10 per well. 5 Cells seeded in 24-well plates at CAR-T cells, normalized for transduction efficiency, were added at an E:T ratio of 1:5. On day 5 of co-culture, cells were harvested and the T cells and residual tumor (CD The frequency of 20+) cells was measured.

[0148] (11) In vitro T cell release assay: 1×10 on scaffolding 6 PBMCs were seeded onto a 40 μm cell mesh (Corning). The mesh was used as a transwell insert and placed in a 6-well plate. The bottom of the well contained complete T cell medium. At the indicated time points, the bottom chamber The cells in the inserts were counted and the scaffolds with inserts were transferred to new wells containing fresh medium. It moved.

[0149] (12) In vivo antitumor activity 10-12 week-old female immunodeficient NSG mice (NOD.Cg-Prkdc scid Il 2rg tm1WJl / SzJ), 1 × 10 6 Daudi cells Four days after injection, each mouse (n=9) was given two MASTERs by subcutaneous injection. Each scaffold was implanted with 1 × 10 6 PBMCs (isolated from human buffy coats, 2 (different donors) and loaded with a gamma retrovirus encoding CD19.CAR, The same day, the mice were subcutaneously implanted with 4 × 10 6 Intravenously inject either non-transduced (NT) T cells 1 × 10 6 MA seeded with PBMCs only (no retrovirus) Mice transplanted with STER were used as negative controls. 4×10 6 Mice injected with CD19 CAR T cells served as positive controls. Tumor burden was monitored using the Xenogen-IVIS imaging system. Monitor the mice for signs of discomfort, including loss of more than 15% of their initial body weight, hind limb weakness, and / or other symptoms. Mice were euthanized when a humane endpoint was reached based on the onset of rheumatoid arthritis or tumor burden. All procedures involving animals were performed by the Institutional Animal Care Center of the University. This was done in accordance with the e and Use Committee.

[0150] To analyze CAR+ cells in blood, bone marrow, or spleen, single cells from these organs The suspension was stained with anti-human CD45, anti-human CD3, and CAR.19 antibodies and analyzed by flow cytometry. The organs were minced through a 70 μm tissue mesh and analyzed by ELISA. A single cell suspension of spleen was obtained by resuspending splenocytes in ACK lysis buffer (Lonza). Lyse red blood cells by flushing the bone with a syringe containing PBS. A single bone marrow cell suspension was obtained by passing the cell suspension through a 70 μm cell mesh. erythrocytes were isolated by resuspending the cells in lysis buffer (BD Pharm Lyse). Similarly, the blood drawn from the cheek was dissolved in lysis buffer (BD Pharm Lyse). After incubation with 0.05% CO, the cells were washed with PBS, passed through a 70 μm cell mesh, and then incubated with antibodies. Due to failure to collect cheek blood on day 22, mouse 1 treated with MASTER scaffold was stained. were excluded.

[0151] 3. Example 3: Physical Characteristics of MASTER To better understand the physical characteristics of the MASTER, cross-sections and cross sections of the MASTER were CT images were taken (Figures 23A and 23B). High density indicated by brighter values. Section and cross-sectional images of the (scaffold) area and the dark areas indicating the air hole areas. CT scans are used to separate pores and color-code pores based on size to determine porosity The relative frequency of the various pore sizes was then calculated (Figure 23). C). Nearly 60% of the pores had an average diameter of 101-200 μm. The majority of the cells were smaller than 100 μm in diameter (approximately 25%). Most of the pores had an elongated shape, as determined by the aspect ratio of the pores. The total surface area inside the MASTER was calculated (Figure 23E). Surface area was plotted as a function of volume. By doing so, the internal surface area of ​​the MASTER was set to approximately 810 mm. The nodes of the pores can be displayed as spheres with lines connecting the pores. The more connections there are, the larger the sphere will appear (Figure 23F). 50% of the pores had less than 3, about 28% had 4-6, and 10% had 7-9. (FIG. 23G). Very few pores (about 6%) have more than nine connections.

[0152] a) Method X-ray computed tomography (CT) scans with the Xradia Versa510 The acquisition parameters seen in the table below were used. [Table 1]

[0153] For each sample, a cylindrical volume of 2.50 × 2 mm was scanned. These volumes were used The porosity and pore size of the sample were calculated using the smaller sub-volumes of 10 × 1 mm. We extracted the data and calculated the connectivity between cells. This reduces the size of the calculation and the results. This was done to enhance visualization.

