Implantable scaffold materials and methods of use

Abstract

The present disclosure provides compositions, systems, and methods for cell transduction. In particular, the present disclosure provides compositions, systems, and methods for implantable macroporous scaffold materials that facilitate rapid and highly efficient cell transduction.

Description

[Technical Field]

[0001] Government support This invention was made with government support under Grant No. CA260223 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 415,806, filed October 13, 2022, and U.S. Provisional Patent Application No. 63 / 441,409, filed January 26, 2023, both of which are incorporated herein by reference in their entirety for all purposes.

[0003] The present disclosure provides compositions, systems, and methods for cell transduction. In particular, the present disclosure provides compositions, systems, and methods for implantable macroporous scaffold materials that facilitate rapid and highly efficient cell transduction. [Background technology]

[0004] Chimeric antigen receptor (CAR) T-cell therapy has achieved groundbreaking clinical success in hematological cancers and has shown promise for a wide range of cancer types. Unfortunately, CAR T-cell therapy has not yet achieved the same efficacy in solid tumors. This challenge stems from several drawbacks of current CAR T-cell products, such as extensive manufacturing procedures, treatment toxicity, limited in vivo persistence, and unachievable long-term therapeutic effects. Because CAR T-cell production is costly and labor-intensive, the successful implementation of CAR T-cell therapy for a variety of hematological and solid tumors presents several challenges. Current CAR T-cell production requires large-scale facilities and a vein-to-vein process that takes several weeks, delaying the initiation of treatment for patients whose disease has already progressed. This long manufacturing time (>2 weeks) and high treatment cost (approximately $350,000) limit the widespread adoption of this therapy. Approaches to simplify the process include automation of manufacturing, the use of allogeneic products, and rapid CAR T-cell production, which eliminates one or more CAR T-cell generation steps. However, these approaches are limited by low product quality, life-threatening toxicity, poor durability, and premature cell differentiation and exhaustion. Generating and expanding tumor-specific CAR T cells in vivo has the potential to alleviate some of these issues by reducing manufacturing time, treatment costs, and producing a highly effective cell product over the long term.

[0005] The in vivo proliferation and long-term persistence of infused CAR T cells are crucial for antitumor function and preventing tumor recurrence. The importance of T cell persistence and long-term function is particularly urgent in solid tumors, where physiological and immunological barriers such as hypoxia and an immunosuppressive tumor microenvironment alter CAR T cell metabolism, promoting CAR T cell exhaustion and limiting therapeutic outcomes. Recent studies suggest that less differentiated CAR T cells may increase cell engraftment and persistence, leading to improved outcomes. Promising strategies for generating less differentiated CAR T cell phenotypes include initial selection of naive cells, optimization of in vitro culture time and cytokine regimens, and addition of small molecule T cell regulators. Despite extensive research, initial selection of naive populations and maintaining them during clinical-grade CAR T cell manufacturing remain technically challenging. A simple, scalable, and tunable CAR T cell manufacturing platform for generating CAR T cells with a less differentiated phenotype would improve long-term persistence and therapeutic efficacy.

[0006] Biomaterials offer the potential to overcome many of the obstacles hindering widespread, safe, and effective CAR T-cell therapy for solid tumors by providing a nurturing microenvironment that optimizes cell growth. Recent efforts have used biomaterials to improve specific individual steps in CAR T-cell manufacturing, including in vitro and in vivo T-cell isolation, T-cell activation, genetic modification, expansion, and delivery. Furthermore, biomaterials have been used to control T-cell proliferation and differentiation, resulting in robust antitumor effects. Finally, biomaterials may enable sustained release of CAR T cells, thereby improving the safety and toxicity profile of CAR T-cell therapy. Recent studies suggest that fractionating the administration of CAR T cells into multiple smaller doses could reduce the toxicity profile and expand the therapeutic window of this therapy. Sustained release allows for improved multiple-dose schedules, providing a means for significant CAR T-cell dosing without the initial acute toxic effects of effector function. Summary of the Invention

[0007] Embodiments of the present disclosure include an implantable macroporous scaffold comprising a crosslinked biopolymer matrix having an average pore size in the range of about 10 μm to about 500 μm and a stiffness in the range of about 1 kPa to about 1000 kPa, where the stiffness of the matrix matches that of a target tissue, and a composition comprising a plurality of cells and a transduction factor. According to such embodiments, the scaffold facilitates transduction of the plurality of cells by the transduction factor.

[0008] In some embodiments, the biopolymer matrix comprises at least one of alginate, hyaluronic acid, collagen, fibrin, polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), gelatin, polyethylene glycol (PEG), chitosan, cellulose, polyglutamic acid, fibrin, silk, agarose, dextran, polyacrylamide, polyvinyl alcohol, poly(N-isopropylacrylamide), poly(2-hydroxyethyl methacrylate), polyurethane, polyethyleneimine, poly(methyl methacrylate), poly(2-oxazoline), polyphosphazene, and any complex, derivative, or combination thereof.

[0009] In some embodiments, the biopolymer matrix comprises alginate having a molecular weight of about 1 kDa to about 500 kDa.

[0010] In some embodiments, the biopolymer matrix comprises an alginate having a G / M ratio of about 0.5 to about 5.0.

[0011] In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 0.1% to about 5.0%.

[0012] In some embodiments, the biopolymer matrix comprises calcium alginate having a calcium concentration in the range of about 0.1% to about 1.0%.

[0013] In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about 0°C to about -80°C.

[0014] In some embodiments, the biopolymer matrix exhibits a stiffness that is about ±25%, about ±50%, about ±75%, about ±100%, about ±125%, about ±150%, about ±175%, about ±200%, about ±225%, or about ±250% of the stiffness of the target tissue.

[0015] In some embodiments, the scaffolding material comprises at least one biological agent. In some embodiments, the biological agent is a small molecule. In some embodiments, the small molecule is selected from the group consisting of a TLR agonist, a checkpoint inhibitor, an IDO inhibitor, a MEK inhibitor, an HDAC inhibitor, a PI3K inhibitor, an immunomodulatory agent, a JAK kinase inhibitor, and an mTOR inhibitor.

[0016] In some embodiments, the at least one biological agent is a protein, peptide, or polypeptide. In some embodiments, the protein, peptide, or polypeptide is selected from the group consisting of a cytokine, an antibody, and a growth factor. In some embodiments, the cytokine comprises at least one of IL-2, IL-15, IL-7, IL-23, TNF-α, and / or IFN-γ.

[0017] In some embodiments, the plurality of cells comprises one or more immune cells. In some embodiments, the one or more immune cells are selected from the group consisting of T cells, B cells, natural killer (NK) cells, NK T cells, macrophages, dendritic cells, tumor infiltrating lymphocytes (TILs), tumor infiltrating NK cells (TINKs), and bone marrow infiltrating lymphocytes (MILs). In some embodiments, the one or more immune cells are activated.

[0018] In some embodiments, the plurality of cells is obtained from a cell culture. In some embodiments, the plurality of cells is obtained from a donor.

[0019] In some embodiments, the transduction agent comprises a viral vector, hi some embodiments, the viral vector is selected from the group consisting of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, a coccivirus, and a baculovirus.

[0020] In some embodiments, the transduction agent comprises a virus-like particle, a cell-mimetic particle, a transposon, an exosome, a nanoparticle, a micelle, and a liposome.

[0021] In some embodiments, the transduction agent comprises a nucleic acid cargo. In some embodiments, the nucleic acid cargo comprises an siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or viral RNA. In some embodiments, the nucleic acid cargo comprises DNA encoding a fusion protein, a chimeric antigen receptor (CAR), a therapeutic peptide or polypeptide, or a combination thereof.

[0022] In some embodiments, the scaffolding material is implanted within or adjacent to a target tissue. In some embodiments, the target tissue is tumor tissue. In some embodiments, the target tissue is solid tumor tissue. In some embodiments, the target tissue comprises at least one of lung tissue, bone tissue, skin tissue, breast tissue, muscle tissue, nerve tissue, brain tissue, lymphatic tissue, prostate tissue, bladder tissue, stomach tissue, intestinal tissue, uterine tissue, ovarian tissue, liver tissue, adipose tissue, cartilage tissue, thyroid tissue, and / or pancreatic tissue.

[0023] In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor with a transduction efficiency of at least 50%.

[0024] In some embodiments, a macroporous scaffolding material of the present disclosure comprises a biopolymer matrix having an average pore size in the range of about 50 μm to about 250 μm. In some embodiments, a macroporous scaffolding material of the present disclosure comprises a biopolymer matrix having an average pore size in the range of about 100 μm to about 200 μm. In some embodiments, a macroporous scaffolding material of the present disclosure comprises a biopolymer matrix having an average pore size in the range of about 50 μm to about 150 μm.

[0025]

[0010] Embodiments of the present disclosure also include methods of treating a subject. According to such embodiments, the method includes implanting a macroporous scaffold material into or adjacent to a target tissue, the scaffold material comprising a crosslinked biopolymer matrix having an average pore size in the range of about 10 μm to about 500 μm and a stiffness in the range of about 1 kPa to about 1000 kPa, where the stiffness of the matrix matches the stiffness of the target tissue, and a composition comprising a plurality of cells and a transduction factor. In some embodiments, the scaffold material facilitates transduction of the plurality of cells with the transduction factor, and the transduced cells treat the subject.

[0026] In some embodiments of the method, the target tissue is tumor tissue. In some embodiments of the method, the target tissue is solid tumor tissue. In some embodiments of the method, the target tissue comprises at least one of lung tissue, bone tissue, skin tissue, breast tissue, muscle tissue, nerve tissue, brain tissue, lymphatic tissue, prostate tissue, bladder tissue, stomach tissue, intestinal tissue, uterine tissue, ovarian tissue, liver tissue, adipose tissue, cartilage tissue, thyroid tissue, and / or pancreatic tissue.

[0027] In some embodiments of the method, the subject has been diagnosed with a disease or condition. In some embodiments of the method, the disease or condition comprises cancer.

[0028] In some embodiments of the methods, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of at least 50%.

[0029] In some embodiments of the method, the biopolymer matrix comprises at least one of alginate, hyaluronic acid, collagen, fibrin, polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), gelatin, polyethylene glycol (PEG), chitosan, cellulose, polyglutamic acid, fibrin, silk, agarose, dextran, polyacrylamide, polyvinyl alcohol, poly(N-isopropylacrylamide), poly(2-hydroxyethyl methacrylate), polyurethane, polyethyleneimine, poly(methyl methacrylate), poly(2-oxazoline), polyphosphazene, and complexes, derivatives, or combinations thereof.

[0030] In some embodiments of the method, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 500 kDa. In some embodiments of the method, the biopolymer matrix comprises an alginate having a G / M ratio of about 0.5 to about 5.0. In some embodiments of the method, the biopolymer matrix comprises alginate at a concentration in the range of about 0.1% to about 5.0%. In some embodiments of the method, the biopolymer matrix comprises calcium alginate with a calcium concentration in the range of about 0.1% to about 1.0%. In some embodiments of the method, the biopolymer matrix is ​​generated at a temperature in the range of about -20°C to about -80°C.

[0031] In some embodiments of the method, the biopolymer matrix exhibits a stiffness that is about ±25%, about ±50%, about ±75%, about ±100%, about ±125%, about ±150%, about ±175%, about ±200%, about ±225%, or about ±250% of the stiffness of the target tissue.

[0032] In some embodiments of the method, the scaffold material comprises at least one biological agent. In some embodiments of the method, the at least one biological agent is a small molecule. In some embodiments of the method, the small molecule is selected from the group consisting of a TLR agonist, a checkpoint inhibitor, an IDO inhibitor, a MEK inhibitor, an HDAC inhibitor, a PI3K inhibitor, an immunomodulator, a JAK kinase inhibitor, and an mTOR inhibitor. In some embodiments of the method, the at least one biological agent is a protein, peptide, or polypeptide. In some embodiments of the method, the protein, peptide, or polypeptide is selected from the group consisting of a cytokine, an antibody, and a growth factor. In some embodiments of the method, the cytokine comprises at least one of IL-2, IL-15, IL-7, IL-23, TNF-α, and / or IFN-γ.

[0033] In some embodiments of the method, the plurality of cells comprises one or more immune cells. In some embodiments of the method, the one or more immune cells are selected from the group consisting of T cells, B cells, natural killer (NK) cells, NK T cells, macrophages, dendritic cells, tumor infiltrating lymphocytes (TILs), tumor infiltrating NK cells (TINKs), and bone marrow infiltrating lymphocytes (MILs). In some embodiments of the method, the one or more immune cells are activated. In some embodiments of the method, the plurality of cells is obtained from cell culture. In some embodiments of the method, the plurality of cells is obtained from a donor.

[0034] In some embodiments of the method, the transduction agent comprises a viral vector, hi some embodiments of the method, the viral vector is selected from the group consisting of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, a coccivirus, and a baculovirus.

[0035] In some embodiments of the method, the transduction agent comprises a virus-like particle, a cell-mimetic particle, a transposon, an exosome, a nanoparticle, a micelle, and a liposome. In some embodiments of the method, the transduction agent comprises a nucleic acid cargo. In some embodiments of the method, the nucleic acid cargo comprises an siRNA, a tasiRNA, an lncRNA, an shRNA, an mRNA, a gRNA, an miRNA, and / or a viral RNA. In some embodiments of the method, the nucleic acid cargo comprises DNA encoding a fusion protein, a chimeric antigen receptor (CAR), a therapeutic peptide or polypeptide, or a combination thereof.

[0036] Other aspects and embodiments of the present disclosure will become apparent in light of the following detailed description and accompanying drawings. [Brief explanation of the drawings]

[0037] [Figure 1] Figures A-G reveal that Drydux scaffolds exhibit a well-connected macroporous structure. A) Schematic showing the synthesis of Drydux scaffolds. B) X-ray CT scan showing vertical (blue) and horizontal (red) cross sections of a Drydux scaffold. In the cross sections showing the scaffold structure, denser scaffold structures are indicated by lighter color values, while less dense (porous) scaffold structures are indicated by darker color values. C) Relative frequency of macropores of different diameters. D) Pore aspect ratio (<1) suggests predominantly elliptical macropores. E) X-ray CT scan of a Drydux scaffold with a calculated porosity of 82.78% showing pore volume as highlighted spaces. F) X-ray CT scan showing well-connected pores. The connectivity map shows the individual connections of each pore, indicated by spheres and connecting lines. The size and color of the spheres indicate the number of connections. G) Quantification of pore connectivity, showing the frequency of pore connections. [Figure 2](A-C) Drydux scaffolds enable efficient static T cell reprogramming. A) Schematic showing the experimental protocol for generating CAR T cells in vitro using Drydux scaffolds. B) GFP expression in primary human T cells followed by reprogramming with conventional or Drydux (unpaired, two-tailed Student's t-test). C) The stability of Drydux scaffolds was measured by storing sealed scaffolds at 4°C and comparing transduction efficiency with that of freshly prepared scaffolds (unpaired, two-tailed Student's t-test). Data represent the mean ± SEM (standard error of the mean) of three independent experiments. [Figure 3] (A-E) Implantable Drydux scaffolds provide improved efficacy against lymphoma. A) Schematic showing the experimental timeline for lymphoma xenograft model generation followed by treatment with implantable scaffolds or conventionally generated CAR T cells (N=5 / group). Scaffolds seeded with untransduced cells were implanted as negative controls (N=3). B) Tumor bioluminescence (BLI) images of lymphoma-bearing NSG mice treated with implantation of untransduced T cells, conventionally generated CAR T cells, or cell- and virus-seeded Drydux scaffolds. C) Kinetics of tumor growth quantified by measuring BLI signal. The bold line represents the mean. D) Percentage weight change of treated mice. Data represent the mean ± SD (standard deviation) of five biologically independent samples (n=3 for untransduced). E) Survival of treated mice (log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test). [Figure 4](A-J) Drydux generates highly functional CAR T cells against solid tumors. A) B7H3.CAR T cells generated using conventional methods and Drydux scaffold material show comparable transduction efficiency (unpaired, two-tailed Student's t-test). Control groups were not exposed to retrovirus encoding B7H3.CAR. B) Representative flow plots showing B7H3.CAR expression. C) Immunophenotypic composition of B7H3.CAR T cells generated using either method at day 14 of in vitro culture (unpaired Student's t-test). D) Immunophenotypic composition of CD8CAR T cells (unpaired Student's t-test). E) Immunophenotypic composition of CD4CAR T cells (unpaired Student's t-test). F) Analysis of exhaustion marker expression on B7H3.CAR T cells (unpaired Student's t-test). G) In vitro expansion of B7H3.CAR T cells transduced using conventional methods or Drydux scaffold material or untransduced T cells. H) Percentage of remaining tumor cells after co-culture of different GFP-expressing tumor cells with B7H3.CAR T cells generated using either method or non-transduced cells at an E:T ratio of 1:5 (one-way ANOVA with Tukey's correction). I) IL-2 quantification and J) IFN-γ release by CAR T cells generated using either method after 24 hours of co-culture with various tumor cells assessed by ELISA (unpaired Student's t-test). Data represent the mean ± SEM from three independent samples. [Figure 5] 1A-B show that implantable Drydux+IL2 scaffolds provide sustained cell release in vitro. A) Schematic showing the experimental setup for the release study. B) Percentage of initially seeded cells released from Drydux+IL2 scaffolds over 21 days (unpaired Student's t-test). Data represent the mean ± SEM from three independent samples. [Figure 6]1A-B show the evaluation of nonspecific transduction and viral leakage outside the Drydux scaffold. A) Schematic showing the experimental setup for evaluating unwanted transduction away from the scaffold around the implantation site. B) GFP expression in fibroblasts after potential retroviral leakage from the Drydux scaffold (unpaired two-tailed Student's t-test). Data represent the mean ± SEM from three independent samples. [Figure 7] (A-G) Implantable Drydux scaffolds outperform conventionally generated CAR T cells against metastatic lung tumors. A) Schematic showing the experimental timeline for generation of a metastatic NSC lung tumor model followed by treatment with untransduced T cells, implantable Drydux scaffolds, or conventionally generated CAR T cells. Donor-matched T cells were used in all groups. B) BLI images of treated tumor-bearing NSG mice. C) Kinetics of tumor growth quantified by measurement of BLI signal. The bold line represents the mean. D) Percent weight change of mice during treatment. Data represent the mean ± SD of five biologically independent samples. E) Survival of treated mice (log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test). Survival includes all deaths, regardless of tumor status. F) Number of circulating B7H3.CAR T cells analyzed at 34 and 99 days after treatment (unpaired, one-tailed Student's t-test). G) Immunophenotypic analysis of circulating CAR T cells 34 days after treatment (unpaired Student's t-test). Data represent the mean ± SEM of four biologically independent samples. [Figure 8](A-E) Implantable Drydux scaffolds generate highly functional CAR T cells against ovarian tumors. A) Schematic showing the experimental timeline for the generation of an intraperitoneal ovarian xenograft model followed by in vivo treatment with donor-matched CAR T cells generated using the implantable Drydux scaffold or conventionally generated CAR T cells. B) BLI images of ovarian tumor-bearing NSG mice treated with CAR T cells. C) Tumor growth kinetics quantified by measuring the BLI signal. D) Mouse weight change over the entire treatment period. Data represent the mean ± SD of five biologically independent samples. E) Survival of treated mice (log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test). [Figure 9](A-L) Implantable Drydux scaffolds generate highly functional and persistent CAR T cells and prevent tumor recurrence in orthotopic pancreatic tumors. A) Schematic showing the experimental timeline for the generation of orthotopic pancreatic tumor models followed by treatment with donor-matched CAR T cells generated using the implantable Drydux scaffold (N=5) or conventionally generated CAR T cells (N=6). Drydux scaffolds seeded with activated PBMCs were used as untransduced controls (N=6). B) BLI images of tumor-bearing NSG mice treated with untransduced T cells, conventionally generated CAR T cells, or implantation of cell- and virus-seeded Drydux scaffolds. C) Tumor growth kinetics quantified by measuring BLI signal. Bold lines represent the mean. D) Percent weight change in mice during treatment. Data represent the mean ± SD of five biologically independent samples. E) Survival of treated mice (log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test). F) Number of circulating B7H3.CAR T cells analyzed at 20, 40, and 123 days after treatment (unpaired two-tailed Student's t-test). G) Immunophenotype analysis of circulating CAR T cells at 40 days after treatment (unpaired Student's t-test), and H) at 123 days after treatment. Only surviving animals treated with Drydux+IL2 scaffolds were alive and analyzed at day 123. I) Number and J) immunophenotype of B7H3.CAR T cells in the bone marrow were assessed at day 123. K) Number and L) immunophenotype of B7H3.CAR T cells in the spleen were assessed at day 123. Data represent the mean ± SEM of five biologically independent samples. [Figure 10] 1A-C show characterization of Drydux scaffolds. A) X-ray CT scan showing the volume of Drydux scaffolds. B) Pore volume color-coded to indicate pores with similar volumes. C) Surface area plotted as a function of volume. [Figure 11]AB show that Drydux supports T cell proliferation and release. A) Proliferation of CFSE-labeled cells within macroporous scaffolds 3 days after cell seeding. B) Percent release of T cells from macroporous scaffolds cultured in vitro in the presence of exogenous cytokines. Data represent the mean ± SEM of three independent samples. [Figure 12] Figure 1 shows that Drydux mediates stable T cell reprogramming. B7H3.CAR expression in conventionally or scaffold-generated T cells during 14 days of in vitro culture (unpaired Student's t-test). Data represent the mean ± SEM of three independent samples. [Figure 13] (A-H) Drydux scaffolds generate highly functional CAR T cells in vitro. A) Expression of B7-H3 antigen in three different solid tumor cell lines (SKOV3-ovarian, A549-lung, and Panc-1-pancreatic) assessed by flow cytometry. B) Representative flow graphs of co-cultures of GFP-expressing Panc-1 cells with untransduced B7H3.CAR T cells, conventionally generated B7H3.CAR T cells, and Drydux-generated B7H3.CAR T cells. C) Percentage of residual tumor cells following co-culture of GFP-expressing Panc-1 with CAR T cells generated using conventional or Drydux methods or untransduced cells at a 1:1 E:T ratio (one-way ANOVA with Tukey's correction). D, E) Quantification of IL-2 and IFN-γ released by CAR T cells generated using either method after 24 hours of coculture with Panc-1 tumor cells at an E:T ratio of 1:1, assessed by ELISA (unpaired two-tailed Student's t-test). F) Percentage of remaining tumor cells after coculture of different GFP-expressing tumor cells with B7H3.CAR T cells generated using either method or untransduced cells at an E:T ratio of 1:10 (one-way ANOVA with Tukey's correction). G, H) Quantification of IL-2 and IFN-γ released by CAR T cells generated using either method after 24 hours of coculture with the indicated tumor cells, assessed by ELISA (unpaired Student's t-test). Data represent the mean ± SEM of three independent samples. [Figure 14] (A-B) Drydux+IL2 promotes T cell proliferation in vitro. A) GFP expression in pre-activated T cells 2 days after transduction seeded in Drydux and Drydux+IL2 (unpaired two-tailed Student's t-test). B) Cell proliferation assessed on the day of seeding (day 0) and 2 days after seeding in Drydux and Drydux+IL2 (unpaired Student's t-test). Data represent the mean ± SEM of three independent samples. [Figure 15] Figures A-G demonstrate that Drydux-generated CAR T cells exhibit superior in vivo persistence in a metastatic lung tumor model. A) Schematic showing experimental details. CAR T cells were generated using donor-matched T cells using either the transplantable Drydux scaffold material or a conventional method (with retronectin and spinoculation followed by in vitro expansion). FFluc-expressing tumor cells were inoculated 14 days before the start of each treatment. B) Representative flow graph showing the gating strategy for determining the number and phenotype of CAR T cells in blood and lymphoid organs. C) Phenotype of CAR T cells in blood 99 days after treatment. D) Number of CAR T cells present in the spleen (unpaired, two-tailed Student's t-test), and E) bone marrow assessed 99 days after treatment (unpaired, two-tailed Student's t-test). F) Phenotype of CAR T cells in the spleen (unpaired, two-tailed Student's t-test) and G) bone marrow 99 days after treatment (unpaired, two-tailed Student's t-test). Data represent the mean ± SEM of two biologically independent samples. [Figure 16](A-F) Figures demonstrate that Drydux-generated CAR T cells exhibit improved in vivo persistence in an intraperitoneal ovarian tumor model. A) Schematic showing experimental details. CAR T cells were generated using implantable Drydux scaffolds or conventional methods (involving retronectin coating, spinoculation, and in vitro expansion) using T cells from the same donor. Scaffolds seeded with cells alone were used as negative controls. FFluc-expressing tumor cells were inoculated 14 days before the start of each treatment. Tumor growth was monitored weekly using IVIS imaging. B) Number of circulating B7H3.CAR T cells in the blood at day 126 after treatment. C) Number of B7H3.CAR T cells in the bone marrow was assessed at day 126. D) Number of B7H3.CAR T cells in the spleen was assessed at day 126. Data represent the mean ± SEM of biologically independent samples. E) Number of circulating B7H3.CAR T cells assessed at day 34 after treatment (unpaired, two-tailed Student's t-test). F) Immunophenotypic analysis of circulating B7H3.CAR T cells (unpaired Student's t test). [Figure 17] (A-B) Drydux-generated CAR T cells improved tumor-free survival and prevented recurrence in an orthotopic pancreatic tumor model. A) Schematic showing experimental details. Donor-matched T cells were used to generate CAR T cells using implantable Drydux scaffolds or conventional methods (involving retronectin and spinoculation followed by in vitro expansion). FFluc-expressing tumor cells were inoculated 12 days before the start of each treatment, and tumor growth was monitored weekly. B) Tumor-free survival of animals inoculated with conventionally generated CAR T cells or in vitro generated CAR T cells with Drydux (log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test). [Figure 18]Figure 1 shows the preparation of a dry macroporous alginate (Drydux) scaffold. An alginate solution is crosslinked with a calcium solution, and the resulting gel is frozen overnight and then lyophilized for 72 hours to produce a dry macroporous scaffold. Activated T cells and viral particles are mixed and seeded onto the scaffold, which is then incubated at 37°C and 5% CO2. EDTA is used to dissolve the scaffold and isolate the transduced T cells. [Figure 19] Figures 25A-25E show the effect of porosity and stiffness on Drydux transduction efficiency at various calcium and alginate concentrations. (A) Photographs of scaffolds with corresponding SEM images and mean pore size. (B) Quantification of retroviral transduction efficiency of primary human PBMCs for each calcium-alginate combination, showing significance between different calcium concentrations. *p<0.0001. All other p-values ​​are shown on the graph. Concentrations used were approximately 5,000 cells / μL and approximately 10,000 virus / μL. n=3 scaffolds per group. Two-way ANOVA with Tukey's correction was used to determine significance. See Figures 25A-25E for significance between different alginate concentrations. (C) Quantification of scaffold pore size using a minimum of 10 pores per scaffold. (D) Spearman correlation between scaffold pore size and transduction efficiency. (E) Quantification of Young's modulus for each scaffold. n=3 scaffolds per group. (F) Spearman correlation between scaffold stiffness and transduction efficiency. Data are shown as mean ± SEM. Statistical analysis was not completed for (C) and (E). [Figure 20]Figures 26A-26D show the effect of porosity and stiffness on Drydux transduction efficiency at various freezing temperatures and alginate concentrations. (A) Photographs of scaffolds with corresponding SEM images and mean pore size. (B) Quantification of retroviral transduction efficiency of primary human PBMCs for each alginate-temperature combination, showing significance across different temperatures. *p<0.0001. All other p-values ​​are shown on the graph. Concentrations used were approximately 5,000 cells / μL and approximately 10,000 virus / μL. n=3 scaffolds per group. Two-way ANOVA with Tukey's correction was used to determine significance. See Figures 26A-26D for significance across different alginate concentrations. (C) Quantification of scaffold pore size using a minimum of 10 pores per scaffold. (D) Spearman correlation between scaffold pore size and transduction efficiency. (E) Quantification of Young's modulus for each scaffold. n=3 scaffolds per group. (F) Spearman correlation between scaffold stiffness and transduction efficiency. Data are shown as mean ± SEM. Statistical analysis was not completed for (C) and (E). [Figure 21] Figures A-G show the effect of seeding volume on transduction with Drydux. (A) Live image of a scaffold absorbing 20 μL of cell-virus solution. (B) Images of a scaffold 24 hours after absorbing different volumes of cell-virus solution. (C) Quantification of transduction efficiency for each seeding volume. (D) Kinetics of absorption for each seeding volume. (E) Spearman correlation between absorption rate and transduction efficiency. (F) Volumetric flux calculated for different seeding volumes. (G) Spearman correlation between volumetric flux and transduction efficiency. Data are shown as mean ± SEM. Concentrations used were approximately 2000 cells / μL and approximately 4000 virus / μL. n=3 scaffolds per group. One-way ANOVA was used to determine significance. [Figure 22]A-D are diagrams illustrating a computational model of flow through scaffold pores. (A) Schematic showing activated T cells and virus seeded together in a dry macroporous scaffold. (B) Particle position at statistical equilibrium for uniform unrestricted flow (top) and flow inside scaffold pores at a volumetric flux of 30 μL / min / cm2 (bottom). (C) Flow velocity distribution at the midplane of the scaffold model showing flow acceleration and deceleration corresponding to changes in model geometry. (D) Quantification of the number of collisions per μL per minute for unrestricted flow, unrestricted flow, and scaffold pore flow at different volumetric fluxes. [Figure 23] AB show representative flow cytometry results and gating strategy for non-transduced cells (A) and representative flow cytometry results and gating strategy for GFP+ cells (B). [Figure 24] Figure 1 shows quantification of transduction efficiency for various MOI values ​​(ratio of viral particles to activated T cells). Data are shown as mean ± SEM. The concentration used was approximately 5000 cells / μL. n=2 scaffolds per group. [Figure 25] Figures A-E show further characterization of biomaterial scaffolds synthesized with various calcium and alginate concentrations. (A-C) Stress-strain curves obtained from compression tests of different calcium-alginate scaffolds. (D) Quantification of transduction efficiency for each calcium-alginate combination (significance across different alginate concentrations is shown). Data are shown as mean ± SEM. Concentrations used were approximately 5,000 cells / µL and approximately 10,000 virus / µL. n = 3 scaffolds per group. Two-way ANOVA with Tukey's correction was used to determine significance. [Figure 26]Figures A-D show further characterization of biomaterial scaffolds synthesized at various alginate concentrations and freezing temperatures. (A-C) Stress-strain curves obtained from compression tests of different alginate-temperature scaffolds. (D) Quantification of transduction efficiency for each alginate-temperature combination (significance between different alginate concentrations is shown. Data are shown as mean ± SEM. Concentrations used were approximately 5,000 cells / μL and approximately 10,000 virus / μL. n = 3 scaffolds per group. Two-way ANOVA with Tukey's correction was used to determine significance). [Figure 27] Figure 1 shows quantification of cell and viral particle concentration versus transduction efficiency. Data are shown as mean ± SEM. n=4 scaffolds per group. One-way ANOVA with Tukey's correction was used to determine significance. [Figure 28] Figures A-C show preliminary experiments testing the effect of seeding volume and corresponding absorption rate on transduction efficiency. (A) Kinetics of absorption for different seeding volumes. (B) Quantification of transduction efficiency for each seeding volume. (C) Spearman correlation between absorption rate of cell-virus solution and transduction efficiency. Data are shown as mean ± SEM. Concentrations used were approximately 2000 cells / μL and approximately 4000 virus / μL. n=4 scaffolds per group. One-way ANOVA with Tukey's correction was used to determine significance. [Figure 29] A-B show the effect of surface area on transduction efficiency. (A) Scaffolds were fabricated in 6-well plates and seeded with primary human T cells and concentrated GFP retrovirus at an MOI of 4. The cell-virus solution was either spread over the entire surface of the scaffold or seeded at a single location on the scaffold. (B) Quantification of transduction efficiency for each group. Data are shown as mean ± SEM. n = 3 per group. An unpaired t-test with Welch's correction was used to determine significance. DETAILED DESCRIPTION OF THE INVENTION

