Compositions and methods for promoting cardiac and pulmonary repair

Abstract

The present disclosure relates to implantable constructs designed to deliver therapeutic agents to a subject to induce tissue regeneration in damaged or diseased lung and heart tissue. In certain aspects, the constructs are designed to degrade over time or upon specific signaling, thereby providing control over the length of time that the therapeutic agent is delivered to the subject. TIFF2025526484000009.tif127128

Description

[Technical Field]

[0001] STATEMENT REGARDING U.S. GOVERNMENT-SPONSORED RESEARCH This invention was made with government support under Grant No. R01DK120459 awarded by the National Institutes of Health and Grant No. 1842494 awarded by the National Science Foundation. The government has certain rights in this invention.

[0002] Priority claim This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 394,481, filed August 2, 2022, the entire contents of which are incorporated herein by reference.

[0003] I. Field The present disclosure relates to the fields of biology, pharmacology, biotechnology, and medical devices. More particularly, it relates to the development and use of implantable constructs designed to deliver therapeutic agents to a subject and protect the subject from unwanted immune responses. In particular, the implantable constructs are designed to deliver cytokines and other agents to induce tissue repair after long-term cardiac injury. [Background technology]

[0004] II. Related Technologies Advances in biomedical research have provided methods for localized and targeted therapy to treat diseases. However, in many cases, the percentage of patients who respond to these approaches remains low (Park et al., Sci. Transl. Med. 10(433)2018). One approach involves the use of implantable devices to deliver therapeutic agents. A fundamental barrier to successful device-based therapy is the lack of biocompatible implantable devices. Therefore, there is a significant medical need to develop biomaterials that overcome major challenges in this field. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Park et al.,Sci.Transl.Med.10(433)2018 Summary of the Invention

[0006] The present disclosure provides, at least in part, a method of treating tissue damage and / or inducing tissue regeneration in a subject, the method comprising: providing encapsulated modified cells expressing a cytokine; and administering the encapsulated cells to the subject. The modified cells can be any type of cell, e.g., Chinese hamster ovary (CHO) cells, retinal pigment epithelial (ARPE-19) cells, human mammary epithelial (MCF-10a and MCF-7) cells, human embryonic kidney (HEK) cells, mesenchymal stem cells (MSCs), human umbilical vein endothelial cells (HUVECs), NIH / 3T3 cells, BJ fibroblasts, or human renal mixed epithelial cells (HRECs), where the cells are modified for regulated expression of the cytokine. In some embodiments, the cytokine can be an anti-inflammatory or pro-inflammatory cytokine. In some embodiments, the cytokine is IL-10. The material encapsulating the modified cells can be degradable. The material encapsulating the modified cells may be a natural or synthetic polymer. For example, the material may be a polysaccharide, e.g., alginate. The material encapsulating the modified cells may further comprise a compound, e.g., a compound comprising a triazole moiety. The tissue may be cardiac or pulmonary tissue, such as cardiac tissue damaged by ischemia due to coronary heart disease and / or myocardial infarction. The pulmonary tissue of the method may be damaged by infection, e.g., by a viral infection. Administration may include administration directly to the damaged tissue.

[0007] The use of the terms "a" or "an," when used in connection with the term "comprising" in the claims and / or specification, can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more." The term "about" means ±5% of a constant.

[0008] It is contemplated that any method or composition described herein can be practiced in conjunction with 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 specific embodiments of the present disclosure, are given by way of illustration only, since 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]

[0009] 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.

