Encapsulated cells expressing IL-12 and uses thereof

Abstract

The present disclosure relates to implantable constructs (encapsulated cells) designed to deliver IL-12 and / or therapeutic reagents such as IL-12, and methods of using same to treat conditions such as cancer. TIFF2025503454000020.tif164135

Description

[Technical Field]

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 291,129, filed December 17, 2021, which is incorporated herein by reference in its entirety.

[0002] II. REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTINGS This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on December 9, 2022, is named "258717_000802_Seq.XML" and is 17,695 bytes in size.

[0003] III. FIELD OF THE INVENTION The present disclosure relates to the fields of biology, medicine, biotechnology, and medical devices. More particularly, the present invention relates to the development and use of implantable constructs designed to deliver antigenic therapeutic reagents to a subject and protect them from an immune response generated by the host. In particular, the constructs are designed to degrade over time or in response to specific signals, thereby providing control over the length of time that the therapeutic substance is delivered to the subject. [Background technology]

[0004] IV. Related Technologies Advances in biomedical research have led to local and targeted therapy methods for the treatment of diseases such as cancer. However, in many instances, the proportion of patients who respond to these approaches remains modest (Park et al., Sci. Transl. Med. 10(433)2018).

[0005] One approach involves the use of implantable devices to deliver therapeutic agents, but a fundamental barrier to successful device-based therapy is the inability to deliver sustained amounts of therapeutic agents without systemic toxic effects to the subject. Thus, there is a need to identify new compositions and methods for enhancing the delivery, distribution, and / or efficacy of therapeutic agents. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Park et al., Sci.Transl.Med.10(433)2018 Summary of the Invention

[0007] overview In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding an IL-12 polypeptide. In some embodiments, the IL-12 polypeptide is a native human IL-12 polypeptide, a recombinant IL-12 polypeptide, or an IL-12 mutein polypeptide. In some embodiments, the native human IL-12 polypeptide is a heterodimeric complex comprising a native human IL-12(p35) polypeptide and a native human IL-12(p40) polypeptide. In some embodiments, the plurality of oligonucleotide molecules encode a native human IL-12(p35) polypeptide and a native human IL-12(p40) polypeptide. In some embodiments, the expressed IL-12(p35) polypeptide and the expressed IL-12(p40) polypeptide form a heterodimeric complex. In some embodiments, the oligonucleotide encoding the native human IL-12(p35) polypeptide comprises the sequence of SEQ ID NO: 1. In some embodiments, the oligonucleotide encoding the native human IL-12(p40) polypeptide comprises the sequence of SEQ ID NO: 2. In some embodiments, the IL-12(p40) mutein polypeptide comprises a mutation selected from N222L or N222Q compared to SEQ ID NO:4.

[0008] In some embodiments, a pharmaceutical composition is provided comprising a population of encapsulated cells provided herein.

[0009] In some embodiments, provided are methods of treating a tumor, such as a pancreatic tumor, in a subject, comprising implanting into the intraperitoneal space of the subject a pharmaceutical composition comprising a plurality of encapsulated cells (e.g., capsules) provided herein to treat the cancer.

[0010] In some embodiments, provided are methods of reducing a tumor burden, such as a pancreatic tumor, in a subject, the method comprising implanting into the intraperitoneal space of the subject a pharmaceutical composition comprising a plurality of encapsulated cells (e.g., capsules) provided herein to treat cancer.

[0011] In some embodiments, provided are methods of treating a tumor, such as a melanoma tumor, in a subject, comprising implanting into the subcutaneous space of the subject a pharmaceutical composition comprising a plurality of encapsulated cells (e.g., capsules) provided herein to treat the cancer.

[0012] In some embodiments, provided are methods of reducing a tumor burden, such as a melanoma tumor, in a subject, the method comprising implanting into the subcutaneous space of the subject a pharmaceutical composition comprising a plurality of encapsulated cells (e.g., capsules) provided herein to treat cancer.

[0013] In some embodiments, provided herein are methods of treating a tumor in a subject by generating memory immunity, hi some embodiments, the methods comprise implanting a pharmaceutical composition comprising a population of encapsulated cells provided herein.

[0014] In some embodiments, methods are provided for selectively activating CD8-positive effector T cells, hi some embodiments, the methods comprise implanting a pharmaceutical composition comprising a population of encapsulated cells provided herein.

[0015] In some embodiments, methods are provided for increasing interferon gamma (IFN-γ) in a subject, hi some embodiments, the methods comprise implanting or administering a pharmaceutical composition comprising a population of encapsulated cells provided herein.

[0016] In some embodiments, methods are provided for preventing or inhibiting fibrosis of capsules encapsulating cells implanted in a subject. In some embodiments, the methods include expressing IL-12 in the implanted encapsulated cells or exposing the implanted encapsulated cells to IL-12 at the implantation site. In some embodiments, the inhibition or prevention of fibrosis is complete. In some embodiments, the inhibition or prevention of fibrosis of the capsules is partial. In some embodiments, the fibrosis that occurs is less than the fibrosis that would occur in the absence of IL-12 expression from the capsules or exposure of the capsules to IL-12. In some embodiments, the fibrosis that occurs is not sufficient to completely block secretion of a protein of interest or a heterologous protein from the encapsulated cells. In some embodiments, the protein of interest is IL-12. In some embodiments, an anti-fibrotic effect is achieved for capsules encapsulating cells that express or are expressed by cells other than IL-12, such as IL-2 or another cytokine. Non-limiting examples are provided herein. This antifibrotic effect can be said to occur "cis" with respect to the same capsules that express IL-12, but can also act "trans" with respect to capsules that do not express IL-12.

[0017] In some embodiments, a method for preparing encapsulated cells that produce a recombinant protein is provided, comprising dispensing through a coaxial needle a first composition comprising a polymer hydrogel and a second composition comprising cells to be encapsulated suspended in the polymer hydrogel to form encapsulated cells dropwise into a crosslinking solution, the crosslinking solution comprising a sugar alcohol, a buffer, a metal salt, and a surfactant.

[0018] In some embodiments, a suspension of encapsulated cells is provided, in some embodiments, the suspension comprises a population of encapsulated cells provided herein, the encapsulated cells are encapsulated by a polymer hydrogel, and the suspension comprises a crosslinking solution comprising a sugar alcohol, a buffer, a metal salt, and a surfactant.

[0019] The use of the words "a" or "an" when used in conjunction with the word "comprising" in the claims and / or specification can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." The word "about" means ±5% of the stated number.

[0020] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present disclosure, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1]Illustrated are survival curves (upper panel) and tumor growth curves (lower panel) for melanoma B16F10 mice challenged subcutaneously or intraperitoneally with encapsulated cells as disclosed herein or sham surgery. [Figure 2] 1 shows tumor growth curves for melanoma B16F10 mice subcutaneously challenged with encapsulated cells as disclosed herein or sham surgery. [Figure 3A] The upper panel shows quantification of total flux from IVIS images taken 6 days after injection of Pan02-fluc cells in all mice (n = 5-6 per group) before treatment. The lower panel shows luminescence images of mice 5 days after control (untreated), RPE, RPE-mIL2, or RPE-mIL12 treatment. [Figure 3B] The top panel shows t-SNE plots of CD8α gene expression in individual cells separated by treatment (untreated, RPE, RPE-mIL2, RPE-mIL12) collected from the IP space after 1 week of treatment. The bottom panel shows t-SNE plots of IFNg gene expression in individual cells separated by treatment (untreated, RPE, RPE-mIL2, RPE-mIL12) collected from the IP space at 1 week of treatment. [Figure 4A] Luminescence images of mice over time after treatment with various doses of RPE-mIL12 are shown. The scale bar minimum and maximum values ​​for mean radiance (photons / sec / cm / ser) are 5x10 and 5x10, respectively. [Figure 4B] Individual animal weights over time are shown for animals treated with 3 or 10 RPE-mIL12 capsules. [Figure 4C] Macroscopic and H&E images of the IP space and pancreas, respectively, after 8 weeks of treatment with RPE-mIL12 or RPE-mIL12. H&E images are at 20x magnification. A indicates acinar cells, D indicates ducts, and I indicates islets of Langerhans. [Figure 5]Luminescence images of explanted abdominal organs from mice over 25 days after sham surgery, RPE, or RPE-mIL12 treatment are shown. Scale bar minimum and maximum values ​​for mean radiance (photons / sec / cm / ser) are 5x10 and 5x10, respectively. [Figure 6A] Shown are macroscopic images of mice bearing metastatic melanoma in the IP space one week after treatment with or without RPE-mIL12. [Figure 6B] Splenic cellularity after 1 week of treatment with or without RPE-mIL12 is shown. [Figure 7] 1 shows survival curves for animals bearing melanoma tumors after treatment with RPE or RPE-mIL12 capsules. [Figure 8] 1 shows survival curves for pancreatic tumor-bearing animals after sham treatment or treatment with RPE or RPE-mIL12 capsules. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description The present disclosure features implantable constructs for delivering native human IL-12 to a subject in a controlled-release manner, and related methods of use thereof, embodiments of which are described in more detail below.

[0024] A.Definition "Cell," as used herein, refers to an individual cell. In some embodiments, the cell is a primary cell or is derived from a cell culture. In some embodiments, the cell is a stem cell or is derived from a stem cell. The cell may be xenogeneic, autologous, or allogeneic. In some embodiments, the cell is engineered (e.g., genetically engineered) or unengineered (e.g., non-genetically engineered). In some embodiments, the cell is an APRE-19 cell. In some embodiments, the cell expresses native human IL-12 protein.

[0025] As used herein, "prevention," "prevent" and "preventing" refer to the administration or application of a treatment, e.g., a treatment that includes administering an implantable construct (e.g., as described herein) comprising a therapeutic substance (e.g., a therapeutic substance described herein) prior to the onset of a disease or condition to prevent the physical manifestation of the disease or condition. In some embodiments, "prevention," "prevent" and "preventing" require that signs or symptoms of the disease or condition have not yet developed or been observed. In some embodiments, treatment includes prevention, and in other embodiments, does not include prevention. In some embodiments, prevention is the prevention of recurrence of a disease, such as a tumor (cancer), after the tumor or cancer has been eradicated by initial treatment.

[0026] As used herein, "subject" refers to a recipient of an implantable construct described herein. A subject may include humans and / or other non-human animals, such as mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cows, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and / or turkeys). In certain embodiments, the animal is a mammal. The animal may be male or female and at any stage of development (e.g., male or female, of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or an adult subject (e.g., young adult, middle-aged adult, or elderly). The non-human animal may be a transgenic animal.

[0027] As used herein, "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset, or inhibiting the progression of one or more symptoms, signs, or underlying causes of a disease or condition (e.g., as described herein), e.g., by administering or applying a therapy, e.g., by administering an implantable construct comprising a therapeutic substance (e.g., a therapeutic substance described herein). In some embodiments, treating includes reducing, reversing, alleviating, delaying the onset, or inhibiting the progression of a symptom of a disease, disorder, or condition. In some embodiments, treating includes reducing, reversing, alleviating, delaying the onset, or inhibiting the progression of a disease or condition. In some embodiments, treating includes reducing, reversing, alleviating, reducing, or delaying the onset of an underlying cause of a disease or condition. In some embodiments, "treatment," "treat," and "treating" require that a sign or symptom of a disease or condition has occurred or is observed. In other embodiments, a therapy may be administered in the absence of signs or symptoms of a disease or condition, e.g., in prophylactic treatment. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., taking into account the history of symptoms and / or taking into account genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent recurrence. Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent recurrence. In some embodiments, treatment includes prevention, and in other embodiments does not include prevention.

[0028] B. Cell The implantable constructs described herein can contain cells, e.g., engineered cells. The cells can be derived from any mammalian organ or tissue, including brain, nerves, ganglia, spine, eyes, heart, liver, kidneys, lungs, spleen, bone, thymus, lymphatic system, skin, muscle, pancreas, stomach, intestine, blood, ovaries, uterus, or testes. In some embodiments, the cells are APRE-19 cells. The implantable constructs can be referred to as capsules. A capsule is a "shell" that encapsulates a population of cells. As provided herein, cells can be engineered to express proteins expressed from heterologous nucleic acid molecules (exogenously introduced into the cells).

[0029] The cells may be derived from a donor (e.g., allogeneic cells), from the subject (e.g., autologous cells), or from another species (e.g., xenogeneic cells). In some embodiments, the cells may be grown in cell culture, prepared from an established cell culture line, or derived from a donor (e.g., a living donor or a cadaver). In some embodiments, the cells are genetically engineered. In other embodiments, the cells are not genetically engineered. The cells may include stem cells, such as reprogrammed stem cells or induced pluripotent cells. Exemplary cells include mesenchymal stem cells (MSCs), fibroblasts (e.g., primary fibroblasts), HEK cells (e.g., HEK293T), Jurkat cells, HeLa cells, retinal pigment epithelial (RPE) cells, HUVEC cells, NIH3T3 cells, CHO-K1 cells, COS-1 cells, COS-7 cells, PC-3 cells, HCT 116 cells, A549MCF-7 cells, HuH-7 cells, U-2 OS cells, HepG2 cells, Neuro-2a cells, and SF9 cells. In some embodiments, the cells for use in the implantable construct are RPE cells.

[0030] The cells contained in the implantable construct may produce or secrete a therapeutic agent, such as native human IL-12. In some embodiments, the native human IL-12 is a heterodimeric complex comprising native human IL-12(p35) and native human IL-12(p40). In some embodiments, the therapeutic agent is recombinant IL-12. In some embodiments, the recombinant IL-12 is a heterodimeric complex comprising recombinant IL-12(p35) and recombinant IL-12(p40). In some embodiments, the recombinant IL-12 is a heterodimeric complex comprising native human IL-12(p35) and recombinant IL-12(p40). In some embodiments, the recombinant IL-12 is a heterodimeric complex comprising recombinant IL-12(p35) and native human IL-12(p40). In some embodiments, the recombinant IL-12 is a heterodimeric complex comprising recombinant IL-12(p35) and native human IL-12(p40). In some embodiments, the therapeutic agent is an IL-12 mutein. In some embodiments, the IL-12 mutein is a heterodimeric complex comprising an IL-12(p35) mutein and an IL-12(p40) mutein. In some embodiments, the IL-12 mutein is a heterodimeric complex comprising a native human IL-12(p35) mutein and an IL-12(p40) mutein. In some embodiments, the IL-12 mutein is a heterodimeric complex comprising an IL-12(p35) mutein and a native human IL-12(p40). In some embodiments, the IL-12 mutein is as provided herein. In some embodiments, the cells contained in the implantable construct may produce or secrete a single type of therapeutic agent or multiple therapeutic agents. In some embodiments, the multiple therapeutic agents include IL-12, as provided herein, and IL-2, as provided herein. In some embodiments, the implantable construct may comprise cells transduced or transfected with a nucleic acid (e.g., a vector) comprising an expression sequence for a therapeutic agent. For example, cells can be transduced or transfected with a lentivirus. The nucleic acid introduced into the cell (e.g., by transduction or transfection) can be incorporated into a nucleic acid delivery system, such as a plasmid, or can be delivered directly.In some embodiments, the nucleic acid introduced into the cell (e.g., as part of a plasmid) can include regions that enhance expression and / or direct targeting or secretion of the therapeutic substance, such as promoter sequences, activator sequences, or cell signaling or export peptides. Exemplary promoters include EF-1α, CMV, Ubc, hPGK, VMD2, and CAG. Exemplary activators include the TET1 catalytic domain, P300 core, VPR, rTETR, Cas9 (e.g., from S. pyogenes or S. aureus), and Cpfl (e.g., from L. bacterium).