[0154] CT data was uploaded to Dragonfly [Dragonfly 2020.1 [Compu The analysis was performed using Object R esearch Systems (ORS) Inc., Montreal, Canada, 2018 To split the samples, we used histogram thresholding and masking techniques to I created a sample training dataset manually. I created the training data Then, a deep learning image segmentation model called U-net is used. The resulting model was then used to train all the samples. To calculate the connectivity between the pores, we used a method called openPNM. We used an open source package that can

[0155] 4. Example 4: MASTER enhances the immunization of CAR T cells in a tumor rechallenge model Improved tumor control compared with intravenous injection. To see the effect of MASTER on tumor control, we performed lymphoma xenograft Using a single-cell rechallenge model, MASTER was compared to intravenous infusion of CAR T cells. NSG mice were implanted with FFLuc-labeled CD19+ human Daudi tumor cells. Four days after the initial injection, the mice were implanted with MASTER or intravenously infused with CAR T cells. Mice were rechallenged 29 days after transplantation (Figure 24A). , NSG mice treated with conventional CAR-T cells or control non-transduced (NT) cells In vivo tumor bioluminescence imaging (BLI) of NT cells (Figure 24C) Individual mice injected with MASTER (Figure 24D) or CAR T cells (Figure 24E) Tumor growth curves were plotted. Each line represents one individual animal. Finally, the survival rate of each group was The IL-10 expression was plotted over time (Figure 24F). By day 30 post-engraftment, 100% of NT mice had Died (i.e., did not survive long enough to be eligible for rechallenge). All mice that received either an intravenous infusion of MASTER or CAR T cells However, after rechallenge, efficacy of the two groups was comparable. By day 60 (30 days after rechallenge), 50% of the patients in the intravenous injection group had Only 10% of patients survived until day 80, and fewer than 20% survived until day 80. The rapid decline in survival rate of mice was observed for at least 80 days after engraftment (50 days after rechallenge). This is in stark contrast to the MASTER group, which continued to show a 100% survival rate.

[0156] 5. Example 5: Biocompatibility of MASTER The present inventors also investigated the M We attempted to evaluate the biocompatibility of ASTER and its components. + Mouse PBMC + GFP-encoding gamma retrovirus, and untreated control, C57Bl6 / Tissue samples were collected 4 weeks after subcutaneous implantation in immunocompetent mice, and H& The mice's blood was further analyzed (Figure 26). No differences were observed between any of the treatment groups shown.

[0157] 6. Example 6: PBMCs and retrovirus-loaded MASTERs induce proliferation in surrounding host cells Does not transduce cells Next, the PBMCs and retrovirus-loaded MASTER are used to transduce host cells. In vitro Transwell Imaging an In Vivo System Using the model, GFP expression was observed in fibroblasts seeded on the bottom of a transwell plate. Therefore, when MASTER was co-inoculated with PBMC and virus, It was shown that no leakage of PBMCs from the scaffolds occurred (MOI 2). The virus-loaded MASTER does not transduce the host cells.

[0158] 7. Example 7: Lentiviral transduction of adherent cells. To analyze the ability of lentiviral transduction on adherent cells, 1 million cells were placed on dry scaffolds. HEK293T cells were seeded with lentivirus encoding GFP (MOI 2) ( (Figure 28). After 72 hours, cells were isolated from the scaffolds and GFP expression was measured. HEK293 41.3% of T cells expressed GFP. E. References Agarwal S,Weidner T,Thalheimer FB,Buchho lz CJ.In vivo generated human CAR T cell s eradicate tumor cells.Oncoimmunology.2 019;8:e1671761. Andersen T,Auk-Emblem P,Dornish M.3D Cel l Culture in Alginate Hydrogels.Microarr ays(Basel).2015;4:133-161. Andreadis ST,Palsson BO.Kinetics of retr ovirus mediated gene transfer:the import ance of intracellular half-life of retro viruses.J Theor Biol.1996;182:1-20. 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Claims

1. A method for transducing a cell, comprising: a) administering to the cell an exogenous gene, an siRNA, a tasi RNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miR a) obtaining a vector encoding DNA encoding the nucleic acid; and b) obtaining said cell and said vector. and applying a vector to the dry scaffold.

2. The scaffold and the cells are mixed together at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 １、１２、１３、１４、１５、１６、１７、１８、１９、２０、２１、２２、２３、２４ 10. The method of claim 1, further comprising incubating the cells for 10 hours, 48 ​​hours, or 72 hours. How to install.