[0010] [Figure 1] Schematic representation of a platform for developing engineered cells to produce various anti-inflammatory cytokines. These engineered cells are then encapsulated in alginate hydrogels, referred to as "cytokine factories." [Figure 2] Schematic representation of the IL-10 and IL-1 signaling pathways. [Figure 3]Anti-inflammatory cytokine factories are encapsulated in biocompatible polymers to maintain cell viability in vivo. Retinal pigment epithelial cells engineered to produce human IL-10 were encapsulated in these hydrogels and evaluated for sustained viability and function. Live / dead staining of encapsulated RPE-hIL10 cells demonstrates no significant loss of viability after encapsulation. To assess changes in cytokine production after encapsulation, cells were 2D plated or encapsulated and incubated for 24 hours. Supernatants were collected and analyzed for hIL10 production via ELISA. Results demonstrate no significant loss of cytokine production after encapsulation. [Figure 4] One major advancement of this technology over recombinant cytokine administration is its ability to localize therapy to target tissues. To demonstrate the ability of RPE-IL10 capsules to maintain local viability for extended periods, encapsulated cells modified with a firefly luciferase reporter were implanted into the pericardial sac of Sprague-Dawley rats. Cell localization over time was demonstrated using an in vivo imaging system (IVIS). Results show that cells maintain a luminescent signal 3 weeks after implantation and are localized to the left pleural cavity (n = 3, 0.5 mL capsules / n). To demonstrate the ability of our cytokine factory to achieve high local concentrations in the absence of significant effects on systemic cytokine concentrations, we used ELISA to quantify hIL10 in pleural fluid compared to plasma. Results showed that pleural fluid IL-10 was significantly greater than that in plasma (approximately 30,000-fold), highlighting the safety profile of this platform. [Figure 5]Figures 5A-C. The inventors have previously demonstrated the therapeutic mechanism of IL-10 treatment. In general, IL-10 capsules administered after acute MI increased M2-like macrophages and regulatory T cells. CyTOF immunomapping of nine rat hearts using 40 protein markers identified 27,874 immune cells. Eight immunophenotypes of CD45+ immune cells were identified based on representative phenotypic surface markers. Compared to wild-type rat hearts, MI and IL-10 treatment induced dramatic changes in macrophage and CD4 T cell populations. Furthermore, IL-10 treatment induced a significant increase in CD163(+) macrophages (M2-like macrophages) and regulatory T cells. In contrast, CD163(-)CD206(-) macrophages (M1-like macrophages) were significantly decreased after IL-10 treatment. [Figure 6] Functional CyTOF demonstrated cytokine production in key cell types after IL-10 capsule administration: Ki67-expressing activated Tregs released IL-10 and IL-17 to induce macrophage differentiation, as measured after 12 hours of Golgistop protein transport inhibitor treatment. [Figure 7] Figure 7A-B. IL-10 treatment caused a significant increase in IL-10 in infarcted hearts compared to untreated infarcts. Higher intracellular IL-10 was measured in myofibroblasts, fibroblasts, macrophages, and regulatory T cells. MMI = mean mass intensity. [Figure 8]The inventors have previously demonstrated the potential for therapeutic applications of this platform in the pleural cavity. Specifically, encapsulation of mesenchymal stem cells demonstrated significant improvement in myocardial function after acute MI. Prior to efficacy experiments, production of various MSC-specific paracrine factors was assessed via ELISA. As previously described, cells were 2D plated and factor production was compared with encapsulated MSCs. Results demonstrated no significant reduction in paracrine factor production after encapsulation. After verifying sustained MSC factor production after encapsulation, MSCs were transplanted into the pericardial cavity via left anterior descending artery ligation after acute MI. The encapsulated MSCs were compared with MSCs directly injected into the pericardium and a control (blank) capsule group (n = 5). Results showed a significant improvement in left ventricular ejection fraction (LVEF) after MSC injection (*; p < 0.05) and MSC capsules (**; p < 0.05) compared with those after infarction. Furthermore, the percent change in EF for individual subjects showed the greatest improvement in the MSC capsule group compared to the MSC injection group (41% vs. 16%; p<0.05). Finally, analysis of Masson's Trichrome-stained myocardial sections 28 days after MI and treatment showed a significant reduction in fibrotic tissue after treatment with encapsulated MSCs. *p<0.05, **p<0.01 [Figure 9] CD4+ subsets regulate fibroblast ECM profile and angiogenic chemokine secretion. (Left) Fibroblasts (P3) treated with conditioned medium from activated lymphocytes (n=3) show upregulation of ECM turnover genes (Collagen3a1, MMP8, and MMP9) by CD4+ (non-Treg or CD4+CD25+ Treg) cells. (Right) SDF1 levels in adult fibroblasts (AFB) in the presence or absence of lenti-IL10 transduction (4 days post-transduction). *p<0.05. [Figure 10]RPE-IL10 improves LV function after MI. LV EF before and after coronary vessel ligation and treatment with 20 capsules of naive RPE or RPE-IL10 28 days later (naive RPE vs. RPE-IL10; n = 3 / group; p < 0.05). Representative (Masson's Trichrome) sections of myocardium 28 days after infarction. Naive RPE capsules (left) and RPE-IL10 (right) show a significant reduction in the area of fibrosis (13 ± 3% vs. 23 ± 4%, p < 0.05). *p < 0.05. [Figure 11] Figures 11A-E. Development and validation of encapsulated anti-inflammatory cytokine factories in vitro and in vivo. (Figures 11A-B) Encapsulated cells engineered to produce rat interleukin-1 receptor antagonist (RIL1Ra) and rat interleukin-10 (RIL10) and human interleukin-1 receptor antagonist (HIL1Ra) and human interleukin-10 (HIL10) demonstrate sustained, high cytokine production. (Figure 11C) Viability assessment of encapsulated cells via live / dead staining demonstrates greater than 90% cell viability. (Figures 11D-E) Transplantation of encapsulated rat IL1Ra and IL10 cells into the pleural cavity demonstrates elevated local cytokine concentrations, along with up to a 100-fold reduction in systemic circulating concentrations at days 1, 3, 7, and 28 post-transplant. (FIG. 11F) Dark-field microscopy images of explanted capsules demonstrate the ability of capsules producing anti-inflammatory cytokines to alleviate fibrosis for a duration of up to 28 days. [Figure 12] Figures 12A-B. Lung histological scoring in the presence or absence of therapeutic capsule treatment. (Figure 12A) Histological scores from n=5 rats on days 1, 7, 14, and 28 after treatment with LPS alone or LPS plus therapeutic IL-10 and IL1Ra capsules. (Figure 12B) Representative histological images of lung sections illustrating the infiltration of different immune cells over time and improved lung tissue in treatment groups by day 14. [Figure 13]Figures 13A-E. (Figure 13A) Fully transfected RPE cells transiently express mCherry and increase in number throughout antibiotic selection. Before selection (left) and 5 days after selection (right). (Figure 13B) Cells treated with DOX for 48 hours produce significantly higher concentrations of anti-inflammatory cytokines compared to untreated cells (n=3). (Figure 13C) Exponential relationship between DOX exposure time and anti-inflammatory cytokine X production (n=3). (Figure 13D) Representative images of capsules synthesized using various potentials show that increasing applied voltage results in a decrease in capsule diameter. (Figure 13E) Quantification of various capsule diameters shows good reproducibility within groups (n=10). DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description The present disclosure features implantable constructs for delivering cells expressing one or more therapeutic agents to a subject in a sustained manner, and related methods of use. The implantable constructs disclosed herein may be formulated into different shapes (e.g., spheres, rods, tubes) and prepared using a variety of materials. Each of these embodiments is described in more detail below.

[0012] A.Definition An "antigenic agent," as used herein, is a substance that induces, activates, or elicits, for example, an immune response in a subject.

[0013] "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 modified (e.g., genetically modified) or unmodified (e.g., genetically unmodified).

[0014] "Degradable," as used herein, refers to a structure that, upon modulation, e.g., cleavage, reduces the ability of the implantable construct to prevent contact between host immune effectors and the engineered cells. For example, a degradable entity can include a site that is cleavable by an enzyme, e.g., an endogenous host enzyme, or an administered enzyme. Typically, the degradable entity mediates a physical property, e.g., thickness, degree of cross-linking, or permeability, of the encapsulated engineered cells that prevents passage of host agents (e.g., host immune components, e.g., host immune cells).

[0015] "Prevention," "prevent," and "preventing," as used herein, refer to the administration or application of a treatment prior to the onset of a disease or condition, e.g., treatment comprising administering an implantable construct (e.g., as described herein) comprising a therapeutic agent (e.g., a therapeutic agent described herein), to eliminate physical signs of said 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 recognized. In some embodiments, treatment includes prevention; in other embodiments, treatment does not include prevention.

[0016] "Subject," as used herein, 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 important mammals such as cows, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially important birds such as chickens, ducks, geese, and / or turkeys). In certain embodiments, the animal is a mammal. The animal is male or female at any stage of development (e.g., male or female at any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults). The non-human animal may be a transgenic animal.

[0017] "Treatment," "treat," and "treating," as used herein, refer to ameliorating, alleviating, delaying the onset of, 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 agent (e.g., a therapeutic agent described herein). In certain embodiments, treating includes reducing, alleviating, relieving, delaying the onset, or inhibiting the progression of a symptom of a disease, disorder, or condition. In certain embodiments, treating includes reducing, alleviating, relieving, delaying the onset, or inhibiting the progression of a symptom of a disease or condition. In certain embodiments, treating includes reducing, alleviating, 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 be present or observed. In other embodiments, treatment 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., based on a history of the condition and / or based on 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, treatment does not include prevention.

