Patent Application
20240374507
A number of variety of diseases and conditions involve pathogenic or undesired antigen-specific immune responses, including autoimmune diseases, allergies, and the development of anti-drug antibody (ADA) responses to biopharmaceuticals. Traditional therapeutic approaches rely on administering broad acting immunosuppressants, which are not specific for the pathologic immune responses that exacerbate each disease or condition and often increase the risks of infections and malignancies. Immune tolerance is a state of unresponsiveness of the immune system to a group of related antigens, including self-tolerance to autoantigens and oral tolerance to food antigens, or to a particular antigen, thus displaying strict antigen-specificity. Therapies that can achieve antigen-specific immune tolerance induction (ITI) have the potential to provide more effective therapies for treating allergic and autoimmune diseases, while maintaining immune reactivity to other antigens, as well as increase the utility of biopharmaceuticals that have been approved for treating a variety of diseases such as hemophilia, rheumatoid arthritis, and cancers.
The present disclosure provides a therapeutic immunization, or vaccination, approach for inducing immune tolerance in which one or more antigens (e.g., immunogenic molecules such as peptides, proteins, DNA) are delivered to a subject in a manner that inhibits the generation of immune responses specific for the delivered antigen(s), such as the development of antigen-specific antibodies.
Described herein is an implantable device that (i) contains cells (e.g., allogenic to the subject) genetically modified to express and secrete one or more antigens and (ii) is configured to deliver, after implant into a recipient, at least one immunomodulatory agent that facilitates tolerance induction, e.g., by promoting an antigen-specific regulatory T cell (Treg) response or by inhibiting an antigen-specific effector T cell response (Teff) response. The device is configured to shield the antigen-secreting cells from the recipient's immune system, mitigate the recipient's foreign body response (FBR, as defined herein) to the implanted device, and prevent the antigen-secreting cells from exiting the device while allowing continuous delivery (e.g., exit) of the secreted antigen(s) and immunomodulatory agent(s) from the device in amounts and for a time period that are sufficient to induce tolerance to the antigen(s).
In an embodiment, each antigen delivered by the device is a protein that comprises the same amino acid sequence as a target protein antigen or is an immunogenic fragment thereof, e.g., a peptide that comprises a T cell epitope present in the target protein antigen.
In an embodiment, the immunomodulatory agent is an immunosuppressant compound, e.g., an mTOR inhibitor such as rapamycin, which optionally is provided as an extended release formulation contained in the device. In an embodiment, the immunomodulatory agent is an immunomodulatory protein (e.g., interleukin-10, interleukin-22, soluble CTLA-4, or soluble FLT3-ligand) that is expressed and secreted by genetically modified cells contained in the device, which may be by the antigen-secreting cells or by different cells. In an embodiment, the device is configured to deliver two or more immunomodulatory agents, e.g., a combination of an mTOR inhibitor and an immunomodulatory protein.
In an embodiment, the surface of the device comprises a compound or polymer that mitigates the FBR (as defined herein) to the device (e.g., an afibrotic compound or afibrotic polymer). In an embodiment, an afibrotic polymer comprises a biocompatible, zwitterionic polymer, e.g., as described in WO 2017/218507, WO 2018/140834, or Liu et al., Zwitterionically modified alginates mitigate cellular overgrowth for cell encapsulation, Nature Communications (2019) 10:5262. In an embodiment, the compound is a compound of Formula (I):
or a pharmaceutically acceptable salt thereof, wherein the variables A, L1, M, L2, P, L3, and Z, as well as related subvariables, are defined herein.
In an embodiment, the device is configured as a two-compartment hydrogel capsule in which an inner compartment comprising the genetically modified cells, and optionally immunomodulatory small-molecule compound, is completely surrounded by a barrier compartment. In some embodiments, the barrier compartment comprises a polymer covalently modified with a compound that mitigates the recipient's foreign body response (FBR) to the device.
In an embodiment, a device described herein, or a plurality of the device, is combined with a pharmaceutically acceptable excipient to prepare a device preparation or a composition which may be administered to a subject (e.g., into the intraperitoneal cavity) in need of tolerance induction to the antigen(s) produced by the device. In an embodiment, the device preparation or composition is administered to prevent the development of a disease or condition, e.g., the subject is genetically predisposed for developing an autoimmune disease, or to induce tolerance to a biopharmaceutical prior to its planned administration to the subject. In an embodiment the device preparation or composition is administered to a subject for treating an active disease or condition, e.g., to slow the progression of, or improve the clinical symptoms, of the disease or condition.