[0031] The implantable constructs described herein may contain one cell or multiple cells. In the case of multiple cells, the concentration and total cell number may vary depending on several factors, such as the cell type, implantation location, and the expected lifespan of the implantable construct. In some embodiments, the total number of cells contained in the implantable construct is greater than about 2, 4, 6, 8, 10, 20, 30, 40, 50, 75, 100, 200, 250, 500, 750, 1000, 1500, 2000, 5000, 10,000, or more. In some embodiments, the implantable construct contains about 10,000 to about 50,000 cells. In some embodiments, the implantable construct contains about 10,000 to about 40,000 cells. In some embodiments, the implantable construct contains about 10,000 to about 30,000 cells. In some embodiments, the implantable construct contains about 10,000 to about 20,000 cells. In some embodiments, the implantable construct comprises about 20,000 to about 40,000 cells. In some embodiments, the implantable construct comprises about 25,000 to about 35,000 cells. In some embodiments, the implantable construct comprises about 30,000 cells. In some embodiments, the implantable construct comprises about 40,000 cells. In some embodiments, the implantable construct comprises about 50,000 cells. In some embodiments, the implantable construct comprises about 10,000 cells. In some embodiments, the implantable construct comprises about 20,000 cells. In some embodiments, the total number of cells contained in the implantable construct is about 1.0 x 10 2 , 1.0×10 3 , 1.0×10 4 , 1.0×10 5 , 1.0×10 6 , 1.0×10 7 , 1.0×10 8 , 1.0×10 9 , 1.0×10 10In some embodiments, the total number of cells contained in the implantable construct is less than about 50,000, 40,000, 30,000, 20,000, 10,000, 5,000, 2,500, 2,000, 1,500, 1,000, 750, 500, 250, 200, 100, 75, 50, 40, 30, 20, 10, 8, 6, 4, 2, or less. In some embodiments, the total number of cells contained in the implantable construct is less than about 1.0 x 10 10 , 1.0×10 9 , 1.0×10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 In some embodiments, the plurality of cells is present as an aggregate. In some embodiments, the plurality of cells is present as a cell dispersion.

[0032] Certain characteristics of the cells contained within the implantable construct can be determined, for example, before and / or after incorporation into the implantable construct. For example, cell viability, cell density, or cell expression levels can be assessed. In some embodiments, cell viability, cell density, and cell expression levels can be determined using standard techniques, such as cell microscopy, fluorescence microscopy, histology, or biochemical assays.

[0033] C. Therapeutic substances The implantable constructs described herein may contain a therapeutic agent produced or secreted by cells, such as, for example, native human IL-12. In some embodiments, the therapeutic agent is recombinant IL-12. In some embodiments, the therapeutic agent is an IL-12 mutein. In some embodiments, the therapeutic agent is native human IL-12(p35). In some embodiments, the therapeutic agent is native human IL-12(p40). In some embodiments, the therapeutic agent is an IL-12(p35) mutein. In some embodiments, the therapeutic agent is an IL-12(p40) mutein. In some embodiments, the therapeutic agent is recombinant IL-12(p35). In some embodiments, the therapeutic agent is recombinant IL-12(p40). In some embodiments, the therapeutic agent is recombinant IL-12. In some embodiments, the therapeutic agent is an IL-12 mutein. In some embodiments, the implantable constructs described herein may contain multiple therapeutic agents produced or secreted by a population of cells, such as, for example, native human IL-12(p35) and native human IL-12(p40). Therapeutic agents may include nucleic acids (e.g., RNA, DNA, or oligonucleotides) encoding proteins, proteins (e.g., antibodies, enzymes, cytokines, hormones, receptors) secreted from cells, and the like. In some embodiments, the implantable construct comprises a cell or multiple cells genetically engineered to produce or secrete a therapeutic agent. In some embodiments, the implantable construct comprises a cell or multiple cells genetically engineered to produce or secrete multiple therapeutic agents.

[0034] In some embodiments, native human IL-12 refers to IL-12(p70), or a heterodimeric complex comprising native human IL-12(p35) and native human IL-12(p40). Without wishing to be bound by any particular theory, when the IL-12(p35) and IL-12(p40) subunits are expressed, they interact with each other to form a heterodimeric complex comprising IL-12(p70), or what is referred to herein as IL-12. In some embodiments, native human IL-12(p35) is It refers to a protein encoded by a nucleic acid sequence comprising TIFF2025503454000002.tif128140.

[0035] In some embodiments, the native human IL-12(p40) is It refers to a protein encoded by a nucleic acid sequence comprising TIFF2025503454000003.tif209140.

[0036] In some embodiments, the native human protein produced by the cell is a first polypeptide comprising the sequence of TIFF2025503454000004.tif23138; and and a second polypeptide comprising the sequence of TIFF2025503454000005.tif32138, wherein the first and second polypeptides form a heterodimeric complex.

[0037] In some embodiments, the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed at a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed at a ratio of 1:1. In some embodiments, the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed at a ratio of 1:2. In some embodiments, the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed at a ratio of 1:3. In some embodiments, the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed at a ratio of 1:4. In some embodiments, the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed at a ratio of 1:5. In some embodiments, the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed at a ratio of 1:6. In some embodiments, the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed at a ratio of 1:7. In some embodiments, the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed at a ratio of 1:8. In some embodiments, the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed at a ratio of 1:9. In some embodiments, the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed at a ratio of 1:10. In some embodiments, IL-12(p35) polypeptide and IL-12(p40) polypeptide expressed at a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 interact with each other to form a heterodimeric complex. In some embodiments, IL-12(p35) polypeptide and IL-12(p40) polypeptide expressed at a ratio of 1:1 interact with each other to form a heterodimeric complex.In some embodiments, an IL-12(p35) polypeptide and an IL-12(p40) polypeptide expressed at a ratio of 1:2 interact with each other to form a heterodimeric complex. In some embodiments, an IL-12(p35) polypeptide and an IL-12(p40) polypeptide expressed at a ratio of 1:3 interact with each other to form a heterodimeric complex. In some embodiments, an IL-12(p35) polypeptide and an IL-12(p40) polypeptide expressed at a ratio of 1:4 interact with each other to form a heterodimeric complex. In some embodiments, an IL-12(p35) polypeptide and an IL-12(p40) polypeptide expressed at a ratio of 1:5 interact with each other to form a heterodimeric complex. In some embodiments, an IL-12(p35) polypeptide and an IL-12(p40) polypeptide expressed at a ratio of 1:6 interact with each other to form a heterodimeric complex. In some embodiments, an IL-12(p35) polypeptide and an IL-12(p40) polypeptide expressed at a ratio of 1:7 interact with each other to form a heterodimeric complex. In some embodiments, an IL-12(p35) polypeptide and an IL-12(p40) polypeptide expressed at a ratio of 1:8 interact with each other to form a heterodimeric complex. In some embodiments, an IL-12(p35) polypeptide and an IL-12(p40) polypeptide expressed at a ratio of 1:9 interact with each other to form a heterodimeric complex. In some embodiments, an IL-12(p35) polypeptide and an IL-12(p40) polypeptide expressed at a ratio of 1:10 interact with each other to form a heterodimeric complex.

[0038] In some embodiments, the IL-12 is an IL-12 mutein, recombinant IL-12, or a modified IL-12 molecule, fusion protein, or antibody acting on the IL-12 pathway. In some embodiments, the IL-12(p35) is an IL-12(p35) mutein, recombinant IL-12(p35), or a modified IL-12(p35) molecule, fusion protein, or antibody. In some embodiments, the IL-12(p40) is an IL-12(p40) mutein, recombinant IL-12(p40), or a modified IL-12(p40) molecule, fusion protein, or antibody. In some embodiments, IL-12(p35) and IL-12(p40) interact with each other to form a heterodimeric complex.

[0039] In some embodiments, an IL-12 mutein can be prepared by mutating one or more residues of IL-12. In some embodiments, the mutation is a deletion, substitution, or insertion. In some embodiments, the mutation is a substitution. In some embodiments, the substitution is a naturally occurring amino acid. In some embodiments, an IL-12(p35) mutein can be prepared by mutating one or more residues of IL-12(p35). In some embodiments, an IL-12(p40) mutein can be prepared by mutating one or more residues of IL-12(p40). In some embodiments, an IL-12 mutein molecule comprises a mutation in the polypeptide sequence at amino acid 220. In some embodiments, an IL-12 mutein molecule comprises a mutation in the polypeptide sequence at amino acid 222. In some embodiments, an IL-12 mutein molecule comprises a mutation in the polypeptide sequence at amino acid 220 and / or 222. In some embodiments, the IL-12 comprises an N222L mutation. In some embodiments, the IL-12 comprises an N222L mutation in the IL-12(p40) amino acid sequence. In some embodiments, the IL-12 comprises an N222L mutation in the IL-12(p40) amino acid sequence compared to SEQ ID NO: 4. In some embodiments, the IL-12(p40) mutein comprises an N222L mutation corresponding to SEQ ID NO: 4. In some embodiments, the IL-12 comprises an N222Q mutation. In some embodiments, the IL-12 comprises an N222Q mutation in the IL-12(p40) amino acid sequence compared to SEQ ID NO: 4. In some embodiments, the IL-12(p40) mutein comprises an N222Q mutation corresponding to SEQ ID NO: 4. In some embodiments, the IL-12 comprises an N220L mutation. In some embodiments, the IL-12 comprises an N220L mutation in the IL-12(p40) amino acid sequence. In some embodiments, the IL-12 comprises a N220L mutation in the IL-12(p40) amino acid sequence compared to SEQ ID NO:5.In some embodiments, the IL-12(p40) mutein comprises an N220L mutation corresponding to SEQ ID NO:5. In some embodiments, the IL-12(p40) mutein can be prepared by mutating one or more residues of IL-12(p40). In some embodiments, the IL-12(p35) comprises a mutation at any position compared to SEQ ID NO:3. In some embodiments, the IL-12(p40) comprises a mutation at any position compared to SEQ ID NO:4. In some embodiments, the IL-12(p40) mutein comprises a mutation at any position compared to SEQ ID NO:5. In some embodiments, the IL-12(p40) mutein comprises an asparagine mutation at position 222 compared to SEQ ID NO:4. In some embodiments, the IL-12(p40) mutein comprises a substitution at position N222 compared to SEQ ID NO:4. In some embodiments, the IL-12(p40) mutein comprises an N222L mutation compared to SEQ ID NO:4. In some embodiments, the IL-12(p40) mutein comprises an N222Q mutation compared to SEQ ID NO:4. In some embodiments, the IL-12(p40) mutein comprises murine IL-12(p40) as set forth in SEQ ID NO: 5. In some embodiments, the murine IL-12(p40) mutein comprises a N220L mutation compared to SEQ ID NO:5. TIFF2025503454000006.tif35138

[0040] In some embodiments, the IL-12 mutein, IL-12(p35) mutein, and IL-12(p40) mutein are as provided in PCT Publication Nos. WO2001068802 and WO2017201350, respectively, which are incorporated by reference herein in their entireties.

[0041] The cells may also be modified to produce or secrete additional proteins or molecules in addition to native human IL-12, hi some embodiments, the cells are modified to produce or secrete native human IL-2.

[0042] In some embodiments, the native human IL-2 is It refers to a protein encoded by a nucleic acid sequence comprising TIFF2025503454000007.tif42140.

[0043] In some embodiments, the nucleic acid coding sequence encoding native human IL-2 is codon-optimized. In some embodiments, the nucleic acid coding sequence encoding native human IL-2 is codon-optimized for expression in mammalian cells. Codon-optimized sequences can be generated using commercially available algorithms, such as GeneOptimizer (ThermoFisher Scientific), OptimumGene™ (GenScript, Piscataway, NJ USA), GeneGPS® (ATUM, Newark, CA USA), Java Codon Adaptation Tool (JCat, http: / / www.jcat.de; Grote, A. et al., Nucleic Acids Research, Vol. 33, Issue suppl_2, pp. W526-W531 (2005); IDT Codon Optimization Tool (Integrated DNA Technologies); VectorBuilder Codon Optimization tool (VectorBuilder Inc.); Codon Optimization OnLine (COOL, http: / / bioinfo.bti.a-star.edu.sg / COOL / ; Chin JX et al., Bioinformatics, Vol. 30, Issue 1 (2005)). 15, pp. 2210-2212 (2014)), or ExpOptimizer (NovoPro, Shanghai, China). Examples of codon-optimized nucleic acid coding sequences encoding native human IL-2 include: Examples include, but are not limited to, TIFF2025503454000008.tif186138.

[0044] In some embodiments, the codon-optimized nucleic acid coding sequence encoding native human IL-2 comprises the nucleic acid sequence set forth in SEQ ID NOs: 7-10. In some embodiments, the codon-optimized nucleic acid coding sequence encoding native human IL-2 comprises the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the codon-optimized nucleic acid coding sequence encoding native human IL-2 comprises a nucleic acid sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NOs: 7-10. In some embodiments, the codon-optimized nucleic acid coding sequence encoding native human IL-2 comprises a nucleic acid sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 7.

[0045] In some embodiments, the native human protein produced by the cell is It is formed from the amino acid sequence of TIFF2025503454000009.tif13139.

[0046] In some embodiments, the additional protein or molecule is native human IL-2. In some embodiments, the additional protein or molecule is an IL-2 mutein or modified IL-2 molecule, a fusion protein, or an antibody that acts on the IL-2 pathway. In some embodiments, the IL-2 is a pegylated IL-2 molecule. In some embodiments, the pegylated IL-2 molecule has a wild-type sequence. In some embodiments, the pegylated IL-2 has a mutant IL-2 sequence. In some embodiments, the therapeutic agent is selected from NKTR-214, THOR-707, ALKS 4230, Nemvaleukin alpha, TransCon IL-2 beta / gamma, BNT151, BNT153, CLN-617, CUE-101, CUE-102, CUE-103, Anktiva® (N-803), KY1043, MDNA11, NL-201, SO-C101, RO6874281, Simlukafusp alpha, RG7461, WTX-124, WTX-330, XTX202, or XTX401, or any combination thereof. The additional proteins may be, for example, about 100 Da, 200 Da, 250 Da, 500 Da, 750 Da, 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa The protein may be of any size, including but not limited to, greater than 100 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 125 kDa, 150 kDa, 200 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, or more. In some embodiments, the protein is composed of a single subunit or multiple subunits (e.g., dimers, trimers, tetramers, etc.). Proteins produced or secreted by cells may be modified, for example, by glycosylation, methylation, or other known natural or synthetic protein modifications.The protein may be produced or secreted as a preprotein or in an inactive form and may require further modification to convert it to an active form.