[0018] B. Implantable Constructs The implantable constructs described herein include materials that reduce or inhibit a response (e.g., an immunomodulatory response) induced by engineered cells disposed therein. For example, the implantable constructs may include materials that shield the engineered cells from exposure to the surrounding environment, e.g., host tissues, host cells, or host cell products. In certain embodiments, the implantable constructs minimize the effect of a host response (e.g., an immune response) induced by engineered cells disposed therein, e.g., compared to similar cells not disposed within the implantable construct.

[0019] The implanted construct can take any shape: the surface can be flat or curved, and can take on a variety of more complex forms such as spheres, tubes (e.g., the inside or outside of a tube), beads, rods, wires, or even more complex three-dimensional structures such as medical devices.

[0020] The implantable construct may comprise a permeable, semi-permeable, or impermeable material, for example, to control the flow of solutions into and out of the implantable construct and / or to assume the shape or size of its surroundings. For example, the material may be permeable or semi-permeable to allow the free passage of small molecules, such as nutrients and waste products, into and out of the construct. Additionally, the material may be permeable or semi-permeable to allow the transport of cytokines out of the implantable construct. Exemplary materials include polymers, metals, ceramics, and combinations thereof.

[0021] In some embodiments, the implantable construct comprises a polymer (e.g., a natural or synthetic polymer). For example, the polymer may include polystyrene, polyester, polycarbonate, polyethylene, polypropylene, polyfluorocarbon, nylon, polyacetylene, polyvinyl chloride (PVC), polyolefin, polyurethane, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polymethyl methacrylate, poly(2-hydroxyethyl methacrylate), polysiloxane, polydimethylsiloxane (PDMS), polyhydroxyalkanoate, PEEK®, polytetrafluoroethylene, polyethylene glycol, polysulfone, polyacrylonitrile, collagen, cellulose, cellulosic polymers, polysaccharides, polyglycolic acid, poly(L-lactic acid) (PLLA), poly(lactic-glycolic acid) (PLGA), polydioxanone (PDA), poly(lactic acid), hyaluronic acid, agarose, alginic acid, chitosan, or mixtures or copolymers thereof. In some embodiments, the implantable construct comprises a polysaccharide (e.g., alginate, cellulose, hyaluronic acid, or chitosan). In some embodiments, the implantable construct comprises hyaluronic acid. In some embodiments, the implantable construct comprises alginate. In some embodiments, the average molecular weight of the polymer is about 2 kDa to about 500 kDa (e.g., about 2.5 kDa to about 175 kDa, about 5 kDa to about 150 kDa, about 10 kDa to about 125 kDa, about 12.5 kDa to about 100 kDa, about 15 kDa to about 90 kDa, about 17.5 kDa to about 80 kDa, about 20 kDa to about 70 kDa, about 22.5 kDa to about 60 kDa, or about 25 kDa to about 50 kDa). The implantable construct may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of a polymer, e.g., a polymer described herein.

[0022] In some embodiments, the implantable construct comprises a polysaccharide, such as hyaluronic acid or alginic acid, a natural polymer containing β-(1-4) linked mannuronic and guluronic acid residues that, as a result of its high density of negatively charged carboxylates, can crosslink with certain cations to form larger structures, such as hydrogels. The alginate polymers described herein can have an average molecular weight of about 2 kDa to about 500 kDa (e.g., about 2.5 kDa to about 175 kDa, about 5 kDa to about 150 kDa, about 10 kDa to about 125 kDa, about 12.5 kDa to about 100 kDa, about 15 kDa to about 90 kDa, about 17.5 kDa to about 80 kDa, about 20 kDa to about 70 kDa, about 22.5 kDa to about 60 kDa, or about 25 kDa to about 50 kDa). In some embodiments, the implantable construct comprises at least 0.5%, 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of an alginate polymer, hi some embodiments, the alginate is ultra-pure alginate (e.g., SLG20 alginate).

[0023] In some embodiments, the implantable construct comprises a metal or metal alloy. Exemplary metals or metal alloys include titanium (e.g., nitinol, nickel-titanium alloys, thermal memory alloy materials), platinum, platinum-based alloys, stainless steel, tantalum, palladium, zirconium, niobium, molybdenum, nickel-chromium, cobalt, tantalum, chromium-molybdenum alloys, nickel-titanium alloys, and cobalt-chromium alloys. In some embodiments, the implantable construct comprises a stainless steel grade. The implantable construct may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of a metal or metal alloy, such as a metal or metal alloy described herein.

[0024] In some embodiments, the implantable construct comprises a ceramic. Exemplary ceramics include carbide, nitride, silica, or oxide materials (e.g., titanium oxide, hafnium oxide, iridium oxide, chromium oxide, aluminum oxide, and zirconium oxide). The implantable construct may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of a ceramic, such as a ceramic described herein.

[0025] In some embodiments, the implantable construct may comprise glass. The implantable construct may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more glass.

[0026] The material within the implantable construct can be further modified, for example, by chemical modification. For example, the material can be coated or derivatized with a chemical modification that provides a particular characteristic, such as immunomodulatory or antifibrotic properties. Exemplary chemical modifications include small molecules, peptides, proteins, nucleic acids, lipids, or oligosaccharides. The implantable construct can comprise 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 that is chemically modified, for example, bearing a chemical modification described herein.

[0027] In some embodiments, the material is chemically modified with a specific density of modifications. 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 the material in, on, or within the implantable constructs described herein can be 0.01, 0.1, 0.5, 1, 5, 10, 15, 20, 50, 75, 100, 200, 400, 500, 750, 1,000, 2,500, or 5,000 chemical modifications per square micrometer or square millimeter.

[0028] In certain embodiments, the chemical modification of the material may include a linker or other binding moiety. These linkers may include crosslinkers, amine-containing linkers, ester-containing linkers, photolabile linkers, peptide-containing linkers, disulfide-containing linkers, amide-containing linkers, phosphoryl-containing linkers, or combinations thereof. The linker may be labile (e.g., hydrolyzable). Exemplary linkers or other binding moieties are described in Bioconjugate Techniques (3 rd ed. Greg T. Hermanson, Waltham, MA: Elsevier, Inc., 2013), which is incorporated herein by reference in its entirety.