In another aspect, the present disclosure features an implantable device for delivering to a subject (e.g., a human subject) at least one therapeutic substance (e.g., a therapeutic protein or peptide) secreted by cells contained in the device. The device is configured to deliver, after implant in the subject, at least one immunomodulatory agent (e.g., an immunosuppressant compound or immunosuppressive cytokine, each as defined herein) in an amount and for a time period sufficient to induce tolerance by the subject's immune system to the delivered therapeutic substance. The device also features a configuration that prevents the cells from exiting the device while allowing exit of the therapeutic substance(s) and immunomodulatory agent(s) from the device, shields the genetically modified cells from the recipient's immune system, and mitigates the FBR (as defined herein) to the implanted device. In an embodiment, delivery of the immunomodulatory agent(s) by the implanted device results in a lower anti-drug antibody (ADA) response (e.g., as defined herein) in a subject than an implanted reference device that does not deliver any immunomodulatory agent. The implantable device is useful in a method of providing a therapeutic substance to a subject which comprises administering (e.g., implanting in) the subject the device, a composition comprising the device or a composition comprising a plurality of the device. In an embodiment, the subject has a blood clotting disorder (e.g., Hemophilia A), a lysosomal storage disorder (e.g., Fabry Disease, MPS-1), an endocrine disorder, diabetes, or a neurodegenerative disease.
Also provided herein is a genetically modified mammalian (e.g., human) cell that expresses and secretes one of more of the immunomodulatory proteins described herein. In an embodiment, the immunomodulatory protein is encoded by an exogenous coding sequence inserted into the cell genome at one or more locations. In an embodiment, the exogenous coding sequence is a codon-optimized to achieve higher expression by the cell. In an embodiment, the cell is derived from a human RPE cell, e.g., an ARPE-19 cell.
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.
The present disclosure features an implantable device capable of continuous delivery of antigens and at least one immunomodulatory agent to a subject in an amount and for a time period sufficient to induce antigen-specific immune tolerance in the subject. The antigens are expressed and secreted by living cells contained in the device. The device also delivers to the subject an immunomodulatory agent(s) such as an immunosuppressant compound or an immunomodulatory protein for a period sufficient to induce tolerance. A variety of device configurations and their use for inducing immune tolerance in preventing and treating a variety of diseases and conditions are contemplated by the present disclosure. Various embodiments will be described below.
Throughout the detailed description and examples of the disclosure the following abbreviations will be used.
So that the disclosure may be more readily understood, certain technical and scientific terms used herein are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.
As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.
“About” or “approximately” means when used herein to modify a numerically defined parameter (e.g., a physical description of a hydrogel capsule such as diameter, sphericity, number of cells encapsulated therein, the number of capsules in a preparation), means that the recited numerical value is within an acceptable functional range for the defined parameter as determined by one of ordinary skill in the art, which will depend in part on how the numerical value is measured or determined, e.g., the limitations of the measurement system, including the acceptable error range for that measurement system. For example, “about” can mean a range of 20% above and below the recited numerical value. As a non-limiting example, a hydrogel capsule defined as having a diameter of about 1.5 millimeters (mm) and encapsulating about 5 million (M) cells may have a diameter of 1.2 to 1.8 mm and may encapsulate 4 M to 6 M cells. As another non-limiting example, a preparation of about 100 devices (e.g., hydrogel capsules) includes preparations having 80 to 120 devices. In some embodiments, the term “about” means that the modified parameter may vary by as much as 15%, 10% or 5% above and below the stated numerical value for that parameter.
“Acquire” or “acquiring”, as used herein, refer to obtaining possession of a value, e.g., a numerical value, or image, or a physical entity (e.g., a sample), by “directly acquiring” or “indirectly acquiring” the value or physical entity. “Directly acquiring” means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity. “Indirectly acquiring” refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value). Directly acquiring a value or physical entity includes performing a process that includes a physical change in a physical substance or the use of a machine or device. Examples of directly acquiring a value include obtaining a sample from a human subject. Directly acquiring a value includes performing a process that uses a machine or device, e.g., using a fluorescence microscope to acquire fluorescence microscopy data.
“Administer”, “administering”, or “administration”, as used herein, refer to implanting, absorbing, ingesting, injecting, or otherwise introducing into a subject, an entity described herein (e.g., a device or a preparation of devices), or providing such an entity to a subject for administration.