[0047] The protein produced or secreted by the cell can include an antibody or antibody fragment, such as the Fc region or variable region of an antibody. Exemplary antibodies include anti-PD-1, anti-PD-L1, anti-CTLA4, anti-TNFα, and anti-VEGF antibodies. The antibody can be monoclonal or polyclonal. Other exemplary proteins include lipoproteins, adhesion proteins, hemoglobin, enzymes, proenkephalin, growth factors (e.g., EGF, IGF-1, VEGFα, HGF, TGFβ, bFGF), or cytokines.

[0048] Proteins produced or secreted by cells can also include hormones, including growth hormone, growth hormone-releasing hormone, prolactin, luteinizing hormone (LH), antidiuretic hormone (ADH), oxytocin, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), thyroxine, calcitonin, parathyroid hormone, aldosterone, cortisol, epinephrine, glucagon, insulin, estrogen, progesterone, and testosterone.

[0049] Proteins produced or secreted by cells may include other cytokines. Cytokines may be pro-inflammatory or anti-inflammatory cytokines. Examples of cytokines include IL-1, IL-1α, IL-1β, IL-1RA, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12a, IL-12b, IL-13, IL-14, IL-15, IL-16, IL-17, G-CSF, GM-CSF, IL-20, IL-23, IFN-α, IFN-β, IFN-γ, CD154, LT-β, ​​CD70, CD153, CD178, TRAIL, TNF-α, TNF-β, SCF, M-CSF, MSP, 4-1BBL, LIF, and OSM. For example, the cytokine may include any cytokine described in M. J. Cameron and D. Kelvin, Cytokines, Chemokines, and Their Receptors (2013), Landes Biosciences, which is incorporated herein by reference in its entirety.

[0050] The implantable constructs provided herein may include cells that express a single type of therapeutic substance, e.g., a single protein or nucleic acid, or may express more than one type of therapeutic substance, e.g., multiple proteins or nucleic acids. In some embodiments, the implantable construct includes cells that express two types of therapeutic substances (e.g., two types of proteins or nucleic acids). In some embodiments, the implantable construct includes cells that express three types of therapeutic substances (e.g., three types of proteins or nucleic acids). In some embodiments, the implantable construct includes cells that express four types of therapeutic substances (e.g., four types of proteins or nucleic acids).

[0051] In some embodiments, the implantable construct comprises cells that express a single type of nucleic acid (e.g., DNA or RNA), or may express more than one type of nucleic acid, e.g., multiple nucleic acids (e.g., DNA or RNA). In some embodiments, the implantable construct comprises cells that express two types of nucleic acids (e.g., DNA or RNA). In some embodiments, the implantable construct comprises cells that express three types of nucleic acids (e.g., DNA or RNA). In some embodiments, the implantable construct comprises cells that express four types of nucleic acids (e.g., DNA or RNA).

[0052] In some embodiments, the implantable construct comprises cells that express a single type of protein, or may express more than one type of protein, e.g., multiple proteins. In some embodiments, the implantable construct comprises cells that express two types of proteins. In some embodiments, the implantable construct comprises cells that express three types of proteins. In some embodiments, the implantable construct comprises cells that express four types of proteins.

[0053] In some embodiments, the implantable construct comprises cells that express a single type of enzyme, or may express more than one type of enzyme, e.g., multiple enzymes. In some embodiments, the implantable construct comprises cells that express two types of enzymes. In some embodiments, the implantable construct comprises cells that express three types of enzymes. In some embodiments, the implantable construct comprises cells that express four types of enzymes.

[0054] In some embodiments, the implantable construct comprises cells that express a single type of antibody or antibody fragment, or may express more than one type of antibody or antibody fragment, e.g., multiple antibodies or antibody fragments. In some embodiments, the implantable construct comprises cells that express two types of antibodies or antibody fragments. In some embodiments, the implantable construct comprises cells that express three types of antibodies or antibody fragments. In some embodiments, the implantable construct comprises cells that express four types of antibodies or antibody fragments.

[0055] In some embodiments, the implantable construct comprises cells that express a single type of hormone, or may express more than one type of hormone, e.g., multiple hormones. In some embodiments, the implantable construct comprises cells that express two types of hormones. In some embodiments, the implantable construct comprises cells that express three types of hormones. In some embodiments, the implantable construct comprises cells that express four types of hormones.

[0056] In some embodiments, the implantable construct comprises cells that express a single type of enzyme, or may express more than one type of enzyme, e.g., multiple enzymes. In some embodiments, the implantable construct comprises cells that express two types of enzymes. In some embodiments, the implantable construct comprises cells that express three types of enzymes. In some embodiments, the implantable construct comprises cells that express four types of enzymes.

[0057] In some embodiments, the implantable construct comprises cells that express a single type of cytokine, or may express more than one type of cytokine, e.g., multiple cytokines. In some embodiments, the implantable construct comprises cells that express two types of cytokines. In some embodiments, the implantable construct comprises cells that express three types of cytokines. In some embodiments, the implantable construct comprises cells that express four types of cytokines.

[0058] D. Characteristics of the implantable construct The implantable constructs described herein may take any suitable shape or form. For example, the implantable construct may be a sphere, spheroid, tube, cord, string, ellipsoid, disk, cylinder, sheet, torus, cube, stadium, cone, pyramid, triangle, rectangle, square, or rod. The implantable construct may include curved or flat portions. In some embodiments, the implantable construct may be prepared through the use of a mold, resulting in a custom shape.

[0059] Implantable constructs can vary in size, depending, for example, on the use or site of implantation. For example, implantable constructs can have an average diameter or size greater than 0.1 mm, e.g., greater than 0.25 mm, 0.5 mm, 0.75 mm, 1 mm, 1.5 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, or more. In some embodiments, implantable constructs can have sections or regions with an average diameter or size greater than 0.1 mm, e.g., greater than 0.25 mm, 0.5 mm, 0.75 mm, 1 mm, 1.5 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, or more. In some embodiments, the implantable construct may have an average diameter or size of less than 1 cm, e.g., less than 50 mm, 40 mm, 30 mm, 20 mm, 10 mm, 7.5 mm, 5 mm, 2.5 mm, 1 mm, 0.5 mm, or less. In some embodiments, the implantable construct may have sections or regions with an average diameter or size of less than 1 cm, e.g., less than 50 mm, 40 mm, 30 mm, 20 mm, 10 mm, 7.5 mm, 5 mm, 2.5 mm, 1 mm, 0.5 mm, or less.

[0060] In some embodiments, the implantable construct comprises at least one zone that can prevent exposure of the encapsulated antigenic or therapeutic substance to the external environment, for example, host effector cells or tissues. In some embodiments, the implantable construct comprises an inner zone (IZ). In some embodiments, the implantable construct comprises an outer zone (OZ). In some embodiments, either the inner zone (IZ) or the outer zone (OZ) may be erodible or degradable. In some embodiments, the zone (IZ) is erodible or degradable. In some embodiments, the outer zone (OZ) is erodible or degradable. In some embodiments, the implantable construct comprises both an inner zone (IZ) and an outer zone (OZ), either of which may be erodible or degradable. In some embodiments, the implantable construct comprises both an inner zone (IZ) and an outer zone (OZ), and the outer zone is erodible or degradable. In some embodiments, the implantable construct comprises both an inner zone (IZ) and an outer zone (OZ), and the outer zone is erodible or degradable. In some embodiments, the implantable construct comprises both an inner zone (IZ) and an outer zone (OZ), and the inner zone is erodible or degradable. The thickness of either of the zones, e.g., the inner or outer zone, can correlate with the length or duration of the "shielding" phase during which the encapsulated antigenic or therapeutic agent is protected or shielded from the external environment, e.g., host effector cells or tissues.

[0061] In some embodiments, the implantable construct can be a polymer matrix mixed with cells expressing a molecule of interest. For example, the implantable construct can be prepared according to the methods provided herein or known to those skilled in the art or otherwise provided herein to create an implantable construct comprising a layer of hydrogel (e.g., alginate) surrounding a population of cells expressing a molecule of interest, such as IL-12. In some embodiments, the layer is an acellular layer. In some embodiments, what is considered an outer layer comprises cells distributed in the outer region of the implantable construct.

[0062] Thus, the implantable construct may include a zone containing the majority of cells, but they may also be distributed throughout the construct.

[0063] As used herein, the term "encapsulated," when used in reference to an implantable construct, refers to cells surrounded by a matrix or hydrogel polymer (e.g., alginate). The term "encapsulated" can refer to a construct having an acellular outer layer or an outer layer with cells distributed therein. Encapsulation is a general term and generally refers to cells mixed with the polymer, but does not imply the absence of cells at or near the surface of the construct. As provided herein, this can also refer to capsules encapsulating a population of cells. The phrase "encapsulated population of cells" should be understood to refer to one or more capsules encapsulating a population of cells. A composition, such as a pharmaceutical composition, can contain multiple capsules. A composition can also contain capsules that are different in that it includes capsules encapsulating cells heterologously expressing a first protein of interest and a second capsule encapsulating cells heterologously expressing a second protein of interest. In some embodiments, the first protein of interest is IL-2 and the second protein of interest is IL-12. In some embodiments, the capsules contain a population of cells that express IL-2 and IL-12, either from the same cells or different cells encapsulated in the same capsule.

[0064] In some embodiments, a zone (e.g., an inner zone or an outer zone) or layer of an implantable construct may comprise a degradable entity, e.g., a degradable entity. The degradable entity may comprise an enzymatic cleavage site, a photolabile site, a pH-sensitive site, or other labile region that may erode or be included over time. In some embodiments, the degradable entity is preferentially degraded upon exposure to a first condition (e.g., exposure to a first environment, e.g., a first pH or a first enzyme) compared to a second condition (e.g., exposure to a second environment, e.g., a second pH or a second enzyme). In one embodiment, the degradable entity is degraded at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, or 100 times faster upon exposure to a first condition compared to a second condition. In some embodiments, the degradable entity is an enzymatic cleavage site, e.g., a proteolytic site. In some embodiments, the degradable entity is a polymer (e.g., a synthetic polymer or a natural polymer, e.g., a peptide or polysaccharide). In some embodiments, the degradable entity is a substrate for an endogenous host component, e.g., a degradative enzyme, e.g., a remodeling enzyme, e.g., a collagenase or a metalloprotease. In some embodiments, the degradable entity comprises a cleavable linker or cleavable segment embedded in the polymer.

[0065] In some embodiments, the implantable construct comprises pores or openings that allow passage of entities such as small molecules (e.g., nutrients or waste products), proteins, or nucleic acids. For example, pores in or on the implantable construct can be greater than 0.1 nm and less than 10 μm. In some embodiments, the implantable construct comprises pores or openings having a size range of 0.1 μm to 10 μm, 0.1 μm to 9 μm, 0.1 μm to 8 μm, 0.1 μm to 7 μm, 0.1 μm to 6 μm, 0.1 μm to 5 μm, 0.1 μm to 4 μm, 0.1 μm to 3 μm, or 0.1 μm to 2 μm.

[0066] The implantable constructs described herein can include chemical modifications in or on any encapsulating material. Exemplary chemical modifications include small molecules, peptides, proteins, nucleic acids, lipids, or oligosaccharides. The implantable constructs can include at least 0.5%, 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the material chemically modified, e.g., with a chemical modification described herein. The implantable constructs can be partially coated with the chemical modification, e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% coated with the chemical modification.

[0067] In some embodiments, the implantable construct is formulated to adjust the duration of release of the antigenic substance and / or therapeutic substance. For example, the implantable construct can be configured in a specific manner to release a specific amount of antigenic substance or therapeutic substance over time, e.g., in a sustained or controlled manner. In some embodiments, the implantable construct includes a degradable zone (e.g., an inner or outer zone) that controls the duration of therapeutic agent release from the construct by gradually terminating the immune protection of the encapsulated cells or by causing a sustained release of the antigenic substance. In some embodiments, the implantable construct is configured so that the release level of the antigenic substance or therapeutic substance is sufficient to regulate the proportion of host effector cells, e.g., host T cells. In some embodiments, the implantable construct is configured so that the release level of the antigenic substance or therapeutic substance is sufficient to activate host cells (e.g., host T effector cells or host NK cells) or increase the levels of certain host cells (e.g., host T effector cells or host NK cells). In some embodiments, the implantable construct is configured such that the level of release of the antigenic or therapeutic substance is not sufficient to activate or increase the levels of host regulatory cells (e.g., host T regulatory cells).

[0068] In some embodiments, the implantable construct comprises a zone that is targeted by the host's or subject's natural foreign body response (FBR), e.g., over a period of time. In some embodiments, the implantable construct, upon administration to a subject, e.g., over a period of time, becomes coated with fibrous hyperplasia. Fibrotic hyperplasia on the surface of the implantable construct can result in a decrease in the function of the implantable construct. For example, the decrease in function can include a decrease in release of antigenic or therapeutic substances over time, a decrease in pore size, or a decrease in the diffusion rate of oxygen and other vital nutrients to the encapsulated cells, resulting in cell death. In some embodiments, the rate of fibrotic hyperplasia can be tailored to design a dosing regimen. For example, fibrous hyperplasia on the surface of an implantable construct may result in a decrease in the function of the implantable construct about 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 2.5 weeks, 3 weeks, 4 weeks, or 6 weeks after administration (e.g., injection or implantation) to a subject.

[0069] In some embodiments, the implantable construct is chemically modified at a specific modification density. The specific density of chemical modifications can be described as the average number of chemical modifications attached per given area. For example, the density of chemical modifications on or in the implantable construct can be 0.01, 0.1, 0.5, 1, 5, 10, 15, 20, 50, 75, 100, 200, 400, 500, 750, 1,000, 2500, or 5000 chemical modifications per square micrometer or square millimeter.

[0070] The implantable construct may be formulated or configured for implantation into any organ, tissue, cell, or portion of a subject. For example, the implantable construct may be implanted or placed into the intraperitoneal space of a subject. In some embodiments, the implantable construct may be implanted or placed into the subcutaneous space of a subject. The implantable construct may be implanted or placed into a tumor or other growth in a subject, or may be implanted or placed about 0.1 mm, 0.5 mm, 1 mm, 0.25 mm, 0.5 mm, 0.75 mm, 1 mm, 1.5 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 1 cm, 5 cm, 10 cm, or further from a tumor or other growth in a subject. The implantable construct may be configured for, implanted in, or positioned on or in the skin, under the skin, a mucosal surface, a body cavity, the central nervous system (e.g., brain or spinal cord), an organ (e.g., heart, eye, liver, kidney, spleen, lung, ovary, breast, uterus), lymphatic system, vascular system, oral cavity, nasal cavity, gastrointestinal tract, bone, muscle, adipose tissue, or other area.

[0071] The implantable construct can be formulated for use for any period of time. For example, the implantable construct can be used for 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or longer. The implantable construct can be configured for limited exposure (e.g., less than 2 days, e.g., less than 2 days, 1 day, 24 hours, 20 hours, 16 hours, 12 hours, 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, or less). Implantable constructs can be configured for long-term exposure (e.g., at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years or more). Implantable constructs can be configured for permanent exposure (e.g., at least 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years or more).