[0038] Embodiments of the present disclosure provide compositions, systems, and methods for cell transduction. In particular, the present disclosure provides compositions, systems, and methods for implantable macroporous scaffolds that facilitate rapid and highly efficient cell transduction. According to these embodiments, as evidenced by the present disclosure, implantable macroporous scaffolds can be tailored to incorporate cell proliferation and release cues to efficiently reprogram T cells and release CAR T cells for the treatment of solid tumors. The compositions and systems described herein are designed to minimize the need for ex vivo manipulation and be implantable within three days of T cell isolation, thereby providing sufficient time for clinically necessary preconditioning and lymphodepletion of patient T cells. As further described herein, the implantable macroporous scaffolds of the present disclosure mediate T cell reprogramming and promote CAR T cell proliferation and release in vitro and in vivo. Such scaffolds have been highly effective in animal models of systemic lymphoma, intravascularly metastatic lung cancer, and intraperitoneally metastatic ovarian cancer, as well as in orthotopic pancreatic cancer. These solid tumors have a poor prognosis, and despite advances in various treatment options, improvements in 5-year survival rates have been limited. The implantable macroporous scaffold dramatically reduced the time and labor required to generate CAR T cells. Furthermore, the implantable macroporous scaffold improved the persistence of CAR T cells, resulting in improved efficacy compared to the same number of CAR T cells generated using conventional methods.

[0039] Furthermore, as evidenced by the results and data presented herein, dry macroporous alginate ("Drydux") scaffolds can improve viral transduction of T cells, as well as other difficult-to-transduce cells. As further described herein, experiments were conducted to elucidate the mechanism behind the function of Drydux scaffolds through studies of the effects of pore size, scaffold stiffness, virus concentration, and uptake on transduction efficiency. Results revealed that scaffold pore size has a complex effect on transduction efficiency, while scaffold stiffness does not affect Drydux transduction. More concentrated virus suspensions were found to lead to higher transduction efficiencies, suggesting that future cell-virus solutions should be as concentrated as possible for optimal transduction efficiency. Interestingly, a strong correlation was found between uptake rate and transduction efficiency. Finally, the experimental results were validated using a computational model of cell-virus collisions flowing through porous scaffolds. Based on the results, absorption rate and volumetric flux appear to be important factors in Drydux transduction.

[0040] Furthermore, the results showed that the stiffness of the scaffold material did not appear to significantly contribute to Drydux transduction. Scaffold stiffness has been shown to affect cell migration and differentiation within the scaffold and cell infiltration into host tissue. Studies have shown that a softer matrix improves T cell proliferation and mechanotransduction, which are necessary for T cell receptor signaling. This is because softer scaffold materials typically have higher porosity and interconnectivity, which promotes more interactions between cells and leads to higher T cell transduction. However, in these examples, the stiffness of the scaffold material was altered by using cell adhesion peptides such as RGD to induce adhesion of cells. In the case of Drydux scaffolds, unmodified alginate does not provide adhesion ligands for T cells, which are themselves non-adherent cells. This is likely why the stiffness of the scaffold material did not affect Drydux transduction.

[0041] The effect of pore size on transduction was not so straightforward. SEM imaging was used to determine pore size because it is readily available and has high throughput. However, SEM only reveals surface porosity, and future studies may focus on more rigorous porosity quantification methods, including microCT, Brunauer-Emmett-Teller (BET) surface area analysis, or porosimetry. Smaller pores are likely to result in higher transduction efficiency. This is because scaffold materials with smaller pores typically have higher porosity and interconnectivity, which allows for greater cell-virus interaction and improved nutrient and oxygen diffusion. When calcium concentration was varied, a significant correlation was observed between smaller pore size and transduction; however, this correlation disappeared when pore size was controlled by freezing temperature. Therefore, pore size may still play an important role in cell transduction.

[0042] Overall, Drydux transduction is a robust process that maintains its efficiency over a wide range of alginate concentrations, calcium concentrations, and freezing temperatures. The robust nature of this system further supports the potential use of the Drydux scaffold for cell therapy and the possibility that it may be particularly beneficial for CAR T cell therapy against solid tumors. In such therapies, tailoring the structure of the scaffold to that of the implanted tissue may improve the success of the treatment. This is crucial when treating certain solid tumors, such as glioblastoma. For example, matching the stiffness of the scaffold to that of the brain can affect cell viability, migration, and infiltration into surrounding tissues.

[0043] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below; however, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. The phrase "in one embodiment," as used herein, may refer to the same embodiment, but does not necessarily refer to the same embodiment. Further, the phrase "in another embodiment," as used herein, may refer to a different embodiment, but does not necessarily refer to a different embodiment. Thus, as described below, various embodiments of the present invention may be readily combined without departing from the scope or spirit of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0044] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude additional acts or structures. The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements set forth herein, whether or not explicitly stated.

[0045] When numerical ranges are recited herein, each numerical value within that range, to the same degree of precision, is specifically contemplated. For example, for the range of 6 to 9, the numbers 7 and 8 are specifically contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are specifically contemplated. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment also includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value also constitutes another embodiment. It will also be understood that each endpoint of a range has meaning not only in relation to the other endpoint, but also independently of the other endpoint. It is also apparent that, although several values ​​are disclosed herein, each value is also disclosed herein as "about" that particular value in addition to the particular value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also apparent that, when a value is disclosed, "less than or equal to" that value, "greater than or equal to" that value, and possible ranges between values ​​are also disclosed, as would be appropriately understood by one of ordinary skill in the art. For example, if the value "10" is disclosed, then "less than or equal to 10" and "greater than or equal to 10" are also disclosed.

[0046] Additionally, throughout this application, data are presented in several different formats; such data are construed as endpoints and starting points, and ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is clear that values ​​greater than or equal to 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15 are also considered to be disclosed, as are values ​​between 10 and 15. Also, each unit amount between two specified unit amounts is also considered to be disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0047] As used herein, "nucleic acid" or "nucleic acid sequence" refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (see Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, as well as any chemical variants thereof (e.g., methylated, hydroxymethylated, or glycosylated versions of these bases). The polymer or oligomer can be heterogeneous or homogeneous in composition and can be isolated from natural sources or produced artificially or synthetically. Furthermore, the nucleic acid can be DNA or RNA, or a mixture thereof, and can exist permanently or transiently in single- or double-stranded form, including homoduplexes, heteroduplexes, and hybrid states. In some embodiments, the nucleic acid or nucleic acid sequence comprises other types of nucleic acid structures, such as, for example, a DNA / RNA helix, a peptide nucleic acid (PNA), a morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14):4503-4510 (2002), and U.S. Patent No. 5,034,506), a locked nucleic acid (LNA, see Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97:5633-5638 (2000)), a cyclohexenyl nucleic acid (see Wang, J. Am. Chem. Soc., 122:8595-8602 (2000)), and / or a ribozyme. Thus, the term "nucleic acid" or "nucleic acid sequence" can also encompass chains that include non-natural nucleotides, modified nucleotides, and / or non-nucleotide building blocks (e.g., "nucleotide analogs") that can perform the same function as natural nucleotides.Furthermore, as used herein, the term "nucleic acid sequence" refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, as well as DNA or RNA of genomic or synthetic origin, which may be single- or double-stranded and may represent the sense or antisense strand. The terms "nucleic acid," "polynucleotide," "nucleotide sequence," and "oligonucleotide" are used interchangeably. They refer to a polymer of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0048] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an "insert," can be attached or incorporated so as to bring about the replication of the attached segment in a cell.

[0049] When exogenous DNA, such as a recombinant expression vector, has been introduced into a cell, the cell has been "genetically modified," "transduced," "transformed," or "transfected" by that DNA. The presence of the exogenous DNA results in a permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the cell's genome. For example, in prokaryotes, yeast, and mammalian cells, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has integrated into a chromosome and is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones, which comprise a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that can be stably grown in vitro for many generations.

[0050] "Optional" or "optionally" means that the event or circumstance described after the term may or may not be present, and that the description encompasses the presence of the event or circumstance and the absence of the event or circumstance.

[0051] "Increase" can refer to any change that results in an increase in a symptom, disease, property, condition, or activity. The increase can be any individual, median, or average increase in a disease, symptom, activity, or property by a statistically significant amount. Thus, an increase can include a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, as long as it is statistically significant.

[0052] "Reduction" can refer to any change that results in a decrease in a symptom, disease, trait, condition, or activity. A substance is also considered to reduce the genetic production of a gene if the genetic production of that gene product with the substance is less than the genetic production of that gene product without the substance. A reduction can also be, for example, a change in the symptoms of a disorder, such that the symptoms of the disorder are less than those previously observed. A reduction can be any individual, median, or average decrease in a disease, symptom, activity, or trait by a statistically significant amount. Thus, a reduction can include a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% reduction, as long as it is statistically significant.

[0053] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% decrease in the activity, response, condition, or disease compared to the original or control level. Thus, the decrease can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% decrease, or any amount therebetween, compared to the original or control level.

[0054] "Decrease" or other forms of "reduction" (e.g., "reduce" or "reducing") generally refer to a decrease in an event or characteristic (e.g., tumor growth). As will be apparent, this is usually relative to some standard or expected value, in other words, relative, although reference to a standard or relative value is not necessarily required. For example, "reducing tumor growth" means reducing the rate of tumor growth compared to a standard or control.

[0055] Other forms of "prevention" or "prevention" (e.g., "prevent" or "preventing") mean to stop a particular event or characteristic, to stabilize or delay the occurrence or progression of a particular event or characteristic, or to reduce to the greatest extent possible the chance of a particular event or characteristic occurring. Prevention is usually more absolute than, for example, reduction, and does not require comparison to a control. As used herein, some things can be reduced while others cannot be prevented, and some things can be both reduced and prevented. Similarly, some things can be prevented while others cannot be reduced, and some things can be either prevented or reduced. As will be apparent, when one of reduce or prevention is used, the use of the other is expressly disclosed unless expressly stated otherwise.

[0056] The term "subject" refers to any individual who is the object of administration or treatment. The subject may be a vertebrate, such as a mammal. In one embodiment, the subject may be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. The subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject may be a human or animal patient. The term "patient" refers to a subject receiving treatment from a clinician (e.g., a physician).

[0057] The term "therapeutically effective" refers to an amount of a composition used that is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration requires only a reduction or alteration, not necessarily elimination.

[0058] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder. This term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, condition, or disorder, as well as causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, condition, or disorder. Additionally, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, condition, or disorder. This term also includes preventative treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the onset of the associated disease, condition, or disorder, as well as supportive treatment, i.e., treatment used to complement another specific therapy aimed at ameliorating the associated disease, condition, or disorder.

[0059] "Biocompatible" generally refers to the material and any metabolic or decomposition products thereof being generally non-toxic to the recipient and causing no significant adverse effects in the subject.

[0060] As used herein, the term "comprising" is intended to mean that a composition, method, etc., includes the recited elements while not excluding other elements. When used to define compositions and methods, "consisting essentially of" is intended to mean including the recited elements but excluding other elements that are essential to the combination. Thus, a composition consisting essentially of elements as defined herein does not exclude trace amounts of contaminants from separation and purification methods, nor does it exclude pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, etc. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transitional phrases are within the scope of this disclosure.

[0061] A "control" is a surrogate subject or sample used in an experiment for comparison purposes. Controls can be "positive" or "negative."

[0062] An "effective amount" of an agent refers to an amount of the agent sufficient to produce a desired effect. The amount of an agent that is "effective" varies from subject to subject, depending on many factors, such as the subject's age and general condition, the specific agent or agents, and other factors. Therefore, it is not always possible to identify a quantified "effective amount." However, an appropriate "effective amount" for any given patient can be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, the "effective amount" of an agent may refer to an amount that encompasses both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of an agent required to achieve a therapeutic effect may vary depending on factors such as the subject's age, sex, and weight. Dosage regimens can be adjusted to produce an optimal therapeutic response. For example, multiple divided doses may be administered daily, or the dose may be proportionally reduced as dictated by the exigencies of the therapeutic situation.

[0063] A "pharmaceutically acceptable" ingredient means a substance that is not biologically undesirable or otherwise undesirable. That is, the substance can be incorporated into the pharmaceutical formulations provided herein and administered to a subject without causing significant undesirable biological effects or interacting in a deleterious manner with any other components of the formulation in which it is included. When used in reference to human administration, the term generally means that the ingredient has met the necessary standards of toxicology and manufacturing testing, or that it is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0064] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") refers to a carrier or additive useful in preparing pharmaceutical or therapeutic compositions, including carriers that are generally safe and non-toxic and acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water emulsions or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any additive, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art for use in pharmaceutical formulations and those further described herein.

[0065] "Pharmacologically active" (or simply "active"), as used in "pharmacologically active" derivative or analog, can refer to a derivative or analog (e.g., salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) that has the same type and approximately the same degree of pharmacological activity as the parent compound.

[0066] A "therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects (e.g., treatment of a disorder or other undesirable physiological condition) and prophylactic effects (e.g., prevention of a disorder or other undesirable physiological condition, e.g., non-immunogenic cancer). These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein. Such derivatives include, but are not limited to, salts, esters, amides, enhancers, precursors of the agent, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a particular agent is specifically identified, the term should be interpreted to include the agent itself, as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, precursors of the agent, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0067] A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition comprising an agent) refers to an amount effective to achieve a desired therapeutic result. In some embodiments, the desired therapeutic result is control of type 1 diabetes. In some embodiments, the desired therapeutic result is control of obesity. The therapeutically effective amount of a given therapeutic agent will typically vary with factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term may also refer to the amount of therapeutic agent, or the rate of delivery of the therapeutic agent (e.g., amount over time), effective to promote a desired therapeutic effect, such as pain relief. The specific desired therapeutic effect will vary depending on the disease being treated, the subject's tolerance, the agent and / or formulation being administered (e.g., potency of the therapeutic agent, agent concentration in the formulation, etc.), and various other factors recognized by those skilled in the art. In some cases, the desired biological or medical response is achieved after multiple administrations of the composition to the subject over a period of days, weeks, or years.

[0068] As used herein, "alginate" generally refers to a salt or ester of alginic acid. Alginates are linear copolymers with homopolymeric blocks of (1-4)-linked β-D-mannuronic acid (M) residues and its C-5 epimer α-L-guluronic acid (G) residues, each covalently linked together in a different sequence or block. These monomers can exist in consecutive G residues (G blocks), consecutive M residues (M blocks), homopolymeric blocks of alternating M and G residues (MG blocks), or randomly arranged blocks. The relative amount of each block type varies depending on both the source of the alginate and the concentration of G and M acids (the "G / M ratio"), thus contributing to various structural and biocompatibility properties. For example, alternating blocks form the most flexible chains and are more soluble at lower pH than other blocks. G blocks form rigid chain elements, and two G blocks of more than six residues each can withstand divalent cations (e.g., Ca, among others). 2+ , Mg 2+ , Ba 2+ , Sr 2+ ) to form stable cross-linked junctions resulting in a three-dimensional gel network.

[0069] As used herein, "Young's modulus" or "Young's elastic modulus" is a mechanical property that measures the stiffness of a material and can be expressed, for example, in kilopascals (kPa) of pressure. As further described herein, the stiffness of specific tissues varies throughout the human body, and this property can elicit various biochemical and cellular responses. Embodiments of the present disclosure include the production of an implantable macroporous scaffold material comprised of a biopolymer matrix that can be tailored to match or adapt to the stiffness of any target tissue.

[0070] Specific methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0071] 2. Composition Embodiments of the present disclosure include compositions, systems, and methods related to cell transduction. In particular, the present disclosure provides compositions, systems, and methods related to implantable macroporous scaffolds that facilitate rapid and highly efficient cell transduction. According to such embodiments, the present disclosure includes an implantable macroporous scaffold comprising a crosslinked biopolymer matrix and a composition comprising a plurality of cells and a transduction factor. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 500 μm and a stiffness in the range of about 1 kPa to about 1000 kPa. In some embodiments, the stiffness of the matrix matches the stiffness of the target tissue, and the scaffold facilitates transduction of a plurality of cells by the transduction factor.