“Afibrotic”, as used herein, means a compound or material that mitigates the foreign body response (FBR). For example, the amount of FBR in a biological tissue that is induced by implant into that tissue of a device (e.g., hydrogel capsule) comprising an afibrotic compound (e.g., a hydrogel capsule comprising a polymer covalently modified with a compound listed in Table 4) is lower than the FBR induced by implantation of an afibrotic-null reference device, i.e., a device that lacks any afibrotic compound, but is of substantially the same composition (e.g., same cell type(s)) and structure (e.g., size, shape, no. of compartments). In an embodiment, the degree of the FBR is assessed by the immunological response in the tissue containing the implanted device (e.g., hydrogel capsule), which may include, for example, protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis, using assays known in the art, e.g., as described in WO 2017/075630, or using one or more of the assays/methods described Vegas, A., et al., Nature Biotechnol (supra), (e.g., subcutaneous cathepsin measurement of implanted capsules, Masson's trichrome (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, cellular staining and confocal microscopy for macrophages (CD68 or F4/80), myofibroblasts (alpha-muscle actin, SMA) or general cellular deposition, quantification of 79 RNA sequences of known inflammation factors and immune cell markers, or FACS analysis for macrophage and neutrophil cells on retrieved devices (e.g., capsules) after 14 days in the intraperitoneal space of a suitable test subject, e.g., an immunocompetent mouse. In an embodiment, the FBR is assessed by measuring the levels in the tissue containing the implant of one or more biomarkers of immune response, e.g., cathepsin, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, or CCL4. In some embodiments, the FBR induced by a device of the invention (e.g., a hydrogel capsule comprising an afibrotic compound disposed on its outer surface), is at least about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% lower than the FBR induced by an FBR-null reference device, e.g., a device that is substantially identical to the test or claimed device except for lacking the means for mitigating the FBR (e.g., a hydrogel capsule that does not comprise an afibrotic compound but is otherwise substantially identical to the claimed capsule. In some embodiments, the FBR (e.g., level of a biomarker(s)) is measured after about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or longer.
“Acid alpha-glucosidase protein”, “acid maltase protein”, “alpha-1,4-glucosidase protein” and “GAA protein” may be used interchangeably herein and refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GAA gene or any fragment, mutant, variant or derivative thereof that has GAA enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature GAA protein, as measured by any art-recognized GAA activity assay. The GAA enzyme catalyzes the hydrolysis of alpha (1,4) and alpha (1,6) linkages in glycogen, yielding free glucose and shortened glycogen polymers. GAA enzymatic activity can be measured using any art-recognized assay. The wild-type human GAA gene encodes a 952 amino acid precursor pro-polypeptide, of which the N-terminal 27 amino acids constitute a signal peptide, and amino acids 28-69 constitute a pro-peptide (UniProtKB—P10253). In some embodiments, the mature human GAA amino acid sequence in a GAA therapeutic protein produced by a device described herein consists essentially of amino acids 70-952 or 28-952 of the precursor sequence shown in UniProtKB—P10253.
“Alpha-galactosidase A protein”, “α-Gal A protein”, and “GLA protein” may be used interchangeably herein and refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GLA gene or any fragment, mutant, variant or derivative thereof that has GLA enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature GLA protein, as measured by any art-recognized GLA activity assay. The GLA enzyme hydrolyzes the terminal alpha-D-galactose residues in glycosphingolipids, particularly in globotriaosylceramide (Gb3). GLA enzymatic activity can be measured using any art-recognized assay. The wild-type human GLA gene encodes a 429-amino acid polypeptide, of which the N-terminal 31 amino acids constitute a signal peptide (UniProtKB—P06280). In some embodiments, the mature human GLA amino acid sequence in a GLA therapeutic protein produced by a device described herein consists essentially of amino acids 32-429 of the precursor sequence shown in UniProtKB—P06280.
“Alpha-L-iduronidase protein” and “IDUA protein” may be used interchangeably herein and refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) IDUA gene or any fragment, mutant, variant or derivative thereof that has IDUA enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature IDUA protein, as measured by any art-recognized IDUA activity assay (e.g., hydrolysis of the substrate 4-methylumbelliferyl-α-L-iduronide (4MU-iduronide), see, e.g., Ou, L. et al., Mol Genet Metab. 2014 February: 111 (2): 113-115). IDUA protein hydrolyzes nonreducing terminal alpha-L-iduronic acid residues in glycosaminoglycans (GAGs) (e.g., dermatan sulfate and heparan sulfate). The wild-type human IDUA gene encodes a 653 amino acid precursor protein, of which the N-terminal 26 or 27 amino acids constitute a signal peptide (GenBAnk Accession No. AAA81589.1, GenBAnk Accession No. AAA51698.1, UniProtKB—P35475). In some embodiments, the mature human IDUA amino acid sequence in an IDUA therapeutic protein produced by a device described herein consists essentially of amino acid 26, 27 or 28 to amino acid 653 of the precursor sequence shown in GenBAnk Accession No. AAA81589.1, GenBAnk Accession No. AAA51698.1 or UniProtKB—P35475.