[0072] In some embodiments, the degradable zone comprises a polymer hydrogel, such as, but not limited to, chitosan, cellulose, hyaluronic acid, or alginate. In some embodiments, the alginate is SLG20. In some embodiments, the alginate is unmodified. In some embodiments, the alginate is not modified with an anti-fibrotic molecule. For clarity, cells expressing molecules that may have an anti-fibrotic effect are not modifications of the alginate or hydrogel that are considered anti-fibrotic.

[0073] Thus, in some embodiments, a population of encapsulated cells is provided comprising oligonucleotide molecules encoding native human IL-12 polypeptides. In some embodiments, a population of encapsulated cells is provided comprising oligonucleotide molecules encoding recombinant IL-12 polypeptides. In some embodiments, a population of encapsulated cells is provided comprising oligonucleotide molecules encoding IL-12 mutein polypeptides. In some embodiments, a population of encapsulated cells is provided comprising oligonucleotide molecules encoding native human IL-12(p35) polypeptides. In some embodiments, a population of encapsulated cells is provided comprising oligonucleotide molecules encoding native human IL-12(p40) polypeptides. In some embodiments, a population of encapsulated cells is provided comprising oligonucleotide molecules encoding recombinant IL-12(p35) polypeptides. In some embodiments, a population of encapsulated cells is provided comprising oligonucleotide molecules encoding recombinant IL-12(p40) polypeptides. In some embodiments, a population of encapsulated cells is provided comprising oligonucleotide molecules encoding IL-12(p35) mutein polypeptides. In some embodiments, a population of encapsulated cells is provided comprising oligonucleotide molecules encoding IL-12(p40) mutein polypeptides.

[0074] In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding native human IL-12 polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding recombinant IL-12 polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding IL-12 mutein polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding native human IL-12(p35) polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding native human IL-12(p40) polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding recombinant IL-12(p35) polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding recombinant IL-12(p40) polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding IL-12(p35) mutein polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding IL-12(p40) mutein polypeptides. In some embodiments, a population of encapsulated cells is provided that comprises a plurality of oligonucleotide molecules encoding native human IL-12(p35) polypeptides and native human IL-12(p40) polypeptides. In some embodiments, a population of encapsulated cells is provided that comprises a plurality of oligonucleotide molecules encoding recombinant IL-12(p35) polypeptides and recombinant IL-12(p40) polypeptides. In some embodiments, a population of encapsulated cells is provided that comprises a plurality of oligonucleotide molecules encoding IL-12(p35) mutein polypeptides and IL-12(p40) mutein polypeptides.In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding native human IL-12(p35) polypeptides and recombinant IL-12(p40) polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding recombinant IL-12(p35) polypeptides and native human IL-12(p40) polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding native human IL-12(p35) polypeptides and IL-12(p40) mutein polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding IL-12(p35) mutein polypeptides and native human IL-12(p40) polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding recombinant IL-12(p35) polypeptides and IL-12(p40) mutein polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding IL-12(p35) polypeptides and IL-12(p40) mutein polypeptides. In some embodiments, a population of encapsulated cells is provided comprising a plurality of oligonucleotide molecules encoding IL-12(p35) mutein polypeptides and recombinant IL-12(p40) polypeptides.

[0075] In some embodiments, the oligonucleotides encoding native human IL-12 polypeptides comprise the sequences of SEQ ID NO: 1 and SEQ ID NO: 2. In some embodiments, the oligonucleotides encoding native human IL-12(p35) polypeptides comprise the sequence of SEQ ID NO: 1. In some embodiments, the oligonucleotides encoding native human IL-12(p40) polypeptides comprise the sequence of SEQ ID NO: 2. In some embodiments, the cells produce recombinant IL-12 protein. In some embodiments, the cells produce recombinant IL-12(p35) protein. In some embodiments, the cells produce recombinant IL-12(p40) protein. In some embodiments, the cells produce an IL-12 mutein protein. In some embodiments, the cells produce an IL-12(p35) mutein protein. In some embodiments, the cells produce an IL-12(p40) mutein protein. In some embodiments, the IL-12(p35) mutein is as provided herein. In some embodiments, the IL-12(p40) mutein is as provided herein.

[0076] In some embodiments, the population of encapsulated cells remains viable for at least 1 to 180 days. In some embodiments, the population of encapsulated cells remains viable for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least In some embodiments, the encapsulated cell population remains viable for at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, or at least 180 days. In some embodiments, the encapsulated cell population remains viable for at least 6 days. In some embodiments, the encapsulated cell population remains viable for at least 14 days. In some embodiments, the encapsulated cell population remains viable for at least 21 days. In some embodiments, the encapsulated cell population remains viable for at least 40 days. In some embodiments, the encapsulated cell population remains viable for at least 120 days.

[0077] Also provided herein are pharmaceutical compositions comprising the encapsulated cells.

[0078] E. Treatment method Described herein are therapeutic methods or uses of encapsulated cells for the preparation of a pharmaceutical composition (or medicament) for the treatment of a tumor or disease.

[0079] In some embodiments, the disease is a proliferative disease. In some embodiments, the proliferative disease is cancer. The cancer can be an epithelial, mesenchymal, or hematological malignancy. Cancer includes primary malignant cells or tumors (e.g., those in which cells have not migrated to a site in the subject's body other than the site of the original malignant tumor or tumor) and secondary malignant cells or tumors (e.g., those resulting from metastasis, the movement of malignant cells or tumor cells to a secondary site different from the site of the original tumor). In some embodiments, the cancer is a solid tumor (e.g., carcinoid, carcinoma, or sarcoma), a soft tissue tumor (e.g., a hemolytic malignancy), or a metastatic lesion, e.g., a metastatic lesion of any of the cancers disclosed herein. In some embodiments, the cancer is a fibrous or desmoplastic solid tumor. In some embodiments, the tumor is a pancreatic tumor.

[0080] Thus, in some embodiments, methods are provided for treating a tumor, such as a pancreatic tumor, in a subject, hi some embodiments, the methods include implanting a pharmaceutical composition comprising a population (e.g., capsules) of a plurality of encapsulated cells provided herein into the intraperitoneal space of the subject to treat the cancer.

[0081] In some embodiments, the pharmaceutical composition comprises a plurality of populations of encapsulated cells expressing IL-12 provided herein. In some embodiments, the pharmaceutical composition comprises a plurality of populations of encapsulated cells expressing IL-12 and IL-2 provided herein.

[0082] A method of providing systemic treatment to a subject having cancer, the method comprising implanting a pharmaceutical composition comprising a population of encapsulated cells (e.g., capsules) provided herein into the intraperitoneal space of the subject, whereby the pharmaceutical composition stimulates activation of immune cells in the intraperitoneal space and the activated immune cells migrate from the intraperitoneal space to a distal region of the subject, thereby systemically treating the cancer in the subject.

[0083] In some embodiments, methods of providing systemic treatment to a subject with cancer are provided. In some embodiments, the methods include implanting a pharmaceutical composition comprising a plurality of encapsulated cell populations (e.g., capsules) provided herein into the intraperitoneal space of the subject. In some embodiments, the pharmaceutical composition activates immune cells within the IP space. In some embodiments, the activated immune cells migrate out of (away from) the intraperitoneal space to treat cancer in the subject at a site not within the IP space. In some embodiments, the activated immune cells migrate out of (away from) the intraperitoneal space to treat cancer in the subject at a site distal to the IP space. In some embodiments, the site is another organ or tissue, such as the pancreas, breast, brain, lung, bone, or as otherwise provided herein.

[0084] In some embodiments, the subject receives a dose of about 0.01 μg / kg / day to about 20 μg / kg / day, about 0.1 μg / kg / day to about 20 μg / kg / day, about 1 μg / kg / day to about 20 μg / kg / day, about 2 μg / kg / day to about 20 μg / kg / day, about 5 μg / kg / day to about 20 μg / kg / day, about 7.5 to about 20 μg / kg / day, about 9 μg / kg / day to about 20 μg / kg / day, about 10 μg / kg / day to about 20 μg / kg / day, about 11 μg / kg / day to about 20 μg / kg / day, or about 15 μg / kg / day to about 20 μg / kg / day. kg / day ~ approx. 20 μg / kg / day, approx. 12 μg / kg / day ~ approx. 20 μg / kg / day, approx. 13 μg / kg / day ~ approx. 20 μg / kg / day, approx. 14 μg / kg / day ~ approx. 15 μg / kg / day, approx. 15 μg / kg / day ~ approx. 20 μg / kg / day, about 10μg / kg / day to about 15μg / kg / day, about 11μg / kg / day to about 15μg / kg / day, about 12μg / kg / day to about 15μg / kg / day, about 13μg / kg / day to about 15μg / kg / day, about 14μg / kg / day to about 15μg / kg / day, about 16μg / kg / day to about 20μg / kg / day, about 17μg / kg / day to about 20μg / kg / day, about 18μg / kg / day to about 20μg / kg / day, about 0.01μg / k g / day, approximately 0.05μg / kg / day, approximately 0.1μg / kg / day, approximately 0.5μg / kg / day, approximately 1μg / kg / day, approximately 2μg / kg / day, approximately 3μg / kg / day, approximately 4μg / kg / day, approximately 5μg / kg / day, approximately 6μg / k The patient may be administered (e.g., implanted) about 7 μg / kg / day, about 8 μg / kg / day, about 9 μg / kg / day, about 10 μg / kg / day, about 11 μg / kg / day, about 12 μg / kg / day, about 13 μg / kg / day, about 14 μg / kg / day, about 15 μg / kg / day, about 16 μg / kg / day, about 17 μg / kg / day, about 18 μg / kg / day, about 19 μg / kg / day, or about 20 μg / kg / day of the encapsulated cells provided herein.

[0085] As described herein, encapsulated cells producing recombinant native human IL-12 can be used to generate memory immunity against tumors. Thus, in some embodiments, the methods provided herein can be used to prevent or reduce the likelihood of tumor recurrence, either at the initial site of the tumor or at a site distal to the tumor's origin. In some embodiments, the tumor is a pancreatic tumor. In some embodiments, a method of treating tumors by generating (inducing) memory immunity comprises implanting a pharmaceutical composition comprising a population of encapsulated cells provided herein.

[0086] In some embodiments, methods are provided for selectively activating CD8 and / or CD4 positive effector T cells. Without being bound by theory, the CD8 and / or CD4 positive effector cells are activated to induce an immune response against tumors, which may be initiated or enhanced by the secretion of natural human IL-12 in the IP space from the encapsulated cells provided herein. In some embodiments, the methods include implanting a pharmaceutical composition comprising a population of encapsulated cells provided herein.

[0087] In some embodiments, effector T cells are selectively activated and expanded relative to Tregs (CD4+CD25+FOXp3+). In some embodiments, the selectively activated T cells secrete IFN-γ.

[0088] In some embodiments, encapsulated cells implanted or administered to a subject do not cause significant fibrosis in the subject. In some embodiments, encapsulated cells expressing IL-12 implanted or administered to a subject do not cause significant fibrosis in the subject. In some embodiments, encapsulated IL-12-expressing cells implanted or administered to a subject do not cause significant fibrosis in the subject over time.

[0089] Exemplary cancers that can be treated by the methods provided herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. In some embodiments, the cancer affects a body system, such as the nervous system (e.g., the peripheral nervous system (PNS) or central nervous system (CNS)), vascular system, skeletal system, respiratory system, endocrine system, lymphatic system, reproductive system, or gastrointestinal tract. In some embodiments, the cancer affects a part of the body, such as the blood, eyes, brain, skin, lungs, stomach, mouth, ears, legs, feet, hands, liver, heart, kidneys, bones, pancreas, spleen, large intestine, small intestine, spinal cord, muscles, ovaries, uterus, vagina, or penis. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0090] Other examples of cancer include, but are not limited to, acute childhood lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) hepatocellular carcinoma, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignancies, anal cancer, astrocytoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, cancer of the pelvis and urinary tract, cancer of the central nervous system (primary lymphoma, central nervous system lymphoma, cerebellar astrocytoma, brain astrocytoma, cervical cancer, childhood (primary) hepatocellular carcinoma, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood brain astrocytoma, childhood extracranial germ cell tumor, childhood Hodgkin's disease, childhood Hodgkin's lymphoma, childhood hypothalamic and visually impaired glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin's lymphoma, childhood pineal and supratentorial primitive neuroectodermal tumor, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic tumor Neuroglioma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, cutaneous T-cell lymphoma, pancreatic endocrine islet cell carcinoma, endometrial cancer, ependymoma, epithelial carcinoma, esophageal cancer, Ewing's sarcoma and related tumors, exocrine pancreatic cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, female breast cancer, Gaucher's disease, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal tumors, germ cell tumors, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancer, intraocular melanoma, islet cell carcinoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity Cancer, liver cancer, lung cancer, lymphoproliferative disorders, macroglobulinemia, male breast cancer, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, metastatic occult primary squamous cell carcinoma of the neck, metastatic primary squamous cell carcinoma of the neck, metastatic squamous cell carcinoma of the neck, multiple myeloma, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, myeloid leukemia, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma during pregnancy, non-melanoma skin cancer, non-small cell lung cancer, occult primary metastatic squamous cell carcinoma, oropharyngeal cancer, bone / malignant fibrous sarcoma, osteosarcoma / malignant fibrous histiocytoma,Osteosarcoma / malignant fibrous histiocytoma, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low-grade malignant potential tumor, pancreatic cancer, parapapilloma, purpura, parathyroid carcinoma, penile cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, pelvic and urinary tract cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoidosis sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, cervical squamous cell carcinoma, Gastric cancer, supratentorial primitive neuroectodermal tumor and pineal tumor, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional renal pelvic and urinary tract cancer, trophoblastic tumor, uterine and renal pelvic cell carcinoma, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and any other hyperproliferative disease other than neoplasia located within the organ systems listed above.

[0091] In some embodiments, the implantable constructs (encapsulated cells) described herein can be used in methods of modulating (e.g., upregulating) an immune response in a subject. For example, when administered to a subject, the implantable construct (or antigenic and / or therapeutic substances disposed therein) can modulate (e.g., upregulate) the level of a component of the immune system in the subject (e.g., increase or decrease the level of an immune system component). Exemplary immune system components that may be modulated by the implantable constructs or related methods described herein include stem cells (hematopoietic stem cells), NK cells, T cells (e.g., adaptive T cells (e.g., helper T cells, cytotoxic T cells, memory T cells, or regulatory T cells) or innate-like T cells (e.g., natural killer T cells, mucosal-associated invariant T cells, or γδ T cells), B cells, antibodies or fragments thereof, or other additional components. In some embodiments, modulation includes increasing or decreasing the activation of T cells or other immune system components (e.g., by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to a control). In some embodiments, the encapsulated cells (implantable constructs) can be used to activate CD4+ and / or CD8+ immune cells.

[0092] The implantable constructs described herein can be used to modulate immune responses in a subject over a specific period of time. For example, administration of the implantable construct (or antigenic and / or therapeutic substances disposed therein) can activate an immune response (e.g., by increasing the level of immune system components) in a subject for at least 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, 1 day, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 1.5 weeks, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, or more. In some embodiments, administration of the implantable construct activates an immune response (e.g., by increasing the level of immune system components) in a subject for 1 hour to 1 month, 1 hour to 3 weeks, 1 hour to 2 weeks, 1 hour to 1 week, 6 hours to 1 week, or 6 hours to 3 days. In some embodiments, implantation of an implantable construct (e.g., an implantable construct described herein) results in upregulation of T cells in the subject, for at least one day, as measured, for example, by a blood test.