[0072] As further described herein, the implantable macroporous scaffold material of the present disclosure is comprised of a crosslinked biopolymer matrix, the stiffness of which is designed to match that of the target tissue. Based on this disclosure, one of skill in the art will understand that the stiffness of a particular target tissue (e.g., as measured using Young's modulus) is an important factor to consider when generating and implanting a biomaterial into a subject. For example, in some embodiments, the target tissue is neural tissue (e.g., brain, spinal cord, sciatic nerve, ulnar nerve, etc.) having a stiffness in the range of about 0.4 kPa to about 7 kPa, and the scaffold material of the present disclosure can be configured to have a stiffness that matches without compromising transduction efficiency. In some embodiments, the target tissue is connective tissue (e.g., tibia, femur, articular cartilage, adipose tissue, patellar tendon, ligament, etc.) having a stiffness in the range of about 2 kPa to about 21 GPa, and the scaffold material of the present disclosure can be configured to have a stiffness that matches without compromising transduction efficiency. In some embodiments, the target tissue is muscle tissue (e.g., smooth muscle, cardiac muscle, skeletal muscle, etc.) having a stiffness in the range of about 2 kPa to about 800 kPa, and the scaffold material of the present disclosure can be configured to have a stiffness that matches without compromising transduction efficiency. In some embodiments, the target tissue is endothelial and epithelial tissue (e.g., skin, lung, intestine, etc.) having a stiffness in the range of about 1 kPa to about 14 MPa, and the scaffold material of the present disclosure can be configured to have a stiffness that matches without compromising transduction efficiency. In some embodiments, the target tissue is an internal organ (e.g., kidney, spleen, liver, thymus, thyroid, pancreas, bladder, etc.) having a stiffness in the range of about 0.1 kPa to about 300 kPa, and the scaffold material of the present disclosure can be configured to have a stiffness that matches without compromising transduction efficiency. In some embodiments, the target tissue is an ocular tissue (e.g., cornea, lens, etc.) having a stiffness in the range of about 4 kPa to about 4 MPa, and the scaffold material of the present disclosure can be configured to have a stiffness that is compatible therewith without compromising transduction efficiency. As will be appreciated by those skilled in the art, the scaffold material of the present disclosure can be configured to have a stiffness that is compatible with any target tissue without compromising transduction efficiency.In some embodiments, the target tissue is or comprises tissue that exhibits characteristics consistent with a disease or condition, including, but not limited to, cancerous tissue (e.g., solid tumor tissue).

[0073] According to such embodiments, the stiffness of a crosslinked biopolymer matrix of the present disclosure can be in the range of about 1 kPa to about 1000 kPa. In some embodiments, the stiffness of a crosslinked biopolymer matrix is ​​about 1 kPa to about 900 kPa. In some embodiments, the stiffness of a crosslinked biopolymer matrix is ​​about 1 kPa to about 800 kPa. In some embodiments, the stiffness of a crosslinked biopolymer matrix is ​​about 1 kPa to about 700 kPa. In some embodiments, the stiffness of a crosslinked biopolymer matrix is ​​about 1 kPa to about 600 kPa. In some embodiments, the stiffness of a crosslinked biopolymer matrix is ​​about 1 kPa to about 500 kPa. In some embodiments, the stiffness of a crosslinked biopolymer matrix is ​​about 1 kPa to about 400 kPa. In some embodiments, the stiffness of a crosslinked biopolymer matrix is ​​about 1 kPa to about 300 kPa. In some embodiments, the stiffness of a crosslinked biopolymer matrix is ​​about 1 kPa to about 200 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 1 kPa to about 100 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 1 kPa to about 50 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 10 kPa to about 1000 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 50 kPa to about 1000 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 100 kPa to about 1000 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 200 kPa to about 1000 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 300 kPa to about 1000 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 400 kPa to about 1000 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 500 kPa to about 1000 kPa. In some embodiments, the stiffness of the crosslinked biopolymer matrix is ​​from about 600 kPa to about 1000 kPa.In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 700 kPa to about 1000 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 800 kPa to about 1000 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 900 kPa to about 1000 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 50 kPa to about 500 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 100 kPa to about 500 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 250 kPa to about 750 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 300 kPa to about 600 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 500 kPa to about 800 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 600 kPa to about 900 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 25 kPa to about 650 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 0.2 kPa to about 65 kPa.

[0074] In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 0.1 kPa to about 10 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 0.1 kPa to about 9 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 0.1 kPa to about 8 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 0.1 kPa to about 7 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 0.1 kPa to about 6 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 0.1 kPa to about 5 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 0.1 kPa to about 4 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 0.1 kPa to about 3 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 0.1 kPa to about 2 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 0.1 kPa to about 1 kPa. In some embodiments, the stiffness of the crosslinked biopolymer matrix is ​​about 0.1 kPa to about 0.5 kPa. In some embodiments, the stiffness of the crosslinked biopolymer matrix is ​​about 0.5 kPa to about 9 kPa. In some embodiments, the stiffness of the crosslinked biopolymer matrix is ​​about 1 kPa to about 9 kPa. In some embodiments, the stiffness of the crosslinked biopolymer matrix is ​​about 2 kPa to about 9 kPa. In some embodiments, the stiffness of the crosslinked biopolymer matrix is ​​about 3 kPa to about 9 kPa. In some embodiments, the stiffness of the crosslinked biopolymer matrix is ​​about 4 kPa to about 9 kPa. In some embodiments, the stiffness of the crosslinked biopolymer matrix is ​​about 5 kPa to about 9 kPa. In some embodiments, the stiffness of the crosslinked biopolymer matrix is ​​about 6 kPa to about 9 kPa. In some embodiments, the stiffness of the crosslinked biopolymer matrix is ​​about 7 kPa to about 9 kPa. In some embodiments, the stiffness of the crosslinked biopolymer matrix is ​​about 8 kPa to about 9 kPa.In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 1 kPa to about 8 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 2 kPa to about 6 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 3 kPa to about 5 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 2 kPa to about 4 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 4 kPa to about 6 kPa. In some embodiments, the crosslinked biopolymer matrix has a stiffness of about 5 kPa to about 7 kPa.

[0075] The stiffness or elasticity (e.g., Young's modulus) of various tissues are described above. Thus, in some embodiments, the implantable macroporous scaffold material of the present disclosure can exhibit a Young's modulus that is compatible with the tissue into which it is implanted. This can improve compatibility between the implantable scaffold material and the host target tissue, while the scaffold material still exhibits desirable transduction efficiency. For example, the macroporous scaffold material of the present disclosure, when hydrated with a composition comprising a plurality of cells and transduction factors, can exhibit a stiffness that is about ±25%, about ±50%, about ±75%, about ±100%, about ±125%, about ±150%, about ±175%, about ±200%, about ±225%, or about ±250% of the stiffness of the target tissue.

[0076] In such embodiments, the scaffolding material is implanted within or adjacent to a target tissue. In some embodiments, the target tissue is tumor tissue. In some embodiments, the target tissue is solid tumor tissue. In some embodiments, the target tissue comprises at least one of lung tissue, bone tissue, skin tissue, breast tissue, muscle tissue, nerve tissue, brain tissue, lymphatic tissue, prostate tissue, bladder tissue, stomach tissue, intestinal tissue, uterine tissue, ovarian tissue, liver tissue, adipose tissue, cartilage tissue, thyroid tissue, and / or pancreatic tissue.

[0077] In some embodiments, the scaffolding material facilitates transduction of a plurality of cells (e.g., immune cells) with a transduction factor (e.g., a viral vector comprising a polynucleotide encoding a protein of interest) with a transduction efficiency of at least 50% (measured in vivo or ex vivo). In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor with a transduction efficiency of at least 60%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor with a transduction efficiency of at least 70%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor with a transduction efficiency of at least 80%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor with a transduction efficiency of at least 90%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor with a transduction efficiency of about 50% to about 90%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 60% to about 90%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 70% to about 90%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 50% to about 80%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 50% to about 70%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 60% to about 80%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 70% to about 90%.

[0078] In addition to stiffness, as further described herein, the average pore size of the crosslinked biopolymer matrix of the implantable macroporous scaffold material of the present disclosure is another important factor to consider when generating a biomaterial sufficient to facilitate cellular transduction and implant it into a subject. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 500 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 450 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 400 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 350 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 300 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 250 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 200 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 150 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 100 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 50 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 10 μm to about 25 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 25 μm to about 500 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 50 μm to about 500 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 100 μm to about 500 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 150 μm to about 500 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 200 μm to about 500 μm.In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 250 μm to about 500 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 300 μm to about 500 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 350 μm to about 500 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 400 μm to about 500 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 450 μm to about 500 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 50 μm to about 400 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 100 μm to about 300 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 200 μm to about 400 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 150 μm to about 350 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 200 μm to about 300 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 50 μm to about 250 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 100 μm to about 200 μm. In some embodiments, the crosslinked biopolymer matrix has an average pore size in the range of about 50 μm to about 150 μm.

[0079] The implantable macroporous scaffold materials of the present disclosure can be composed of a variety of different stiffness, semi-rigidity, flexibility, gel, self-assembly, liquid crystal, or fluid compositions. Such compositions include, but are not limited to, peptide polymers, polysaccharides, synthetic polymers, ceramics (e.g., calcium phosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans, metals, and metal alloys. The compositions can be fabricated using methods known in the art, such as injection molding, freeze-drying of preformed structures, printing, self-assembly, phase inversion, solvent casting, melt processing, gas foaming, fiber formation / processing, particulate leaching, or combinations thereof.

[0080] In some embodiments, the implantable macroporous scaffold materials disclosed herein can be fabricated using any suitable biodegradable polymer. A "polymer" refers to a relatively high molecular weight organic compound, natural or synthetic, whose structure can be represented by repeating small units (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 a polymer formed from two or more different repeating units (monomer residues). By way of example and without limitation, a copolymer can be an alternating copolymer, a random copolymer, a block copolymer, or a graft copolymer. In certain aspects, it is also contemplated that the various block segments of a block copolymer may themselves comprise copolymers. The term "polymer" encompasses all forms of polymers, including, but not limited to, natural polymers, synthetic polymers, homopolymers, heteropolymers or copolymers, addition polymers, etc.

[0081] Exemplary materials that may be used to form the implantable macroporous scaffold material of the present disclosure include, but are not limited to, polylactic acid, polyglycolic acid, polylactide-glycolide copolymers (PLG), alginate and alginate derivatives, gelatin, collagen, fibrin, fibronectin, methacrylamide, acrylamide, decellularized tissue, hyaluronic acid, laminin-rich gel, agarose, natural and synthetic polysaccharides, polyamino acids, polypeptides, polyesters, polyanhydrides, polyphosphazines, poly(vinyl alcohol), poly(alkylene oxide), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, pluronic polyols, poloxamers, poly(uronic acid), poly(vinylpyrrolidone), and copolymers or graft copolymers of any of the above. In some embodiments, the biopolymer matrix comprises at least one of alginate, hyaluronic acid, collagen, fibrin, polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), gelatin, polyethylene glycol (PEG), chitosan, cellulose, polyglutamic acid, fibrin, silk, agarose, dextran, polyacrylamide, polyvinyl alcohol, poly(N-isopropylacrylamide), poly(2-hydroxyethyl methacrylate), polyurethane, polyethyleneimine, poly(methyl methacrylate), poly(2-oxazoline), polyphosphazene, and any complex, derivative, or combination thereof.

[0082] In some embodiments, the hydrogel comprises an RGD-modified alginate. Accordingly, further disclosed herein is a macroporous scaffold material comprising a cross-linked hydrogel, such as alginate, and / or a cross-linked biopolymer. In some embodiments, the macroporous scaffold material comprises a cross-linked polymer, such as cross-linked alginate, cross-linked gelatin, or derivatives thereof, such as methacrylated ones.

[0083] In some embodiments, the macroporous scaffold material may comprise a biocompatible polymer (e.g., alginate, etc.). Such polymers may also aid in the slow release of CAR T cells, CAR NK cells, TILs, and / or MILs into the tissue. As used herein, biocompatible polymers include, but are not limited to: Polysaccharides, hydrophilic polypeptides, poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine, polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO), poly(oxyethylated polyols), poly(olefin alcohols), polyvinylpyrrolidone, poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(hydroxy acids), poly(vinyl alcohol), poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid), poly(3-hydroxybutyrate) or poly(4-hydroxybutyrate), Examples of biocompatible polymers include polyhydroxyalkanoates such as acrylates, polycaprolactones, poly(orthoesters), polyanhydrides, poly(phosphazenes), poly(lactide-caprolactone) copolymers, polycarbonates such as tyrosine polycarbonate, polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids), polyesteramides, polyesters, poly(dioxanones), poly(alkylene alkylates), hydrophobic polyethers, polyurethanes, polyetheresters, polyacetals, polycyanoacrylates, polyacrylates, polymethyl methacrylates, polysiloxanes, poly(oxyethylene) / poly(oxypropylene) copolymers, polyketals, polyphosphates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(maleic acid), and copolymers thereof.Polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohol (PVA), methacrylate PVA (m-PVA), polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and their copolymers, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, polymers of acrylic and methacrylic acid esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, sodium cellulose sulfate, 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(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohol), poly(vinyl acetate), polyvinyl chloride, polystyrene, and polyvinylpyrrolidone, their derivatives, their linear, branched, and block copolymers, and blends thereof. Exemplary biodegradable polymers include polyesters, poly(orthoesters), poly(ethyleneamines), poly(caprolactones), poly(hydroxybutyric acid), poly(hydroxyvaleric acid), polyanhydrides, poly(acrylic acid), polyglycolides, poly(urethanes), polycarbonates, polyphosphate esters, polyphospriazenes, their derivatives, their linear, branched, and block copolymers, and blends thereof.

[0084] In some embodiments, the particles contain biocompatible and / or biodegradable polyesters or polyanhydrides, such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid). The particles may contain the following polyesters: homopolymers containing glycolic acid units (referred to herein as "PGA"), homopolymers containing lactic acid units (e.g., poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide (collectively referred to herein as "PLA")), homopolymers containing caprolactone units (e.g., poly(e-caprolactone)) (collectively referred to herein as "PCL"), and copolymers containing lactic acid and glycolic acid units (e.g., various forms of poly(lactic-co-glycolic acid) and poly(lactide-co-glycolide) copolymers characterized by the ratio of lactic acid to glycolic acid (referred to herein as "PLGA"). (collectively referred to as "PEG") and polyacrylates, and derivatives thereof. Exemplary polymers also include copolymers of polyethylene glycol (PEG) with the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers." In certain embodiments, the PEG region can be covalently attached to the polymer by a cleavable linker to produce a "PEGylated polymer." In one aspect, the polymer comprises at least 60, 65, 70, 75, 80, 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent acetal pendant groups.

[0085] The triblock copolymers disclosed herein may comprise a core polymer, such as polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), poly(vinylpyrrolidone-vinyl acetate) copolymer, polymethacrylate, polyoxyethylene alkyl ether, polyoxyethylene castor oil, polycaprolactam, polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid), poly(lactic acid-glycolic acid) copolymer (PLGA), or a cellulose derivative (e.g., hydroxymethylcellulose, hydroxypropylcellulose, etc.).

[0086] As mentioned above, one material for the implantable macroporous scaffold material of the present disclosure is alginate or modified alginate material. Alginate is a versatile polysaccharide-based polymer that can be formulated for specific applications by controlling the molecular weight, degradation rate, and scaffold formation method. Alginate molecules are composed of (1-4) linked β-D-mannuronic acid (M unit) and αL-guluronic acid (G unit) monomers, which can vary in proportion and sequential distribution along the polymer chain. Alginate polysaccharides are capable of withstanding divalent cations (e.g., Ca). +2 , Mg +2 , Ba +2), forming a stable scaffold when exposed to these molecules. See Martinsen A., et al., Biotech. & Bioeng., 33 (1989) 79-89. For example, calcium-crosslinked alginate scaffolds are useful in the methods described herein. For example, the polymer of the hydrogel, e.g., alginate, is 0-100% crosslinked (e.g., at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more crosslinked). In other embodiments, the polymer of the scaffold, e.g., alginate, is not crosslinked. In some examples, the polymer of the scaffold, e.g., alginate, contains less than 50% crosslinks (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%, or less than crosslinks).

[0087] Alginate can be chemically modified to impart new properties. For example, alginate can be oxidized to increase its biodegradation rate. Alternatively, alginate can be reduced to improve biocompatibility. Alginate can also be chemically modified to alter its crosslinking behavior. For example, alginate can be modified with bioorthogonal click groups to enable click crosslinking. In another example, alginate can be modified with acrylic groups to enable radical polymerization crosslinking. In another example, alginate can be modified using host-guest chemistry to enable host-guest crosslinking.

[0088] Alginate polymers can be formed into a variety of scaffold types. Alginate scaffolds can be formed from alginates with molecular weights ranging from 1,000 Da to 500,000 Da. Alginate scaffolds can be formed from alginates with G / M ratios between 0.5 and 5. Different hydrogel formulations control the degradation rate of the scaffold. The release rate of pharmaceutical compositions (e.g., small molecules, morphogens, or other bioactive agents) from alginate macroporous scaffolds can be controlled by the scaffold formulation, which delivers the pharmaceutical composition in a spatially and temporally controlled manner. This controlled release eliminates systemic side effects and the need for multiple injections. Useful polysaccharides other than alginate include, but are not limited to, agarose and microbial polysaccharides (e.g., fungal pullulan, scleroglucan, chitin, chitosan, elsinan, bacterial xanthan gum, curdlan, dextran, gelatin, levan, emulsan, cellulose, hyaluronic acid, etc.).

[0089] According to the above embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 500 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 450 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 400 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 350 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 300 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 350 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 300 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 300 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 250 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 200 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 150 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 100 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 50 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 50 kDa to about 500 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 100 kDa to about 500 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 150 kDa to about 500 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 200 kDa to about 500 kDa. In some embodiments, the biopolymer matrix comprises alginate having a molecular weight of about 250 kDa to about 500 kDa.In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 300 kDa to about 500 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 350 kDa to about 500 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 400 kDa to about 500 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 450 kDa to about 500 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 100 kDa to about 400 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 200 kDa to about 400 kDa. In some embodiments, the biopolymer matrix comprises an alginate having a molecular weight of about 100 kDa to about 300 kDa. In some embodiments, the biopolymer matrix comprises alginate having a molecular weight of about 150 kDa to about 350 kDa.

[0090] According to various embodiments described herein, the biopolymer matrix of the implantable macroporous scaffold material of the present disclosure can comprise an alginate having a G / M ratio of about 0.5 to about 5.0. In some embodiments, the biopolymer matrix comprises an alginate matrix having a G / M ratio of about 0.5 to about 4.0. In some embodiments, the biopolymer matrix comprises an alginate matrix having a G / M ratio of about 0.5 to about 3.0. In some embodiments, the biopolymer matrix comprises an alginate matrix having a G / M ratio of about 0.5 to about 2.0. In some embodiments, the biopolymer matrix comprises an alginate matrix having a G / M ratio of about 0.5 to about 1.0. In some embodiments, the biopolymer matrix comprises an alginate matrix having a G / M ratio of about 1.0 to about 5.0. In some embodiments, the biopolymer matrix comprises an alginate matrix having a G / M ratio of about 2.0 to about 5.0. In some embodiments, the biopolymer matrix comprises an alginate matrix having a G / M ratio of about 3.0 to about 5.0. In some embodiments, the biopolymer matrix comprises an alginate matrix having a G / M ratio of about 4.0 to about 5.0. In some embodiments, the biopolymer matrix comprises an alginate matrix having a G / M ratio of about 1.0 to about 4.0. In some embodiments, the biopolymer matrix comprises an alginate matrix having a G / M ratio of about 2.0 to about 3.0.

[0091] According to various embodiments described herein, the biopolymer matrix of the implantable macroporous scaffold material of the present disclosure may comprise alginate at a concentration ranging from about 0.1% to about 5.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 0.1% to about 4.5% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 0.1% to about 4.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 0.1% to about 3.5% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 0.1% to about 3.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 0.1% to about 2.5% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 0.1% to about 2.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 0.1% to about 1.5% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 0.1% to about 1.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 0.1% to about 0.5% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 0.5% to about 5.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 1.5% to about 5.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 2.0% to about 5.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 2.5% to about 5.0% (w / v), hi some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 3.0% to about 5.0% (w / v).In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 3.5% to about 5.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 4.0% to about 5.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 4.5% to about 5.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 1.5% to about 3.5% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 2.0% to about 4.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 3.0% to about 4.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration ranging from about 0.5% to about 2.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 0.5% to about 1.5% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 0.5% to about 1.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 1.0% to about 2.0% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 1.0% to about 1.5% (w / v). In some embodiments, the biopolymer matrix comprises alginate at a concentration in the range of about 1.5% to about 2.0% (w / v).

[0092] According to various embodiments described herein, the biopolymer matrix of the implantable macroporous scaffold material of the present disclosure can comprise calcium alginate having a calcium concentration in the range of about 0.1% to about 1.0% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.1% to about 0.9% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.1% to about 0.8% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.1% to about 0.7% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.1% to about 0.6% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.1% to about 0.5% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.1% to about 0.4% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.1% to about 0.3% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.1% to about 0.2% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.2% to about 0.9% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.3% to about 0.9% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.4% to about 0.9% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.5% to about 0.9% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.6% to about 0.9% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.7% to about 0.9% (w / v).In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.8% to about 0.9% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.2% to about 0.8% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.3% to about 0.6% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.4% to about 0.8% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.3% to about 0.7% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.2% to about 0.5% (w / v). In some embodiments, the biopolymer matrix has a calcium concentration in the range of about 0.3% to about 0.7% (w / v).

[0093] Embodiments of the present disclosure also include methods of manufacturing any of the macroporous scaffold materials disclosed herein. According to such embodiments, the method includes cross-linking alginate chains using calcium to form an alginate hydrogel, cryogelating the hydrogel to form an alginate cryogel, lyophilizing the cryogel to form a macroporous scaffold material, mixing a retrovirus with freshly isolated immune cells, and seeding the retrovirus and immune cell mixture onto the macroporous scaffold material. In some embodiments, the method includes activating the immune cells (e.g., with anti-CD3 and / or anti-CD28 antibodies) before mixing with the retrovirus. In some embodiments, the method further includes adding a biological agent (e.g., a cytokine (e.g., IL-2)) to the macroporous scaffold material prior to or consecutively with seeding the immune cells and retrovirus onto the scaffold material.

[0094] In some embodiments, the macroporous scaffold material of the present disclosure can be hydrated by adding a composition (e.g., an aqueous sample) comprising a plurality of cells to be transduced. In some embodiments, the composition further comprises a transduction factor (e.g., a viral vector comprising a nucleic acid cargo) to be introduced into the plurality of cells. Prior to adding the composition to hydrate the scaffold material, the scaffold material can be a dry macroporous scaffold material (e.g., as described in PCT / US2021 / 026805, filed April 12, 2021 (internationally published as WO2021 / 207724) and USSN 17 / 917,770, each of which is incorporated herein by reference in its entirety).

[0095] In accordance with such embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about 0°C to about -80°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about 0°C to about -70°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about 0°C to about -60°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about 0°C to about -50°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about 0°C to about -40°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about 0°C to about -30°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about 0°C to about -20°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about 0°C to about -10°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about -10°C to about -80°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about -20°C to about -80°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about -30°C to about -80°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about -40°C to about -80°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about -50°C to about -80°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about -60°C to about -80°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about -70°C to about -80°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about -10°C to about -70°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about -20°C to about -60°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about -30°C to about -50°C. In some embodiments, the biopolymer matrix is ​​generated at a temperature ranging from about -40°C to about -60°C.

[0096] In some embodiments, the biopolymer matrix comprising a macroporous scaffolding material can be a "dry scaffolding material." As used herein, "dry scaffolding material" refers to any scaffolding material having 10% or less water by weight. In other words, a dry scaffolding material contains less than 20% water by weight. In some embodiments, a dry scaffolding material contains about 18% or less water by weight. In some embodiments, a dry scaffolding material contains about 16% or less water by weight. In some embodiments, a dry scaffolding material contains about 14% or less water by weight. In some embodiments, a dry scaffolding material contains about 12% or less water by weight. In some embodiments, a dry scaffolding material contains about 10% or less water by weight. In some embodiments, a dry scaffolding material contains about 8% or less water by weight. In some embodiments, a dry scaffolding material contains about 6% or less water by weight. In some embodiments, a dry scaffolding material contains about 4% or less water by weight. In some embodiments, a dry scaffolding material contains about 2% or less water by weight. In some embodiments, a dry scaffolding material contains about 5% to about 15% water by weight. In some embodiments, a dry scaffolding material comprises about 10% to about 15% water by weight. In some embodiments, a dry scaffolding material comprises about 15% to about 20% water by weight. In some embodiments, a dry scaffolding material comprises about 5% to about 10% water by weight. In some embodiments, a dry scaffolding material comprises about 10% to about 20% water by weight.