“Alpha-N-acetyl-glucosaminidase protein”, N-acetyl-alpha-glucosaminidase” and “NAGLU protein” may be used interchangeably herein to refer to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) NAGLU gene or any fragment, mutant, variant or derivative thereof that has enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature NAGLU protein, as measured by any art-recognized NAGLU assay. NAGLU catalyzes the hydrolysis of terminal non-reducing N-acetyl-D-glucosamine residues in N-acetyl-alpha-D-glucosaminides. The wild-type human NAGLU gene encodes a 743 amino acid precursor polypeptide, of which the N-terminal 23 amino acids constitute a signal peptide (UniProtKB—P54802). In some embodiments, the mature human NAGLU amino acid sequence in a NAGLU therapeutic protein produced by a device described herein consists essentially of amino acids 24-743 of the precursor sequence shown in UniProtKB—P54802.
“Anti-drug antibody response” and “ADA response” may be used interchangeably herein to refer to the post-implant generation of antibodies that bind to a therapeutic substance (e.g., protein or peptide) delivered by implantation of a device described herein. The antibodies may include antibodies that bind to the therapeutic substance with any degree of affinity, e.g., low affinity antibodies and high affinity antibodies. In an embodiment, an ADA response means the presence of low affinity ADAs in the plasma of an implanted subject (e.g., a human subject). In an embodiment, an ADA response means the presence of high affinity ADAs in the plasma of an implanted subject (e.g., a human subject). Low affinity ADAs and high affinity ADAs are typically detectable in serum samples collected from the subject at one week and three weeks after administration (e.g., implantation) of the device, respectively. The existence and extent of an ADA response in a subject implanted with a device described herein may be determined using any art-recognized method (e.g., ELISA) to assay serum samples collected from the subject prior to (e.g., the day of) and at one, two or more time points after administration of the device, e.g., at day 7 and any combination of 14, 21, 28, 35, 42 and 48 days. In an embodiment, the amount of ADAs to a therapeutic substance delivered by administration of a device claimed herein (e.g., a hydrogel capsule comprising an extended-release formulation of an immunosuppressant compound) are at least about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% lower than the amount of ADAs induced by an reference device, e.g., a device that is substantially identical to the claimed device except for delivering any immunomodulatory agent (e.g., a hydrogel capsule that does not contain or deliver any immunosuppressant compound but is otherwise substantially identical to the claimed capsule). In an embodiment, the reduction in the amount of ADAs induced by a claimed device in comparison to a reference device is based on ADA results obtained for two populations of substantially similar subjects implanted with the claimed device or the reference device.
“Antigen”, as used herein, refers to a substance capable of binding to an antigen binding region of an immunoglobulin molecule (or antibody) or a T cell receptor (TCR). Non-limiting examples of antigens are antigenic determinants, haptens, and immunogens which may be proteins, polypeptides, peptides, small molecules (including oligopeptide mimics (i.e., organic compounds that mimic the antibody binding properties of the antigen)), carbohydrates, polysaccharides, lipids, nucleic acids, or combinations thereof. Antigen polypeptides may comprise an amino acid sequence of a native target antigen, or an immunogenic variant thereof, e.g., have one or more amino acid substitutions.
Arylsulfatase B protein” and “ARSB protein” may be used interchangeably herein and refer to a protein comprising the amino acid sequence of a mature, wild-type mammalian ARSB or any fragment, mutant, variant or derivative thereof that has enzyme activity (e.g., sulfatase activity) that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature ARSB protein, as measured by an art-recognized ARSB activity assay. ARSB hydrolyses sulfates in the body by metabolizing the sulfate moiety of dermatan sulfate and chondroitin sulfate. The wild-type human ARSB gene encodes a 533 amino acid precursor polypeptide, of which the N-terminal 36 or 38 amino acids constitute a signal peptide. In some embodiments, the mature human ARSB amino acid sequence in an ARSB therapeutic protein produced by a device described herein consists essentially of amino acids 37 to 533 or amino acids 39 to 533 of the human precursor ARSB amino acid sequence shown in UniProtKB P15848-1.
“Beta-glucoronidase protein” and “GUSB protein” may be used interchangeably herein to refer to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GUSB gene or any fragment, mutant, variant or derivative thereof that has enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature GUSB protein, as measured by any art-recognized GUSB assay. GUSB catalyzes the hydrolysis of beta-D-glucoronoside into an alcohol and d-glucoronate. The wild-type human GUSB gene encodes a 651 amino acid precursor polypeptide, of which the N-terminal 22 amino acids constitute a signal peptide (UniProtKB PO8236). In some embodiments, the mature human GUSB amino acid sequence in a GUSB therapeutic protein produced by a device described herein consists essentially of amino acids 23-651 of the precursor sequence shown in UniProtKB PO8236.