[0093] The implantable constructs described herein may further comprise an additional pharmaceutical agent, such as an antiproliferative agent, an anticancer agent, an anti-inflammatory agent, an immunomodulatory agent, or an analgesic agent, for example, for use in combination therapy. The additional pharmaceutical agent may be disposed in or on the implantable construct or may be produced by cells disposed in or on the implantable construct. In some embodiments, the additional pharmaceutical agent is a small molecule, protein, peptide, nucleic acid, oligosaccharide, or other drug.

[0094] In some embodiments, the additional pharmaceutical agent is an anti-cancer agent. In some embodiments, the anti-cancer agent is a small molecule, a kinase inhibitor, an alkylating agent, a vascular disrupting agent, a microtubule targeting agent, a mitotic inhibitor, a topoisomerase inhibitor, an anti-angiogenic agent, or an antimetabolite. In some embodiments, the anti-cancer agent is a taxane (e.g., paclitaxel, docetaxel, larotaxel, or cabazitaxel). In some embodiments, the anti-cancer agent is an anthracycline (e.g., doxorubicin). In some embodiments, the anti-cancer agent is a platinum-based agent (e.g., cisplatin or oxaliplatin). In some embodiments, the anti-cancer agent is a pyrimidine analog (e.g., gemcitabine). In some embodiments, the anti-cancer agent is selected from camptothecin, irinotecan, rapamycin, FK506, 5-FU, leucovorin, or a combination thereof. In other embodiments, the anti-cancer agent is a protein biologic (eg, an antibody molecule) or a nucleic acid therapy (eg, an antisense or inhibitory double-stranded RNA molecule).

[0095] In some embodiments, the additional pharmaceutical agent is an immunomodulatory agent, such as one or more of an activator of a costimulatory molecule, an inhibitor of an immune checkpoint molecule, or an anti-inflammatory agent. In some embodiments, the immunomodulatory agent is an inhibitor of an immune checkpoint molecule (e.g., an inhibitor of PD-1, PD-L1, LAG-3, TIM-3, or CTLA4, or any combination thereof). In some embodiments, the immunomodulatory agent is a cancer vaccine.

[0096] In some embodiments, the immunomodulatory agent is an inhibitor of PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD73, CD160, 2B4, and / or TGFRbeta. In one embodiment, the inhibitor of an immune checkpoint molecule inhibits PD-1, PD-L1, LAG-3, TIM-3, or CTLA4, or any combination thereof. Inhibition of the inhibitory molecule can be achieved at the DNA, RNA, or protein level. In some embodiments, inhibitory nucleic acids (e.g., dsRNA, siRNA, or shRNA) can be used to inhibit expression of the inhibitory molecule. In other embodiments, the inhibitor of an inhibitory signal is a polypeptide that binds to an inhibitory molecule, e.g., a soluble ligand (e.g., PD-1-Ig or CTLA-4 Ig), or an antibody or antigen-binding fragment thereof, such as an antibody or fragment thereof that binds to PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD73, CD160, 2B4, and / or TGFR beta, or a combination thereof. In some embodiments, the immunomodulatory agent is an anti-inflammatory agent, e.g., an anti-inflammatory agent described herein. In some embodiments, the anti-inflammatory agent is an agent that blocks, inhibits, or reduces inflammation or signaling from an inflammatory signaling pathway. In some embodiments, the anti-inflammatory agent inhibits or reduces the activity of any one or more of the following immune components of a subject: In some embodiments, the anti-inflammatory agent is an IL-1 or IL-1 receptor antagonist, such as anakinra, rilonacept, or canakinumab. In some embodiments, the anti-inflammatory agent is an IL-6 or IL-6 receptor antagonist, e.g., an anti-IL-6 antibody or an anti-IL-6 receptor antibody, e.g., tocilizumab (ACTEMRA®), olokizumab, clazakizumab, sarilumab, sirumab, silutuximab, or ALX-0061.In some embodiments, the anti-inflammatory agent is a TNF-α antagonist, e.g., an anti-TNF-α antibody, e.g., infliximab (REMICADE®), golimumab (SIMPONI®), adalimumab (HUMIRA®), certolizumab pegol (CIMZIA®), or etanercept. In one embodiment, the anti-inflammatory agent is a corticosteroid, e.g., as described herein.

[0097] F. Composition and Administration of Implantable Constructs The present disclosure also provides a pharmaceutical composition comprising an implantable construct provided herein and, optionally, a pharmaceutically acceptable excipient. In some embodiments, the implantable construct is provided in an effective amount in the pharmaceutical composition. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is a prophylactically effective amount.

[0098] In some embodiments, the effective amount is the amount that produces an effective amount of native human IL-12. In some embodiments, the effective amount is the amount that produces an effective amount of recombinant IL-12. In some embodiments, the effective amount is the amount that produces an effective amount of an IL-12 mutein. In some embodiments, the effective amount is the amount that produces an effective amount of native human IL-12(p35). In some embodiments, the effective amount is the amount that produces an effective amount of native human IL-12(p40). In some embodiments, the effective amount is the amount that produces an effective amount of recombinant IL-12(p35). In some embodiments, the effective amount is the amount that produces an effective amount of recombinant IL-12(p40). In some embodiments, the effective amount is the amount that produces an effective amount of an IL-12(p35) mutein. In some embodiments, the effective amount is the amount that produces an effective amount of an IL-12(p40) mutein. In some embodiments, the effective amount is the amount that produces an effective amount of IL-2.

[0099] The pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such methods include the step of bringing into association the implantable construct with the carrier and / or one or more other accessory ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into the desired single- or multi-dose unit.

[0100] Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the implantable construct can generally be equal to the dosage of the antigenic and / or therapeutic agent administered to a subject, and / or a convenient fraction of such a dosage (e.g., one-half or one-third of such a dosage).

[0101] In some embodiments, the pharmaceutical composition is frozen or cryopreserved. In some embodiments, the pharmaceutical composition is not frozen or not cryopreserved.

[0102] The relative amounts of implantable construct, pharmaceutically acceptable excipient, and / or any additional components in the pharmaceutical compositions of the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100% (w / w) of any component.

[0103] Implantable constructs and pharmaceutical compositions thereof may be administered or implanted orally, parenterally (including subcutaneously, intramuscularly, intravenously, and intradermally), by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. In some embodiments, provided compounds or compositions are administrable intravenously and / or orally. In some embodiments, the implantable construct is injected subcutaneously. In some embodiments, the implantable construct is injected into the intraperitoneal space. In some embodiments, the implantable construct is delivered to the subject using a device, such as a cannula or catheter.

[0104] The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraocular, intravitreal, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intraperitoneal, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, subcutaneously, intraperitoneally, or intravenously. Sterile injectable forms of the compositions of the present disclosure may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media.

[0105] For ophthalmic use, the provided compounds, compositions, and devices may be formulated as a micronized suspension or in an ointment such as petrolatum.

[0106] In some embodiments, the release of the antigenic substance, therapeutic substance, or additional pharmaceutical agent is sustained. To prolong the effect of certain drugs, it is often desirable to delay the absorption of the drug from injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends on its dissolution rate, which may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle.

[0107] Although the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for administration to humans, one of ordinary skill in the art will understand that such compositions are generally suitable for administration to animals of all kinds. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and an ordinarily skilled veterinary pharmacologist can design and / or perform such modifications using routine experimentation.

[0108] The implantable constructs provided herein are typically formulated in unit dosage forms, such as single-unit dosage forms, for ease of administration and uniformity of dosage. However, it is understood that the total daily usage amount of the compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend on various factors, including: the severity of the disease or disorder being treated, the activity of the specific compound used; the specific composition used, the age, weight, general health, sex, and diet of the subject; the administration time, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or simultaneously with the specific therapeutic substance used; and similar factors well known in the medical field.

[0109] The exact amount of compound required to achieve an effective dose will vary from subject to subject, depending, for example, on the subject's species, age, and general condition, the severity of any side effects or disorders, the identity of the particular compound(s), the mode of administration, etc. The desired dosage can be delivered three times daily, twice daily, once daily, every other day, every third day, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations).

[0110] The therapeutic agent to be administered may be at a dosage level sufficient to deliver about 0.00001 mg / kg to about 100 mg / kg, about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 50 mg / kg, preferably about 0.1 mg / kg to about 40 mg / kg, preferably about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, more preferably about 0.001 mg / kg to about 1 mg / kg of subject body weight per day, one or more times per day, to obtain the desired therapeutic effect.

[0111] It will be understood that the dosage ranges described herein provide guidance for administering the provided pharmaceutical compositions to adults. For example, the amount administered to a child or adolescent can be determined by a physician or person skilled in the art and can be lower than or the same as the amount administered to an adult.

[0112] Constructs (e.g., encapsulated cells) can be prepared according to any known method. For example, in some embodiments, methods for preparing encapsulated cells that produce recombinant proteins are provided. In some embodiments, the methods include dispensing a first composition comprising a polymer hydrogel and a second composition comprising encapsulated cells suspended in the polymer hydrogel through a coaxial needle to form encapsulated cells, the crosslinking solution comprising a sugar alcohol, a buffer, a metal salt, and a surfactant.

[0113] In some embodiments, the encapsulated cells comprise an oligonucleotide molecule encoding native human IL-12. In some embodiments, the encapsulated cells comprise an oligonucleotide molecule encoding recombinant IL-12. In some embodiments, the encapsulated cells comprise an oligonucleotide molecule encoding an IL-12 mutein. In some embodiments, the encapsulated cells comprise an oligonucleotide molecule encoding native human IL-12(p35). In some embodiments, the encapsulated cells comprise an oligonucleotide molecule encoding native human IL-12(p40). In some embodiments, the encapsulated cells comprise an oligonucleotide molecule encoding recombinant IL-12(p35). In some embodiments, the encapsulated cells comprise an oligonucleotide molecule encoding recombinant IL-12(p40). In some embodiments, the encapsulated cells comprise an oligonucleotide molecule encoding an IL-12(p35) mutein. In some embodiments, the encapsulated cells comprise an oligonucleotide molecule encoding an IL-12(p40) mutein. In some embodiments, the encapsulated cells comprise multiple oligonucleotide molecules encoding native human IL-12(p35) and native human IL-12(p40). In some embodiments, the encapsulated cells comprise multiple oligonucleotide molecules encoding recombinant IL-12(p35) and recombinant IL-12(p40). In some embodiments, the encapsulated cells comprise multiple oligonucleotide molecules encoding IL-12(p35) muteins and IL-12(p40) muteins. In some embodiments, the encapsulated cells comprise oligonucleotide molecules encoding native human IL-2.

[0114] In some embodiments, the oligonucleotide encoding native human IL-12(p35) comprises the sequence of SEQ ID NO: 1. In some embodiments, the oligonucleotide encoding native human IL-12(p40) comprises the sequence of SEQ ID NO: 2. In some embodiments, native human IL-12(p35) and native human IL-12(p40) interact with each other to form a heterodimeric complex. In some embodiments, the heterodimeric complex is native human IL-12. In some embodiments, the cell produces native human IL-12 protein. In some embodiments, the native human IL-12 protein is formed from the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4. In some embodiments, the cell produces recombinant IL-12 protein. In some embodiments, the recombinant IL-12 protein comprises recombinant IL-12(p35) protein and recombinant IL-12(p40) protein. In some embodiments, the cell produces an IL-12 mutein protein. In some embodiments, the IL-12 mutein protein comprises an IL-12(p35) mutein protein and an IL-12(p40) mutein protein. In some embodiments, the oligonucleotide encoding native human IL-2 comprises the sequence of SEQ ID NO:6.

[0115] The cell can be any type of cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an epithelial cell. In some embodiments, the cell is an RPE cell. In some embodiments, the cell is an ARPE-19 cell, an ARPE-19-SEAP-2-neo cell, an RPE-J cell, or an hTERT RPE-1 cell. In some embodiments, the cell is an engineered RPE cell. In some embodiments, the engineered cell is derived from the ARPE-19 cell line. In some embodiments, the cell is as provided herein. In some embodiments, the surfactant is TWEEN 20 (polysorbate 20). In some embodiments, the buffer is a HEPES buffer. In some embodiments, the sugar alcohol is mannitol. In some embodiments, the metal salt is barium chloride.

[0116] In some embodiments, the methods include washing the encapsulated cells produced according to the methods provided herein in the buffer solution in which they were produced, hi some embodiments, the washing step removes substantially all or all of the free barium or barium chloride.

[0117] In some embodiments, encapsulated cells prepared according to the methods provided herein are stored in a storage buffer, such as DMEM / F12 cell culture medium. In some embodiments, the stored cells retain viability for at least 10 days, 20 days, or 30 days. In some embodiments, the storage buffer is substantially free of Plasmalyte buffer.

[0118] Also provided herein are populations of encapsulated cells prepared according to the methods provided herein.

[0119] In some embodiments, a suspension of encapsulated cells is provided. In some embodiments, the suspension comprises a population of encapsulated cells provided herein. In some embodiments, the encapsulated cells are encapsulated by a polymer hydrogel, and the suspension comprises a crosslinking solution comprising a sugar alcohol, a buffer, a metal salt, and a surfactant. In some embodiments, the cells are ARPE-19 cells. In some embodiments, the surfactant is TWEEN 20 (polysorbate 20). In some embodiments, the buffer is HEPES buffer. In some embodiments, the sugar alcohol is mannitol. In some embodiments, the metal salt is barium chloride.

[0120] In some embodiments, a suspension of encapsulated cells is provided, the suspension comprising a population of encapsulated cells provided herein, the encapsulated cells being encapsulated by a polymer hydrogel and a storage buffer, such as DMEM / F12 cell culture medium.

[0121] In some embodiments, the suspensions provided herein are substantially free of Plasmalyte buffer.