3. The cells include immune cells, and the scaffold comprises a ligand or an antibody for an immune cell receptor. The method of transducing a cell according to claim 1 or 2, further comprising:

4. The immune cells include T cells, natural killer (NK) cells, NKT cells, tumor-infiltrating lymphocytes, and the like. lymphocytes (TIL), tumor infiltrating NK cells (TINK), and / or bone marrow infiltrating lymphocytes (BMIL) The method of claim 3, comprising transducing a cell, the cell comprising a human IL or a macrophage.

5. The immune cells include naive T cells, NK cells, NKT cells, macrophages, TILs, TINK, or MIL, and the scaffold is for co-stimulation of T cells, NK cells, or NKT cells. The method according to claim 3 or 4, further comprising administering to the patient a ligand or an antibody that induces signal transduction via the receptor. The method of transducing immune cells described herein.

6. Prior to inducing signal transduction via a costimulatory receptor in T cells, NK cells, or NKT cells The ligand or antibody may be anti-CD28, B7-1, B7-2, anti-induced costimulatory agent (ICOS), ), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, anti-CD4 0L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TIM-2 , anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript-1 (LLT-1), Anti-CD137, CD48, MICA, anti-2B4, and anti-glucocorticoid-induced tumor destruction 6. The immune cell transduction of claim 5, comprising the death factor receptor-related protein (GITR). method.

7. The ligand or antibody is selected from the group consisting of anti-CD3, CD1d, Fc fragment of immunoglobulin, and anti-F The phenotype of the cell according to any one of claims 3 to 6, comprising the Fcγ receptor (FcγRIII). How to install.

8. The cell of any one of claims 1 to 7, wherein the vector comprises a viral vector. Transduction methods.

9. The viral vector may be a lentivirus, a retrovirus, an adenovirus, or an adenovirus. The method of transduction of a cell according to claim 8, comprising a deno-associated virus.

10. The method according to any one of claims 1 to 9, wherein the exogenous gene comprises a chimeric antigen receptor. Methods for transducing cells.

11. 11. Any one of claims 1, 2, or 8 to 10, wherein the cell is a primary cell or a cell line. A method for transducing cells according to claim 1.

12. A method for producing chimeric antigen receptor (CAR) immune cells, comprising: a) obtaining immune cells; and b) applying said immune cells to a scaffold, said scaffold comprising: i) a T cell receptor or a ligand or antibody for an NK cell receptor, and ii) a vector encoding a chimeric antigen receptor. A method for producing a chimeric antigen receptor (CAR) immune cell, comprising:

13. The scaffold and the immune cells are mixed together at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, １１、１２、１３、１４、１５、１６、１７、１８、１９、２０、２１、２２、２３、２ The CAR of claim 12, further comprising incubating for 4, 48, or 72 hours. How to make immune cells.

14. The CAR cell is a CAR T cell, a CAR NK cell, or a CAR NKT cell. or the method for producing a CAR immune cell according to claim 12 or 13, which comprises a CAR macrophage. Law.

15. The ligand or antibody is an anti-CD3, anti-CD28, CD1d, Fc fragment of immunoglobulin or anti-Fcγ receptor (FcγRIII), A method for producing the described CAR immune cells.

16. The cells are naive cells, and the scaffold is a T cell, a NK cell, or a NKT cell.

13. The method of claim 12, further comprising administering to said subject a ligand or antibody that induces signaling through a costimulatory receptor. A method for producing a CAR immune cell according to any one of claims 1 to 15.

17. Prior to inducing signal transduction via a costimulatory receptor in T cells, NK cells, or NKT cells The ligand or antibody may be anti-CD28, B7-1, B7-2, anti-induced costimulatory agent (ICOS), ), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, anti-CD4 0L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TIM-2 , anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript-1 (LLT-1), Anti-CD137, CD48, MICA, anti-2B4, and anti-glucocorticoid-induced tumor destruction The method according to any one of claims 12 to 16, comprising administering to the patient a death factor receptor-related protein (GITR). The method for producing the CAR immune cells described above.

18. The vector according to any one of claims 12 to 17, wherein the vector comprises a viral vector. Methods for producing CAR immune cells.

19. The viral vector may be a lentivirus, a retrovirus, an adenovirus, or an adenovirus.

20. The method of producing a CAR immune cell of claim 18, comprising a deno-associated virus.

20. The scaffold according to any one of claims 12 to 19, wherein the scaffold comprises a hydrogel matrix. A method for producing CAR immune cells.

21. The CAR immunocytoplasmic reticulum of any one of claims 12 to 19, wherein the scaffold comprises a dry scaffold. How to make cells.

22. The CAR immunosuppressant according to any one of claims 12 to 21, wherein the scaffold comprises alginate. How to make immune cells.