[0029] C. Triazole In one embodiment, the surface is made of a material that is autoimmunogenic. In other embodiments, the surface is coated with a different material that is either immunogenic or immunoevasive. The inventors have developed a library of compounds such as those represented by the following formula: formula: AL-R1(I) or a pharmaceutically acceptable salt thereof, During the ceremony, A is a polymer, L is a group having the formula: NR a X1(CH2CH2O) o (In the formula, R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; o is 2, 3, 4, or 5; and X1 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) is) or a linker of the formula: NR b (CH2) p X2 (In the formula, R b is hydrogen, alkyl(C≦6) , or substituted alkyl (C≦6) and; p is 1, 2, or 3; and X2 is an array (C≦12) or substituted arenediyl (C≦12) is) is a linker for R1 is cycloalkyl (C≦12) ;Haloaryl (C≦12) ;S-containing heteroaryl (C≦12) ;Substituted S-containing heteroaryl (C≦12) ;Alkyl (C≦6) , haloalkyl (C≦6) , alkenyl (C≦6) , or Alkynye (C≦6) Substituted Aryl (C≦12) ;Aralkyl (C≦12) ;Substituted aralkyl (C≦12) ;Heterocycloalkyl (C≦12) ;substituted heterocycloalkyl (C≦12) ;2-Pyridinyl;3-Aminophenyl;4-Alkoxy (C≦6) Substituted Aryl (C≦12) or the formula: X3OR2 (In the formula, X3 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) and; R2 is aryl (C≦12) or substituted aryl (C≦12) is) is the basis of

[0030] The compound is AL-R1(I) or a pharmaceutically acceptable salt thereof; During the ceremony, A is a polymer, L is a group having the formula: NR a X1(CH2CH2O) m is a linker for During the ceremony, R a is hydrogen, alkyl (C≦6), or substituted alkyl (C≦6) and; m is 2, 3, 4, or 5; and X1 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) and R1 is cycloalkyl (C≦12) ;Haloaryl (C≦12) ;S-containing heteroaryl (C≦12) ;Substituted S-containing heteroaryl (C≦12) ;Alkyl (C≦6) , haloalkyl (C≦6) , alkenyl (C≦6) , or Alkynye (C≦6) Substituted Aryl (C≦12) ;3-aminophenyl;4-alkoxy (C≦6) Substituted Aryl (C≦12) or the formula: X3OR2 (In the formula, X3 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) and; R2 is aryl (C≦12) or substituted aryl (C≦12) is) is the basis of

[0031] The compound is AL-R1(I) or a pharmaceutically acceptable salt thereof; During the ceremony, A is a polymer, L is a group having the formula: NR a X1(CH2CH2O) m (In the formula, R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; m is 2, 3, 4, or 5; and X1 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) is) or a linker of the formula: NR b (CH2) n X2 (In the formula, R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; n is 1, 2, or 3; and X2 is an array (C≦12) or substituted arenediyl (C≦12) is) is a linker for R1 is haloaryl (C≦12) ;Aralkyl (C≦12) ;Substituted aralkyl (C≦12) ;Heterocycloalkyl (C≦12) ;substituted heterocycloalkyl (C≦12) ;2-pyridinyl;3-aminophenyl.

[0032] The polymer may comprise one or more saccharide repeat units. The repeat unit may be of the formula: TIFF2025526484000002.tif30170, During the ceremony, R3 or R4 are each independently hydrogen or hydroxy; R5 is hydroxy, alkoxy (C≦8) , substituted alkoxy (C≦8) or a covalent bond to a linker; and m is a number of repeating units having a molecular weight of about 50,000 daltons to about 500,000 daltons.

[0033] The polymer has the formula: TIFF2025526484000003.tif37170, During the ceremony, R3, R3', R4, or R 4’ are each independently hydrogen or hydroxy; R5 is hydroxy, alkoxy (C≦8) , substituted alkoxy (C≦8) or a covalent bond to a linker; R5' is a covalent bond to the linker; and m and n result in some repeating units having a molecular weight of about 50,000 daltons to about 500,000 daltons.

[0034] The polymer may be an acrylate polymer, for example a methacrylic acid polymer.

[0035] In particular, the following molecules are useful in the disclosed assays: TIFF2025526484000004.tif179170TIFF2025526484000005.tif233170TIFF20255264840 00006.tif198170TIFF2025526484000007.tif189170TIFF2025526484000008.tif197170

[0036] The methods may use human or non-human animal model hosts. The materials / surfaces may be implanted subcutaneously, intramuscularly, or intraperitoneally, implanted in the brain or other organ, or inserted into a body orifice such as the mouth, urethra, or rectum. The implanted / inserted materials / surfaces may remain in situ for about 24 hours or longer, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 3 weeks, or 4 weeks.

[0037] D. Cell The implantable constructs described herein may contain cells, e.g., modified cells, which may 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, intestines, blood, ovaries, uterus, or testes.

[0038] The cells may be derived from a donor (e.g., allogeneic cells), a subject (e.g., autologous cells), or from another species (e.g., xenogeneic cells). In some embodiments, the cells may be expanded 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 modified. In other embodiments, the cells are not genetically modified. The cells may comprise stem cells, e.g., 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, HCT116 cells, A549MCF-7 cells, HuH-7 cells, U-2 OS cells, HepG2 cells, Neuro-2a cells, and SF9 cells. In one embodiment, the cells used in the implantable construct are RPE cells.

[0039] Cells contained in the implantable construct may produce or secrete a therapeutic agent. In some embodiments, cells contained in the implantable construct may produce or secrete a single type of therapeutic agent or multiple therapeutic agents. In some embodiments, the implantable construct may include cells that have been transduced or transfected with a nucleic acid (e.g., a vector) containing an expression sequence for a therapeutic agent. For example, the cells may be transduced or transfected with a lentivirus. The nucleic acid introduced into the cells (e.g., by transduction or transfection) may be incorporated into a nucleic acid delivery system, e.g., a plasmid, or may be delivered directly. In some embodiments, the nucleic acid introduced into the cells (e.g., as part of a plasmid) may enhance expression of the therapeutic agent and / or include a region for directing targeting or secretion, for example, a promoter sequence, an activator sequence, or a cell signaling peptide or a cell transport peptide. Exemplary promoters include EF-1a, 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 Cpf1 (e.g., from L. bacterium).

[0040] The implantable constructs described herein may comprise a single 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, 10000, or more. In some embodiments, the total number of cells contained in the implantable construct is greater than about 1.0 x 10 2 Super, 1.0×10 3 , 1.0×10 4 , 1.0×10 5 , 1.0×10 6 , 1.0×107 , 1.0×10 8 , 1.0×10 9 , 1.0×10 10 In some embodiments, the total number of cells contained in the implantable construct is less than about 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 Less than 1.0 x 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 aggregate.

[0041] 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 certain 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.