“Beta-gludosidase protein”, “acid beta-glucocerebrosidase protein”, “glucocerebrosidase protein”, “glucosylceramidase protein”, “lysosomal acid glucosylceramidase protein”, “lysosomal acid GCase protein”, and “GBA protein” may be used interchangeably herein to refer to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GBA gene or any fragment, mutant, variant or derivative thereof that has enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature GBA protein, as measured by any art-recognized GBA assay. GBA, within the lysosomal compartment, catalyzes the breakdown of the glycolipid glucosylceramide (GlcCer) to ceramide and glucose. The wild-type human GBA gene encodes a 536 amino acid precursor polypeptide, of which the N-terminal 39 amino acids constitute a signal peptide (UniProtKB P04062.3). In some embodiments, the mature human GBA amino acid sequence in a GBA therapeutic protein described herein consists essentially of amino acids 40-536 of the precursor sequence shown in UniProtKB P04062.3.
“Cell,” as used herein, refers to a genetically modified cell or a cell that is not genetically modified. In an embodiment, a cell is an immortalized cell or a genetically modified cell derived from an immortalized cell. In an embodiment, the cell is a live cell, e.g., is viable as measured by any technique described herein or known in the art.
“Conservatively modified variants” or conservative substitution”, as used herein, refers to a variant of a reference peptide or polypeptide that is identical to the reference molecule, except for having one or more conservative amino acid substitutions in its amino acid sequence. In an embodiment, a conservatively modified variant consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the reference amino acid sequence. A conservative amino acid substitution refers to substitution of an amino acid with an amino acid having similar characteristics (e.g., charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) and which has minimal impact on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution tables of functionally similar amino acids are well known in the art, and exemplary substitutions grouped by functional features are set forth in Table 1 below.
“Consists essentially of”, and variations such as “consist essentially of” or “consisting essentially of” as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified molecule, composition, device, or method. As a non-limiting example, an immunomodulatory protein that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions in the recited amino acid sequence, of one or more amino acid residues, which do not materially affect the relevant biological activity of the immunomodulatory protein.
“CTLA-4” and “CTLA4” refers to Cytotoxic T-Lymphocyte Antigen 4, also known as CD 152 (Cluster of differentiation 152), a protein that binds via its extracellular domain to costimulatory ligands B7-1 (CD80) and B7-2 (CD86) on the surface of antigen presenting cells (APCs). CLTA-4 inhibits immune response in two principal ways: it competes with CD28 for binding to B7-1 and B7-2 and thereby blocks co-stimulation, and it negatively signals to inhibit T cell activation.
“Soluble CTLA-4” and “sCTLA-4”, as used herein, refer to a secreted protein (e.g., lacks an operable transmembrane domain), which comprises the CTLA-4 extracellular domain (ECD) or portions thereof that bind B7-1 and/or B7-2. In an embodiment, the CTLA-4 ECD is from a mammalian CTLA-4 protein (e.g., human CTLA-4). In an embodiment, a soluble CTLA-4 protein comprises the ECD of human CTLA-4 (e.g., amino acids 38-161 of
“sCTLA-4 fusion protein”, as used herein, refers to an sCTLA-4 protein operably linked to all or a portion of a heterologous protein that confers a beneficial property, e.g., higher expression, better stability, longer half-life in vivo. Examples of sCTLA-4 fusion proteins are described in United States patent application publication US 2014/0147418 A1.
“CTLA-4-Ig fusion protein”, as used herein, is a sCTLA-4 fusion protein in which the heterologous component of the fusion comprises a mammalian immunoglobulin protein (IgG) or a portion thereof (e.g., Fc region). In an embodiment, the CTLA-4 amino acid sequence is linked to the amino acid sequence of the IgG Fc region via a linker, e.g., a single glutamine residue (as described in EP3029062A1) or any of the linkers described in WO201103584.
In some embodiments, the cells in a device described herein are genetically modified to express a precursor CTLA-4-Ig protein that comprises a signal sequence operably linked to the N-terminus of the mature CTLA-4 component. The signal sequence may be any sequence that will permit secretion of the precursor fusion protein. In an embodiment, the signal sequence is from a mammalian CTLA-4 (e.g., a human CTLA-4, e.g., amino acids 1-37 of
“Derived from”, as used herein with respect to cells, refers to cells obtained from tissue, cell lines, or cells, which optionally are then cultured, passaged, differentiated, induced, etc. to produce the derived cells. For example, mesenchymal stem cells can be derived from mesenchymal tissue and then differentiated into a variety of cell types.
“Device”, as used herein, refers to any implantable object (e.g., a particle, a hydrogel capsule, an implant, a medical device), which contains a cell or cells (e.g., live cells) capable of expressing and secreting an antigen and optionally an immunomodulatory protein following implant of the device, and has a configuration that supports the viability of the cells by allowing cell nutrients to enter the device.
“Differential volume,” as used herein, refers to a volume of one compartment within a device described herein that excludes the space occupied by another compartment(s). For example, the differential volume of the second (e.g., outer) compartment in a 2-compartment device with inner and outer compartments, refers to a volume within the second compartment that excludes space occupied by the first (inner) compartment.