23. The scaffold further comprises a cytokine, the cytokine being IL-2, IL-15, Any of claims 1 to 22, comprising IL-7, IL-23, TNF-α, or IFN-γ. A method for producing a CAR immune cell according to one aspect.

24. The immune cells are obtained from an autologous, allogeneic, or haploidentical donor source. A method for producing a CAR immune cell according to any one of claims 1 to 23.

25. Contains one or more ligands or antibodies specific for T cell receptors and / or NK cell receptors , a hydrogel matrix and / or a dry scaffold.

26. The ligand or antibody specific for the T cell receptor and / or the NK cell receptor is anti-CD3 , CD1d, Fc fragment of immunoglobulin, or anti-Fcγ receptor (FcγRIII) 26. The hydrogel matrix and / or dry scaffold of claim 25.

27. Exogenous genes, siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or the siRNA, tasiRNA, lncRNA, s Vectors encoding DNA encoding hRNA, mRNA, gRNA, and miRNA 27. The hydrogel matrix and / or dry scaffold of claim 25 or 26, further comprising:

28. 28. The hydrogel matrix of claim 27, wherein the exogenous gene comprises a chimeric antigen receptor. and / or dry scaffolding.

29. The vector according to any one of claims 25 to 28, wherein the vector comprises a viral vector. A hydrogel matrix and / or a dry scaffold.

30. The viral vector may be a lentivirus, a retrovirus, an adenovirus, or an adenovirus.

30. The hydrogel matrix of claim 29, comprising a deno-associated virus and / or dry foot Place.

31. T cells, natural killer (NK) cells, NKT cells, macrophages, tumor-infiltrating lymphocytes tumor infiltrating lymphocytes (TIL), tumor infiltrating NK cells (TINK), or bone marrow infiltrating lymphocytes (MIL) The method of claim 1, further comprising administering to said patient one or more antibodies, cytokines, and / or costimulatory molecules that activate said Item 31. The hydrogel matrix and / or dry scaffold according to any one of items 25 to 30.

32. The hydrogel matrix binds to a costimulatory receptor on T cells, NK cells, or NKT cells.

32. Any of claims 25 to 31, further comprising a ligand or antibody that induces signal transduction via the 3. The hydrogel matrix and / or dry scaffold according to claim 1 .

33. Prior to inducing signal transduction via a costimulatory receptor in T cells, NK cells, or NKT cells The ligand or antibody may be anti-CD28, B7-1, B7-2, anti-induced costimulatory agent (ICOS), ), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, anti-CD4 0L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TIM-2 , anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript-1 (LLT-1), Anti-CD137, CD48, MICA, anti-2B4, and anti-glucocorticoid-induced tumor destruction 33. The hydrogel matrix of claim 32, comprising death factor receptor-related protein (GITR). and / or dry scaffolding.

34. 25 to 3, wherein the cytokine comprises IL-2, TNF-α, or IFN-γ.

4. The hydrogel matrix and / or dry scaffold according to any one of claims 3.

35. The hydrogel matrix according to any one of claims 25 to 34, further comprising a chemotherapeutic agent. and / or dry scaffolding.

36. A method of treating cancer in a subject, comprising administering to the subject any one of claims 25 to 35. Administering a hydrogel matrix and / or a dry scaffold according to any one of claims 1 to 4.

37. A method of treating cancer in a subject, comprising administering to the subject any one of claims 25 to 35. Administering cells isolated from the hydrogel matrix and / or dry scaffold according to any one of claims 1 to 5. Includes, a method.

38. Treating, reducing, inhibiting, ameliorating, decreasing, and / or preventing cancer and / or metastasis in a subject The method includes: a) obtaining immune cells; and b) applying the immune cells to a scaffold. The scaffold comprises: i) a ligand or antibody for a T cell receptor, or a NK cell receptor; and i) a vector encoding a chimeric antigen receptor; and c) applying said scaffold. and administering to the subject together with the immune cells.

39. The immune cells are mixed with the hydrogel and / or the dry scaffold in at least one, two, three or four infusions. 、４５、６、７、８、９、１０、１１、１２、１３、１４、１５、１６、１７、１８、１ and further incubating for 9, 20, 21, 22, 23, 24, 48, or 72 hours.

40. The method of claim 38, comprising:

40. 39. The method of claim 38, wherein the scaffold with the immune cells is administered without incubation. A method for treating cancer.

41. 40. The method of treating cancer of claim 38, wherein the scaffold comprises a hydrogel matrix. 。

42. 40. The method of treating cancer according to claim 38, wherein the scaffold comprises a dry scaffold.