[0122] G. Enumerated Embodiments 1. A population of encapsulated cells comprising a plurality of oligonucleotide molecules encoding an IL-12 polypeptide. 2. The population of encapsulated cells of embodiment 1, wherein the IL-12 polypeptide is a native human IL-12 polypeptide, a recombinant IL-12 polypeptide, or an IL-12 mutein polypeptide. 3. The population of encapsulated cells of embodiment 2, wherein the native human IL-12 polypeptide is a heterodimeric complex comprising a native human IL-12(p35) polypeptide and a native human IL-12(p40) polypeptide. 4. The population of encapsulated cells of any one of embodiments 1 to 3, wherein the plurality of oligonucleotide molecules encodes a native human IL-12(p35) polypeptide and a native human IL-12(p40) polypeptide. 5. The oligonucleotide encoding the native human IL-12 (p35) polypeptide is A population of encapsulated cells described in any one of embodiments 1 to 4, comprising the sequence TIFF2025503454000010.tif77166. 6. The oligonucleotide encoding the native human IL-12 (p40) polypeptide is A population of encapsulated cells described in any one of embodiments 1 to 5, comprising the sequence TIFF2025503454000011.tif125166. 7. The population of encapsulated cells of any one of embodiments 1 to 6, wherein the expressed IL-12(p35) polypeptide and the expressed IL-12(p40) polypeptide form a heterodimeric complex. 8. The population of encapsulated cells of embodiment 2, wherein the recombinant IL-12 is a heterodimeric complex comprising a recombinant IL-12(p35) polypeptide and a recombinant IL-12(p40) polypeptide. 9. The population of encapsulated cells of embodiment 2, wherein the IL-12 mutein is a heterodimeric complex comprising an IL-12(p35) mutein polypeptide and an IL-12(p40) mutein polypeptide. 10. The population of encapsulated cells of embodiment 9, wherein the plurality of oligonucleotide molecules encodes an IL-12(p35) mutein polypeptide and an IL-12(p40) mutein polypeptide. 11. The population of encapsulated cells of any one of embodiments 9 or 10, wherein the IL-12(p40) mutein polypeptide comprises a mutation selected from N220L, N222L or N222Q compared to SEQ ID NO: 4. 12. The population of encapsulated cells of any one of embodiments 9 or 10, wherein the IL-12(p40) mutein polypeptide comprises the following mutation compared to SEQ ID NO:4: N222L. 13. The population of encapsulated cells of any one of embodiments 9 or 10, wherein the IL-12(p40) mutein polypeptide comprises the mutation N222Q compared to SEQ ID NO:4. 14. The population of encapsulated cells of any one of embodiments 9 or 10, wherein the IL-12(p40) mutein polypeptide comprises the following mutation compared to SEQ ID NO:5: N220L. 15. The population of encapsulated cells of any one of embodiments 1 to 14, wherein the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. 16. The population of encapsulated cells of any one of embodiments 1 to 15, wherein the cells produce a native human IL-12 heterodimeric protein, a recombinant IL-12 heterodimeric protein, or an IL-12 mutein heterodimeric protein. 17. The native human IL-12 protein expressed by a cell is a first polypeptide comprising the sequence of TIFF2025503454000012.tif11165; a second polypeptide comprising the sequence of TIFF2025503454000013.tif19166; and 17. The population of encapsulated cells of embodiment 16, comprising a polypeptide comprising: 18. The population of encapsulated cells of any one of embodiments 1-17, wherein the population of cells produces about 10 to about 50, about 10 to about 30, about 10 to about 20, or about 20 nanograms / cell / day of native human IL-12. 19. The population of encapsulated cells of any one of embodiments 1 to 18, wherein the cells are as provided herein. 20. The population of encapsulated cells of any one of embodiments 1-19, wherein the cells are ARPE-19 cells, ARPE-19-SEAP-2-neo cells, RPE-J cells, hTERT RPE-1 cells, or any combination thereof. 21. A population of encapsulated cells according to any one of embodiments 1 to 20, wherein the cells are encapsulated in a polymer hydrogel. 22. The population of encapsulated cells of any one of embodiments 1 to 21, wherein the polymer hydrogel comprises chitosan, cellulose, hyaluronic acid, or alginate. 23. The population of encapsulated cells of any one of embodiments 1 to 22, wherein the polymer hydrogel comprises alginate. 24. The population of encapsulated cells of any one of embodiments 1 to 23, wherein the alginate comprises SLG20. 25. The population of encapsulated cells of any one of embodiments 1 to 24, wherein the cells remain viable for at least 5, 10, 15, 20, 40, 120, or 180 days. 26. A population of encapsulated cells according to any one of embodiments 1 to 25, wherein the encapsulated cells do not proliferate. 27. The population of encapsulated cells of any one of embodiments 1 to 26, wherein the encapsulated cells do not exhibit fibrotic hyperplasia over time after transplantation into a subject. 28. The population of encapsulated cells of any one of embodiments 1 to 27, wherein the encapsulated cells exhibit minor fibrotic hyperplasia over time after transplantation into a subject. 29. A pharmaceutical composition comprising a population of encapsulated cells according to any one of embodiments 1 to 28. 30. The pharmaceutical composition of embodiment 29, further comprising a population of encapsulated cells comprising an oligonucleotide molecule encoding native human IL-2. 31. An oligonucleotide encoding native human IL-2 is 31. The pharmaceutical composition of embodiment 30, comprising the sequence of TIFF2025503454000014.tif27169. 32. The pharmaceutical composition of embodiment 31, wherein the oligonucleotide encoding native human IL-2 comprises a codon-optimized sequence. 33. The pharmaceutical composition of embodiment 32, wherein the codon-optimized oligonucleotide encoding native human IL-2 comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7. 34. The pharmaceutical composition according to any one of embodiments 30 to 33, wherein the cells produce recombinant native human IL-2 protein. 35. The recombinant native human IL-2 protein expressed by a cell is A pharmaceutical composition described in any one of embodiments 30 to 34, comprising the amino acid sequence of TIFF2025503454000015.tif11165. 36. The pharmaceutical composition of any one of embodiments 30 to 35, wherein the population of cells produces about 1 to about 10, about 1 to about 5, or about 2 to about 4 PCD (picograms / cell / day) of native human IL-2. 37. A method of treating a tumor, such as a pancreatic tumor, in a subject, comprising implanting into the intraperitoneal space of the subject a pharmaceutical composition comprising a plurality of encapsulated cells (e.g., capsules) of any one of embodiments 1-28 to treat the cancer. 38. A method of reducing tumor burden, such as a pancreatic tumor, in a subject, comprising implanting into the intraperitoneal space of the subject a pharmaceutical composition comprising a plurality of encapsulated cells (e.g., capsules) of any one of embodiments 1-28 to treat cancer. 39. A subject is administered about 0.01 μg / kg / day to about 20 μg / kg / day, about 0.1 μg / kg / day to about 20 μg / kg / day, about 1 μg / kg / day to about 20 μg / kg / day, about 2 μg / kg / day to about 20 μg / kg / day, about 5 μg / kg / day to about 20 μg / kg / day, about 7.5 to about 20 μg / kg / day, about 9 μg / kg / day to about 20 μg / kg / day, about 10 μg / kg / day to about 20 μg / kg / day, or about 11 μg / kg / day to about 20 μg / kg / day. Approximately 20μg / kg / day, approximately 12μg / kg / day to approximately 20μg / kg / day, approximately 13μg / kg / day to approximately 20μg / kg / day, approximately 14μg / kg / day to approximately 15μg / kg / day, approximately 15μg / kg / day to approximately 20μg / day kg / day, about 10μg / kg / day to about 15μg / kg / day, about 11μg / kg / day to about 15μg / kg / day, about 12μg / kg / day to about 15μg / kg / day, about 13μg / kg / day to about 15μg / kg / day, about 14μg / kg / day to about 15μg / kg / day, about 16μg / kg / day to about 20μg / kg / day, about 17μg / kg / day to about 20μg / kg / day, about 18μg / kg / day to about 20μg / kg / day, about 0.01μ g / kg / day, approximately 0.1μg / kg / day, approximately 1μg / kg / day, approximately 2μg / kg / day, approximately 3μg / kg / day, approximately 4μg / kg / day, approximately 5μg / kg / day, approximately 6μg / kg / day, approximately 7μg / kg / day, approximately 8μg / day

[0039] The method of embodiment 37 or 38, wherein about 9 μg / kg / day, about 10 μg / kg / day, about 11 μg / kg / day, about 12 μg / kg / day, about 13 μg / kg / day, about 14 μg / kg / day, about 15 μg / kg / day, about 6 μg / kg / day, about 17 μg / kg / day, about 18 μg / kg / day, about 19 μg / kg / day, or about 20 μg / kg / day of the encapsulated cells of any one of embodiments 1-28 are administered. 40. A method of treating a tumor, such as a melanoma tumor, in a subject, comprising implanting into the subcutaneous space of the subject a pharmaceutical composition comprising a plurality of encapsulated cells (e.g., capsules) of any one of embodiments 1-28 to treat the cancer. 41. A method of reducing tumor burden, such as a melanoma tumor, in a subject, comprising implanting into the subcutaneous space of the subject a pharmaceutical composition comprising a plurality of encapsulated cells (e.g., capsules) of any one of embodiments 1-28, to treat cancer. 42. A subject is administered about 0.01 μg / kg / day to about 20 μg / kg / day, about 0.1 μg / kg / day to about 20 μg / kg / day, about 1 μg / kg / day to about 20 μg / kg / day, about 2 μg / kg / day to about 20 μg / kg / day, about 5 μg / kg / day to about 20 μg / kg / day, about 7.5 to about 20 μg / kg / day, about 9 μg / kg / day to about 20 μg / kg / day, about 10 μg / kg / day to about 20 μg / kg / day, or about 11 μg / kg / day to about 20 μg / kg / day. Approximately 20μg / kg / day, approximately 12μg / kg / day to approximately 20μg / kg / day, approximately 13μg / kg / day to approximately 20μg / kg / day, approximately 14μg / kg / day to approximately 15μg / kg / day, approximately 15μg / kg / day to approximately 20μg / day kg / day, about 10μg / kg / day to about 15μg / kg / day, about 11μg / kg / day to about 15μg / kg / day, about 12μg / kg / day to about 15μg / kg / day, about 13μg / kg / day to about 15μg / kg / day, about 14μg / kg / day to about 15μg / kg / day, about 16μg / kg / day to about 20μg / kg / day, about 17μg / kg / day to about 20μg / kg / day, about 18μg / kg / day to about 20μg / kg / day, about 0.01μ g / kg / day, approximately 0.1μg / kg / day, approximately 1μg / kg / day, approximately 2μg / kg / day, approximately 3μg / kg / day, approximately 4μg / kg / day, approximately 5μg / kg / day, approximately 6μg / kg / day, approximately 7μg / kg / day, approximately 8μg / day 42. The method of embodiment 40 or 41, wherein about 9 μg / kg / day, about 10 μg / kg / day, about 11 μg / kg / day, about 12 μg / kg / day, about 13 μg / kg / day, about 14 μg / kg / day, about 15 μg / kg / day, about 6 μg / kg / day, about 17 μg / kg / day, about 18 μg / kg / day, about 19 μg / kg / day, or about 20 μg / kg / day of the encapsulated cells of any one of embodiments 1-28 are administered. 43. A method for treating a tumor in a subject by generating memory immunity, comprising transplanting a pharmaceutical composition comprising a population of encapsulated cells according to any one of embodiments 1 to 28. 44. A method for reducing tumor burden in a subject by generating memory immunity, comprising transplanting a pharmaceutical composition comprising a population of encapsulated cells according to any one of embodiments 1 to 28. 45. The method of embodiment 43 or 44, wherein the tumor is a pancreatic tumor or a melanoma tumor. 46. ​​A method for selectively activating CD8-positive effector T cells, comprising implanting a pharmaceutical composition comprising a population of encapsulated cells according to any one of embodiments 1 to 28. 47. The method of embodiment 46, wherein effector T cells are selectively activated and expanded compared to Tregs (CD4+CD25+FoxP3+). 48. The method of embodiment 47, wherein the selectively activated effector T cells secrete IFN-γ. 49. A method for increasing interferon gamma (IFN-γ) in a subject, comprising implanting or administering a pharmaceutical composition comprising a population of encapsulated cells according to any one of embodiments 1 to 28. 50. A method of treating melanoma in a subject, comprising implanting or administering a pharmaceutical composition comprising a population of encapsulated cells according to any one of embodiments 1 to 28. 51. The method of any one of embodiments 37-50, wherein the encapsulated cells implanted or administered to a subject do not cause significant fibrosis in the subject. 52. The method of embodiment 51, wherein the encapsulated cells implanted or administered to a subject do not cause fibrosis in the subject. 53. A method for preventing or inhibiting fibrosis of encapsulated cells transplanted into a subject, comprising causing the transplanted encapsulated cells to express IL-12 or exposing the transplanted encapsulated cells to IL-12 at the site of transplantation. 54. The method of embodiment 53, wherein the transplanted encapsulated cells comprise a population of encapsulated cells described in any one of embodiments 1 to 28, or are transplanted into an environment comprising a population of encapsulated cells that express IL-12, or are transplanted into an environment where IL-12 is present. 55. Dispensing a first composition comprising a polymer hydrogel and a second composition comprising cells to be encapsulated suspended in the polymer hydrogel through a coaxial needle to dropwise drop into a crosslinking solution to form encapsulated cells, wherein the crosslinking solution comprises a sugar alcohol, a buffer, a metal salt, and a surfactant. A method for preparing encapsulated cells that produce a recombinant protein, comprising: 56. The method of embodiment 55, wherein the encapsulated cells comprise a plurality of oligonucleotide molecules encoding IL-12 polypeptides. 57. The method of embodiment 56, wherein the IL-12 polypeptide is a native human IL-12 polypeptide, a recombinant IL-12 polypeptide, or an IL-12 mutein polypeptide. 58. The method of any one of embodiments 56 or 57, wherein the native human IL-12 polypeptide is a heterodimeric complex comprising a native human IL-12(p35) polypeptide and a native human IL-12(p40) polypeptide. 59. The method of any one of embodiments 56 to 58, wherein the plurality of oligonucleotide molecules encodes a native human IL-12(p35) polypeptide and a native human IL-12(p40) polypeptide. 60. An oligonucleotide encoding a native human IL-12 (p35) polypeptide, 60. The method of any one of embodiments 56 to 59, comprising the sequence TIFF2025503454000016.tif75166. 61. An oligonucleotide encoding a native human IL-12 (p40) polypeptide, The method of any one of embodiments 56 to 60, comprising the sequence TIFF2025503454000017.tif123166. 62. The method of any one of embodiments 56 to 61, wherein the expressed IL-12(p35) polypeptide and the expressed IL-12(p40) polypeptide form a heterodimeric complex. 63. The method of embodiment 57, wherein the recombinant IL-12 is a heterodimeric complex comprising a recombinant IL-12(p35) polypeptide and a recombinant IL-12(p40) polypeptide. 64. The method of embodiment 63, wherein the plurality of oligonucleotide molecules encodes a recombinant IL-12(p35) polypeptide and a recombinant IL-12(p40) polypeptide. 65. The method of embodiment 57, wherein the IL-12 mutein is a heterodimeric complex comprising an IL-12(p35) mutein polypeptide and an IL-12(p40) mutein polypeptide. 66. The method of embodiment 65, wherein the plurality of oligonucleotide molecules encodes an IL-12(p35) mutein polypeptide and an IL-12(p40) mutein polypeptide. 67. The method of any one of embodiments 65 or 66, wherein the IL-12(p40) mutein polypeptide comprises a mutation selected from N220L, N222L or N222Q compared to SEQ ID NO: 4. 68. The method of any one of embodiments 65 or 66, wherein the IL-12(p40) mutein polypeptide comprises the mutation N222L compared to SEQ ID NO:4. 69. The method of any one of embodiments 65 or 66, wherein the IL-12(p40) mutein polypeptide comprises the mutation N222Q compared to SEQ ID NO:4. 70. The method of any one of embodiments 65 or 66, wherein the IL-12(p40) mutein polypeptide comprises the mutation N220L compared to SEQ ID NO:5. 71. The method of any one of embodiments 55 to 70, wherein the IL-12(p35) and IL-12(p40) polypeptides are expressed in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. 72. The method according to any one of embodiments 55 to 71, wherein the IL-12(p35) and IL-12(p40) polypeptides are expressed in a ratio of 1:3. 73. The method of any one of embodiments 55-72, wherein the cells produce recombinant native human IL-12 heterodimeric protein. 74. Recombinant native human IL-12 protein expressed by a cell a first polypeptide comprising the sequence of TIFF2025503454000018.tif12165; a second polypeptide comprising the sequence of TIFF2025503454000019.tif21166; and wherein the first and second polypeptides form a heterodimeric complex. 75. The method of any one of embodiments 37-74, wherein the cells are ARPE-19 cells, ARPE-19-SEAP-2-neo cells, RPE-J cells, hTERT RPE-1 cells, or any combination thereof. 76. The method of any one of embodiments 37-75, wherein the surfactant is TWEEN® 20 (Polysorbate 20). 77. The method of any one of embodiments 37-76, wherein the buffer is a HEPES buffer. 78. The method of any one of embodiments 37-77, wherein the sugar alcohol is mannitol. 79. The method of any one of embodiments 37-78, wherein the metal salt is barium chloride. 80. The method of any one of embodiments 37-79, further comprising washing the encapsulated cells in a buffer solution. 81. The method of any one of embodiments 37 to 80, further comprising storing the encapsulated cells in a storage buffer, such as DMEM / F12 cell culture medium. 82. The method of embodiment 81, wherein the preserved cells retain viability for at least 30 days. 83. The method of embodiment 81 or 82, wherein the storage buffer is substantially free of Plasmalyte buffer. 84. A population of encapsulated cells prepared according to the method of any one of embodiments 55 to 83. 85. A suspension of encapsulated cells, the suspension comprising a population of encapsulated cells according to any one of embodiments 1 to 28, wherein the encapsulated cells are encapsulated by a polymer hydrogel, and the suspension is a crosslinked solution comprising a sugar alcohol, a buffer, a metal salt, and a surfactant. 86. The suspension of embodiment 85, wherein the cells are ARPE-19 cells, ARPE-19-SEAP-2-neo cells, RPE-J cells, hTERT RPE-1 cells, or any combination thereof. 87. The suspension of embodiment 85 or 86, wherein the surfactant is TWEEN® 20 (Polysorbate 20). 88. The suspension according to any one of embodiments 85 to 87, wherein the buffer is a HEPES buffer. 89. A suspension according to any one of embodiments 85-88, wherein the sugar alcohol is mannitol. 90. The suspension of any one of embodiments 85-89, wherein the metal salt is barium chloride. 91. A suspension of encapsulated cells, wherein the suspension comprises a population of encapsulated cells according to any one of embodiments 1 to 28, and the encapsulated cells are encapsulated by a polymer hydrogel and a storage buffer, such as DMEM / F12 cell culture medium. 92. The suspension of embodiment 91, wherein the suspended encapsulated cells retain viability for at least 30 days. 93. The suspension according to any one of embodiments 85-92, wherein the suspension buffer is substantially free of Plasmalyte buffer. [Example]