“Endogenous nucleic acid” as used herein, is a nucleic acid that occurs naturally in a subject cell.
“Endogenous polypeptide,” as used herein, is a polypeptide that occurs naturally in a subject cell.
“Exogenous nucleic acid,” as used herein, is a nucleic acid that does not occur naturally in a subject cell.
“Exogenous polypeptide,” as used herein, is a polypeptide that does not occur naturally in a subject cell, e.g., genetically modified cell. Reference to an amino acid position of a specific sequence means the position of said amino acid in a reference amino acid sequence, e.g., sequence of a full-length mature (after signal peptide cleavage) wild-type protein (unless otherwise stated), and does not exclude the presence of variations, e.g., deletions, insertions and/or substitutions at other positions in the reference amino acid sequence.
“Extended-release formulation”, as used herein, means a formulation that releases an immunosuppressant compound (as defined herein) from a device in a sustained-release (SR) or controlled-release (CR) profile. SR maintains release of the immunosuppressant over a sustained period but not at a constant rate. CR maintains release of the immunosuppressant over a sustained period at a nearly constant rate. In some embodiments, extended-release means SR over a period of at least 30 days, at least 45 days, at least any of 50, 60, 70, 80, 90, 100, 110, 120 days or more.
“FLT3-Ligand”, “FLT3-L” and “FLT3L”, as used herein, refer to Fms-like tyrosine kinase-3 ligand, an alpha-helical cytokine that binds to Fms-like tyrosine kinase-3 (Flt3, also known as CD135), resulting in proliferation, differentiation, development and mobilization of multiple hematopoietic cell lineages. FLT3-L is expressed as a noncovalently-linked dimer by T cells and bone marrow and thymic fibroblasts. Each precursor polypeptide chain in human FLT3-L is 235 amino acids in length (e.g., the sequence of
“Soluble FLT3-L” and sFLT3-L”, as used herein, refer to a secreted protein that comprises the FLT3-L ECD or portions thereof that bind to Flt3. In an embodiment, the FLT3-L ECD is from a mammalian FLT3-L protein (e.g., human FLT3-L). In an embodiment, a soluble FLT3-L protein comprises amino acids 27 to 179 of
In some embodiments, the cells in a device described herein are genetically modified to express a precursor sFLT3-L protein that comprises a signal sequence operably linked to the mature FLT3-L amino acid sequence. The signal sequence may be any sequence that will permit secretion of the precursor protein. In an embodiment, the signal sequence is from a mammalian FLT3-L (e.g., human FLT3-L, e.g., amino acids 1-26 of
“sFTL3-L fusion protein”, as used herein, refers to an sFLT3-L protein operably linked to all or a portion of a heterologous protein that confers a beneficial property, e.g., higher expression, better stability, longer half-life in vivo. In an embodiment, the heterologous component of the fusion comprises a mammalian immunoglobulin protein (IgG) or a portion thereof (e.g., Fc region). In an embodiment, the FLT3-L amino acid sequence is linked to the amino acid sequence of the IgG Fc region via a linker.
“Genetically-modified cell,” as used herein, is a cell (e.g., an RPE cell) having a non-naturally occurring alteration, and typically comprises a nucleic acid sequence (e.g., an exogenous DNA or RNA) or a polypeptide not present (or present at a different level than) in an otherwise similar cell under similar conditions that is not genetically modified (e.g., lacks the exogenous nucleic acid sequence). In an embodiment, a genetically modified cell comprises an exogenous nucleic acid (e.g., a vector or an altered chromosomal sequence). In an embodiment, a genetically modified cell comprises an exogenous polypeptide. In an embodiment, a genetically modified cell comprises an exogenous nucleic acid sequence, e.g., a sequence, e.g., DNA or RNA, not present in a similar cell that is not genetically modified. In an embodiment, the exogenous nucleic acid sequence is chromosomal, e.g., the exogenous nucleic acid sequence is an exogenous sequence disposed in endogenous chromosomal sequence. In an embodiment, the exogenous nucleic acid sequence is chromosomal or extra chromosomal, e.g., a non-integrated vector. In an embodiment, the exogenous nucleic acid sequence comprises an RNA sequence, e.g., an mRNA. In an embodiment, the exogenous nucleic acid sequence comprises a chromosomal or extra-chromosomal exogenous nucleic acid sequence that comprises a sequence which is expressed as RNA, e.g., mRNA or a regulatory RNA. In an embodiment, the exogenous nucleic acid sequence comprises a chromosomal or extra-chromosomal nucleic acid sequence, which comprises a sequence that