43. The immune cells include T cells, natural killer (NK) cells, NKT cells, macrophages, tumor infiltrating lymphocytes (TIL), tumor infiltrating NK cells (TINK), or bone marrow infiltrating lymphocytes The method of treating cancer according to any one of claims 38 to 42, comprising a sphere (MIL).

44. The hydrogel further comprises a vector encoding a chimeric antigen receptor (CAR). A method for treating cancer according to any one of claims 37 to 43.

45. The immune cells include naive T cells, NK cells, NKT cells, macrophages, TILs, TINK, or MIL, and the scaffold is for co-stimulation of T cells, NK cells, or NKT cells. Claims 38 to 44, further comprising a ligand or antibody that induces signal transduction via the receptor.

2. A method for treating cancer according to any one of the preceding claims.

46. Prior to inducing signal transduction via a costimulatory receptor in T cells, NK cells, or NKT cells The ligand or antibody may be anti-CD28, B7-1, B7-2, anti-induced costimulatory agent (ICOS), ), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, anti-CD4 0L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TIM-2 , anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript-1 (LLT-1), Anti-CD137, CD48, MICA, anti-2B4, and anti-glucocorticoid-induced tumor destruction 46. ​​The method of claim 45, comprising administering to a patient a death factor receptor-associated protein (GITR). Law.

47. 47. The method according to claim 38, wherein the hydrogel matrix further comprises an immunoblocking inhibitor. A method for treating cancer according to any one of claims 1 to 5.

48. 48. Any of claims 38 to 47, wherein the hydrogel matrix further comprises a chemotherapeutic agent. A method of treating cancer according to any one of claims 1 to 4.

49. The vector transduces the immune cells, and the transduced immune cells The method of any one of claims 38 to 48, wherein the cancer therapeutic agent is released from a hydrogel into the cancer. How to treat.

50. The immune cells are released from the hydrogel about 1 week to about 12 weeks after administration of the hydrogel. The method of any one of claims 38 to 49, wherein the compound is released.

51. Methods for Treating, Reducing, Inhibiting, Alleviating, Reducing, and / or Preventing a Disease or Condition in a Subject A) a cell, and an exogenous gene, siRNA, tasiRNA, lncRNA , shRNA, mRNA, gRNA, miRNA, and / or the siRNA, tasi DNA encoding RNA, lncRNA, shRNA, mRNA, gRNA, miRNA A vector encoding the A gene; and b) applying the cells and the vector to a scaffold. and c) administering the scaffold to the subject together with the cells.

52. 52. The method of claim 51 , wherein the cells comprise primary cells.

53. Mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), dendritic cells, neural stem cells, induced pluripotency 53. The method of claim 52, wherein the cell is a stem cell or a pancreatic islet cell.

54. The disease or condition is selected from the group consisting of graft-versus-host disease (GVHD), myocardial infarction, ischemia, bone / cartilage damage, Osteoarthritis, epilepsy, hemophilia, severe osteogenesis imperfecta, Wiskott-Aldrich syndrome syndrome, X-linked severe combined immunodeficiency, β-thalassemia, adrenoleukodystrophy (ALD) D) Metachromatic leukodystrophy (MLD), cancer, or Parkinson's disease.

51. The method according to claim 51.

55. Methods for Treating, Reducing, Inhibiting, Alleviating, Reducing, and / or Preventing a Disease or Condition in a Subject A) a cell, and an exogenous gene, siRNA, tasiRNA, lncRNA , shRNA, mRNA, gRNA, miRNA, and / or the siRNA, tasi DNA encoding RNA, lncRNA, shRNA, mRNA, gRNA, miRNA A vector encoding the A gene; and b) applying the cells and the vector to a scaffold. and c) culturing said cells within said scaffold for 1, 2, 4, 8, 12, 24, 48, or 72 hours. and d) isolating the cells from the scaffold. and e) administering to a patient.

56. The cells are mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), dendritic cells, neural stem cells, 56. The method of claim 55, comprising primary cells such as human induced pluripotent stem cells, or pancreatic islet cells.

57. The disease or condition is selected from the group consisting of graft-versus-host disease (GVHD), myocardial infarction, ischemia, bone / cartilage damage, Osteoarthritis, epilepsy, hemophilia, severe osteogenesis imperfecta, Wiskott-Aldrich syndrome syndrome, X-linked severe combined immunodeficiency, β-thalassemia, adrenoleukodystrophy (ALD) D) Metachromatic leukodystrophy (MLD), cancer, or Parkinson's disease. The method described in 55.

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