[0123] H. Working Example The following examples are included to demonstrate preferred embodiments. It should be understood by those of skill in the art that the techniques disclosed in the examples below represent techniques discovered by the inventors to function well in the practice of the embodiments, and therefore can be considered to constitute preferred modes for that practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0124] The following examples are included to demonstrate preferred embodiments of the present disclosure. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0125] Example 1. Encapsulated cells are non-tumorigenic Transplantable cells can become tumorigenic, so it is important to demonstrate that encapsulated cells are unable to become tumorigenic or divide uncontrollably. The cells used, ARPE-19 cells, were chosen because they are non-tumorigenic, exhibit contact-inhibited growth characteristics in 2D culture, are amenable to genetic modification, and have been shown to be safe in previous clinical studies. To assess the fate of ARPE-19 cells after encapsulation, various assays were performed to analyze the viability and proliferation of encapsulated ARPE-19 cells within the capsules. These studies demonstrated that encapsulated ARPE-19 cells exhibited desirable characteristics, including viability in culture for at least 4 weeks, proliferation in 2D culture, and contact inhibition upon encapsulation, preventing further cell proliferation within the capsules. Briefly, ARPE-19 viability within encapsulated cells was assessed in vitro over time using a LIVE / DEAD viability / cytotoxicity kit. Live cells were rapidly distinguished from dead cells by simultaneous staining with green fluorescent staining, indicating intracellular esterase activity (live cells), and red fluorescent ethidium homodimer-1, indicating loss of plasma membrane integrity (dead cells). Cell proliferation within the capsules was measured using a Click-iT EdU imaging kit. A 1X working solution of EdU diluted in culture medium was added to each capsule. At 0, 24, 72 hours, and 7 days, the medium was removed, and the capsules were fixed in 4% PFA. The capsules were washed with 3% BSA and permeabilized with 0.5% Triton X-100. To stain the cells, 0.2 ml of Click-iT reaction cocktail was added to each capsule and incubated for 30 minutes. The capsules were then washed with 3% BSA. Finally, cell nuclei were stained with Hoechst 33343 (NucBlu). Capsules were imaged using an EVOS XL microscope at 4x magnification.

[0126] First, we assessed the viability of encapsulated ARPE-19 cells using live / dead staining by fluorescence microscopy. We observed no difference in the number of viable encapsulated ARPE-19 cells in vitro over a 28-day period. Next, we investigated the proliferation status of ARPE-19 cells. To compare the proliferation of encapsulated ARPE cells with that of HEK cells, cells known to exhibit continuous in vitro proliferation within alginate hydrogels, we performed an in vitro evaluation of the capsules. ARPE cells (or HEK cells as a control) were encapsulated in 1.5 mm alginate capsules and imaged in vitro over time for up to 7 days. At separate time points, capsules were randomly removed from the main population and qualitatively assayed using DAPI staining to visualize encapsulated cells and EdU (GFP) as a proliferation marker. Images were taken at 4x magnification. Only HEK cells stained with the GFP marker, confirming that ARPE-19 cells did not proliferate after encapsulation within alginate hydrogels (data not shown). This observation was confirmed using PCR analysis. In conclusion, encapsulated ARPE-19 cells remained viable in vitro for at least 28 days. Although the encapsulated ARPE-19 cell line was able to grow in 2D culture, it exhibited contact inhibition upon encapsulation and therefore did not continue to proliferate inside the capsule. This characteristic is important for adjusting the cytokine secretion dose per capsule after administration.

[0127] Example 2. Preparation of encapsulated cells Polyclonal ARPE-19 cells were expanded and transfected at a 5:1 (transposase:transposon) ratio using a Lipofectamine protocol to generate cells expressing human native IL-12. Transfected cells were cultured and plated at 0.5 cells / well for single cell growth; however, concentrations may vary if polyclonal samples of cells are used. IL-12 production of selected clones was approximately 3.8 picograms per cell per day (PCD). Clones were grown in cell flasks / stacks for up to two weeks before being harvested into cell pellets and suspended in alginate (SLG20) for encapsulation. The encapsulation process involves loading two syringes: one with SLG20 and one with the cell pellet (10,500,000 cells / mL) suspended in alginate (SLG20). Using a power source (electric current), a syringe was provided with a coaxial needle, allowing droplets to fall into a crosslinking bath containing mannitol, barium chloride, HEPES buffer, and Tween 20, where capsules formed. After 5 minutes in the bath, the capsules were retrieved from the bath and washed eight times (2 minutes per wash) with a 1:25 ratio of capsules to HEPES buffer to aid in the removal of loosely bound barium. The encapsulated cells were stored in DMEM / F12 cell culture medium at ambient temperature in containers such as conical tubes, biotainer bottles, or other sterile containers.

[0128] Example 3. RPE-mIL12 capsules eradicate pancreatic cancer in less than 10 days of treatment PAN02-Fluc cells (5 × 10 6) was injected into the IP space of B6 albino mice to establish IP PAN02 tumors. Seven days after injection, mice were randomly divided into groups of eight and treated with 20 RPE capsules (approximately 30k cells / cap), sham surgery, or 20 RPE-mIL12 capsules (approximately 30k cells / cap, approximately 400ng mIL-12 / day). Twenty-five days after treatment, mice were sacrificed, and the liver, kidney, abdominal wall, spleen, and tumors were examined ex vivo using IVIS imaging. Analysis showed no positive IVIS signals in RPE-mIL12-treated mice at 10 or 25 days after treatment.

[0129] Example 4. RPE-mIL12 capsules show reduced fibrosis over time Pan02-Fluc cells (5 × 10) suspended in HBSS were injected into the IP space of albino B6 mice (mixed sex). Six days after IP injection, mice were imaged by IVIS and stratified into four groups of 5–6 mice. Seven days after IP injection, mice were treated with either (1) untreated, (2) 200 RPE capsules, (3) 200 RPE-mIL2 capsules (approximately 7000 ng mIL2 / day), or (4) 10 RPE-mIL12 capsules (approximately 200 ng mIL12 / day). All capsules were administered by surgical implantation. Five days after capsule implantation, mice were imaged by IVIS to visualize the extent of IP tumor burden. Seven days after capsule implantation, mice were sacrificed, and IP cells were collected using a 10 mL PBS wash. The cell suspension was filtered using a 70 μm filter and spun down using a centrifuge (2000 rpm, 4 min). The supernatant was discarded, and 3 mL of RBC lysis buffer was added for 10 min at room temperature. 10 mL of PBS + 2% FBS was added to each tube. The cells were spun down (5000 rpm, 10 min). The supernatant was discarded, and the cell pellet was resuspended in 2 mL of complete medium (DME / F12 + 10% FBS + 1% AntiAnti). Individual samples from each group were pooled. The cells were spun down (2000 rpm, 10 min). The supernatant was discarded, and the cells were resuspended in FC block on ice for 10 min, then spun down (2000 rpm, 10 min). The cells were stained with CD45 antibody on ice for 30 min, then spun down (2000 rpm, 10 min). The supernatant was discarded, and the cells were resuspended in 4 mL of complete medium. Cells were stained with ReadiDrop propidium iodide and sorted (PI-CD45+). Sorted cells were processed, libraries prepared, and sequenced at the BCM core. Visual analysis and analysis of CSF1R and FN1 macrophage markers indicated that RPE-mIL12 capsules showed minimal fibrotic hyperplasia at 7 days posttreatment, whereas RPE and RPE-mIL2 capsules showed extensive hyperplasia. Thus, the expression of IL-12 from the capsules, the only difference between the capsules, appears to inhibit fibrotic hyperplasia. This was a surprising result, as it was achieved without modifying the alginate with antifibrotic compounds or modifications.

[0130] Example 5. Treatment with RPE-mIL12 capsules results in increased expression of CD8α, PRF1, and IFN-γ Pan02-Fluc cells (5 × 10) suspended in HBSS were injected into the IP space of albino B6 mice (mixed sex). Six days after IP injection, mice were imaged by IVIS and stratified into four groups of 5–6 mice. Seven days after IP injection, mice were treated with either (1) untreated, (2) 200 RPE capsules, (3) 200 RPE-mIL2 capsules (approximately 7000 ng mIL2 / day), or (4) 10 RPE-mIL12 capsules (approximately 200 ng mIL12 / day). All capsules were administered by surgical implantation. Five days after capsule implantation, mice were imaged by IVIS to visualize the extent of IP tumor burden. Seven days after capsule implantation, mice were sacrificed, and IP cells were collected using a 10 mL PBS wash. The cell suspension was filtered using a 70 μm filter and spun down using a centrifuge (2000 rpm, 4 min). The supernatant was discarded, and 3 mL of RBC lysis buffer was added for 10 min at room temperature. 10 mL of PBS + 2% FBS was added to each tube. The cells were spun down (5000 rpm, 10 min). The supernatant was discarded, and the cell pellet was resuspended in 2 mL of complete medium (DME / F12 + 10% FBS + 1% AntiAnti). Individual samples from each group were pooled. The cells were spun down (2000 rpm, 10 min). The supernatant was discarded, and the cells were resuspended in FC block on ice for 10 min, then spun down (2000 rpm, 10 min). The cells were stained with CD45 antibody on ice for 30 min, then spun down (2000 rpm, 10 min). The supernatant was discarded, and the cells were resuspended in 4 mL of complete medium. Cells were stained with ReadiDrop propidium iodide and sorted (PI-CD45+). Sorted cells were processed, libraries prepared, and sequenced at the BCM core. Data showed that mice treated with RPE-mIL12 exhibited increased expression of CD8α, PRF1, and IFN-γ compared with untreated or RPE-treated mice. Data further showed that topical high-dose RPE-mIL2 did not increase FoxP3 expression, a marker of Tregs, whereas RPE-mIL12 treatment did increase IFN-γ expression.

[0131] Example 6. Subcutaneous or intraperitoneal administration of RPE-mIL12 delays subcutaneous tumor growth in the B16F10 melanoma model B16F10 (5 × 10) cells suspended in HBSS 5 ) cells were injected subcutaneously into the right flank of B6 mice (mixed sex). Six days after IP injection, tumors were measured using digital calipers and stratified into four groups of 3–4 mice. Seven days after IP injection, mice were treated with either (1) sham surgery, (2) 10 RPE-mIL12 capsules implanted subcutaneously, (3) 10 RPE-mIL12 capsules implanted intraperitoneally, or (4) 10 RPE-mIL12 capsules implanted intraperitoneally plus five frozen-thawed B16F10 capsules (antigen). All capsules were administered by surgical implantation. To prepare the antigen, B16F10 melanoma cells were encapsulated in alginate capsules and subjected to three rounds of freeze / thaw cycles at -80°C to disrupt the cells.

[0132] At the time of administration, the antigen capsules were thawed and administered intraperitoneally along with 10 RPE-mIL12 capsules to mice bearing subcutaneous B16F10 tumors. After capsule administration, tumors were measured every 2–4 days until they reached 15 mm in either direction. Treatment with RPE-mIL12 capsules or RPE-mIL12 capsules co-administered with antigen increased survival and delayed tumor growth, as shown in Figure 1.

[0133] Example 7. Co-administration of RPE-mIL2 and RPE-mIL12 delays subcutaneous tumor growth in the B16F10 melanoma model B16F10 (5 × 10) cells suspended in HBSS 5) cells were injected subcutaneously into the right flank of male B6 mice. Six days after subcutaneous injection, tumors were measured using digital calipers and stratified into three groups. Seven days after subcutaneous injection, mice were treated with either RPE-mIL2 capsules, RPE-mIL2 capsules co-administered with RPE-mIL12 capsules, or RPE-mIL12 capsules. Treatment with RPE-mIL2 capsules, RPE-mIL2 capsules co-administered with RPE-mIL12 capsules, or RPE-mIL12 capsules delayed tumor growth, as shown in Figure 2.