[0097] In some embodiments, a dry scaffolding material contains about 1.0% (e.g., by weight) or less crosslinker. This includes embodiments of dry scaffolding materials without detectable crosslinker. In some embodiments, a dry scaffolding material contains about 0.9% or less crosslinker. In some embodiments, a dry scaffolding material contains about 0.8% or less crosslinker. In some embodiments, a dry scaffolding material contains about 0.7% or less crosslinker. In some embodiments, a dry scaffolding material contains about 0.6% or less crosslinker. In some embodiments, a dry scaffolding material contains about 0.5% or less crosslinker. In some embodiments, a dry scaffolding material contains about 0.4% or less crosslinker. In some embodiments, a dry scaffolding material contains about 0.3% or less crosslinker. In some embodiments, a dry scaffolding material contains about 0.2% or less crosslinker. In some embodiments, a dry scaffolding material contains about 0.1% or less crosslinker. In some embodiments, a dry scaffolding material contains less than about 0.1% crosslinker. In some embodiments, a dry scaffolding material comprises about 0.1% to about 1.0% crosslinker. In some embodiments, a dry scaffolding material comprises about 0.2% to about 0.8% crosslinker. In some embodiments, a dry scaffolding material comprises about 0.4% to about 0.6% crosslinker. In some embodiments, a dry scaffolding material comprises about 0.001% to about 0.1% crosslinker. In some embodiments, a dry scaffolding material comprises about 0.01% to about 0.1% crosslinker.

[0098] In some embodiments, the implantable macroporous scaffold material of the present disclosure can be configured in a variety of geometric shapes and dimensions (e.g., discs, beads, pellets), niches, and planar layers (e.g., thin sheets). For example, discs ranging from about 0.1 millimeter to about 50 centimeters in diameter, such as 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, 70, 80, and 90 millimeters, and 10, 15, 20, 25, 30, 35, 40, 45, and 50 centimeters in diameter, can be generated and implanted. Discs can have thicknesses ranging from 0.1 to 10 millimeters, e.g., 1, 2, and 5 millimeters. Discs can be compressed and / or lyophilized for implantation into a subject. Multi-component devices (e.g., those including scaffold materials) are optionally constructed with concentric layers, each characterized by different physical properties (% polymer, % crosslinking of polymer, chemical composition of dry scaffold material, pore size, porosity and pore structure, stiffness, toughness, ductility, viscoelasticity, and / or pharmaceutical composition).

[0099] The macroporous scaffold materials disclosed herein can be fabricated in or inserted into wells of multiwell plates, including 384-well plates, 96-well plates, 48-well plates, 24-well plates, 12-well plates, or 6-well plates. The scaffold materials can be fabricated in or inserted into flasks, including T25, T75, T175, or T225 flasks. The scaffold materials can be fabricated in or inserted into culture dishes, including 35 mm, 60 mm, 100 mm, and 150 mm dishes. The scaffold materials can be fabricated in or inserted into cell culture tubes, including tubes with a capacity of 3 mL, 5 mL, 7 mL, 8 mL, 12 mL, 14 mL, 15 mL, 16 mL, 19 mL, 21 mL, or 50 mL. The scaffold material can be of any shape and size, and can be fabricated in or inserted into molds, including molds ranging in size from 1 mm to 0.1 m. The scaffold material can be fabricated in or inserted into cell culture bags with volumes of 50 mL, 100 mL, 200 mL, 300 mL, 500 mL, 1000 mL, 2000 mL, 5000 mL, or 10,000 mL.

[0100] The scaffold materials disclosed herein can be fabricated in wells of a multi-well plate or in a culture dish, and thus can have a disk shape with a diameter of 1 mm to 50 cm and a thickness of 1 mm to 50 cm. The scaffold materials can be fabricated in square or rectangular molds with a side length of 1 mm to 50 cm and a thickness of 1 mm to 50 cm. Furthermore, the scaffold materials can be fabricated in regular or irregular molds, and can have regular shapes, such as triangular, pentagonal, hexagonal, star, or diamond shapes, or can be irregular. The regular or irregular molds can have a surface area of ​​1 mm or less. 2 ~2500cm 2, the thickness can be 1mm to 50cm.

[0101] Scaffolding materials can be composed of a collection of individual particles. These microparticles can be produced by spray drying, electrospinning, extrusion, emulsification / gelation, chopping, spin drying, or other known particle-making techniques. Examples of particles that make up scaffolding materials can be microspheres with diameters of 50 μm, 100 μm, 200 μm, 500 μm, 1000 μm, 2000 μm, or 5000 μm. Scaffolding materials can also be composed of sections cut from a larger whole product. The dimensions of the original whole can range from 0.1 meters square to 1,000 meters square.

[0102] The scaffold structure may comprise microporous or macroporous pores. Pore sizes may include 10 μm, 20 μm, 50 μm, 100 μm, 200 μm, 500 μm, and 1000 μm. In one aspect, 50-70% of the pores have a diameter of 100-200 mm (e.g., a scaffold has 60% of the pores having a diameter of 100-200 mm). The pore pattern is optionally uniform, non-uniform, aligned, repeating, or random. In some embodiments, 100-200 mm diameter pores occupy at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% (e.g., 82.78%) of the scaffold volume.

[0103] Embodiments of the present disclosure also relate to methods and compositions relating to implantable macroporous scaffold materials comprising at least one biological agent. The biological agent can be included in the compositions of the present disclosure and / or incorporated into the biopolymer matrix of the scaffold material. In some embodiments, the biological agent is included to promote the growth, proliferation, survival, and / or differentiation of a plurality of cells. The biological agent can be included to enhance compatibility of the plurality of cells and / or the scaffold material with a host target tissue. The biological agent can also be included to improve one or more mechanical properties of the scaffold material.

[0104] In some embodiments, the biological agent is a small molecule. In some embodiments, the small molecule is selected from the group consisting of a TLR agonist, a checkpoint inhibitor, an IDO inhibitor, a MEK inhibitor, an HDAC inhibitor, a PI3K inhibitor, an immunomodulator, a JAK kinase inhibitor, and an mTOR inhibitor. In some embodiments, at least one biological agent is a protein, peptide, or polypeptide. In some embodiments, the protein, peptide, or polypeptide is selected from the group consisting of a cytokine, an antibody, and a growth factor. In some embodiments, the cytokine comprises at least one of IL-2, IL-15, IL-7, IL-23, TNF-α, and / or IFN-γ.

[0105] In some embodiments, the composition applied to the implantable macroporous scaffold material may further comprise: receptors (epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR)), ligands (for example, but not limited to, epidermal growth factor (EGF), platelet-derived growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), Fibroblast growth factors (FGFs), insulin-like growth factor (IGF) 1 (IGF-1), and / or IGF-2), bone morphogenetic proteins (BMPs), ephrins (A1, A2, A3, A4, A5, B1, B2, B3), erythropoietin, fibroblast growth factors (FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15) , FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23), glial cell line-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), neurotrophins (BDNF, NGF, NT-3, NT-4), T cell growth factor (TCGF), transforming growth factors (TGF-α, TGF-β), tumor necrosis factor α (TNF-α), Wnt signaling pathway, integrins (e.g., VLA-1, VLA-2, VLA-3, VLA-4), A-4, VLA-5, VLA-6, FLJ25220, RLC, PRO827, HsT18964, FLJ39841, HUMINAE, LFA1A, MAC-1, VNRA, MSK8, GPIIb, cadherins (e.g., E-cadherin), and / or immune stimulatory and / or immune sustaining antibodies, chemokines, and cytokines (e.g., IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-21, IL-23, TNF-α, or IFN-γ).

[0106] As will be appreciated by those skilled in the art based on the present disclosure, a costimulatory signal may be required to fully activate immune cells. Thus, in one embodiment, the composition comprises one or more costimulatory molecules that activate: T cells, natural killer (NK) cells, NK T cells, macrophages, tumor-infiltrating lymphocytes (TIL), tumor-infiltrating NK cells (TINK), or bone marrow-infiltrating lymphocytes (MIL) (e.g., without limitation, anti-CD28, B7-1, B7-2, anti-inducible costimulatory factor (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-induced tumor necrosis factor receptor-related protein (GITR). Accordingly, the compositions applied to the implantable macroporous scaffold materials described herein may also include T cells, NK cells, or NK Ligands or antibodies that induce signaling through T cell costimulatory receptors, for example, but not limited to, anti-CD28, B7-1, B7-2, anti-inducible costimulatory factor (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-induced tumor necrosis factor receptor-related protein (GITR).

[0107]

[0010] Embodiments of the present disclosure also include applying a composition comprising a plurality of cells and at least one transduction factor to a biopolymer matrix to produce a scaffold material described herein. According to such embodiments, the plurality of cells can comprise one or more immune cells. In some embodiments, the one or more immune cells are selected from the group consisting of T cells, B cells, natural killer (NK) cells, NK T cells, macrophages, dendritic cells, tumor-infiltrating lymphocytes (TILs), tumor-infiltrating NK cells (TINKs), and bone marrow-infiltrating lymphocytes (MILs).

[0108] In some embodiments, the target cell is a commercially available (e.g., immortal) cell line or a primary cell line. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is selected from the group consisting of T cells, natural killer (NK) cells, NK T cells, macrophages, tumor-infiltrating lymphocytes (TILs), tumor-infiltrating NK cells (TINKs), and bone marrow-infiltrating lymphocytes (MILs). In some embodiments, the cell is a stem cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is an engineered cell. In certain embodiments, the engineered cell is derived from tissue sources including, but not limited to, adipose tissue, skin tissue, muscle tissue, blood, bone marrow, nervous tissue, liver tissue, pancreatic tissue, cartilage tissue, lung tissue, intestinal tissue, ovarian tissue, testicular tissue, umbilical cord tissue, placental tissue, synthetic and biomimetic scaffolds, and any derivatives thereof. In some embodiments, the cell can be of any cell type. In certain embodiments, the cell type can be a prokaryotic cell, a eukaryotic cell, a human-specific cell, an immune cell, a stem cell, a cancer cell, a microbial cell, a specialized cell, and any derivative thereof. In some embodiments, the cell can be any cell type. In certain embodiments, the cell type can be a prokaryotic cell, a eukaryotic cell, a human-specific cell, an immune cell, a stem cell, a cancer cell, a microbial cell, a specialized cell, and any derivative thereof.

[0109] In some embodiments, the transduction agent is a vector used to deliver a target nucleic acid to a cell. In some embodiments, the vector is selected from the group consisting of lentivirus, retrovirus, adenovirus, herpes simplex virus (HSV), vesicular stomatitis virus (VSV), Sendai virus, adeno-associated virus, coccivirus, and baculovirus. In some embodiments, the transduction agent comprises a virus-like particle, a cell-mimetic particle, a transposon, an exosome, a nanoparticle, a micelle, a liposome, a modified vaccinia ankara (MVA), a plasmid, and any derivative thereof.

[0110] As further described herein, embodiments of the present disclosure facilitate the delivery of a target nucleic acid to a cell. As will be apparent to one of skill in the art based on this disclosure, the target nucleic acid can be any nucleic acid. In some embodiments, the target nucleic acid comprises RNA. For example, the target nucleic acid can be viral RNA, including siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and any combination and / or derivative thereof. In other embodiments, the target nucleic acid comprises DNA, including any derivative or variant thereof. In some embodiments, the target nucleic acid is DNA encoding RNA. In some embodiments, the RNA encoded by the DNA is siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and viral RNA. In some embodiments, the target nucleic acid encodes a protein (e.g., a chimeric antigen receptor, or CAR).

[0111] In some embodiments, the dry scaffolding material comprises at least one biological agent. In some embodiments, the at least one biological agent is a small molecule. In some embodiments, small molecules include, but are not limited to, TLR agonists, checkpoint inhibitors, IDO inhibitors, MEK inhibitors, HDAC inhibitors, PI3K inhibitors, immunomodulators, JAK, and mTOR inhibitors, and any combination thereof. In some embodiments, the at least one biological agent is a protein, peptide, or polypeptide. In some embodiments, proteins, peptides, or polypeptides include, but are not limited to, cytokines, antibodies, and growth factors, and any combination thereof.

[0112] As further described herein, the present disclosure provides methods for transducing cells (e.g., immune cells) using the implantable macroporous scaffold materials of the present disclosure. In some embodiments, the cells may include, for example, T cells, B cells, natural killer (NK) cells, NK T cells, macrophages, tumor-infiltrating lymphocytes (TILs), tumor-infiltrating NK cells (TINKs), or bone marrow-infiltrating lymphocytes (MILs), or any combination thereof. In some embodiments, the one or more cells are obtained from an autologous donor source, an allogeneic donor source, and / or a haploidentical donor source. In some embodiments, the one or more cells include non-immune cells (e.g., mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), dendritic cells, neural stem cells, induced pluripotent stem cells, or islet cells). In some embodiments, the one or more cells include primary immune cells and primary non-immune cells, as well as immune and non-immune cell lines.

[0113] In some embodiments, the implantable macroporous scaffold material of the present disclosure facilitates the transduction of a plurality of cells with a transduction factor to treat a disease or disorder in a target tissue, the transduction factor being, for example, a therapeutic cargo (including, but not limited to, a polynucleotide encoding a fusion protein, a chimeric antigen receptor (CAR) (e.g., CAR T cells targeting CD19, CD33, IL-13 receptor alpha chain 2 (IL13Rα2), B7-H3, neural / glial antigen 2 (NG2), disialoganglioside GD2, EGFRvIII, MUC1, PSMA, mesothelin, HER2, or CEA), a CAR NK cell, a CAR NK The viral vector (e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus, virus-like particle, transposon, or liposome) encoding a target gene (e.g., a target cell ...

[0114] In some embodiments, to facilitate transduction of cells with a therapeutic nucleic acid molecule, a composition comprising a plurality of cells and a transduction factor is applied to a macroporous scaffold material of the present disclosure for a certain incubation period prior to administration / implantation into a subject, hi some embodiments, the cells are incubated with the scaffold material for at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 minutes. In some embodiments, the cells are incubated with the scaffold material for 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, 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, or 96 hours. In some embodiments, the cells are incubated with the scaffold material for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, a composition comprising a plurality of cells and a transduction factor is applied to a macroporous scaffold material of the present disclosure without prior incubation (i.e., implanted into a subject without prior incubation) to facilitate transduction of the cells with a therapeutic nucleic acid molecule.

[0115] As noted above, the cells can be from a commercially available cell line. Exemplary cells that can be transduced include, but are not limited to: NCI-H295R, 5637, HT-1376, J82, SW780, T24, T24-Luc-Neo, T24P, BT142, D54-Luc, DBTRG (tumor), DBTRG-05MG, Gli36-DsRed-R-Luc (rescue), LN-18, LN-229, L N-827(pMMP-LucNeo), M059K, SF-295, SF-539, SF-767, SNB-19, U-251, U-251-Luc-mCh-Puro, U-87MG, U-87MG-Luc, CaSki, HeLa, KB, C2BBe1, Caco-2, COLO 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, HEL92.1.7, HEL92.1.7-Luc-Neo, HEL-Luc-Neo, TF-1a-Luc-Neo, OE33, A4573, Hs 895.T, NHDF (normal human dermal fibroblast), TE 353.Sk, TE 354.T, GIST-T1, NCI-N87, NUGC-4, SNU-5, CAL 27, FaDu, L1210, M-NFS-60, HL-60, EOL-1, Kasumi-1, Kasumi-3, Kasumi-3-Luc-mCh-Puro, KG-1-Luc-mCh-Puro, MOLM-13, MV-4-11, MV-4-11-Luc-mCh-Puro, NOMO-1, THP-1, NALM6 , NALM6-Luc-MCh-Puro, Reh, Reh(pMMP-Luc-Neo), K-562, K-562-Luc2, ARH-77, CCRF-CEM, DND-41-Luc-mCh-Puro, Jurkat, Jurkat-clone E6-1, MOLT-4, MOLT-4-Luc-MCh-Puro, Hep 3B2.1-7, HepG2、LL、LL / 2、LL / 2-Luc-M38、NCI-H596、Calu-6、NCI-H322M、A549、A549-Luc-C8、Calu-1、C alu-3、HCC827、HCC827-Luc-mCh-Puro、NCI-H125、NCI-H125-Luc、NCI-H1299、NCI-H1650、N CI-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, SK-MES-1, RL, DB, DB / M2, GRANTA-519, Farange, B-Daudi-JAB, Daudi -Luc-mCh-Pure、NAMALWA、Raji、Raji-Luc、Ramos、Ramos-Luc、HuT78、HT、SU-DHL-6、SU-DHL-6-Luc-mCh-Pure、OCI-Ly1 LN、OCI-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-Puro、SU-DHL-8、TMD8、Toledo-Luc-Neo、WSU-DLCL2、WSU-FSCCL、WSU-FSCCL-CMV-Luc-Puro、WSU-FSCCL-MSCV-Luc-Co-Luc-Puro、WSU-FSCCL-MSCV-Luc-Co-Puro-KARPMI 299、BT-20、BT-474、HCC1395、HCC70、Hs 578Bst、Hs 578T、MCF10A, MCF-7, MCF7-Luc-mCh-Puro, MDA-MB-231, MDA-MB-231-2LMP, MDA-MB-231-Luc-D3H1, MDA-MB-231-Luc-D3H2, MDA-MB-231-Luc-D3H2LN, MDA-MB-231-Luc-D3H3, MDA-MB-361, MDA-MB-453, MDA-MB-468, MX-1, MX-1-Luc, SK-BR-3, T47D, UISO-BCA-1, ZR-75-1, A2058, A375, COLO 829, G-361, LOX-IMVI, M14, MDA-MB-435S, OCM-1, OCM-1-Luc-mCh-Puro, 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 8226, U266B1, SK-N-AS, SK-N-FI, SK-N-SH, MKL-1, A2780, A2780-Luc, IGROV1, IGROV1-Luc-Mch-Puro, 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 1990, 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 (local), PC-3M-Luc-C6 (tibial), PC-3M-Luc-C6 (SC-Axilla), VCaP, 769-P, 786-O, 786-O-Luc-Neo (rescue), A-498, ACHN, Cki-!1, TK-10, A-673, HT-1080, MG-63, Saos-2, SJSA-1, SW872, MB-1, TT, SK-LMS-1, 293T, HEK293, or HeLa cells. As will be apparent and as contemplated herein, the transduced cells can be adherent cells (e.g., HEK cells) or non-adherent cells (e.g., T cells), or adherent or non-adherent cell lines.

[0116] Embodiments of the present disclosure also include applying a composition comprising a plurality of cells and at least one transduction factor to a biopolymer matrix to produce a scaffold material described herein. According to such embodiments, the implantable macroporous scaffold material of the present disclosure facilitates transduction of a plurality of cells with the transduction factor. Transduction of cells (e.g., immune cells) can be achieved by any means known in the art based on the present disclosure. In some embodiments, transduction of cells can be achieved via a vector encoding a transgene (e.g., a CAR). Thus, the scaffold material disclosed herein can include a vector (e.g., a lentivirus, retrovirus, adenovirus, adeno-associated virus, virus-like particle, liposome, transposon, or the like) encoding a transgene (e.g., a chimeric antigen receptor (CAR) or the like).

[0117] There are many compositions and methods that can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can be broadly categorized into two types: viral-based delivery systems and non-viral-based delivery systems. For example, nucleic acids can be delivered via several direct delivery systems, such as electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, virus-like particles (VLPs), transposons (e.g., class II transposable elements, including Sleeping Beauty transposase, Frog Prince, piggyBac, Tol2, and other Tc1 / mariner transposases), zinc finger nucleases, meganucleases, transcription activator-like effectors (e.g., TALENs), triplexes, mediators of epigenetic modifications, and CRISPR and rAAV methods), or nucleic acids can be delivered via a transfer or carrier of genetic material into a cell (e.g., a virus-like particle, a cell-mimetic particle, a transposon, an exosome, a nanoparticle, a micelle, or a liposome). Suitable means for transfection, including vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described, for example, in Wolff, JA, et al., Science, 247, 1465-1468, (1990) and Wolff, JANature, 352, 815-818, (1991). Such methods are well known in the art and can be easily adapted for use with the compositions and methods described herein. In some cases, the methods are modified to specifically act using large DNA molecules. Furthermore, these methods can be used to target specific diseases and cell populations by using the targeting properties of carriers.

[0118] Retroviruses are animal viruses belonging to the Retroviridae virus family, including any type, subfamily, genus, or tropism. Retroviral vectors are commonly referred to as Verma, IM, and retroviral vectors for gene transfer. Examples of retroviruses that can be used as vectors include, but are not limited to, human T-lymphotrophic virus (HTLV)-1 (HTLV-1), HTLV-2, HTLV-3, HTLV-4, simian foamy virus, human foamy virus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and Rous sarcoma virus. Retroviruses are essentially packages that contain nucleic acid cargo. The nucleic acid cargo carries a packaging signal with it. The packaging signal ensures that replicated daughter molecules are efficiently packaged within the package coat. In addition to the packaging signal, several molecules required for cis replication and packaging of the replicated virus are present. Retroviral genomes typically contain the gag, pol, and env genes, which are involved in generating the protein coat. Typically, the gag, pol, and env genes are replaced by the foreign DNA introduced into the target cell. Retroviral vectors usually contain the following: a packaging signal for incorporation into the package coat; a sequence signaling the initiation of the gag transcription unit; elements required for reverse transcription, including a primer-binding site that binds to the tRNA primer for reverse transcription; terminal repeats that guide RNA strand switching during DNA synthesis; a purine-rich sequence 5' to the 3' LTR that serves as a priming site for second-strand DNA synthesis; and specific sequences near the end of the LTR that allow the retroviral DNA form to be inserted into the host genome. Removal of the gag, pol, and env genes allows approximately 8 kb of foreign sequence to be inserted into the viral genome, which can be reverse-transcribed and packaged into new retroviral particles during replication. This amount of nucleic acid is sufficient to deliver one to many genes, depending on the size of each transcript.In some embodiments, positive and / or negative selectable markers can be included in the insert along with other genes.

[0119] Because the replication machinery and packaging proteins (gag, pol, and env) have been removed from most retroviral vectors, the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line that has been transfected or transformed with a retrovirus that contains the replication and packaging machinery but lacks a packaging signal. When a vector with the appropriate DNA is transfected into such a cell line, the vector containing the gene of interest is replicated and packaged into new retroviral particles by machinery provided in cis by the helper cells. The genome of the machinery is not packaged because it lacks the necessary signals.

[0120] Lentiviral vectors (LV), including but not limited to human immunodeficiency virus (HIV) vectors and simian immunodeficiency virus (SIV) vectors, are versatile vectors for in vivo gene transfer into dividing and non-dividing cells or cell culture. This system has the advantage of being flexible for transduction into various lung cancer cells without the need for time-consuming selection for stable expression. Replication-deficient VSV G pseudotyped lentiviral vectors (custom-made by GeneCopoeia) can be used to transduce cells.

[0121] The construction of replication-deficient adenoviruses has been described (Berkner et al., J. Virology 61:1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987); Zhang, "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis," BioTechnologies 15:868-872 (1993)). The advantage of using these viruses as vectors is that they can replicate within the initially infected cells but are unable to form new infectious viral particles, limiting their ability to spread to other cell types.Recombinant adenoviruses have been shown to achieve highly efficient gene transfer after direct in vivo delivery to airway epithelia, hepatocytes, vascular endothelium, CNS parenchyma, and several other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moulier, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993), Zabner, Nature Genetics 6:75-83 (1994), Guzman, Circulation Research 73:1201-1207 (1993), Bout, Human Gene Therapy 5:3-10 (1994), Zabner, Cell 75:207-216 (1993), Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993), and RaGot, J. Gen. Virology 74:501-507 (1993)).Recombinant adenoviruses achieve gene transfer by binding to specific cell surface receptors, and the virus is then internalized by receptor-mediated endocytosis, just like wild-type or replication-deficient adenoviruses (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell. 73:309-319 (1993). The viral vector can be based on an adenovirus from which the E1 gene has been deleted, and such viral vectors are produced in cell lines such as the human 293 cell line. In another embodiment, both the E1 and E3 genes are deleted from the adenoviral genome.