[0042] E. Modified Cells In some embodiments, the implantable construct comprises a single cell or multiple cells that have been genetically modified to produce or secrete a therapeutic agent. In some embodiments, the implantable construct comprises cells that produce or secrete a protein. The protein can be any size, for example, greater than 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, or any other size. The molecular weight of the protein may be 55 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 certain 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. Proteins may be produced or secreted as preproteins or in an inactive form and may require further modification to convert them to an active form.

[0043] The protein produced or secreted by the cell may be an antibody or antibody fragment, e.g., an 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 may be monoclonal or polyclonal. Other exemplary proteins include lipoproteins, adhesion proteins, blood clotting factors (e.g., Factor VII, Factor VIII, Factor IX, GCG, or VWF), hemoglobin, enzymes, proenkephalin, growth factors (e.g., EGF, IGF-1, VEGF alpha, HGF, TGF beta, bFGF), or cytokines.

[0044] Proteins produced or secreted by the cells may 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.

[0045] The protein produced or secreted by the cell may include a cytokine. The cytokine may be a pro-inflammatory cytokine or an anti-inflammatory cytokine. Examples of cytokines include IL-1, IL-1α, IL-1β, IL-1RA, IL-2, 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-16, IL-17, G-CSF, GM-CSF, IL-20, IFN-α, IFN-β, IFN-γ, CD154, LT-β, CD70, CD153, CD178, TRAIL, TNF-α, TNF-β, SCF, M-CSF, MSP, 4-1BBL, LIF, OSM, and others. For example, the cytokine may include any cytokine described in MJ Cameron and DJ Kelvin, Cytokines, Chemokines, and Their Receptors (2013), Landes Biosciences, which is incorporated herein by reference in its entirety.

[0046] The implantable construct may include cells that express a single type of therapeutic agent, e.g., a single protein or nucleic acid, or that can express more than one type of therapeutic agent, e.g., multiple proteins or nucleic acids. In some embodiments, the implantable construct includes cells that express two types of therapeutic agents (e.g., two types of proteins or nucleic acids). In some embodiments, the implantable construct includes cells that express three types of therapeutic agents (e.g., three types of proteins or nucleic acids). In some embodiments, the implantable construct includes cells that express four types of therapeutic agents (e.g., four types of proteins or nucleic acids).

[0047] In some embodiments, the implantable construct comprises cells that express a single type of nucleic acid (e.g., DNA or RNA) or that can 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).

[0048] In some embodiments, the implantable construct comprises cells that express a single type of protein or that can 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.

[0049] In some embodiments, the implantable construct comprises cells that express a single type of enzyme or that can 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.

[0050] In some embodiments, the implantable construct comprises cells that express a single type of antibody or antibody fragment, or that can 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.

[0051] In some embodiments, the implantable construct comprises cells that express a single type of hormone, or that can 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.

[0052] In some embodiments, the implantable construct comprises cells that express a single type of enzyme or that can 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.

[0053] In some embodiments, the implantable construct comprises cells that express a single type of cytokine or that can 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.

[0054] F. 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, chord, ellipsoid, disc, cylinder, sheet, torus, cube, stadium, pyramid, cone, triangle, rectangle, square, or rod. The implantable construct may comprise a curved or flat section. In some embodiments, the implantable construct may be prepared through the use of a mold that results in a custom shape.

[0055] The implantable construct may vary in size, for example, depending on the use or site of implantation. For example, the implantable construct may have an average diameter or size of greater than 0.1 mm, e.g., greater than 0.25 mm, 0.5 mm, 0.75, 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 certain embodiments, the implantable construct may have sections or regions of an average diameter or size of greater than 0.1 mm, e.g., greater than 0.25 mm, 0.5 mm, 0.75, 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 of 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.

[0056] In some embodiments, the implantable construct comprises pores or openings that allow passage of objects such as small molecules (e.g., nutrients or waste products), proteins, or nucleic acids. For example, pores in or on the implantable construct may be greater than 0.1 nm and less than 10 μm. In some embodiments, the implantable construct comprises pores or openings in the size ranges 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.

[0057] The implantable constructs described herein may include chemical modifications in or on any encapsulated material. Exemplary chemical modifications include small molecules, peptides, proteins, nucleic acids, lipids, or oligosaccharides. The implantable construct may comprise 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 that is chemically modified, e.g., bearing a chemical modification described herein. The implantable construct may 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.

[0058] In some embodiments, the implantable construct is chemically modified with a specific density of modifications. A 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 within 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, 2,500, or 5,000 chemical modifications per square micrometer or square millimeter.

[0059] The implantable construct can be formulated or designed for implantation in any organ, tissue, cell, or portion of a subject. For example, the implantable construct may be implanted or placed in the intraperitoneal cavity of a subject. The implantable construct may be implanted into or placed on top of a tumor or other growth in a subject, or may be implanted into or placed within about 0.1 mm, 0.5 mm, 1 mm, 0.25 mm, 0.5 mm, 0.75, 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 more from a tumor or other growth in a subject. The implantable construct may be designed for, or implanted or placed on, or within the skin, mucosal surfaces, body cavities, the central nervous system (e.g., brain or spinal cord), organs (e.g., heart, eye, liver, kidney, spleen, lung, ovary, breast, uterus), lymphatic system, vasculature, oral cavity, nasal cavity, gastrointestinal tract, bone, muscle, adipose tissue, skin, or other areas.

[0060] The implantable construct may be formulated for use over any period of time. For example, the implantable construct may 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 more. The implantable construct may be designed 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 designed for extended 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 designed for sustained 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).

[0061] G. Treatment Methods Described herein are implantable constructs that include encapsulation of modified cells, and related methods of use. In some embodiments, the implantable constructs are used to treat diseases, such as those described herein.

[0062] The implantable constructs described herein may further comprise an additional pharmaceutical 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.

[0063] In some embodiments, the additional pharmaceutical agent is an immunomodulatory agent, e.g., 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.

[0064] 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 carried out 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, e.g., a soluble ligand (e.g., PD-1-Ig or CTLA-4 Ig), or an antibody or antigen-binding fragment thereof, that binds to an inhibitory molecule; for example, 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 certain embodiments, the anti-inflammatory agent is an agent that blocks, inhibits, or reduces inflammation or signaling from an inflammatory signaling pathway. In certain embodiments, the anti-inflammatory agent inhibits or reduces the activity of one or more of the following immune components of a subject: In certain embodiments, the anti-inflammatory agent is an IL-1 or IL-1 receptor antagonist, e.g., 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 anti-IL-6 receptor antibody, e.g., tocilizumab (ACTEMRA®), olokizumab, clazakizumab, sarilumab, sirukumab, siltuximab, 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, for example, as described herein.