encodes a polypeptide, or which is expressed as a polypeptide. In an embodiment, the exogenous nucleic acid sequence comprises a first chromosomal or extra-chromosomal exogenous nucleic acid sequence that modulates the conformation or expression of a second nucleic acid sequence, wherein the second amino acid sequence can be exogenous or endogenous. For example, a genetically modified cell can comprise an exogenous nucleic acid that controls the expression of an endogenous sequence. In an embodiment, a genetically modified cell comprises a polypeptide present at a level or distribution which differs from the level found in a similar cell that has not been genetically modified. In an embodiment, a genetically modified cell comprises an RPE genetically modified to produce an RNA or a polypeptide. For example, a genetically modified cell may comprise an exogenous nucleic acid sequence comprising a chromosomal or extra-chromosomal exogenous nucleic acid sequence that comprises a sequence which is expressed as RNA, e.g., mRNA or a regulatory RNA. In an embodiment, a genetically modified cell (e.g., an RPE cell) comprises an exogenous nucleic acid sequence that comprises a chromosomal or extra-chromosomal nucleic acid sequence comprising a sequence that encodes a polypeptide, or which is expressed as a polypeptide. In an embodiment, the polypeptide is encoded by a codon optimized sequence to achieve higher expression of the polypeptide than a naturally-occurring coding sequence. The codon optimized sequence may be generated using a commercially available algorithm, e.g., GeneOptimizer (ThermoFisher Scientific), OptimumGene™ (GenScript, Piscataway, NJ USA), GeneGPS® (ATUM, Newark, CA USA), or Java Codon Adaptation Tool (JCat, www.jcat.de, Grote, A. et al., Nucleic Acids Research, Vol 33, Issue suppl_2, pp. W526-W531 (2005)). In an embodiment, a genetically modified cell (e.g., an RPE cell) comprises an exogenous nucleic acid sequence that modulates the conformation or expression of an endogenous sequence. In an embodiment, a genetically modified cell (e.g., RPE cell) is cultured from a population of stably-transfected cells, or from a monoclonal cell line.
“Glucocorticoid”, as used herein, means a naturally occurring or synthetic compound (e.g., hormone, small molecule) which binds to the glucocorticoid receptor expressed by mammalian cells (e.g., human cells), which binding results in up-regulation of the expression of anti-inflammatory proteins (e.g. TGF-beta, interleukin (IL)-1 receptor antagonist, IL-4, IL-10, IL-11, IL-13) and down-regulation of the expression of pro-inflammatory proteins (e.g., interferon gamma, granulocyte-macrophage stimulating factor (GM-CSF), IL-1, IL-12, tumor necrosis factor alpha (TNF-, MCP-1). Non-limiting examples of glucocorticoids include triamcinolone and triamcinolone derivatives (e.g., triamcinolone hexacetonide (TAH), triamcinolone acetonide, triamcinolone benetonide, triamcinolone diacetate), fluticasone and fluticasone derivatives (e.g., fluticasone furoate, fluticasone propionate), mometasone and mometasone derivatives (e.g., mometasone furoate), clobetasol and clobetasol derivatives (e.g., clobetasol propionate), beclomethasone and beclomethasone derivatives (e.g., beclomethasone dipropionate), prednisone, prednisolone, methylprednisolone, hydrocortisone, betamethasone, and dexamethasone. In an embodiment, the glucocorticoid is a compound of Formula (IV), as defined herein. In some embodiments, the glucocorticoid is not dexamethasone, prednisone, methylprednisolone, prednisolone, hydrocortisone, or fludrocortisone. In some embodiments, the glucocorticoid is not triamcinolone, triamcinolone acetonide, or clobetasol propionate.
“Heparan-alpha-glucosaminide N-acetyltransferase protein”, “heparan acetyl-CoA: alpha-glucosaminide N-acetyltransferase protein”, “HGSNAT protein”, and “N-acetyltransferase protein” may be used interchangeably herein to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) gene or any fragment, mutant, variant or derivative thereof that has HGSNAT enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature HGSNAT protein, as measured by any art-recognized HGSNAT assay. HGSNAT catalyzes acetylation of the terminal glucosamine residues of intralysosomal heparan or heparan sulfate, converting it into a substrate for hydrolysis by NAGLU. The wild-type human HGSNAT gene encodes a 663 amino acid polypeptide, which includes a predicted signal sequence that is not cleaved upon translocation into the endoplasmic reticulum (UniProtKB—Q68CP4). In some embodiments, the HGSNAT amino acid sequence in a HGSNAT therapeutic protein produced by a device described herein consists essentially of amino acids 1-663 of UniProtKB—Q68CP4.