[0122] Another type of viral vector is based on adeno-associated virus (AAV). This defective parvovirus can infect many cell types and is nonpathogenic to humans, making it a preferred vector. AAV-type vectors can transport approximately 4-5 kb, and wild-type AAV is known to stably integrate into chromosome 19 (e.g., at AAV integration site 1 (AAVS1)). Vectors with this site-specific integration property can also be used. One embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which may contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene (e.g., a gene encoding green fluorescent protein GFP).

[0123] In another type of AAV virus, the AAV comprises a pair of inverted terminal repeats (ITRs) flanking at least one cassette containing a promoter, which is operably linked to a heterologous gene to direct cell-specific expression. In this context, "heterologous" refers to any nucleotide sequence or gene that is not native to AAV or B19 parvovirus.

[0124] Typically, the coding regions of AAV and B19 are deleted, resulting in a safe, non-cytotoxic vector. The AAV ITRs or modifications thereof confer infectivity and site-specific integration but not cytotoxicity, and the promoter directs cell-specific expression. U.S. Patent No. 6,261,834 is incorporated herein by reference for its content related to AAV vectors.

[0125] Thus, the disclosed vectors provide DNA molecules that are capable of integration into mammalian chromosomes without substantial toxicity. Genes inserted into viruses and retroviruses usually contain a promoter and / or enhancer that help control expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a fixed location relative to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors and may contain upstream elements and response elements.

[0126] Molecular genetic experiments using large human herpesviruses have provided tools for cloning, propagating, and establishing large heterologous DNA fragments in cells permissive to herpesvirus infection (Sun et al., Nature Genes 8:33-41, 1994; Cotter and Robertson, Curr Opin Mol Ther 5:633-644, 1999). Such large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV)) have the potential to deliver fragments of human heterologous DNA exceeding 150 kb to specific cells. EBV recombination can maintain large DNA fragments as episomal DNA within infected B cells. Individual clones harbored human genomic inserts of up to 330 kb, which appeared to be genetically stable. Maintenance of such episomes requires the specific EBV nucleoprotein EBNA1, which is constitutively expressed during EBV infection. Furthermore, such vectors can be used for transfection, which can transiently produce large amounts of protein in vitro. The herpesvirus amplicon system has also been used to package DNA fragments larger than 220 kb and infect cells that can stably maintain the DNA as an episome.

[0127] Other useful systems include, for example, replicating vaccinia virus vectors and host-restricted non-replicating vaccinia virus vectors.

[0128] Nucleic acids delivered to cells typically contain expression control systems. Genes inserted into viral and retroviral systems usually contain a promoter and / or enhancer that help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a fixed location relative to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors and may contain upstream elements and response elements.

[0129] Promoters controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, including, but not limited to, the genomes of viruses such as polyoma virus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and cytomegalovirus, or from heterologous mammalian promoters (e.g., the beta-actin promoter). The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication (Fiers et al., Nature, 273:113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenway, PJ et al., Gene 18:355-360 (1982)). Promoters from host cells or related species are also useful in the present disclosure.

[0130] Enhancers generally refer to DNA sequences that function at different distances from the transcription start site and can be 5' to a transcription unit (Laumins, L. et al., Proc. Natl. Acad. Sci. 78:993 (1981)) or 3' (Lusky, M. L. et al., Mol. Cell Bio. 3:1108 (1983)). Enhancers can also be located within introns (Banerji, J. Lett. et al., Cell 33:729 (1983)) or even within the coding sequence itself (Osborne, T. F. et al., Mol. Cell Bio. 4:1293 (1984)). Enhancers are usually 10-300 bp in length and function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate transcriptional regulation. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of gene expression. Although many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), enhancers from eukaryotic cell viruses are typically used for general expression. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0131] The promoter and / or enhancer can be specifically activated either by light or by specific chemical events that trigger their function. The system can be regulated by drugs such as tetracycline and dexamethasone. There are also methods to enhance viral vector gene expression by exposure to radiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0132] In certain embodiments, the promoter and / or enhancer region may act as a constitutive promoter and / or enhancer, maximizing expression of the transcribed transcription unit region. In certain constructs, the promoter and / or enhancer region is active in all eukaryotic cell types, even if it is only expressed in certain cell types at certain times. One such type of promoter is the CMV promoter (650 bases). Other promoters include the SV40 promoter, cytomegalovirus (full-length promoter), and retroviral vector LTR.

[0133] As will be shown, all of the specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types, such as melanoma cells. The glial fibrillary acidic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

[0134] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells) may further contain sequences necessary for the termination of transcription, which can affect mRNA expression. Such regions are transcribed as polyadenylation segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated region also contains a transcription termination site. In some embodiments, the transcription unit also contains a polyadenylation region. One advantage of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. Homologous polyadenylation signals can be used in transgene constructs. In one particular transcription unit, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of approximately 400 bases. The transcribed unit can also contain other standard sequences, alone or in combination with the above sequences, to improve expression from or stability of the construct.

[0135] Viral vectors can contain nucleic acid sequences encoding marker products that can be used to determine whether a gene has been delivered to a cell and is being expressed after delivery. Marker genes include the E. coli lacZ gene, which encodes β-galactosidase and green fluorescent protein.

[0136] In some embodiments, the marker can be a selectable marker. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. Successful transfer of such selectable markers into mammalian host cells allows the transformed mammalian host cells to survive when placed under selection pressure. There are two widely used distinct categories of selection methods. The first category is based on cellular metabolism and the use of mutant cell lines that lack the ability to grow independent of supplemented media. Two examples are CHO DHFR cells and mouse LTK cells. These cells lack the ability to grow without added nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes required for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes to alter their growth requirements. Individual cells that are not transformed with the DHFR or TK genes cannot survive in unsupplemented medium.

[0137] The second category is dominant selection, which refers to a selection scheme that can be used with any cell type and does not require the use of mutant cell lines. Such schemes typically use a drug that stops the growth of host cells. Cells carrying the novel gene express a protein that confers drug resistance and survive selection. Examples of such dominant selection include those using the drug neomycin (Southern P. and Berg, P., J. Molec. Appl. Genet. 1:327 (1982)), mycophenolic acid (Mulligan, R.C. and Berg, P. Science 209:1422 (1980)), or hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5:410-413 (1985)). These three examples use bacterial genes under eukaryotic control to confer resistance to the appropriate drug, G418, neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively. Others include the neomycin analogue G418 and pramycin.

[0138] 3.How to use

[0010] Embodiments of the present disclosure also include methods of treating a subject. According to such embodiments, and as further described herein, the method includes implanting a macroporous scaffold material into or adjacent to a target tissue, the scaffold material comprising a crosslinked biopolymer matrix having an average pore size in the range of about 10 μm to about 500 μm and a stiffness in the range of about 1 kPa to about 1000 kPa, wherein the stiffness of the matrix matches the stiffness of the target tissue, and a composition comprising a plurality of cells and a transduction factor. In some embodiments, the scaffold material facilitates transduction of the plurality of cells with the transduction factor, and the transduced cells treat the subject.

[0139] In some embodiments of the method, the target tissue is tumor tissue. In some embodiments of the method, the target tissue is solid tumor tissue. In some embodiments of the method, the target tissue comprises at least one of lung tissue, bone tissue, skin tissue, breast tissue, muscle tissue, nerve tissue, brain tissue, lymphatic tissue, prostate tissue, bladder tissue, stomach tissue, intestinal tissue, uterine tissue, ovarian tissue, liver tissue, adipose tissue, cartilage tissue, thyroid tissue, and / or pancreatic tissue. In some embodiments of the method, the subject has been diagnosed with a disease or condition. In some embodiments of the method, the disease or condition comprises cancer.

[0140] In some embodiments, the scaffolding material facilitates transduction of a plurality of cells (e.g., immune cells) with a transduction factor (e.g., a viral vector comprising a polynucleotide encoding a protein of interest) with a transduction efficiency of at least 50% (measured in vivo or ex vivo). In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor with a transduction efficiency of at least 60%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor with a transduction efficiency of at least 70%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor with a transduction efficiency of at least 80%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor with a transduction efficiency of at least 90%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor with a transduction efficiency of about 50% to about 90%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 60% to about 90%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 70% to about 90%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 50% to about 80%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 50% to about 70%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 60% to about 80%. In some embodiments, the scaffolding material facilitates transduction of a plurality of cells with a transduction factor at a transduction efficiency of about 70% to about 90%.

[0141] In some embodiments of the method, the biopolymer matrix comprises at least one of alginate, hyaluronic acid, collagen, fibrin, polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), gelatin, polyethylene glycol (PEG), chitosan, cellulose, polyglutamic acid, fibrin, silk, agarose, dextran, polyacrylamide, polyvinyl alcohol, poly(N-isopropylacrylamide), poly(2-hydroxyethyl methacrylate), polyurethane, polyethyleneimine, poly(methyl methacrylate), poly(2-oxazoline), polyphosphazene, and complexes, derivatives, or combinations thereof.

[0142] As further described above, in some embodiments of the method, the biopolymer matrix comprises an alginate having a molecular weight of about 1 kDa to about 500 kDa. In some embodiments of the method, the biopolymer matrix comprises an alginate having a G / M ratio of about 0.5 to about 5.0. In some embodiments of the method, the biopolymer matrix comprises an alginate at a concentration in the range of about 0.1% to about 5.0%. In some embodiments of the method, the biopolymer matrix comprises an alginate with a calcium concentration in the range of about 0.1% to about 1.0%. In some embodiments of the method, the biopolymer matrix is ​​generated at a temperature in the range of about -20°C to about -80°C.

[0143] Further as described above, in some embodiments of the method, the biopolymer matrix exhibits a stiffness that is about ±25%, about ±50%, about ±75%, about ±100%, about ±125%, about ±150%, about ±175%, about ±200%, about ±225%, or about ±250% of the stiffness of the target tissue.

[0144] In some embodiments of the method, the scaffold material comprises at least one biological agent. In some embodiments of the method, the at least one biological agent is a small molecule. In some embodiments of the method, the small molecule is selected from the group consisting of a TLR agonist, a checkpoint inhibitor, an IDO inhibitor, a MEK inhibitor, an HDAC inhibitor, a PI3K inhibitor, an immunomodulator, a JAK kinase inhibitor, and an mTOR inhibitor. In some embodiments of the method, the at least one biological agent is a protein, peptide, or polypeptide. In some embodiments of the method, the protein, peptide, or polypeptide is selected from the group consisting of a cytokine, an antibody, and a growth factor. In some embodiments of the method, the cytokine comprises at least one of IL-2, IL-15, IL-7, IL-23, TNF-α, and / or IFN-γ.

[0145] In some embodiments of the method, the plurality of cells comprises one or more immune cells. In some embodiments of the method, the one or more immune cells are selected from the group consisting of T cells, B cells, natural killer (NK) cells, NK T cells, macrophages, dendritic cells, tumor infiltrating lymphocytes (TILs), tumor infiltrating NK cells (TINKs), and bone marrow infiltrating lymphocytes (MILs). In some embodiments of the method, the one or more immune cells are activated. In some embodiments of the method, the plurality of cells is obtained from cell culture. In some embodiments of the method, the plurality of cells is obtained from a donor.

[0146] As further described above, in some embodiments of the method, the transduction agent comprises a viral vector. In some embodiments of the method, the viral vector is selected from the group consisting of lentivirus, retrovirus, adenovirus, adeno-associated virus, coccivirus, and baculovirus. As further described above, in some embodiments of the method, the transduction agent comprises a virus-like particle, a cell-mimetic particle, a transposon, an exosome, a nanoparticle, a micelle, and a liposome. In some embodiments of the method, the transduction agent comprises a nucleic acid cargo. In some embodiments of the method, the nucleic acid cargo comprises siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or viral RNA. In some embodiments of the method, the nucleic acid cargo comprises DNA encoding a fusion protein, a chimeric antigen receptor (CAR), a therapeutic peptide or polypeptide, or a combination thereof.

[0147] In accordance with the above embodiments, the present disclosure provides various pharmaceutically acceptable embodiments of the implantable macroporous scaffold material disclosed herein. As noted above, the scaffold material of the present disclosure can also be administered in vivo with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that a substance is not biologically undesirable or otherwise undesirable; i.e., the substance can be administered to a subject together with the nucleic acid or vector without causing undesirable biological effects or interacting in a deleterious manner with any other components of the pharmaceutical composition in which it is included. The carrier will, of course, be selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject, as will be known to those skilled in the art.

[0148] Such agents can be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), or targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of the technique to target tumor tissue with specific proteins (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" liposomes and other antibody-bound liposomes (including lipid-mediated drugs targeting colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-induced tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of technology to target tumor tissue with specific proteins (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biochemicala Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand-induced. Such receptors cluster in clathrin-coated cavities, enter cells via clathrin-coated vesicles, pass through acidifying endosomes where the receptors are sorted and then recycled to the cell surface, stored intracellularly, or degraded in lysosomes.Internalization pathways perform a variety of functions, including nutrient uptake, removal of activated proteins, exclusion of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and regulation of receptor levels. Many receptors follow multiple intracellular pathways depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0149] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Further examples of carriers include sustained-release formulations. Sustained-release formulations are, for example, semipermeable matrices of solid hydrophobic polymers containing an antibody, and such matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. As will be apparent to those skilled in the art, certain carriers may be more suitable depending, for example, on the route of administration and the concentration of the administered composition.

[0150] Pharmaceutical carriers are known to those skilled in the art. Such carriers are most typically standard carriers for administering drugs to humans, including solutions such as sterile water, physiological saline, and buffered solutions at physiological pH. The composition can be administered intramuscularly or subcutaneously. Other compounds are administered according to standard procedures used by those skilled in the art. In addition to the molecule of choice, pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. Pharmaceutical compositions may also include one or more active ingredients, such as antibacterial agents, anti-inflammatory agents, anesthetics, etc.

[0151] Formulations for administration include sterile aqueous or non-aqueous solutions, sterile suspensions, and sterile emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, as well as saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be used. Some compositions may optionally be administered as pharmaceutically acceptable acid or base addition salts. Such acid or base addition salts are formed by reaction with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid), organic acids (e.g., formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid), or inorganic bases (e.g., sodium hydroxide, ammonium hydroxide, potassium hydroxide), or organic bases (e.g., mono-, di-, tri-, and aryl amines and substituted ethanol amines).

[0152] The effective dose and schedule for administering the composition may be determined empirically, and making such a determination is within the skill of one in the art. The dose range for administering the composition is large enough to produce the desired effect of affecting the symptoms of the disease. The dose should not be so large as to cause adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, etc. Generally, the dose will vary depending on the age, condition, sex, extent of the patient's disease, route of administration, or whether other drugs are included in the regimen, and can be determined by one skilled in the art. The dose can be adjusted by an individual physician in the event of any contraindications. The dose can vary and can be administered for one or more days in one or more daily doses. Literature provides guidance on the appropriate dose for a given type of pharmaceutical product. For example, guidance in selecting an appropriate dose of an antibody is provided in literature on the therapeutic use of antibodies, such as Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357, and Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389.

[0153] In accordance with the above embodiments, a scaffolding material of the present disclosure can be implanted within or adjacent to a target tissue. In some embodiments, the target tissue is tumor tissue. In some embodiments, the target tissue is solid tumor tissue. In some embodiments, the target tissue comprises at least one of lung tissue, bone tissue, skin tissue, breast tissue, muscle tissue, nerve tissue, brain tissue, lymphatic tissue, prostate tissue, bladder tissue, stomach tissue, intestinal tissue, uterine tissue, ovarian tissue, liver tissue, adipose tissue, cartilage tissue, thyroid tissue, and / or pancreatic tissue.

[0154] The disclosed scaffold materials can be used to treat any disease in which uncontrolled cell proliferation occurs, for example, cancer. Representative, but non-limiting, cancers that can be treated using the compositions of the present disclosure include lymphomas, such as B-cell lymphoma and T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia (including, but not limited to, acute myeloid leukemia (AML) and / or chronic myeloid leukemia (CML)), bladder cancer, brain cancer, and cancers of the nervous system. head and neck cancer, squamous cell carcinoma of the head and neck, renal cancer, lung cancer such as small cell lung cancer, non-small cell lung cancer (NSCLC), lung squamous cell carcinoma (LUSC), and lung adenocarcinoma (LUAD), neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, pharynx, larynx, and lung, cervical cancer, breast cancer including but not limited to triple-negative breast cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, and colon and rectal cancer.

[0155] In some embodiments, disclosed herein are methods of treating, inhibiting, reducing, reducing, ameliorating, and / or preventing solid cancer tumors and / or metastases (e.g., brain cancer, including but not limited to glioblastoma) in a subject, the method comprising implanting into the subject any of the loaded macroporous scaffold materials disclosed herein, wherein the scaffold material is implanted into tissue, and wherein the scaffold material, when hydrated with biological fluids, exhibits a Young's modulus that is compatible with the tissue. For example, disclosed herein are methods of treating, inhibiting, diminishing, reducing, ameliorating, and / or preventing solid cancer tumors and / or metastases (e.g., brain cancers, including but not limited to glioblastoma) in a subject, the methods comprising administering to a subject a therapeutic cargo (including, but not limited to, a fusion protein, a chimeric antigen receptor (CAR) (e.g., CD19, CD33, IL-13 receptor alpha chain 2 (IL13Rα2), B7-H3, neural / glial antigen 2 (NG2), disialoganglioside GD2, epidermal growth factor receptor vIII (EGFRvIII), MUC1, PSMA, mesothelin, HER2, or CEA-targeting CAR T cells, CAR NK cells, CAR NK cells, or the like) targeting a targeting CAR T cell or CAR NK cell, or the like, to treat a disease or disorder of the tissue. The method includes implanting a supported macroporous scaffold material containing a viral vector (e.g., a lentivirus, retrovirus, adenovirus, adeno-associated virus, virus-like particle, transposon, or liposome) encoding a target cell type (e.g., T cells, or CAR macrophages), a foreign gene, siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or DNA encoding the gene, a therapeutic ligand, or a combination thereof), wherein the supported scaffold material is prepared by a process including incubating a dry macroporous scaffold material having an average pore size in the range of about 10 μm to about 500 μm and a stiffness in the range of about 1 kPa to about 1,000 kPa.

[0156] Also disclosed herein are methods of treating, inhibiting, reducing, ameliorating, and / or preventing cancerous tumors and / or metastasis using the implantable macroporous scaffold material of the present disclosure. In some embodiments, the scaffold material exhibits a stiffness that is ±25%, ±50%, ±75%, ±100%, ±125%, ±150%, ±175%, ±200%, ±225%, or ±250% of the stiffness of the tissue (e.g., cancerous tumor).

[0157] As described further herein, by generating a scaffold to be compatible with the target tissue, the scaffold can be applied directly to any target tissue or tumor, as opposed to administering only cells transduced with the scaffold. Accordingly, disclosed herein are methods of treating, inhibiting, reducing, reducing, ameliorating, and / or preventing cancerous tumors (e.g., solid tumors) and / or metastasis, wherein a scaffold is implanted subcutaneously or directly into the tumor.

[0158] In one embodiment, the CAR can be tailored to target a particular cancer by adjusting the target to which the CAR binds. For example, if the cancer is glioblastoma, the scaffold can include one or more CARs targeting epidermal growth factor receptor vIII (EGFRvIII), HER2, IL-13 receptor alpha chain 2 (IL13Rα2), B7-H3, disialoganglioside GD2, and / or MUC1; if the cancer is leukemia, B-cell acute leukemia, B-cell non-Hodgkin's lymphoma, follicular lymphoma, Mantel cell lymphoma, the scaffold can include one or more CARs targeting CD20, CD22, and / or CD19 (e.g., tisagenlecleucel, axicabtagene ciloleucel, CTL-119, UCART119, JCAR014, JCAR017); if the cancer is multiple myeloma, the scaffold can include one or more CARs targeting BCMA and / or CD138. If the cancer is breast cancer, the scaffold can comprise one or more CARs targeting MET, MUC1, and / or HER2; if the cancer is ovarian cancer or cervical cancer, the scaffold can comprise one or more CARs targeting MUC16 and / or FR; if the cancer is pancreatic cancer, the scaffold can comprise one or more CARs targeting mucin 1 (MUC1), mesothelin (MSLN), and / or CD19; if the cancer is prostate cancer, the scaffold can comprise one or more CARs targeting PSA and / or PSMA; if the cancer is acute myeloid leukemia, the scaffold can comprise one or more CARs targeting CD33 and / or CD123. Alternatively, the scaffold can comprise a CAR targeting a non-cargo such as PD-L1, CLDN6, or a PD1-CD28 fusion.

[0159] In one embodiment, the implantable macroporous scaffold material of the present disclosure is configured to carry a therapeutic cargo (including, but not limited to, fusion proteins, chimeric antigen receptors (CARs) (e.g., CD19, CD30, CD20, Cd171, Cd80 / 86, c-MET, DLL-3, DR5, EpHA2, BCMA, GD2, B7H3, NKR2, NKG2D, CD133, CEA, EGFR, EGFR806, mesothelin, PSCA, PSMA, EpCAM, MUC1, ICAM-1, CD147, EpHA2, HER2, IL13Rα2, FOLR1, MSLN, CLDN18.2, VEGFR2, AFP, nectin4 / FAP, Lewis and viral vectors (e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus, virus-like particle, transposon, or liposome) encoding a target gene (e.g., CAR T cells, CAR NK cells, CAR NK T cells, or CAR macrophages targeting Y, glypican-3, AFP, AXL, DR5, gp100, MAGE-A1 / 3 / 4, LMP1, DLL-3, IL-13 receptor alpha chain 2 (IL13Rα2), B7-H3, neural / glial antigen 2 (NG2), disialoganglioside GD2, epidermal growth factor receptor vIII (EGFRvIII), MUC1, PSMA, mesothelin, HER2, or CEA), a foreign gene, siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or DNA encoding the gene, a therapeutic ligand, or a combination thereof.

[0160] Also disclosed herein are methods for treating, inhibiting, reducing, reducing, ameliorating, and / or preventing brain cancer (e.g., glioblastoma, etc.), the methods comprising implanting any of the macroporous scaffold materials disclosed herein into the brain of a subject in need thereof. For example, disclosed herein are methods of treating, inhibiting, reducing, diminishing, ameliorating, and / or preventing brain cancer (e.g., glioblastoma, etc.), the methods comprising administering to the patient a therapeutic cargo (including, but not limited to, a fusion protein, a chimeric antigen receptor (CAR) (e.g., CD19, CD30, CD20, Cd171, Cd80 / 86, c-MET, DLL-3, DR5, EpHA2, BCMA, GD2, B7H3, NKR2, NKG2D, CD133, CEA, EGFR, EGFR806, mesothelin, PSCA, PSMA, EpCAM, MUC1, ICAM-1, CD147, EpHA2, HER2, IL13Rα2, FOLR1, MSLN, CLDN18.2, VEGFR2, AFP, nectin4 / FAP, Lewis and treating brain cancer by implanting a macroporous scaffold material containing a viral vector (e.g., a lentivirus, retrovirus, adenovirus, adeno-associated virus, virus-like particle, transposon, or liposome) encoding a target gene (e.g., CAR T cells, CAR NK cells, CAR NK T cells, or CAR macrophages targeting Y, glypican-3, AFP, AXL, DR5, gp100, MAGE-A1 / 3 / 4, LMP1, DLL-3, IL-13 receptor alpha chain 2 (IL13Rα2), B7-H3, neural / glial antigen 2 (NG2), disialoganglioside GD2, epidermal growth factor receptor vIII (EGFRvIII), MUC1, PSMA, mesothelin, HER2, or CEA), a foreign gene, siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or DNA encoding the gene, a therapeutic ligand, or a combination thereof.