[0065] H. Composition and Administration of Implantable Constructs The present disclosure features pharmaceutical compositions including an implantable construct and modified cells, and optionally a pharmaceutically acceptable excipient. In some embodiments, the implantable construct is provided in the composition in an effective amount. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is a prophylactically effective amount.

[0066] The compositions described herein can be prepared by any method known in the art of pharmacology. Generally, such methods include the step of bringing the implantable construct into association with the carrier and / or one or more other accessory ingredients, and then, if necessary and / or desirable, forming and / or packaging the formulation into the desired single or multi-dosage unit.

[0067] The 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 may generally be equal to the dose of the cytokine when administered to a subject and / or a convenient fraction of such a dose, such as, for example, one-half or one-third of such a dose.

[0068] The relative amounts of implantable construct, pharmaceutically acceptable excipient, and / or any additional components in the pharmaceutical compositions of the present invention 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 to be administered. By way of example, the composition may contain from 0.1% to 100% (w / w) of any component.

[0069] Implantable constructs and pharmaceutical compositions thereof can 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 can be administered intravenously and / or orally. In some embodiments, the implantable construct is injected subcutaneously. In some embodiments, the implantable construct is injected into the intraperitoneal cavity. In some embodiments, the implantable construct is delivered to the subject using a device, such as a cannula or catheter.

[0070] 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 invention may be aqueous or oily suspensions. These suspensions can be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic saline. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media.

[0071] For ophthalmic use, the provided compounds, compositions, and devices can be formulated as a micronized suspension or in an ointment such as petrolatum.

[0072] In some embodiments, the release of cytokines or additional pharmaceutical agents is sustained. To prolong the effect of certain drugs, it is often desirable to slow the absorption of the drug from injection. This can be achieved by using a suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends on its dissolution rate, which in turn 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.

[0073] While the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to animals of all kinds. Modifications of pharmaceutical compositions suitable for administration to humans to adapt them for administration to a variety of animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or perform such modifications with routine experimentation.

[0074] The implantable constructs provided herein are typically formulated in dosage unit form, e.g., single unit dosage form, for ease of administration and uniformity of dosage. However, it will be understood that the total daily use of the compositions of the present invention 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 a variety of factors, including the severity of the disease or disorder being treated; the activity of the specific active ingredient used; the specific composition used; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and excretion rate of the specific active ingredient used; the duration of treatment; drugs used in combination or concomitantly with the specific therapeutic agent used; and similar factors well known in the medical arts.

[0075] The exact amount of compound required to achieve an effective dose will vary from subject to subject, depending, for example, on the species, age, and general condition of the subject, the severity of the side effect or disorder, the identity of the particular compound, the mode of administration, etc. The desired dose may be delivered three times daily, twice daily, once daily, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dose may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations).

[0076] An effective amount of therapeutic agent released from the implantable construct may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg of therapeutic agent per unit dosage form (e.g., per implantable construct).

[0077] The therapeutic agent to be administered may be at a dosage level sufficient to deliver 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, and more preferably about 1 mg / kg to about 25 mg / kg of subject body weight per day, one or more times per day to obtain the desired therapeutic effect.

[0078] It will be understood that the dosage ranges set forth herein provide guidance for administration of the provided pharmaceutical compositions to adults. For example, the amount to be administered to a child or adolescent can be determined by a physician or person skilled in the art and may be lower or equivalent to that for administration to an adult.

[0079] Enumerated Embodiments 1. A method of treating tissue damage and / or inducing tissue regeneration in a subject, comprising: providing an implantable construct comprising encapsulated modified cells that express a cytokine; administering the implantable construct to a subject; thereby treating tissue damage and / or inducing tissue regeneration in a subject. 2. The method of embodiment 1, wherein said cytokine is an anti-inflammatory cytokine or a pro-inflammatory cytokine. 3. The method of any one of the preceding embodiments, wherein the cytokine comprises IL-10 (e.g., the cytokine is IL-10). 4. The method of any one of the preceding embodiments, wherein the implantable construct is degradable. 5. The method of any one of the preceding embodiments, wherein the implantable construct comprises a polymer. 6. The method of embodiment 5, wherein the polymer is a natural polymer or a synthetic polymer. 7. The method of any one of embodiments 5-6, wherein the polymer is a polysaccharide (e.g., alginate). 8. The method of any one of the preceding embodiments, wherein the implantable construct further comprises a triazole compound. 9. The method of any one of the preceding embodiments, wherein the tissue damage or tissue regeneration occurs in cardiac or pulmonary tissue of the subject. 10. The method of any one of the preceding embodiments, wherein cardiac tissue in the subject is damaged by ischemia, e.g., due to coronary heart disease and / or myocardial infarction. 11. The method of embodiment 9, wherein the lung tissue is damaged by infection, for example, by a viral infection. 12. The method of any one of the preceding embodiments, wherein administering comprises administering directly to the tissue (e.g., by subcutaneous injection). 13. The method of embodiment 12, wherein the tissue is cardiac tissue or lung tissue. 14. The method of embodiment 13, wherein the tissue is damaged. 15. The method of any one of the preceding embodiments, wherein the modified cells are epithelial cells. 16. The method of any one of the preceding embodiments, wherein the modified cells are selected from Chinese hamster ovary (CHO) cells, retinal pigment epithelial (ARPE-19) cells, human mammary epithelial (MCF-10a and MCF-7) cells, human embryonic kidney (HEK) cells, mesenchymal stem cells (MSCs), human umbilical vein endothelial cells (HUVECs), NIH / 3T3 cells, BJ fibroblasts, and human renal mixed epithelial cells (HRECs). 17. The method of any one of the preceding embodiments, wherein the cell is modified for regulated expression of the cytokine in a modified cell, e.g., the cell is modified for regulated expression of the cytokine. 18. The method of any one of the preceding embodiments, wherein the implantable construct is formulated as a pharmaceutical composition. 19. The method of any one of the preceding embodiments, wherein the subject is a mammal (e.g., a human). 20. A method of treating tissue damage in the heart or lung in a subject, comprising: providing an implantable construct comprising encapsulated modified retinal pigment epithelial (RPE) cells that express cytokines; administering the implantable construct to a subject; thereby treating tissue damage in the heart or lung in a subject. 21. A method of treating cardiovascular disease in a subject, comprising: providing an implantable construct comprising encapsulated modified retinal pigment epithelial (RPE) cells that express cytokines; administering the implantable construct to a subject; thereby treating cardiovascular disease in a subject. 22. A method of treating a pulmonary disease in a subject, comprising: providing an implantable construct comprising encapsulated modified retinal pigment epithelial (RPE) cells that express cytokines; administering the implantable construct to a subject; thereby treating a pulmonary disease in a subject. [Example]

[0080] I. 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 which follow represent techniques discovered by the inventors to function well in the practice of the embodiments, and can therefore be considered to constitute preferred modes for their practice. However, those skilled in the art will, in light of the present disclosure, understand that many changes can be made in the specific embodiments which are disclosed, while still obtaining like or similar results, without departing from the spirit and scope of the present disclosure.