[0134] Example 8. RPE-mIL12 capsules caused reduction of KPC pancreatic tumors without recurrence in less than one week of treatment KPC-Fluc cells (1×10 6 ) was injected into the IP space of B6 mice to establish IP KPC tumors. Seven days after injection, 15 mice were stratified into three groups and treated with three RPE-mIL12 capsules (approximately 30k cells / cap, approximately 60ng mIL-12 / day), ten RPE-mIL12 capsules (approximately 30k cells / cap, approximately 200ng mIL-12 / day), or fifteen RPE-mIL12 capsules (approximately 30k cells / cap, approximately 300ng mIL-12 / day). IVIS analysis at days 6, 14, and 21 after treatment showed a reduction in KPC pancreatic tumors. Mice treated with 3 RPE-mIL12 capsules (approximately 30k cells / cap, approximately 60ng mIL-12 / day) and 10 RPE-mIL12 capsules (approximately 30k cells / cap, approximately 200ng mIL-12 / day) were further observed for up to 180 days and showed no tumor recurrence. Analysis of internal organs 180 days after treatment showed no increase in vascularization or discoloration, as observed in untreated KPC tumor control mice.

[0135] Example 9. Cytokine Factors Reduce Pancreatic Cancer Tumor Burden in Less Than 7 Days of Treatment PAN02-Fluc cells (5 × 10 6) was injected into the IP space of B6 albino mice to establish IP PAN02 Fluc tumors. Seven days after injection, mice were stratified into groups of 5–6 and treated with either untreated, 200 RPE capsules (approximately 30k cells / cap), 200 RPE-mIL2 capsules (approximately 30k cells / cap, approximately 7000 ng mIL-2 / day), or 10 RPE-mIL12 capsules (approximately 30k cells / cap, approximately 200 ng mIL-12 / day). Seven days after treatment, mice were sacrificed, and IP fluids were processed for single-cell RNA sequencing. IVIS analysis five days after treatment showed a reduction in KPC pancreatic tumors in mice treated with RPE-mIL2 or RPE-mIL12 capsules. Analysis of immune cell populations by RNAseq showed a relative decrease in the number of B cells and dendritic cells, and an increase in monocytes or NK / T cells, in mice treated with RPE-mIL2 or RPE-mIL12 capsules compared with untreated mice.

[0136] Example 10. Cytokine Factors Reduce Pancreatic Cancer Tumor Burden with 5 Days of Treatment Mice (n = 5–6 per group) were treated with Pan02-fluc cells (5 × 10 6) were injected. As shown in the upper panel of Figure 3A, IVIS imaging demonstrated stable levels of total flux 6 days after injection. Five days after treatment with control (untreated), RPE, RPE-mIL2, or RPE-mIL12, luminescence imaging demonstrated a decrease in tumor burden, as shown in the lower panel of Figure 3A. The scale bar minimum and maximum values ​​for mean radiance (photons / sec / cm² / ser) were 5 x 10 and 5 x 10, respectively. Next, individual cells separated by treatment (untreated, RPE, RPE-mIL2, and RPE-mIL12) were collected from the IP space 1 week after treatment, and CD8α gene expression was assessed. A t-SNE plot embedding of CD8α gene expression shows increased expression after RPE-mIL2 or RPE-mIL12 treatment (Figure 3B, upper panel; heatmap represents log2-scaled expression of CD8α in cells). A t-SNE plot embedding of IFNg gene expression in individual cells separated by treatment (untreated, RPE, RPE-mIL2, RPE-mIL12) collected from the IP space 1 week after treatment showed increased IFNg expression after RPE-mIL2 or RPE-mIL12 treatment (Figure 3B, lower panel; heatmap represents log2-scale expression of IFNg in cells).

[0137] Example 11. RPE-mIL12 capsules resulted in a long-lasting reduction in pancreatic cancer tumor burden KPC cells (1×10 6 ) into the IP space of mice to establish IP KPC tumors. Seven days after injection, mice were stratified into three groups and treated with three RPE-mIL12 capsules, ten RPE-mIL12 capsules, or fifteen RPE-mIL12 capsules. IVIS analysis at days 6, 14, 21, 40, and 120 after treatment demonstrated KPC pancreatic tumor reduction (Figure 4A). Furthermore, a gradual increase in body weight was observed in animals treated with three or ten RPE-mIL12 capsules up to day 180 after treatment (Figure 4B). Macroscopic and hematoxylin and eosin imaging of the pancreas after 8 weeks of treatment with or without RPE-mIL12 demonstrated that the pancreas of animals treated with RPE-mIL12 capsules exhibited preserved cellular morphology and architecture (Figure 4C).

[0138] Example 12. RPE-mIL12 capsules reduced pancreatic cancer tumor burden in the liver, kidney, abdominal wall, and spleen As provided in the examples above, mice were injected with Pan02-fluc cells (5×10 6 ) were injected. Seven days after injection of pancreatic cancer cells, mice were subjected to sham treatment, RPE treatment, or RPE-mIL12 treatment. 25 days after treatment, the liver, kidney, abdominal wall, and spleen were extracted from the mice. Luminescence imaging revealed no pancreatic cancer tumors in the liver, kidney, abdominal wall, or spleen of animals treated with RPE-mIL12 capsules (Figure 5).

[0139] Example 13. Administration of RPE-mIL12 reduced tumor growth and preserved splenic cellularity in a melanoma model Melanoma cells (5 × 10 5 ) cells were injected into the IP space of mice. Seven days after melanoma cell injection, mice were either untreated (control) or treated with RPE-mIL2 capsules. Treatment with RPE-mIL12 capsules resulted in reduced tumor growth, as shown in Figure 6A (n = 3-4 per group). Next, spleens were extracted and dissociated into single-cell suspensions for automated cell counting. Treatment with RPE-mIL12 capsules resulted in preserved splenic cellularity, as shown in Figure 6B.

[0140] Example 14. Administration of RPE-mIL12 increased survival of animals bearing melanoma tumors Melanoma cells (5 × 10 5 ) cells were injected into the IP space of mice. Seven days after melanoma cell injection, mice (n = 4-6 mice per group) were treated with RPE capsules or RPE-mIL2 capsules. Treatment with RPE-mIL12 capsules resulted in an increased probability of survival, as shown in Figure 7.

[0141] Example 15. Administration of RPE-mIL12 increased survival of pancreatic tumor-bearing animals Pan02-fluc cells (5 × 10 6 ) cells were injected into the IP space of mice. Seven days after melanoma cell injection, mice (n = 5-8 mice per group) underwent sham surgery (control), treatment with RPE capsules, or treatment with RPE-mIL2 capsules. Treatment with RPE-mIL12 capsules resulted in an increased probability of survival, as shown in Figure 8, while control or RPE-treated animals showed a lower probability of survival.

[0142] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be made in the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined by the appended claims.

Claims

1. A population of encapsulated cells comprising a plurality of oligonucleotide molecules encoding an IL-12 polypeptide.

2. 2. The population of encapsulated cells of claim 1, wherein the IL-12 polypeptide is a native human IL-12 polypeptide, a recombinant IL-12 polypeptide, or an IL-12 mutein polypeptide.

3. the native human IL-12 polypeptide is a heterodimeric complex comprising a native human IL-12(p35) polypeptide and a native human IL-12(p40) polypeptide; and / or the recombinant IL-12 polypeptide is a heterodimeric complex comprising a recombinant IL-12(p35) polypeptide and a recombinant IL-12(p40) polypeptide; and / or the IL-12 mutein polypeptide is a heterodimeric complex comprising an IL-12 (p35) mutein polypeptide and an IL-12 (p40) mutein polypeptide; A population of encapsulated cells according to claim 2.

4. The population of encapsulated cells described in claim 3, wherein the IL-12 (p40) mutein polypeptide comprises a mutation selected from N220L, N222L, or N222Q compared to the amino acid sequence of SEQ ID NO:

4.

5. 2. The population of encapsulated cells of claim 1, wherein the plurality of oligonucleotide molecules encodes a native human IL-12 (p35) polypeptide and a native human IL-12 (p40) polypeptide.

6. the oligonucleotide encoding the native human IL-12 (p35) polypeptide comprises the sequence of SEQ ID NO: 1 and the oligonucleotide encoding the native human IL-12 (p40) polypeptide comprises the sequence of SEQ ID NO: 2; and / or the expressed IL-12(p35) polypeptide and the expressed IL-12(p40) polypeptide form a heterodimeric complex, and / or the IL-12(p35) polypeptide and the IL-12(p40) polypeptide are expressed in a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (IL-12(p35):IL-12(p40)); A population of encapsulated cells according to claim 5.

7. 10. The population of encapsulated cells of claim 1, wherein the cells produce a native human IL-12 heterodimeric protein, a recombinant IL-12 heterodimeric protein, or an IL-12 mutein heterodimeric protein.

8. 8. The population of encapsulated cells of claim 7, wherein the native human IL-12 protein expressed by the cells comprises a polypeptide comprising a first polypeptide comprising the sequence of SEQ ID NO:3 and a second polypeptide comprising the sequence of SEQ ID NO:4, wherein the first and second polypeptides form a heterodimeric complex.

9. the population of cells produces about 10 to about 50, about 10 to about 30, about 10 to about 20, or about 20 nanograms / cell / day of native human IL-12; and / or the cells are ARPE-19 cells, ARPE-19-SEAP-2-neo cells, RPE-J cells, hTERT RPE-1 cells, or any combination thereof; and / or the cells are encapsulated in a polymer hydrogel; A population of encapsulated cells according to claim 1.

10. The population of encapsulated cells of claim 9 , wherein the polymer hydrogel comprises chitosan, cellulose, hyaluronic acid, or alginate.

11. The population of encapsulated cells of claim 10 , wherein the alginate comprises SLG20.

12. the cells remain viable for at least 5, 10, 15, 20, 40, 120, or 180 days; and / or the encapsulated cells do not proliferate, and / or a capsule encapsulating said population of cells does not cause fibrous hyperplasia or causes partial fibrous hyperplasia after implantation into a subject; A population of encapsulated cells according to claim 1.

13. A pharmaceutical composition comprising a population of encapsulated cells according to any one of claims 1 to 12.

14. and / or a population of encapsulated cells comprising an oligonucleotide molecule encoding native human IL-2. the cells produce recombinant native human IL-2 protein, and / or the recombinant native human IL-2 protein expressed by said cells comprises the amino acid sequence of SEQ ID NO: 11; and / or the population of cells produces about 1 to about 10, about 1 to about 5, or about 2 to about 4 PCD (picograms per cell per day) of native human IL-2; The pharmaceutical composition of claim 13.

15. the oligonucleotide encoding native human IL-2 comprises the sequence of SEQ ID NO: 6, and / or the oligonucleotide encoding native human IL-2 comprises a codon-optimized sequence; The pharmaceutical composition of claim 14.

16. 16. The pharmaceutical composition of claim 15, wherein the codon-optimized oligonucleotide encoding native human IL-2 comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

7.

17. 14. The pharmaceutical composition of claim 13 for use in a method for treating a tumor in a subject, the method comprising implanting the pharmaceutical composition into the intraperitoneal space of the subject to treat cancer; or 14. The pharmaceutical composition of claim 13, for use in a method for reducing tumor burden in a subject, the method comprising implanting the pharmaceutical composition into the intraperitoneal space of the subject to treat cancer.

18. the tumor is a pancreatic tumor or a melanoma tumor, and / or The subject is administered a dose of about 0.01 μg / kg / day to about 20 μg / kg / day, about 0.1 μg / kg / day to about 20 μg / kg / day, about 1 μg / kg / day to about 20 μg / kg / day, about 2 μg / kg / day to about 20 μg / kg / day, about 5 μg / kg / day to about 20 μg / kg / day, about 7.5 to about 20 μg / kg / day, about 9 μg / kg / day to about 20 μg / kg / day, about 10 μg / kg / day to about 20 μg / kg / day, or about 11 μg / kg / day. / day to about 20 μg / kg / day, about 12 μg / kg / day to about 20 μg / kg / day, about 13 μg / kg / day to about 20 μg / kg / day, about 14 μg / kg / day to about 15 μg / kg / day, about 15 μg / kg / day to About 20μg / kg / day, about 10μg / kg / day to about 15μg / kg / day, about 11μg / kg / day to about 15μg / kg / day, about 12μg / kg / day to about 15μg / kg / day, about 13μg / kg / day to about 15μ g / kg / day, about 14 μg / kg / day to about 15 μg / kg / day, about 16 μg / kg / day to about 20 μg / kg / day, about 17 μg / kg / day to about 20 μg / kg / day, about 18 μg / kg / day to about 20 μg / k g / day, about 0.01μg / kg / day, about 0.1μg / kg / day, about 1μg / kg / day, about 2μg / kg / day, about 3μg / kg / day, about 4μg / kg / day, about 5μg / kg / day, about 6μg / kg / day, about 7μ 13. The encapsulated cells of any one of claims 1 to 12 are administered at a dose of about 10 μg / kg / day, about 8 μg / kg / day, about 9 μg / kg / day, about 10 μg / kg / day, about 11 μg / kg / day, about 12 μg / kg / day, about 13 μg / kg / day, about 14 μg / kg / day, about 15 μg / kg / day, about 16 μg / kg / day, about 17 μg / kg / day, about 18 μg / kg / day, about 19 μg / kg / day, or about 20 μg / kg / day.

18. The pharmaceutical composition of claim 17.

19. The pharmaceutical composition of claim 13 for use in a method for treating a tumor or reducing tumor burden in a subject by generating memory immunity, the method comprising transplanting or administering the pharmaceutical composition to the subject.

20. 20. The pharmaceutical composition of claim 19, wherein the tumor is a pancreatic tumor or a melanoma tumor.

21. The pharmaceutical composition of claim 13 for use in a method for selectively activating CD8-positive effector T cells in a subject, the method comprising transplanting or administering the pharmaceutical composition to the subject.

22. 22. The pharmaceutical composition of claim 21, wherein the effector T cells are selectively activated and expanded compared to Tregs (CD4+CD25+FoxP3+).

23. 23. The pharmaceutical composition of claim 22, wherein the selectively activated effector T cells secrete IFN-γ.

24. The pharmaceutical composition of claim 13 for use in a method for increasing interferon gamma (IFN-γ) in a subject, the method comprising implanting or administering the pharmaceutical composition to the subject.

25. 18. The pharmaceutical composition of claim 17, wherein the capsule containing the encapsulated cells implanted or administered to the subject does not undergo fibrosis or undergoes partial fibrosis after implantation or administration.

26. dispensing a first composition comprising a polymer hydrogel and a second composition comprising cells to be encapsulated suspended in the polymer hydrogel through a coaxial needle to dropwise drop into a crosslinking solution to form the encapsulated cells, the crosslinking solution comprising a sugar alcohol, a buffer, a metal salt, and a surfactant; A method for preparing the encapsulated cells of any one of claims 1 to 12, comprising:

27. the cells are ARPE-19 cells, ARPE-19-SEAP-2-neo cells, RPE-J cells, hTERT RPE-1 cells, or any combination thereof; and / or the surfactant is TWEEN® 20 (Polysorbate 20), and / or the buffer is a HEPES buffer, and / or the sugar alcohol is mannitol, and / or the metal salt is barium chloride; 27. The method of claim 26.

28. 28. The method of claim 27, further comprising storing the encapsulated cells in a storage buffer, such as DMEM / F12 cell culture medium.

29. 29. The method of claim 28, wherein the preserved cells retain viability for at least 30 days.

30. 27. A population of encapsulated cells prepared according to the method of claim 26.

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