[0161] As will be understood by those skilled in the art based on the present disclosure, the treatment regimens can be used alone or in combination with any anti-cancer therapeutic agent known in the art, 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, Afinitor (everolimus), Aquinzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), and alectinib. , alemtuzumab, Alimta (pemetrexed disodium), Alicopa (copanlisib hydrochloride), Alkeran injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), amifostine, aminolevulinic acid, anastrozole, aprepitant, Alesia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanon (nelarabine), arsenic trioxide, Arzera (ofatumumab), Erwinia chrysanthemi-derived asparaginase, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Besenum (carmustine), Beleodac (belinostat), belinstat, bendamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, Bexar (tositumomab and iodine I131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, Busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib-S-malate), cabozantinib-S-malate,CAF, Campath (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Carac (topical fluorouracil), carboplatin, carboplatin-taxol, carfilzomib, Carumbris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, Cerbidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin tin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, cobimetinib, Cometriq (cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Kotellic (cobimetinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposomal, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, Darzalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and liposomal cytarabine, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diftitox, denosumab, DepoCyt (liposomal cytarabine), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxolone) Doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), durvalumab, Efudex (topical fluorouracil), Eitek (rasburicase), Ellence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), Emplici (elotuzumab), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH,Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinase (asparaginase from Erwinia chrysanthemi), Ethyol (amifostine), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Evacet (doxorubicin hydrochloride liposomal), everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil- -topical), Fareston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil-topical), fluorouracil injection, fluorouracil-topical, flutamide, Folex (methotrexate), Folex PFS (methotrexate), Forfiri, Forfiri-bevacizumab, Forfiri-cetuximab, Forfirinox, Forfox, Forlotin (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nonvalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamycin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Glia Del (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant HPV nonvalent vaccine, recombinant HPV quadrivalent vaccine, recombinant Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), ibritumomab tiusetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride),Idarubicin hydrochloride, idelalisib, Idhifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide, Ifosfamidum (ifosfamide), IL-2 (aldesleukin), imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), imiquimod, Imlygic (talimogene laherparepvec), Inlyta (axitinib), inotuzumab ozogamicin, interferon alfa-2b, recombinant interleukin-2 (aldesleukin), Intron A (recombinant interferon alpha-2b), iodine I131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposomal, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), JEB, Jevtana (cabazitaxel), Kadcyla (ado-trastuzumab emtansine), Keoxifen (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlecleucel), Cyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, Lartruvo (olaratumab), Levitra Nalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, Leustatin (cladribine), Levran (aminolevulinic acid), Linfolidine (chlorambucil), LipoDox (doxorubicin hydrochloride liposomal), lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lynparza (olaparib), Marqibo (vincristine sulfate liposomal), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna, Mesnex (mesna), Methazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), midostaurin, mitomycin C, mito Xantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlynx (neratinib maleate), netupitant, and palonosetron hydrochloride,Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandrone (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraparib tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiprotim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olaratumab, omacetaxine mepesuxinate, Oncasper (pegaspargase), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposomal), On tak (denileukin diftitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticles, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, peginterferon alfa-2b, PEG-Intron (peginterferon alfa-2b) , pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portraza (necitumumab), pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine), propranolol methylprednisolone hydrochloride, Provenge (sipuleucel-T), Purinetol (mercaptopurine), Prixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonvalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib,Relistor (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hycela (rituximab and human hyaluronidase), rituximab, rituximab and human hyaluronidase, rolapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib camsylate), rucaparib camsylate, ruxolitinib phosphate, Rydapt (midostaurin), sclerosol intrapleural aerosol (talc), siltuximab, sipuleucel-T, Somatuline Depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon alfa-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuxinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Taglisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tese Intrik (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Tolak (fluorouracil - topical), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine I131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP,Varubi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Versar (vinblastine sulfate), vemurafenib, Venclexta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, VIP, vismodegib, Vistogard (uridine triacetate), Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vixeos (daunorubicin hydrochloride and cytarabine liposome), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervo (ipilimumab), Yondelis (trabectedin), Zaltrap (Ziv-aflibercept), Zarxio (filgrastim), Zejula (niraparib tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and / or Zytiga (abiraterone acetate).

[0162] Therapeutic methods can further include checkpoint inhibitors. Such checkpoint inhibitors include, but are not limited to, antibodies that block PD-1 (e.g., nivolumab (BMS-936558 or MDX1106), pembrolizumab, CT-011, MK-3475, etc.), antibodies that block PD-L1 (e.g., atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C, etc.), antibodies that block PD-L2 (e.g., rHIgM12B7, etc.), antibodies that block CTLA-4 (e.g., ipilimumab (MDX-010), tremelimumab (CP-675, 206)), antibodies that block IDO, antibodies that block B7-H3 (e.g., MGA271, MGD009, omburtamab, etc.), antibodies that block B7-H4, antibodies that block B7-H3, antibodies that block Ig and T cell immunoreceptors with ITIM domains (TIGIT) (e.g., BMS-986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO, etc.), antibodies blocking CD96, antibodies blocking B and T lymphocyte attenuator (BTLA), antibodies blocking V-domain Ig suppressor of T cell activation (VISTA) (e.g., JNJ-61610588, CA-170, etc.), TI M3-blocking antibodies (e.g., TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3-blocking antibodies (e.g., BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immunep). [Example]

[0163] 4. Working Example As will be readily apparent to those skilled in the art, other suitable modifications and variations of the disclosed methods described herein can be readily applied and recognized, and can be made using suitable equivalents, without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having described the present disclosure in detail, the present disclosure will be more clearly understood by reference to the following examples. It should be noted that the examples are intended merely to illustrate some aspects and embodiments of the present disclosure and should not be construed as limiting the scope of the present disclosure. The disclosures of all journal articles, U.S. patents, and publications mentioned herein are hereby incorporated by reference in their entirety.

[0164] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

[0165] Example 1 The well-connected Drydux scaffold structure supports efficient T cell reprogramming. Transduction of T cells to generate stably expressing CAR T cells is a critical step in CAR T cell manufacturing. Current technologies utilize transduction-enhancing agents (RetroNectin, Polybrene) and physical forces such as centrifugation (spinoculation) to promote stable viral-mediated gene transfer with high transduction efficiency. However, such multi-step methods increase the time, cost, and complexity of genetic modification.

[0166] To enable single-step static transduction, Drydux scaffolds were synthesized by gentle cryogelation of calcium-crosslinked alginate gels (Figure 1A). Because macroporosity and hygroscopicity were recognized to be important for scaffold-mediated static T cell reprogramming, detailed characterization of the scaffold's porosity and structure was performed. X-ray computed tomography (CT) analysis of Drydux revealed 100-200 μm oval, well-connected pores that accounted for 82.78% of the scaffold's volume (Figures 1B-1E and 10A-10C). This well-connected macroporous structure (Figures 1F-1G) facilitates effective interaction between activated cells and CAR-encoding viral particles, enabling gene transfer and nutrient mass transfer, supporting T cell proliferation (Figure 11A) and release (Figure 11B).

[0167] To test the ability of the Drydux scaffold to reprogram T cells in vitro, we mixed freshly isolated and activated human peripheral blood mononuclear cells (PBMCs) with a retrovirus encoding GFP, seeded them onto the dry macroporous scaffold, and incubated them for 3 days (Figure 2A). A conventional CAR T cell generation method (spinoculation using retronectin-coated plates) was used as a positive control. The Drydux scaffold reprogrammed approximately 80% of GFP. + While conventional spinoculation produced cells with approximately 95% GFP + Both methods resulted in high-quality transduction, although Drydux was slightly less efficient, and the Drydux method was significantly less complicated, eliminating the need for continuous centrifugation.

[0168] Because RetroNectin-coated plates can be stored at 4°C for up to 1–2 months, the shelf life of Drydux scaffolds was tested. Drydux scaffolds stored in sealed bags at 4°C for 6, 12, and 18 months showed similar, if not better, transduction efficiency compared to freshly prepared scaffolds, suggesting excellent shelf life and long-term functionality (Figure 2C).

[0169] Example 2 The implantable Drydux scaffold material provides superior antitumor efficacy against lymphoma. Previous studies have reported that biomaterial scaffolds can generate highly functional CD19.CAR T cells in vitro. Experiments were conducted to determine whether this scaffold platform could generate and release functional CD19-targeted CAR T cells in vivo. The efficacy of implanted Drydux scaffolds was first investigated in vivo using the well-studied Daudi lymphoma model. IL-2-supplemented medium containing activated PBMCs and CD19.CAR-encoding retroviral particles was applied to the Drydux scaffold material. Four days after tumor cell inoculation, the scaffold material was subcutaneously implanted into lymphoma-bearing mice (Figure 3A). Tumor-bearing mice intravenously injected with conventionally generated CAR T cells served as a positive control, while Drydux scaffolds seeded with activated PBMCs but without virus served as a negative control. CD19.CAR T cells generated in vivo using Drydux or conventional methods similarly eradicated tumors, as assessed by measuring tumor bioluminescence intensity (Figures 3B-3C). Tumor regression was not accompanied by any signs of toxicity, as assessed by measuring overall appearance and body weight (Figure 3D), and improved overall survival (Figure 3E). This demonstrated that the implantable Drydux scaffold is capable of generating functional CD19.CAR T cells in vivo with potency similar to conventionally generated CAR T cells, without complex ex vivo manipulations.

[0170] Example 3 Drydux Generates Highly Functional B7H3-Targeted CAR T Cells. To test the ability of the Drydux scaffold to generate CAR T cells for solid tumors, we generated CAR T cells targeting the B7-H3 antigen. PBMCs isolated from healthy donors were activated on αCD3 / αCD28-coated plates, mixed with gammaretrovirus encoding B7H3.CAR, and seeded onto Drydux scaffolds (Figure 2A). After two days, cells were isolated and transduction efficiency was assessed using flow cytometry. T cells reprogrammed using conventional retronectin and spinoculation methods served as a positive control. Activated cells seeded onto Drydux scaffolds without gammaretrovirus served as a non-transduced control. By day 5, Drydux and conventional methods reprogrammed approximately 79% and 82% of B7H3.CAR, respectively. + The CAR T cells generated by Drydux and conventional methods showed comparable transduction efficiencies (Figures 4A-4B). Furthermore, both CAR T cells generated by Drydux and conventional methods showed stable CAR expression over 14 days (Figure 12), suggesting robust and stable reprogramming. CAR T cells generated using either method were CD4 + and CD8 + showed similar percentages of the population (Figure 4C), as well as naive / stem cell memory (CD45RA + CCR7 + ), Central Memory (CD45RA - CCR7 + ), Effector Memory (CD45RA - CCR7 - ), and effector (CD45RA + CCR7 - ) populations within each subset (Figures 4D-4E). Furthermore, CAR T cells generated using either method showed similar expression of exhaustion markers or inhibitory receptors such as PD1, LAG3, and TIM3 (Figure 4F). Furthermore, CAR T cells generated using either method showed comparable and robust cell proliferation kinetics (Figure 4G).

[0171] To examine the functionality of B7H3.CAR T cells in vitro, Drydux- and conventionally generated CAR T cells were cocultured with B7-H3-expressing ovarian (SKOV3), lung (A549), and pancreatic (PANC-1) cell lines at different effector-to-target (E:T) ratios (Figure 13A). Untransduced cells were used as negative controls. CAR T cells generated using conventional or Drydux scaffolds conferred effective tumor growth suppression against ovarian, lung, and pancreatic tumor cells at an E:T ratio of 1:5 (Figure 4H). Furthermore, superior cytotoxicity of B7H3.CAR T cells was observed against pancreatic tumor cells at an E:T ratio of 1:1 (Figures 13B-13C), whereas partial cytolytic activity was observed against ovarian, lung, and pancreatic tumor cells at an E:T ratio of 1:10 (Figure 13F). Cytokine (IL-2 and IFN-γ) release by CAR T cells in response to target tumor cells was also determined (Figures 4I-4J, Figures 13D-13E, and Figures 13G-13H).

[0172] Taken together, the Drydux scaffold simplifies genetic modification and generates CAR T cells with transduction efficiencies comparable to conventional methods. Furthermore, CAR T cells generated with the scaffold expand to clinically relevant doses, retain their effector phenotype, and demonstrate functionality against a variety of solid tumor cell lines in vitro.

[0173] Example 4 Implantable Drydux scaffolds demonstrate interleukin-mediated proliferation in vitro. IL-2 was physically encapsulated in Drydux scaffolds (Drydux+IL2) to promote CAR T cell proliferation and release after subcutaneous implantation in vivo. The transduction efficiencies of Drydux and Drydux+IL2 were comparable (Figure 14A), suggesting that IL-2 does not affect transduction. Furthermore, Drydux+IL2 scaffolds also supported T cell proliferation without the need for additional cytokines in the culture medium (Figure 14B). Furthermore, IL-2 significantly promoted cell release from the scaffolds, as measured by an in vitro cell release assay. Drydux+IL2 scaffolds placed in transwell inserts released cells into the lower chamber over a 21-day period (Figures 5A-5B), demonstrating the potential of Drydux+IL2 scaffolds to promote sustainable release of CAR T cells after in vivo implantation.

[0174] Example 5 The implantable Drydux scaffold material has demonstrated the absence of accidental transduction in vitro. Finally, experiments were performed to test the possibility of retroviral leakage and transduction of surrounding host cells upon implantation of a scaffold carrying activated PBMCs and a CAR-encoding retrovirus. An in vitro transwell experiment was designed in which PBMCs and Drydux carrying a GFP-encoding retrovirus were placed in a transwell insert, and human fibroblasts were seeded in the lower chamber. At predetermined time points, GFP expression in fibroblasts was assessed to confirm retroviral leakage and unwanted transduction (Figure 6A). When only GFP-encoding retrovirus was seeded on Drydux, approximately 17% GFP expression was observed in fibroblasts, indicating viral leakage from the scaffold without co-seeded PBMCs. In contrast, when both PBMCs and a GFP-encoding retrovirus were seeded on Drydux scaffolds, no transduction was observed in fibroblasts, suggesting that the virus encounters and transduces the closest cells (Figure 6B). This provides indirect evidence suggesting that the Drydux scaffold likely does not transduce host cells upon implantation into the subcutaneous space.

[0175] Example 6 The implantable Drydux scaffold outperforms conventionally generated CAR T cells in metastatic lung tumors. Given the Drydux scaffold's ability to generate and deliver tumor-specific CAR T cells in vitro, we conducted experiments to explore the potential of this platform in solid tumors. We hypothesized that in vivo generated CAR T cells would not only reduce generation time and cost, but also generate less differentiated, longer-lasting, and more functional CAR T cells for difficult-to-treat solid tumors. Therefore, we tested the Drydux+IL2 scaffold's ability to generate B7-H3-targeting CAR T cells in vivo in a panel of solid tumor models.

[0176] First, the antitumor activity of Drydux-generated CAR T cells was evaluated in a metastatic model of non-small cell (NSC) lung tumors. Tumors were established in NSG mice by inoculation with FFluc-expressing A549 cells via tail vein injection. Treatment began 14 days after tumor cell inoculation. Each animal was inoculated with either Drydux+IL2 scaffold seeded with donor-matched untransduced T cells, or CAR T cells generated using conventional methods, or Drydux+IL2 scaffold seeded with activated cells and a CAR-encoding retrovirus (approximately 2.5x10 CAR T cells). 6) (Figure 7A, Figure 15A). Tumors grew rapidly in control animals inoculated with untransduced T cells, whereas animals inoculated with conventionally generated CAR T cells or the Drydux+IL2 scaffold effectively suppressed tumor growth until day 14 after treatment. However, after 14 days, animals inoculated with the Drydux+IL2 scaffold showed continued tumor regression, while animals inoculated with conventionally generated CAR T cells showed signs of tumor recurrence. Furthermore, CAR T cells generated in vivo using the Drydux+IL2 scaffold produced long-lasting antitumor effects (Figures 7B-7C). Neither of the two groups inoculated with CAR T cells exhibited weight loss, suggesting that this therapy was well tolerated over the treatment period, with the exception of the late development of GVHD, a common complication of using human cells in mouse models (Figure 7D). Both treatments demonstrated similar survival benefits, with surviving animals treated with Drydux+IL2 remaining tumor-free up to 98 days after treatment (FIG. 7E).

[0177] To assess the ability of CAR T cells to persist in vivo after treatment, analysis of CAR+ cells was performed at defined time points using flow cytometry. 34 days after treatment, the number and immunophenotypic composition of circulating CAR T cells were assessed, revealing a higher number of circulating CAR T cells in the Drydux+IL2 scaffold-inoculated group. + A large number of CAR cells were identified (Figure 7F). + The population exhibits predominantly effector memory and effector phenotypes, along with minorities of circulating stem cell memory, central memory, and exhaustion (PD1 + LAG3 +) cells (Figure 7G). This suggested robust in vivo expansion of CAR T cells while maintaining a highly functional, less differentiated phenotype. At the end of the experiment (day 99), blood, spleen, and bone marrow were collected from surviving animals to assess CAR T cell numbers and their immunophenotypic composition. At day 99, animals treated with Drydux+IL2 scaffolds exhibited circulating CAR T cells with predominantly effector memory and effector phenotypes (Figure 15C). Analysis of CAR T cells from the spleen (Figure 15D) and bone marrow (femur) (Figure 15E) showed a significant presence of effector memory and effector CAR T cells (Figures 15F-15G). This suggested long-lasting antitumor potential of CAR T cells generated with Drydux+IL2 scaffolds.

[0178] Taken together, CAR T cells generated in vivo in the Drydux+IL2 scaffold exhibited higher functionality and improved persistence, resulting in long-term antitumor efficacy and prevention of tumor recurrence in a metastatic lung tumor model.

[0179] Example 7 The implantable Drydux scaffold generates highly functional CAR T cells against ovarian tumors. Following the excellent antitumor efficacy of Drydux-generated CAR T cells in metastatic lung tumors, the potential of this platform was evaluated in an intraperitoneal ovarian xenograft model. FFluc-expressing SKOV3 tumor cells were inoculated intraperitoneally into NSG mice, and treatment began on day 14. Each animal was inoculated with: 2.5 x 10 donor-matched CAR T cells generated using conventional methods; 6PBMCs or Drydux+IL2 scaffolds seeded with PBMCs and a retrovirus encoding CAR. Negative control animals were inoculated with Drydux+IL2 scaffolds containing only PBMCs (Figure 8A, Figure 16A). Tumor growth was monitored for up to 126 days after treatment. Animals inoculated with the control scaffolds showed rapid tumor progression and, interestingly, the development of GVHD within 15 days of treatment. Both conventionally and Drydux+IL2 scaffold-generated B7H3.CAR T cells demonstrated significant tumor eradication, whereas conventionally treated animals showed signs of tumor recurrence within 30 days of treatment. In contrast, animals inoculated with Drydux+IL2 scaffolds demonstrated long-term antitumor effects, with all treated mice remaining tumor-free until the end of the experiment (126 days after treatment) (Figures 8A-8C). Neither group showed significant weight loss. This suggested that both groups tolerated the CAR T cell dose well (Figures 8D-8E). Furthermore, animals inoculated with CAR T cells generated in the Drydux+IL2 scaffold showed significantly improved overall survival compared to conventionally treated animals (Figure 8E).

[0180] To assess the persistence of CAR T cells in the blood, CAR T cells were collected from the blood, bone marrow, and spleen of surviving animals. At day 123, animals treated with scaffold-generated CAR T cells showed circulating B7H3.CAR T cells. +The tumors showed significant B7H3.CAR T cell abundance, demonstrating the ability of in vivo-generated CAR T cells to persist in the circulation even after tumor remission (Figure 16B). CAR T cell analysis from bone marrow (femur) and spleen demonstrated significant B7H3.CAR T cell abundance (Figures 16C-16D). Animals treated with conventionally generated CAR T cells could not be analyzed for CAR T cells because they did not survive at the final data point. To quantify CAR T cells in the blood at an earlier time point, this tumor model was repeated with a second donor, and animals inoculated with CAR T cells generated from the second donor were evaluated at day 34 post-treatment. At day 34, mice treated with the Drydux+IL2 scaffold had significantly higher numbers of circulating CAR T cells compared with animals treated with the same amount of conventionally generated CAR T cells (Figure 16E). Immunophenotypic assessment revealed a predominantly effector memory and effector phenotype. Furthermore, mice treated with Drydux+IL2-generated CAR T cells showed higher circulating numbers of less differentiated stem cell memory and central memory cells (Figure 16F). Taken together, Drydux-generated CAR T cells exhibited superior antitumor efficacy and prevented tumor recurrence compared with conventionally generated CAR T cells, likely due to their improved in vivo persistence.

[0181] Example 8 The implantable Drydux scaffold generates highly functional and persistent CAR T cells and prevents tumor recurrence in orthotopic pancreatic tumors. Finally, the Drydux platform was tested for its ability to generate functional CAR T cells in an aggressive and lethal orthotopic pancreatic tumor model. FFluc-expressing Panc-1 cells were inoculated into the pancreas of NSG mice, and treatment began 12 days later. Each animal was inoculated with: 2.5 x 10 donor-matched untransduced T cells; 6 Drydux+IL2 scaffolds seeded with CAR T cells, or CAR T cells generated using conventional methods, or Drydux+IL2 scaffolds seeded with cells and CAR-encoding retrovirus (CAR T cell abundance was approximately 2.5x10 6) (Figure 9A, Figure 17A). Negative control animals inoculated with Drydux+IL2 and PBMCs but not virus showed rapid tumor growth. Animals receiving intravenous infusions of conventionally generated CAR T cells showed good initial tumor regression but gradually recurred tumors 70–100 days after treatment. In sharp contrast, animals inoculated with the Drydux+IL2 scaffold demonstrated long-term antitumor efficacy, with all treated mice remaining tumor-free until the end of the experiment (123 days after treatment) (Figures 9B–9C). Monitoring of animal weights suggested that treatment was well tolerated throughout, and no obvious toxicity associated with CAR T cell therapy was observed (Figure 9D). Furthermore, animals inoculated with CAR T cells generated with the Drydux+IL2 scaffold demonstrated significantly improved overall (Figure 9E) and tumor-free survival (Figure 17B) compared with animals treated with the same amount of conventionally generated CAR T cells.

[0182] To assess the persistence of CAR T cells in vivo, blood was collected at days 20 and 40 after treatment. Animals inoculated with the Drydux+IL2 scaffold had nearly 24-fold more B7H3.CAR T cells at day 20 compared to conventionally generated CAR T cells. + cells, 10-fold more B7H3.CAR at day 40 +CAR T cells (Figure 9F). Furthermore, immunophenotyping was performed on blood CAR T cells on day 40 to assess the differentiation status of circulating CAR T cells after treatment. Animals treated with Drydux+IL2 scaffolds exhibited higher numbers of naive / stem cell memory, effector memory, and effector populations, while conventionally generated CAR T cells exhibited only a few effector memory cells. This suggested superior persistence and antitumor potential of Drydux-generated CAR T cells. Furthermore, both groups exhibited similar numbers of exhausted cells (Figure 9G). At the end of the experiment (day 123), blood, spleen, and bone marrow of surviving animals were collected, and CAR T cells were quantified and subjected to immunophenotyping. At day 123, animals treated with scaffold-generated CAR T cells exhibited circulating B7H3.CAR T cells with predominantly effector memory and effector phenotypic configurations. + The results showed that circulating naive / stem cell memory cells were largely absent, along with some exhausted populations (Figure 9H). This further demonstrates that in vivo-generated CAR T cells have the ability to persist longer in the circulation after tumor remission. Analysis of CAR T cells from the spleen and bone marrow (femur) revealed the significant presence of effector memory cells, followed by the absence of effector CAR T cells (Figures 9I-9L). None of the animals treated with conventionally generated CAR T cells were tumor-free at the final data point, and therefore could not be analyzed for CAR T cells.

[0183] Taken together, CAR T cells generated in vivo using the Drydux platform demonstrated high functionality against orthotopic pancreatic tumors. Furthermore, the implantable macroporous scaffold provided sustained cell delivery of less differentiated CAR T cells, demonstrating improved in vivo persistence that prevented disease recurrence. Furthermore, the Drydux scaffold was implanted within three days of T cell isolation, reducing the time and complexity associated with CAR T cell generation.