[0081] Example 1 - Methods for cardiac and pulmonary tissue repair Ischemic heart disease is the leading cause of death in the developed world. Heart failure (HF) develops in 20–30% of patients after myocardial infarction (MI) due to extensive scarring exacerbated by a sustained increase in inflammatory macrophages, which increases injury. Cytokines, such as interleukin-10 (IL-10), and cytokine inhibitors, such as interleukin-1 receptor antagonist (IL-1Ra), are potent immunomodulators that can attenuate inflammation, reduce infarct size, and improve ventricular function in animal models of MI. However, poor biodistribution, toxicity, infection due to systemic immunosuppression, and paradoxical pro-inflammatory responses with continuous administration represent critical challenges that hinder the clinical translation of systemic cytokine therapy. In post-MI patients with HF, sustained therapeutic delivery likely requires several months to sufficiently promote tissue repair and result in functional recovery.

[0082] Furthermore, the global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in unprecedented morbidity, mortality, and economic impact. As SARS-CoV-2 is the third coronavirus to cause severe global human disease within the past two decades, urgent solutions are needed to mitigate coronavirus-induced acute lung injury (ALI) and prevent acute respiratory distress syndrome (ARDS). Immunomodulatory therapies represent a therapeutic option to correct this imbalance in the host immune response, reduce ALI, and prevent ARDS.

[0083] The inventors report a new technique for tissue repair after cardiac or pulmonary injury. Their approach uses polymer-encapsulated cells that have been engineered to continuously produce natural interleukin-10 (IL-10) and induce a pro-regenerative immune response, with applications in tissue and wound healing, tissue repair, and organ transplantation.

[0084] The inventors' goal is to develop novel and innovative immunomodulatory cell therapies to treat immune system dysfunction resulting from MI and SARS-CoV-2 ALI / ARDS. These approaches combine hydrogels with cells engineered to produce IL-10 and IL-1Ra with tunable pharmacokinetics for localized targeted delivery to the heart and lungs.

[0085] Here, the inventors describe a hydrogel-based delivery system consisting of cells engineered to produce anti-inflammatory cytokines and agonists that suppress activated immune cells in vivo. The engineered cells and hydrogel-based spheres play an important role in modulating responses from the immune system, and when combined, allow for fine-tuned control of immunomodulatory activity. Figures 1-10 illustrate the contemplated system and method.

[0086] Cytokines: Cell engineering and production of cytokines have been achieved by the inventors. Cytokines are cell signaling proteins produced by various cells in the body in response to specific stimuli. These proteins function to regulate the signaling and activation state of cells of the immune system. The inventors have designed a cell engineering platform using synthetic biology principles to create genetically engineered cell lines that continuously produce defined concentrations of these proteins in animals. There are three major classes of cytokines, including pro-inflammatory cytokines, which function to activate immune cells; anti-inflammatory cytokines, which function to suppress immune cells; and chemokines, which function to initiate immune cell migration. Molecules in these three classes of proteins are similar in size and molecular structure, and therefore, each can be produced with very fine changes in engineering techniques.

[0087] The following cytokines can be produced by the inventor's engineered cells: IL-1, IL-1a, IL-1b, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12a, IL-12 b, IL-13, IL-14, IL-15, IL-16, IL-17, IL-20, IFN-a, IFN-b, IFN-c, TNF-a, TNF-b, TGF-b, CCL-1, CCL-2, CCL-3, CCL-4, CCL-5, CCL-6, CCL-7, CCL-8, CC CCL-9, CCL-10, CCL-11, CCL-12, CCL-13, CCL-14, CCL-15, CCL-16, CCL-17, CCL-18, CCL-19, CCL-20, CCL-21, CCL-22, CCL-23, CCL-24, CCL-25, CCL-26, CCL-27, CCL-28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17. Control of when cytokines are produced by these engineered cells allows for precise coordination of the immune system. To ensure future clinical translatability, mouse and human versions of the IL-10 and IL-1RA cell lines used in this project were generated using the appropriate gene sequences from NCBI. Modifications to this system require only the exchange of the gene of interest, allowing for rapid and easy "plug-and-play" cell engineering.

[0088] The base cell line selection includes Chinese hamster ovary (CHO), retinal pigment epithelium (ARPE-19), human mammary epithelium (MCF-10a and MCF-7), human embryonic kidney (HEK), mesenchymal stem cells (MSC), human umbilical vein endothelial cells (HUVEC), NIH / 3T3 cells, BJ fibroblasts, and human renal mixed epithelial cells (HREC).

[0089] Hydrogel: Different types of alginate can be used. These hydrogel spheres are made using a custom-built, two-fluid, coaxial electrostatic spraying device. The device consisted of a potential generator attached to the tip of a coaxial needle and grounded to a crosslinked bath of 1:4 barium chloride:mannitol. The coaxial needle was fed by two separate syringes containing a 1.4% alginate solution diluted with 0.9% saline.

[0090] The inventors can fine-tune the cytokine dose by varying the number of cells within each sphere and / or the number of spheres administered per dose, according to the necessary constraints on a per patient basis. For example, individual spheres can be filled with 10,000 to 80,000 cells. Spheres can range in size from 50 μm to 3 mm.

[0091] Embodiments: Exemplary embodiments of this technology include the following: 1: Treatment for myocardial repair after a heart attack or associated tissue damage through delivery of IL-10, IL-1Ra, and any combination of cytokines and / or immunosuppressants. IL-10 and cytokine inhibitors, such as interleukin-1 receptor antagonist (IL-1Ra), are potent immune modifiers that can reduce inflammation, infarct size, and improve ventricular function. 2: Treatment for lung repair after SARS-CoV-2 ALI / ARDS or associated tissue damage through delivery of IL-10 and any combination of cytokines and / or immunosuppressants. 3: Organ transplantation: IL-10-secreting spheres can be administered with transplanted organs to prevent immune system activation and the subsequent onset of graft-versus-host disease, which leads to inflammation and organ rejection.

[0092] Example 2 - Method for treating pleural inflammation Pleural inflammation is just one example among various pathologies in which an excessive immune response causes health problems. In this sense, anti-inflammatory cytokines have significant therapeutic potential for pleural inflammation and other autoimmune pathologies by playing an immunosuppressive role. However, long-term immunosuppression is associated with susceptibility to malignancies and infections. Controlling the timing and dosage of cytokine delivery can facilitate clinical translation.