[0184] In these examples, the results demonstrate that the simple, stable, and implantable "Drydux" scaffold generates highly functional CAR T cells in a shorter timeframe. The Drydux scaffold provides a favorable structure for effective T cell reprogramming and delivery. This scaffold serves as an excellent transduction agent for generating highly functional CAR T cells. Furthermore, this platform can be easily tailored for in vivo applications, minimizing the need for ex vivo manipulations and reducing the complexity of current CAR T cell manufacturing protocols. Upon subcutaneous implantation, the Drydux scaffold generated highly functional CAR T cells in animal models of systemic lymphoma, intraperitoneal metastatic ovarian cancer, intravascular metastatic lung cancer, and orthotopic pancreatic cancer. Furthermore, the Drydux scaffold significantly improved CAR T cell persistence, with circulating cells detectable for up to 120 days posttreatment, resulting in greater efficacy compared to the same number of CAR T cells generated using conventional methods. This tunable platform has the potential to broaden the scope and accessibility of costly and time-consuming cell therapies.

[0185] Example 9 The development of next-generation cell therapies relies on rapid, versatile, and efficient cell reprogramming. Novel biomaterials play a central role in this process by providing bioactive signals and scaffolds that determine cell fate and function. Previous studies have reported that dry macroporous alginate scaffolds mediate retroviral transduction of primary T cells with efficiencies comparable to those achieved by the typical clinical spinoculation method, which requires centrifugation on retronectin-coated plates. Transduction with this scaffold requires that the scaffold be both macroporous and dry. Transduction with dry macroporous scaffolds, termed "Drydux transduction," offers a rapid and inexpensive method for transducing cells for cell therapies, including the generation of CAR T cells.

[0186] In these examples, the mechanism of action by which Drydux transduction works was investigated by investigating the effects of pore size, stiffness, virus concentration, and absorption rate on transduction efficiency. Results revealed that Drydux scaffolds with macropores in the 50-230 μm range and Young's modulus in the 25-620 kPa range all effectively transduced primary T cells. While this suggests that these parameters are not central to the mechanism of action, it demonstrates that Drydux scaffolds can be tailored without loss of functionality. Increasing virus concentration led to significantly higher transduction efficiency, demonstrating the need for increased cell-virus interactions for optimal transduction. Finally, the rate at which the cell-virus solution was absorbed into the scaffold was found to closely correlate with viral transduction efficiency, with faster absorption resulting in significantly higher transduction. Computational modeling of fluid flow through porous media indicates that increased fluid flow substantially increases collisions between virus particles and cells in the porous scaffold, thus supporting this finding. Taken together, these data indicate that the fluid flow rate through the scaffold, rather than pore size or stiffness, acts as the central controlling factor for efficient Drydux transduction.

[0187] Scaffold Fabrication. Macroporous scaffolds were fabricated by freeze gelation (FIG. 18). Briefly, equal volumes of calcium solution and alginate solution were vigorously mixed and poured into the wells of a 24-well plate. The samples were then frozen and lyophilized to produce dry macroporous alginate scaffolds, which were designated "Drydux" scaffolds.

[0188] MOI Calibration. Previous publications have reported conditions for high transduction efficiencies of 85–95%. However, these high efficiencies potentially pose a problem, as even minor improvements in scaffold optimization may be overlooked. Therefore, the multiplicity of infection (MOI) of the GFP-encoding gammaretrovirus was adjusted to achieve a transduction efficiency of less than 60% in primary PBMCs isolated from human blood, as this would allow for gradual improvements to be more easily observed. Lowering the MOI resulted in a decrease in the percent transduction (Figure 24). An MOI of 2, which resulted in a transduction efficiency of 59%, was determined to be optimal and was used in all of the following experiments unless otherwise noted.

[0189] Pore ​​size, but not stiffness, correlates with Drydux transduction efficiency when alginate and calcium concentrations are varied. To assess whether calcium or alginate concentration affects Drydux transduction, scaffolds were prepared with various calcium concentrations (0.1%, 0.2%, 0.3%) and alginate concentrations (0.5%, 1.0%, 1.5%, 2.0%) (w / v). The cross-sectional area of ​​the scaffolds was approximately 1.72 cm. 2 The scaffolds were approximately 5.37 mm in height (Figure 19A). All scaffolds yielded transduction efficiencies greater than 50%, demonstrating their high ability to transduce cells (Figure 19B). Scaffolds fabricated with 0.1% calcium had significantly higher transduction efficiencies than those fabricated with 0.2% and 0.3% calcium. Scaffolds fabricated with 0.5% alginate exhibited significantly lower transduction rates than almost all other alginate concentrations. This is likely due to the lack of surface porosity in 0.5% alginate scaffolds compared to the other scaffolds. The average pore size of these scaffolds ranged from 76 to 230 μm (Figure 19C). Spearman correlation revealed a strong and significant correlation (p = 0.0065) between pore size and transduction efficiency (Figure 19D).

[0190] To determine the stiffness of the scaffolds, Drydux scaffolds were subjected to compression tests with a force of 50 N and a compression speed of 0.1 mm / s. Young's modulus was calculated based on the stress-strain curves obtained from the compression tests (Figure 25). Scaffolds containing 0.1% calcium (w / v) were more flexible than those containing 0.2% and 0.3% calcium (w / v), with 1.5% (w / v) alginate forming the stiffest scaffold (Figure 19E). There was no significant Spearman correlation between the Young's modulus of the scaffolds and transduction efficiency (p = 0.3510) (Figure 19F).

[0191] Example 10 Neither pore size nor stiffness correlated with Drydux transduction efficiency when alginate concentration and freezing temperature were varied. Several groups have reported that the freezing temperature during cryogelation determines the pore size of the cryogel. To further evaluate the effect of pore size without the complication of altering the crosslinking concentration, we assessed how varying the freezing temperature affected the pore size and stiffness of the scaffold material and further assessed whether these changes affected the transduction efficiency. Scaffolds were again synthesized by cryogelation at different alginate concentrations (0.5%, 1.0%, 1.5%, and 2.0%) with a constant calcium concentration of 0.2%, as previously reported. The scaffolds were then frozen at -20°C, -40°C, -60°C, or -80°C and lyophilized. The cross-sectional area of ​​the scaffolds was approximately 1.78 cm. 2The average transduction efficiency was approximately 4.86 mm (Figure 20A). All scaffolds showed a transduction efficiency of over 60%, demonstrating that all scaffolds successfully transduced cells (Figure 20B). As shown above, scaffolds containing 0.5% alginate showed significantly lower transduction efficiency than scaffolds made with other alginate concentrations. There was no significant difference between scaffolds with alginate concentrations of 1.0%, 1.5%, and 2.0%. Scaffolds frozen at -40°C and -80°C showed no significant difference in transduction, but both showed significantly higher transduction efficiency than scaffolds frozen at -20°C and -60°C. The average pore size of these scaffolds ranged from 52 to 131 μm (Figure 20C). In contrast to the results shown in Figure 19, application of Spearman correlation did not yield a significant correlation between pore size and transduction efficiency in this experiment (p=0.6669) (Figure 20D).

[0192] Dryudux scaffolds were compressed with a force of 50 N at a compression rate of 0.1 mm / s to measure the stiffness of the scaffolds. Young's modulus was calculated based on the stress-strain curves obtained from the compression tests (Figure 26). Slower freezing rates generally resulted in stiffer scaffolds, with scaffolds with alginate concentrations of 1.0% and 1.5% forming the stiffest scaffolds (Figure 20E). There was no significant Spearman correlation between Young's modulus of the scaffolds and transduction efficiency (p=0.5938) (Figure 20F).

[0193] Example 11 Both virus concentration and seeding volume significantly correlated with Drydux transduction efficiency. Because Drydux transduction did not appear to be dependent on stiffness and had an unpredictable relationship with pore size, experiments were performed to identify other factors that may affect transduction efficiency. Viral transduction depends on the interaction between the virus and the cells. We reasoned that higher virus concentrations should result in higher transduction efficiencies. Approximately 50,000 primary human PBMCs and 100,000 gammaretroviral particles were suspended in 25 μL, 50 μL, 100 μL, or 200 μL, and Drydux transduction of these solutions was assessed. Diluting the virus significantly reduced transduction, confirming our hypothesis (Figure 27).

[0194] In previous experiments, we noticed that larger volumes required significantly more time for absorption into the scaffold material (Figure 21A, Figure 28). It was unclear whether the volume of the solution, and therefore the absorption rate, could affect Drydux transduction. To investigate this possibility, retroviral transduction of primary human PBMCs was evaluated using different solution volumes (10 μL, 25 μL, 50 μL, 100 μL, or 200 μL) at a fixed concentration (Figure 21B). Simultaneously, we measured the rate of liquid absorption into the scaffold material by photographing the absorption process and measuring the time it took for that volume to be completely absorbed into the scaffold material. There was a clear trend in transduction efficiency, with smaller seeding volumes resulting in significantly higher transduction efficiency (Figure 21C). Furthermore, there was a clear trend in absorption rate, with smaller seeding volumes resulting in faster absorption rates (Figure 21D). Applying Spearman correlation to this data, a strong and significant correlation (p<0.0001) was found between the resorption rate of the scaffold and transduction efficiency (Figure 21E). By measuring the area of ​​the scaffold wetted by the droplets viewed from above, different seeding volumes had different resorption areas (Figure 21B). Therefore, the volumetric flux was calculated by dividing the resorption rate by the resorption area (Figure 21F). A strong and significant Spearman correlation (p<0.0001) was found between the volumetric flux and transduction (Figure 21G).

[0195] From these results, we conclude that a smaller seeding volume results in faster absorption into the scaffold material, resulting in increased volumetric flux and higher transduction efficiency. Therefore, it makes sense that spreading the cell-virus solution over a larger surface area would lead to faster absorption and higher transduction. To test this hypothesis, we used a 6-well plate with a cross-sectional area of ​​approximately 8.12 cm. 2 Scaffolds were fabricated and seeded with a solution containing 4,000 cells / μL primary human PBMCs and 16,000 particles / μL retroviral particles (MOI=4) either by spreading the volume over the entire surface area or by seeding the volume at a single location (Figure 29A). Liquid spread over the entire surface of the scaffold was absorbed faster than the same volume added to a single location on the scaffold, resulting in significantly higher transduction efficiency (Figure 29B). These results confirm that the surface area of ​​the scaffold can be used to control the absorption rate of the cell-virus solution and influence Drydux transduction.

[0196] Example 12 Computational modeling confirms the importance of the porous structure for transduction efficiency. The above results suggest that fluid flow through the scaffold during absorption governs Drydux transduction. To better understand the possible mechanism behind this finding, we computationally simulated fluid flow through the scaffold. We hypothesized that fluid flow through the scaffold should increase the number of cell-virus collisions, and that higher flow rates would increase the probability of collisions and therefore improve transduction. Flow in the scaffold was simulated in Ansys Fluent v21 using a discrete particle method and computational fluid dynamics. Virus (radius = 5 × 10) particles were injected into the scaffold. -8 m) and cells (radius = 3.5 × 10 -6 m) were modeled as rigid spheres flowing under three scenarios: quiescent fluid, uniform unrestricted flow (FIG. 22A), and flow through the pores of the scaffold (FIG. 22B). Following previous data collected on pore geometry, the scaffold pore geometry was modeled as 1.3×10 center-to-center. -4 m away from the radius of 7.5 × 10 -5The scaffold was modeled as a set of overlapping interconnected spheres (Figure 22B). Periodic boundaries were applied to a representative base volume of the shape to approximate the numerous pores present in the scaffold. The volumetric flux of the flow was varied (1.5, 3.0, 6.0, and 30.0 μL / min / cm). 2 ), experimental volumetric fluxes of different seeding volumes into the scaffold pores were established as shown in Figure 21F. A 60-second flow solution was calculated by numerically solving the incompressible Navier-Stokes equations. Because particles occupy less than 0.1% of the liquid volume, particle trajectories were initially tracked from a random distribution using one-way coupling with the flow solution. The particle model included drag forces on cell and virus particles, Brownian diffusion, and lift forces under shear. The quiescent fluid was modeled, and no collisions were observed between the virus and T cells. Additionally, a flow rate of 1.5 μL / min / cm was calculated. 2 For unrestricted flow with a volumetric flux of 30 μL / min / cm, no collisions are observed. 2 At a volumetric flux of 100 s, few collisions were observed. However, modeling the flow inside the scaffold pores predicted that the flow velocity would increase fourfold inside the constriction point when compared to the widest cross-section of the pore, such that mass conservation was satisfied (Figure 22C). This increase in flow velocity inside the scaffold pores resulted in a more than 20-fold increase in the number of T cell–virus collisions when compared to unrestricted flow (Figure 22D). Furthermore, the number of collisions inside the scaffold pores consistently increased with volumetric flux. These results reveal the importance of scaffold geometry in facilitating T cell–virus interactions during the transduction process.

[0197] In these examples, the experimental results further shed light on the mechanism behind Drydux transduction by detailing the effects of scaffold pore size, stiffness, virus concentration, seeding volume, and absorption rate on transduction of human primary T cells. Macroporous alginate scaffolds were synthesized with various physical properties by varying alginate concentration, calcium concentration, and freezing temperature. Within the range investigated, pore size was found to have some but unpredictable effects on transduction, while stiffness did not. Diluting the virus reduced transduction efficiency. Surprisingly, reducing the seeding volume on the scaffold without changing the concentration significantly improved cell transduction. Both seeding volume and transduction efficiency were found to correlate well with absorption rate, defined as the time required for the droplet to be completely absorbed into the scaffold. Taken together, these data indicate that the absorption rate of the cell-virus solution likely governs Drydux transduction and suggest specific ways to optimize Drydux scaffolds in future studies.

[0198] 5. Materials and Methods Drydux Preparation. Macroporous alginate scaffolds (Drydux) were prepared as described above. Briefly, 2% w / v ultrapure alginate (Pronova, MVG) was dissolved in sterile-filtered DI water and vigorously stirred. Once the alginate was completely dissolved, an equal volume of 0.4% calcium D-gluconate solution was mixed with the alginate solution and vigorously stirred for 15 minutes. The resulting gel was then cast into a 48-well plate (300 μl / well) and frozen at −20°C overnight. The next day, the cryogel was transferred to a freeze dryer. After 72 hours, the scaffolds were removed and stored at 4°C in vacuum-sealed bags until further use. As will be understood by those of skill in the art based on this disclosure, w / v % = mass of solute (g) / volume of solution (mL) × 100 (e.g., 20 mg (0.02 g) alginate in 1 mL of solution = 0.02 g / 1 mL = 0.02 = 2% w / v). In some embodiments, alginate gels can be made at twice the final concentration. For example, one solution containing 2% w / v alginate can be mixed with an equal volume of 0.4% w / v% calcium solution. The final solution concentration is 1% alginate with 0.2% calcium.

[0199] To prepare macroporous scaffolds (Drydux+IL2) for subcutaneous implantation, recombinant human IL-2 (PeproTech) was introduced into the scaffolds at a concentration of 0.2 μg / mg alginate, followed by cross-linking and cryogelation.

[0200] X-ray CT. To characterize the macroporosity of Drydux, X-ray CT scans were performed on the scaffolds. Scans were performed on an Xradia Versa 510 using Zeiss Scout and Scan version 13 with an 8-second exposure, X4 optical magnification, 2.6 μm pixel size, 1600 projections, no filter, 74 μA current, and 40 kV voltage. A cylindrical volume of 2.50 × 2 mm was scanned in the sample. The data from such a volume was then used to calculate the porosity and pore size of the sample. Smaller subvolumes of 10 × 1 mm were extracted to visualize and calculate connectivity. CT data were analyzed using Dragonfly 2020.1 software (Object Research Systems, http: / / www.theobjects.com / dragonfly). To segment the samples, a training dataset was manually created for each sample using histogram thresholding and masking methods. Once the training data were generated, they were used to train a deep learning image segmentation model called U-net. The resulting model was then used to segment the entire sample into scaffolds and pores. To calculate the connectivity between pores, an open-source package (openPNM 2.8) was used.

[0201] Conventional CAR T cell generation. CAR T cells were generated at the University of North Carolina at Chapel Hill according to the current manufacturing practices and quality control standards used for the production of clinical-grade cell products for clinical trials. Human PBMCs were isolated from buffy coat fractions of healthy donors (Gulf Coast Regional Blood Center) using Lymphoprep density separation (Accurate Chemical and Scientific Corporation). Freshly isolated PBMCs were activated on plates coated with 1 μg / ml of CD3 (Miltenyi Biotec, 30-093-387, clone OKT-3) and CD28 (BD Biosciences, 555725, clone CD28.2) agonist monoclonal antibodies. Retroviral supernatants used for cell transduction were provided by a collaborator. To transduce activated T cells with retroviruses encoding GFP, CD19.CAR, or B7H3.CAR, 24-well plates were coated with Retronectin (Takara Bio). Two days after T cell activation, retroviral supernatant was spun onto retronectin-coated plates at 2000 g for 90 minutes, and then activated T cells were spun onto retrovirus- and retronectin-coated plates at 1000 g for 10 minutes. Plates were incubated for 72 hours, after which CAR T cells were harvested and expanded in complete medium consisting of Click medium (Irvine Scientific) and RPMI 1640 (1:1 v / v), 10% Hyclone FBS (GE Healthcare), 2 mmol / L GlutaMax (Gibco), penicillin (100 U / ml) (Gibco), and streptomycin (100 mg / ml) (Gibco), supplemented with 10 ng / ml IL-7 (PeproTech) and 5 ng / ml IL-15 (PeproTech). Cells were harvested on days 12–14 for in vitro and in vivo experiments.

[0202] Scaffold-Based CAR T Cell Generation. To generate CAR T cells using Drydux scaffolds, human T cells were isolated from buffy coats (Gulf Coast Regional Blood Center) and activated on plates using CD3 (1 μg / ml, Miltenyi Biotec, clone OKT-3) and CD28 (1 μg / ml, BD Biosciences, clone CD28.2) agonist monoclonal antibodies as described above. To prepare retroviral supernatants for transduction, the supernatants of retroviruses encoding GFP, CD19.CAR, or B7H3.CAR were concentrated 10-fold using an Amicon centrifuge (MWCO 100 kDa, Millipore) at 2500 g for 15–20 min. Finally, the activated cells and concentrated retroviral supernatants (MOI 2) were combined in a volume of approximately 100 μl and pipetted onto each dry macroporous scaffold. Control scaffolds were seeded with activated cells alone. For in vivo studies, the seeded scaffolds were incubated at 37°C in 5% CO2 for at least 1 hour before implantation. The scaffolds were then subcutaneously implanted into tumor-bearing NSG mice on the same day of transduction. For in vitro studies, the seeded scaffolds were cultured in excess medium + / - cytokines for 72 hours. After 3 days of culture, the scaffolds were digested with 0.25 M EDTA (a calcium chelator) and washed twice with excess PBS to dissociate the cells. Dissociated cells were analyzed for GFP or B7H3.CAR expression by flow cytometry. More than 95% of the cells were recovered and viable. The remaining cells were cultured in complete medium supplemented with 10 ng / ml IL-7 (PeproTech) and 5 ng / ml IL-15 (PeproTech) for various in vitro experiments.

[0203] Cell lines and culture. Daudi cells were obtained from the American Type Culture Collection (ATCC) and transduced with a retroviral vector encoding FFluc. After transduction, cells were selected in puromycin (Sigma-Aldrich). Cells were maintained in RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco), 2 mmol / L GlutaMax (Gibco), and penicillin (100 U / ml) (Gibco) and streptomycin (100 mg / ml) (Gibco) at 5% CO2 and 37°C.

[0204] The human ovarian cancer cell line SKOV-3 (female source), the human NSCLC cell line A549, and the human pancreatic (PDAC) tumor cell line Panc-1 (male source) were kindly provided by Dr. Dotti's laboratory. These cell lines were originally obtained from ATCC and then transduced with retroviral vectors encoding GFP and firefly luciferase (GFP-FFluc) genes.

[0205] SKOV-3 cells were cultured in McCoy's medium (Corning) supplemented with 10% FBS, 2 mM GlutaMax, and (100 units / mL) penicillin (Gibco) and streptomycin (Gibco). A549 cells were cultured in RPMI 1640 (Gibco) supplemented with 10% FBS and 2 mM GlutaMax. Penicillin (100 units / mL) (Gibco) and streptomycin (100 μg / mL) (Gibco) were added to the cell culture medium. Panc-1 cells were cultured in DMEM (GIBCO) supplemented with 10% FBS, 2 mM GlutaMax, and (100 units / mL) penicillin (Gibco) and streptomycin (Gibco). All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0206] In vitro cytotoxicity. GFP-expressing tumor cells (SKOV3, A549, or Panc-1) were plated at approximately 1 × 10 per well in a 24-well plate. 5CAR T cells generated using conventional or Drydux scaffolding were normalized for transduction efficiency and co-cultured with tumor cells at different effector-to-target ratios (E:T of 1:1, 1:5, and 1:10) without exogenous cytokines. Non-transduced cells were used as a negative control. After 5 days of co-culture, cells were harvested and residual tumor cells were measured by flow cytometry. Dead cells were excluded by gating using Zombie Aqua Dye (Biolegend) staining. Tumor cells (SKOV3, A549, and panc-1) were identified by GFP expression, and T cells were identified by CD3 expression.

[0207] ELISA. CAR T cells were co-cultured with tumor cells (SKOV3, A549, and panc-1) at E:T ratios of 1:1, 1:5, and 1:10 without the addition of exogenous cytokines. After 24 hours, supernatants were collected, and IL-2 and IFN-γ were quantified using specific ELISA kits (R&D Systems) according to the manufacturer's instructions.

[0208] In vitro cell proliferation and release. To assess cell proliferation in Drydux (with and without exogenous cytokines) and Drydux + IL2, activated T cells were labeled with 1.5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen) and seeded onto the scaffolds. After 3 days, cells were detached, counted, and CFSE dilution was measured using flow cytometry.

[0209] To assess cell release, the scaffolds seeded with activated T cells were placed in a 40 μm transwell (Corning). Fresh medium was placed in the lower chamber in contact with the mesh. At designated time points, the medium in the lower chamber was collected, the released cells were counted, and the scaffolds were transferred to a new well containing fresh medium.

[0210] In vitro safety assessment. To assess the possibility of retrovirus leakage from the scaffold during implantation, an in vitro experiment was designed to indirectly verify the safety of the scaffold after implantation. Drydux scaffolds loaded with cells alone or loaded with cells and a retrovirus encoding GFP were placed in transwells (0.4 μm pore size), and human fibroblasts were seeded in the bottom well. Fibroblasts were harvested at predetermined time points and analyzed for GFP expression using flow cytometry.

[0211] In vivo studies. All animal procedures were approved by and conducted in compliance with the North Carolina State University Institutional Animal Care and Use Committee (IACUC). All animals were obtained from the UNC-Chapel Hill Animal Core Facility. Animals were maintained under pathogen-free conditions with regular health monitoring. All treated animals were monitored for signs of discomfort and were euthanized upon loss of more than 15% of their initial body weight, hind limb paralysis or hunched posture, or upon reaching a humane endpoint based on tumor burden. For the lymphoma xenograft tumor model, 10-12 week-old female immunodeficient NSG mice (NOD.Cg-Prkdsscid Il2rgtm1Wjl / SzJ) were injected with 1 x 10 6 FFluc-expressing Daudi cells were injected intravenously. Four days after injection, 1x10 cells were injected intravenously in IL-2-supplemented medium. 6 Two Drydux scaffolds, each loaded with 1 x 10 PBMCs and a CD19.CAR-encoding gammaretrovirus, were subcutaneously implanted into each mouse (N = 5). In the control group (N = 3), 1 x 10 PBMCs were implanted in IL-2-supplemented medium. 6 Two Drydux scaffolds seeded with 2 × 10 PBMCs (non-transduced) were implanted into mice. 6Mice injected with CD19.CAR T cells were used as positive controls (N=5). Tumor burden was monitored weekly using a Xenogen-IVIS imaging system. For solid tumor models (lung, ovarian, and pancreatic), animals were imaged before the start of treatment (day 0), and animals without obvious tumor signals were excluded from the study. Before being assigned to the control or treatment group, animals were randomized based on tumor bioluminescence to ensure equal groups. Animal deaths after treatment were included in the survival study. For the metastatic model of NSLC, 1 × 10 6 FFluc-A549 tumor cells were injected intravenously into 8-10 week-old NSG mice via tail vein injection. Two weeks after tumor cell inoculation, conventionally generated B7H3.CAR T cells (2.5 × 10 6 Each animal was inoculated intravenously (N=4) with 2.5×10 CAR T cells / animal, or with activated PBMCs and two Drydux+IL2 scaffolds (total volume of 2.5×10 CAR T cells / animal) loaded with B7H3.CAR-encoding retrovirus. 6 Two Drydux+IL2 scaffolds (total 2.5 × 10 CAR T cells / animal) seeded with PBMCs alone were subcutaneously implanted into each animal (N = 4). 6 T cells) were transplanted as a control (N=4). Tumor burden was monitored weekly using an IVIS imaging system. After 34 days of treatment, blood was collected via buccal puncture and analyzed for the number and immunophenotypic composition of B7H3.CAR+ cells. At the end of the study (day 99), blood, spleen, and bone marrow were collected from the conventional and Drydux-treated groups to determine the number and immunophenotypic composition of B7H3.CAR+ cells. In the ovarian xenograft tumor model, 5x10 5 FFluc-SKOV3 cells were suspended in 1:1 PBS:Matrigel and inoculated intraperitoneally into 6- to 8-week-old female NSG mice. 14 days later, conventionally generated B7H3.CAR T cells (2.5 × 10 6 CAR T cells / animal) were intravenously infused (N=5) or activated PBMCs and two Drydux+IL2 scaffold materials (total volume 2.5 x 10) carrying B7H3.CAR-encoding retrovirus. 6Treatment was initiated by subcutaneous implantation of two Drydux+IL2 scaffolds (total of 2.5 × 10 CAR T cells / animal) (N = 6). 6 T cells) were transplanted as a control (N=3). Tumor burden was monitored weekly using an AMI imaging system. The study was repeated twice with two different PBMC donors. On day 34 post-treatment, blood was collected via mandibular buccal bleed and analyzed for the number and immunophenotypic composition of B7H3.CAR T cells. On day 126, blood, spleen, and bone marrow were collected from the surviving group to determine the number of B7H3.CAR+ cells. In the orthotopic PDAC tumor model, 2×10 5 FFluc-Panc-1 tumor cells were suspended in 50 μl of 1:1 PBS:Matrigel and surgically implanted into the pancreas of 8-10 week-old female NSG mice. Briefly, the pancreas was exposed through an incision in the left flank, and tumor cells were injected into the posterior part of the pancreas using a 29-gauge needle. The wound was closed in two layers with 4-0 Vicryl and polypropylene sutures. 12 days after tumor cell inoculation, treatment with CAR T cells was initiated. Activated PBMCs and two Drydux+IL2 scaffolds (total volume 2.5 × 10 cells) carrying B7H3.CAR-encoding retrovirus were implanted into the pancreas. 6 Two scaffolds seeded with PBMCs alone (2.5 × 10 total CAR T cells / animal) were implanted subcutaneously (N = 5). 6 T cells) were transplanted as a control (N=6). Conventionally generated B7H3.CAR T cells (2.5 × 10 6 CAR T cells / animal) were injected intravenously as a positive control (N=6). Animals were monitored for weight loss and tumor burden was measured weekly using an IVIS imaging system. Blood was collected by mandibular buccal bleed at 20 and 40 days post-treatment to measure the number of circulating CAR T cells. At day 123, blood, spleen, and bone marrow from survivors were collected to determine the number and immunophenotypic composition of B7H3.CAR+ cells.

[0212] Flow cytometry and antibodies. All samples were captured on a BD LSRII using BD FACSDiva software, acquiring a minimum of 10,000 events per sample. Absolute cell numbers were calculated using CountBright absolute counting beads (C36950, Thermo Fisher Scientific). Samples were analyzed with FlowJo software (version 10.8.1).

[0213] [Table 1]

[0214] Quantification and statistical analysis. Comparisons between two groups were performed using unpaired one-sided or two-sided Student's t-tests with Holm-Sidak correction for multiple comparisons. For multiple comparisons, one-way analysis of variance with Tukey's post hoc analysis was used. All analyses were performed using Graph Pad Prism software, version 9.4.1.

[0215] Preparation of macroporous alginate scaffolds. Scaffolds were prepared as described above. A DI solution of ultrapure alginate (Pronova, MVG) was vigorously mixed with an equal volume of a deionized (DI) solution of calcium D-gluconate for 15 minutes. The final alginate concentrations used ranged from 0.5% to 2%, and the final calcium D-gluconate concentrations ranged from 0.1% to 0.3%. The resulting mixture was poured into a 24-well plate at 1 mL per well and frozen overnight. Freezing temperatures ranged from -20°C to -80°C. All frozen scaffolds were freeze-dried for 72 hours. Scaffolds were stored at 4°C until use.

[0216] Determination of viral titer and MOI. Viral titer was determined by standard flow cytometry assay. Serially diluted viral stocks were added to HEK293T cells. After 48 hours, GFP expression was analyzed using flow cytometry. GFP expression was 10-20%. +The group containing the cells was used to calculate the viral titer. The titer was calculated using the following formula: Titer (TU / mL) = (number of cells used for infection × GFP) + MOI was calculated as the ratio of the number of transducing viral particles to the number of activated T cells. 0.5 × 10 6 MOI values ​​from 0.25 to 4 were tested using activated T cells to determine which MOI gave approximately 60% transduction efficiency. Various volumes of GFP virus stock were concentrated and transduced to 0.5x10 6 Activated primary T cells were mixed with 1000 cells / ml and seeded onto dried macroporous alginate scaffolds. The scaffolds were incubated for 72 hours in 1 mL of complete cell culture medium (45% Click Medium (Irvine Scientific), 45% RPMI-1640, 10% HyClone fetal bovine serum (GE Healthcare), 2 mmol / L GlutaMax (Gibco), penicillin (100 units / mL), and streptomycin (100 mg / mL, Gibco)) supplemented with IL-7 (Peprotech, 5 ng / mL) and IL-15 (Peprotech, 10 ng / mL). After 72 hours, the scaffolds were dissolved in 1 mL of 0.25 M EDTA. The cells were detached, washed twice with PBS, and analyzed for GFP expression using flow cytometry.

[0217] Scanning electron microscopy. Dried macroporous alginate scaffolds were coated with 70 nm of AuPd (60% Au, 40% Pd) at 7 nm / min for 5 min and analyzed with a Hitachi SU-3900 variable pressure SEM. Pore size was quantified using ImageJ, and SEM images were analyzed measuring a minimum of 10 pores per scaffold.

[0218] Compression Test. Dry macroporous alginate scaffolds were compressed using an Instron 5944. The scaffolds were compressed with a force of 50 N at a ramp rate of 0.1 mm / s. Force (N) and displacement (mm) were recorded every 100 ms. Stress was calculated using the formula: force / cross-sectional area. Cross-sectional area was determined using ImageJ, and images of each scaffold were analyzed. Strain was calculated using the formula: displacement / initial length. ImageJ was used to analyze images of each scaffold, determining the initial length. Young's modulus was calculated by determining the slope of the stress-strain curve in the linear region before the inflection point.

[0219] Drydux transduction of activated T cells. GFP retroviral supernatant (5 × 10 6 The concentrated retrovirus (2 × 10 TU / mL) was concentrated using an Amicon centrifugal filter (MWCO 100 kDa, Millipore) at 1,500 g for 10 min in a swing-type rotor. 6 TU) suspended in 50 μL of complete cell culture medium. 6 The cells were mixed with activated primary T cells (MOI = 2) and pipetted onto dry macroporous alginate scaffolds. The seeded scaffolds were incubated for 45 minutes, after which 1 mL of complete cell culture medium supplemented with IL-7 (Peprotech, 5 ng / mL) and IL-15 (Peprotech, 10 ng / mL) was added to each scaffold. After 72 hours of incubation, the scaffolds were dissolved in 1 mL of 0.25 M EDTA. The cells were detached, washed twice with PBS, and then analyzed for GFP expression using flow cytometry.

[0220] Absorption rate and volumetric flux. Different volumes of activated T cells were mixed with concentrated GFP-encoding retroviral supernatant to maintain a constant MOI of 2. This mixture was seeded onto scaffolds to form films. The absorption rate was calculated as the liquid volume divided by the time required for the entire droplet to be absorbed into the scaffold when no liquid was visible above the scaffold. Volumetric flux was calculated by dividing the absorption rate by the area of ​​the scaffold wetted by the droplet as viewed from above.

[0221] Cell lines. Peripheral blood mononuclear cells were isolated from buffy coats (Gulf Coast Regional Blood Center) using Lymphoprep medium (Accurate Chemical and Scientific Corporation) and frozen in freezing medium (50% HyClone fetal bovine serum (GE Healthcare), 40% RPMI-1640, 10% DMSO (Sigma)) until needed. Cells were thawed, resuspended in 9 mL of complete medium, and centrifuged at 400 g for 5 minutes to remove DMSO. Cells were activated on plates coated with 1 μg / mL of CD3 (Miltenyi Biotec, 130-093-387, clone OKT-3) and CD28 (BD Biosciences, 555725, clone CD28.2) agonist monoclonal antibodies. GFP-encoding retroviruses were prepared as described above. All cells were maintained at 37°C, 5% CO2, and 95% humidity.

[0222] Flow cytometry. All samples were analyzed using a BD LSRII, acquiring a minimum of 10,000 events per sample. Cells were gated for live cells, FSC singlets, and GFP-positive cells (Figures 23A-23B). BD FACS Diva 8.0.1 software was used for analysis.

[0223] Statistical Analysis and Spearman Correlation. All statistical analyses were performed using Graph Pad Prism 9 using one-way or two-way ANOVA with Tukey's correction or unpaired t-test with Welch's correction. The specific test used and exact p-values ​​are indicated on the individual figures. Spearman correlations were calculated using Graph Pad Prism 9, and r- and p-values ​​are indicated on the individual figures. * indicates p<0.0001; all other p-values ​​are indicated on the graphs.

Claims

1. 1. An implantable macroporous scaffold material comprising: a crosslinked biopolymer matrix having an average pore size in the range of about 10 μm to about 500 μm and a stiffness in the range of about 1 kPa to about 1000 kPa, wherein the stiffness of the matrix matches the stiffness of the target tissue; a composition comprising a plurality of cells and a transduction factor; The macroporous scaffold material, which facilitates transduction of the plurality of cells by the transduction factor.

2. 10. The macroporous scaffold material of claim 1, wherein the biopolymer matrix comprises at least one of alginate, hyaluronic acid, collagen, fibrin, polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), gelatin, polyethylene glycol (PEG), chitosan, cellulose, polyglutamic acid, fibrin, silk, agarose, dextran, polyacrylamide, polyvinyl alcohol, poly(N-isopropylacrylamide), poly(2-hydroxyethyl methacrylate), polyurethane, polyethyleneimine, poly(methyl methacrylate), poly(2-oxazoline), polyphosphazene, and complexes, derivatives, or combinations thereof.

3. The macroporous scaffold material of claim 1 or claim 2, wherein the biopolymer matrix comprises alginate having a molecular weight of about 1 kDa to about 500 kDa.

4. The macroporous scaffold material according to any one of claims 1 to 3, wherein the biopolymer matrix comprises alginate having a G / M ratio of about 0.5 to about 5.

0.

5. The macroporous scaffold material according to any one of claims 1 to 4, wherein the biopolymer matrix comprises alginate at a concentration in the range of about 0.1% to about 5.0% (w / v).

6. The macroporous scaffold material according to any one of claims 1 to 4, wherein the biopolymer matrix comprises alginate at a concentration in the range of about 0.5% to about 2.0% (w / v).

7. The macroporous scaffold material according to any one of claims 1 to 6, wherein the biopolymer matrix comprises alginate at a concentration in the range of about 0.5% to about 1.5% (w / v).

8. The macroporous scaffold material according to any one of claims 1 to 6, wherein the biopolymer matrix comprises alginate at a concentration in the range of about 0.5% to about 1.0% (w / v).

9. The macroporous scaffold material according to any one of claims 1 to 6, wherein the biopolymer matrix comprises alginate at a concentration in the range of about 1.0% to about 2.0% (w / v).

10. The macroporous scaffold material according to any one of claims 1 to 6, wherein the biopolymer matrix comprises alginate at a concentration in the range of about 1.0% to about 1.5% (w / v).

11. The macroporous scaffold material according to any one of claims 1 to 6, wherein the biopolymer matrix comprises alginate at a concentration in the range of about 1.5% to about 2.0% (w / v).

12. The macroporous scaffold material according to any one of claims 1 to 11, wherein the biopolymer matrix comprises calcium alginate having a calcium concentration in the range of about 0.1% to about 1.0% (w / v).

13. The macroporous scaffold material according to any one of claims 1 to 11, wherein the biopolymer matrix comprises calcium alginate having a calcium concentration in the range of about 0.1% to about 0.3% (w / v).

14. The macroporous scaffold material according to any one of claims 1 to 11, wherein the biopolymer matrix comprises calcium alginate having a calcium concentration in the range of about 0.1% to about 0.2% (w / v).

15. The macroporous scaffold material according to any one of claims 1 to 11, wherein the biopolymer matrix comprises calcium alginate having a calcium concentration in the range of about 0.2% to about 0.3% (w / v).

16. The macroporous scaffold material according to any one of claims 1 to 15, wherein the biopolymer matrix is ​​produced at a temperature in the range of about 0°C to about -80°C.

17. 17. The macroporous scaffold material of any one of claims 1 to 16, wherein the biopolymer matrix exhibits a stiffness that is about ±25%, about ±50%, about ±75%, about ±100%, about ±125%, about ±150%, about ±175%, about ±200%, about ±225%, or about ±250% of the stiffness of the target tissue.

18. The macroporous scaffolding material according to any one of claims 1 to 17, wherein the scaffolding material comprises at least one biological agent.

19. The macroporous scaffolding material of claim 18 , wherein the at least one biological agent is a small molecule.

20. 20. The macroporous scaffold material of claim 19, wherein the small molecule is selected from the group consisting of a TLR agonist, a checkpoint inhibitor, an IDO inhibitor, a MEK inhibitor, an HDAC inhibitor, a PI3K inhibitor, an immunomodulator, a JAK kinase inhibitor, and an mTOR inhibitor.

21. The macroporous scaffolding material of claim 18 , wherein the at least one biological agent is a protein, peptide, or polypeptide.

22. 22. The macroporous scaffold material of claim 21 , wherein the protein, peptide, or polypeptide is selected from the group consisting of a cytokine, an antibody, and a growth factor.

23. The macroporous scaffold material according to claim 22, wherein the cytokines comprise at least one of IL-2, IL-15, IL-7, IL-23, TNF-α, and / or IFN-γ.

24. The macroporous scaffold material according to any one of claims 1 to 23, wherein the plurality of cells comprises one or more immune cells.

25. 25. The macroporous scaffold material of claim 24, wherein the one or more immune cells are selected from the group consisting of T cells, B cells, natural killer (NK) cells, NK T cells, macrophages, dendritic cells, tumor infiltrating lymphocytes (TIL), tumor infiltrating NK cells (TINK), and bone marrow infiltrating lymphocytes (MIL).

26. 25. The macroporous scaffold material of claim 24, wherein the one or more immune cells are activated.

27. The macroporous scaffold material according to any one of claims 1 to 26, wherein the plurality of cells is obtained from a cell culture.

28. The macroporous scaffold material according to any one of claims 1 to 27, wherein the plurality of cells is obtained from a donor.

29. The macroporous scaffold material according to any one of claims 1 to 28, wherein the transduction factor comprises a viral vector.

30. 30. The macroporous scaffold material according to claim 29, wherein the viral vector is selected from the group consisting of lentivirus, retrovirus, adenovirus, adeno-associated virus, coccivirus, and baculovirus.

31. The macroporous scaffold material according to any one of claims 1 to 30, wherein the transduction factors comprise virus-like particles, cell-mimetic particles, transposons, exosomes, nanoparticles, micelles, and liposomes.

32. A macroporous scaffold according to any preceding claim, wherein the transduction factor comprises a nucleic acid cargo.

33. 33. The macroporous scaffold material of claim 32, wherein the nucleic acid cargo comprises siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or viral RNA.

34. 33. The macroporous scaffold material of claim 32, wherein the nucleic acid cargo comprises DNA encoding a fusion protein, a chimeric antigen receptor (CAR), a therapeutic peptide or polypeptide, or a combination thereof.

35. A macroporous scaffold material according to any one of claims 1 to 34, wherein the scaffold material is implanted within or adjacent to the target tissue.

36. The macroporous scaffold material according to any one of claims 1 to 35, wherein the target tissue is a tumor tissue.

37. A macroporous scaffold material according to any one of claims 1 to 36, wherein the target tissue is a solid tumor tissue.

38. 38. A macroporous scaffold material according to any one of claims 1 to 37, wherein the target tissue comprises at least one of lung tissue, bone tissue, skin tissue, breast tissue, muscle tissue, nerve tissue, brain tissue, lymphatic tissue, prostate tissue, bladder tissue, stomach tissue, intestinal tissue, uterine tissue, ovarian tissue, liver tissue, adipose tissue, cartilage tissue, thyroid tissue, and / or pancreatic tissue.

39. 39. A macroporous scaffold according to any one of claims 1 to 38, wherein the scaffold promotes transduction of the plurality of cells by the transduction factor with a transduction efficiency of at least 50%.

40. The macroporous scaffold material according to any one of claims 1 to 39, wherein the biopolymer matrix has an average pore size in the range of about 50 μm to about 250 μm.

41. A macroporous scaffold material according to any one of claims 1 to 39, wherein the biopolymer matrix has an average pore size in the range of about 100 μm to about 200 μm.

42. A macroporous scaffold material according to any one of claims 1 to 39, wherein the biopolymer matrix has an average pore size in the range of about 50 μm to about 150 μm.

43. The macroporous scaffold material according to any one of claims 1 to 42, wherein the biopolymer matrix has a stiffness in the range of about 25 kPa to about 650 kPa.

44. The macroporous scaffold material according to any one of claims 1 to 42, wherein the biopolymer matrix has a stiffness in the range of about 0.2 kPa to about 65 kPa.

45. The macroporous scaffold material according to any one of claims 1 to 42, wherein the biopolymer matrix has a stiffness in the range of about 0.1 kPa to about 10 kPa.

46. 1. A method of treating a subject, comprising: implanting a macroporous scaffold material within or adjacent to a target tissue, said scaffold material comprising: a crosslinked biopolymer matrix having an average pore size in the range of about 10 μm to about 500 μm and a stiffness in the range of about 1 kPa to about 1000 kPa, wherein the stiffness of the matrix matches the stiffness of the target tissue; a composition comprising a plurality of cells and a transduction factor; The method of treating the subject, wherein the scaffolding material facilitates transduction of the plurality of cells with the transduction factor, and the transduced cells treat the subject.

47. 47. The method of claim 46, wherein the target tissue is a tumor tissue.

48. 48. The method of claim 46 or claim 47, wherein the target tissue is a solid tumor tissue.

49. 49. The method of any one of claims 46 to 48, wherein the target tissue comprises at least one of lung tissue, bone tissue, skin tissue, breast tissue, muscle tissue, nerve tissue, brain tissue, lymphatic tissue, prostate tissue, bladder tissue, stomach tissue, intestinal tissue, uterine tissue, ovarian tissue, liver tissue, adipose tissue, cartilage tissue, thyroid tissue, and / or pancreatic tissue.

50. 50. The method of any one of claims 46 to 49, wherein the subject has been diagnosed with a disease or condition.

51. 51. The method of claim 50, wherein the disease or condition comprises cancer.

52. 52. The method of any one of claims 46 to 51, wherein the scaffolding material facilitates the transduction of the plurality of cells by the transduction factor with a transduction efficiency of at least 50%.

53. 53. The method of any one of claims 46-52, wherein the biopolymer matrix comprises at least one of alginate, hyaluronic acid, collagen, fibrin, polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), gelatin, polyethylene glycol (PEG), chitosan, cellulose, polyglutamic acid, fibrin, silk, agarose, dextran, polyacrylamide, polyvinyl alcohol, poly(N-isopropylacrylamide), poly(2-hydroxyethyl methacrylate), polyurethane, polyethyleneimine, poly(methyl methacrylate), poly(2-oxazoline), polyphosphazene, and complexes, derivatives, or combinations thereof.

54. 54. The method of any one of claims 46 to 53, wherein the biopolymer matrix comprises alginate having a molecular weight of about 1 kDa to about 500 kDa.

55. 55. The method of any one of claims 46 to 54, wherein the biopolymer matrix comprises an alginate having a G / M ratio of about 0.5 to about 5.

0.

56. 56. The method of any one of claims 46 to 55, wherein the biopolymer matrix comprises alginate at a concentration in the range of about 0.1% to about 5.0%.

57. 57. The method of any one of claims 31 to 56, wherein the biopolymer matrix comprises calcium alginate having a calcium concentration in the range of about 0.1% to about 1.0%.

58. 58. The method of any one of claims 31 to 57, wherein the biopolymer matrix is ​​generated at a temperature in the range of about -20°C to about -80°C.

59. 59. The method of any one of claims 46-58, wherein the biopolymer matrix exhibits a stiffness that is about ±25%, about ±50%, about ±75%, about ±100%, about ±125%, about ±150%, about ±175%, about ±200%, about ±225%, or about ±250% of the stiffness of the target tissue.

60. 60. The method of any one of claims 46 to 59, wherein the scaffolding material comprises at least one biological agent.

61. 61. The method of claim 60, wherein the at least one biological agent is a small molecule.

62. 62. The method of claim 61 , wherein the small molecule is selected from the group consisting of a TLR agonist, a checkpoint inhibitor, an IDO inhibitor, a MEK inhibitor, an HDAC inhibitor, a PI3K inhibitor, an immunomodulator, a JAK kinase inhibitor, and an mTOR inhibitor.

63. 61. The method of claim 60, wherein the at least one biological agent is a protein, peptide, or polypeptide.

64. 64. The method of claim 63, wherein the protein, peptide, or polypeptide is selected from the group consisting of a cytokine, an antibody, and a growth factor.

65. 65. The method of claim 64, wherein the cytokine comprises at least one of IL-2, IL-15, IL-7, IL-23, TNF-α, and / or IFN-γ.

66. 66. The method of any one of claims 46 to 65, wherein the plurality of cells comprises one or more immune cells.

67. 67. The method of claim 66, wherein the one or more immune cells are selected from the group consisting of T cells, B cells, natural killer (NK) cells, NK T cells, macrophages, dendritic cells, tumor infiltrating lymphocytes (TILs), tumor infiltrating NK cells (TINKs), and bone marrow infiltrating lymphocytes (MILs).

68. 68. The method of claim 67, wherein the one or more immune cells are activated.

69. 69. The method of any one of claims 46 to 68, wherein the plurality of cells is obtained from a cell culture.

70. 70. The method of any one of claims 46 to 69, wherein the plurality of cells is obtained from a donor.

71. 71. The method of any one of claims 46 to 70, wherein the transduction agent comprises a viral vector.

72. 72. The method of claim 71, wherein the viral vector is selected from the group consisting of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, a coccivirus, and a baculovirus.

73. 73. The method of any one of claims 46 to 72, wherein the transduction agent comprises a virus-like particle, a cell-mimetic particle, a transposon, an exosome, a nanoparticle, a micelle, and a liposome.

74. 74. The method of any one of claims 46 to 73, wherein the transduction factor comprises a nucleic acid cargo.

75. 75. The method of claim 74, wherein the nucleic acid cargo comprises siRNA, tasiRNA, lncRNA, shRNA, mRNA, gRNA, miRNA, and / or viral RNA.

76. 75. The method of claim 74, wherein said nucleic acid cargo comprises DNA encoding a fusion protein, a chimeric antigen receptor (CAR), a therapeutic peptide or polypeptide, or a combination thereof.

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