[0093] Here, the inventors investigate small-molecule-inducible, cell-based delivery of cytokines to the retinal pigment epithelium (RPE). Using VectorBuilder®, the inventors modified RPE cells with a construct containing a tetracycline response element (TRE), a cytokine gene of interest, and neomycin as a selectable marker. The vector was delivered to RPE cells using Lipofectamine® 3000 (ThermoFisher). Modified cells were selected with neomycin, and then selected cells were expanded. Doxycycline hydrochloride (DOX) treatment (2 μg / ml) induces the Tet-On system in the cells, delivering the cytokine of interest. Cytokine production was assessed by ELISA on medium from DOX-treated cells. Figures 13A-E show that cells can be externally regulated to modulate the production of anti-inflammatory cytokines. This allows for time- and dose-specific production of anti-inflammatory cytokines, reducing vulnerabilities associated with long-term immunosuppression (e.g., infections, malignancies), and is translatable to the treatment of various inflammatory and autoimmune conditions. The inventors contemplated encapsulating cells in hydrogels for improved delivery and cell survival.

[0094] Example 3 - In vivo validation of implantable constructs containing cytokine-containing cells In this experiment, implantable constructs were prepared containing cells capable of producing anti-inflammatory cytokines, namely, rat interleukin-1 receptor antagonist (RIL1Ra), rat interleukin-10 (RIL10), human interleukin-1 receptor antagonist (HIL1Ra), and human interleukin-10 (HIL10). The implantable constructs were first evaluated to demonstrate their ability to continuously produce therapeutic cytokines (Figures 11A-B). Notably, Figure 11C shows that over 90% of the encapsulated cells maintained viability, indicating their potential for long-term functionality. These implantable constructs were then implanted into the pleural cavity in a mouse model, and local cytokine concentrations were observed to increase, while systemic circulating concentrations were significantly reduced by up to 100-fold at various time points (days 1, 3, 7, and 28 after implantation, Figures 11D-E). This localization effect suggests that encapsulated cells could enable targeted cytokine delivery and minimize potential systemic side effects. Furthermore, we investigated the potential of capsules producing anti-inflammatory cytokines for up to 28 days to alleviate fibrosis, as observed in dark-field microscopy images of explanted capsules (Figure 11F). This indicates that encapsulated cells can effectively suppress fibrosis and contribute to the extended therapeutic effect of anti-inflammatory cytokine treatment.

[0095] These studies were also performed in lung tissue, where implantable constructs were implanted into rats treated in the absence or presence of LPS and the cytokines IL-10 and IL1Ra produced locally from the implantable construct. Histological scores from rats treated with LPS alone or LPS plus therapeutic agents are shown in Figures 12A-B, showing that the treatment groups exhibited improved lung tissue compared to the LPS-only group by day 14 (Figure 12B). The presence of immune cell infiltrates over time suggests that the encapsulated cells have the potential to modulate immune responses within lung tissue, which may contribute to the observed therapeutic effects.

[0096] Taken together, these studies demonstrate that implantable constructs capable of producing the anti-inflammatory cytokines described herein have great potential to provide therapeutic outcomes in a variety of inflammatory conditions.

[0097] All of the compositions and 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 modifications may be applied to the compositions and methods, and in the 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 which 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 substitutions 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. 1. A method of treating tissue damage and / or inducing tissue regeneration in a subject, comprising: providing an implantable construct containing encapsulated modified cells that express a cytokine; administering the implantable construct to the subject; thereby treating tissue damage and / or inducing tissue regeneration in said subject. The method.

2. 10. The method of claim 1, wherein the cytokine is an anti-inflammatory cytokine or a pro-inflammatory cytokine.

3. The method of claim 1, wherein the cytokine comprises IL-10 (eg, the cytokine is IL-10).

4. The method of claim 1 , wherein the implantable construct is degradable.

5. The method of claim 1 , wherein the implantable construct comprises a polymer.

6. The method of claim 5 , wherein the polymer is a natural polymer or a synthetic polymer.

7. The method of any one of claims 4 to 5, wherein the polymer is a polysaccharide (e.g., alginic acid).

8. The method of claim 1 , wherein the implantable construct further comprises a triazole compound.

9. 10. The method of claim 1, wherein the tissue damage or tissue regeneration occurs in cardiac or pulmonary tissue of the subject.

10. 10. The method of claim 9, wherein the cardiac tissue in the subject is damaged by ischemia, e.g., due to coronary heart disease and / or myocardial infarction.

11. 10. The method of claim 9, wherein the lung tissue is damaged by infection, for example by a viral infection.

12. 10. The method of claim 1, wherein administering comprises administering directly to the tissue (e.g., by subcutaneous injection).

13. 13. The method of claim 12, wherein the tissue is cardiac tissue or lung tissue.

14. The method of claim 13 , wherein the tissue is damaged.

15. The method of claim 1 , wherein the modified cells are epithelial cells.

16. 2. The method of claim 1, wherein the modified cells are selected from Chinese hamster ovary (CHO) cells, retinal pigment epithelial (ARPE-19) cells, human mammary epithelial (MCF-10a and MCF-7) cells, human embryonic kidney (HEK) cells, mesenchymal stem cells (MSCs), human umbilical vein endothelial cells (HUVECs), NIH / 3T3 cells, BJ fibroblasts, and human renal mixed epithelial cells (HRECs).

17. The method of claim 1 , wherein the modified cell, e.g., the cell, is modified for regulated expression of the cytokine.

18. The method of claim 1 , wherein the implantable construct is formulated as a pharmaceutical composition.

19. The method of claim 1 , wherein the subject is a mammal (e.g., a human).

20. 1. A method of treating tissue damage in the heart or lung in a subject, comprising: providing an implantable construct comprising encapsulated modified retinal pigment epithelial (RPE) cells that express a cytokine; administering the implantable construct to the subject; thereby treating tissue damage in the heart or lung in the subject. The method.

21. 1. A method of treating cardiovascular disease in a subject, comprising: providing an implantable construct comprising encapsulated modified retinal pigment epithelial (RPE) cells that express a cytokine; administering the implantable construct to the subject; thereby treating cardiovascular disease in said subject. The method.

22. 1. A method of treating a pulmonary disease in a subject, comprising: providing an implantable construct comprising encapsulated modified retinal pigment epithelial (RPE) cells that express cytokines; administering the implantable construct to the subject; thereby treating a pulmonary disease in said subject. The method.

Images