“Iduronate-2-sulfatase protein”, “IDS protein”, “I2S protein”, and “Alpha-L-iduronate sulfate sulfatase protein” may be used interchangeably herein to refer to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) IDS gene or any fragment, mutant, variant or derivative thereof that has IDS enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature IDS protein, as measured by any art-recognized IDS assay. IDS hydrolyzes the 2-sulfate groups of the L-iduronate 2-sulfate units of dermatan sulfate, heparan sulfate and heparan. The wild-type human IDS gene encodes a 550 amino acid precursor pro-polypeptide, of which the N-terminal 25 amino acids constitute a signal peptide, and the remaining amino acids constitute a pro-polypeptide that is processed into the mature polypeptide by removal of the pro-peptide of amino acids 26-33 and then cleavage into two chains formed by amino acids 34-455 and amino acids 456-550. (UniProtKB—P22304). In some embodiments, the mature human IDS amino acid sequence in an IDS therapeutic protein produced by a device described herein comprises amino acids 34-550 of UniProtKB—P22304 or consists essentially of amino acids 26-550 of the precursor sequence shown in UniProtKB—P22304.
“Immunosuppressant compound” or “immunosuppressant” as used herein, refers to a compound other than a protein that inhibits or prevents an activity of the immune system. Non-limiting examples of immunosuppressants that may be released by devices and compositions described herein include: (i) compounds that act on immunophilins (e.g., cyclosporine, everolimus, rapamycin, tacrolimus, zotarolimus); (ii) corticosteroids, including synthetic glucocorticoids (e.g., TAH, fluticasone furoate, fluticasone propionate, mometasone furioate); and (iii) cytostatics (e.g., azathioprine, mercaptopurine, cyclophosphamide, methotrexate).
“Immunosuppressive cytokine”, as used herein, refers to a naturally-occurring cytokine that exhibits one or more immunosuppressive activities in the regulation of the immune system, as well as variants thereof (e.g., modified amino acid sequence, fusion proteins) that have substantially the same immunosuppressive activities of the naturally-occurring cytokine. In an embodiment, the immunosuppressive cytokine is a cytokine normally expressed by Th2 cells, with non-limiting examples including interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-22 (IL-22), interleukin-25 (IL-25), interleukin-27 (IL-27), interleukin-35 (IL-35) and transforming growth factor-beta (TGF-β). In an embodiment, the immunosuppressive cytokine is IL-10 or IL-22.
“Interleukin-10 protein” and “IL-10 protein”, as used herein, refer to a dimeric protein comprising two, non-covalently joined monomers, which becomes biologically inactive upon disruption of the non-covalent interactions between the two monomers. In some embodiments, each monomer in an IL-10 protein produced by a device described herein comprises an amino acid sequence that is identical to the amino acid sequence of a mammalian IL-10 monomer, e.g., human IL-10. In an embodiment, the IL-10 amino acid sequence in one or both of the monomers is a variant of a mammalian IL-10 amino acid sequence, provided that the resulting homodimer exhibits an activity that is comparable to or greater than the corresponding activity exhibited by the wild-type mammalian IL-10 protein. In an embodiment, a device described herein produces a high affinity variant of hIL-10, e.g., in which each of the monomers comprises amino acids 19-170 shown in
In some embodiments, the cells in a device described herein are genetically modified to encode a precursor IL-10 monomer, which comprises a signal sequence operably linked to the N-terminus of a mammalian mature IL-10 amino acid sequence of 160 amino acids (or a variant thereof) which includes two pairs of cysteine residues that form two intramolecular disulfide bonds. The wild-type human IL-10 (hIL-10) monomer comprises the mature amino acid sequence set forth in
“Interleukin-22 protein” or “IL-22 protein”, as used herein, refers to a protein comprising the amino acid sequence of a mammalian precursor IL-22 or a variant thereof. The active, secreted form of wild-type human IL-22 is a 146 amino acid monomer protein which signals through a heterodimeric receptor comprised of IL-10R2 subunit and IL-22R1 subunit. The wild-type precursor human IL-22 has the 179 amino acid sequence shown in
In some embodiments, the cells in a device described herein are genetically modified to express a precursor IL-22 protein that comprises a signal sequence operably linked to the N-terminus of a mammalian mature IL-22 amino acid sequence (e.g., amino acids 34-179 of
“mTOR inhibitor” as used herein, includes the neutral tricyclic compound rapamycin and other rapamycin compounds, including, e.g., rapamycin derivatives, rapamycin analogues and other macrolide compounds which are thought to have the same mechanism of action (e.g., inhibition of mTOR activity).
“N-acetylgalactosamine-6-sulfatase protein”, “Glucosamine N-acetyl-6-sulfatase protein” and “GNS protein” may be used interchangeably herein to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GNS gene or any fragment, mutant, variant or derivative thereof that has GNS enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature GNS protein, as measured by any art-recognized GNS enzymatic assay. GNS catalyzes the hydrolysis of the 6-sulfate groups of the N-acetyl-D-glucosamine 6-sulfate units of heparan sulfate and keratan sulfate. The wild-type human GNS gene encodes a 552 amino acid precursor polypeptide, of which the N-terminal 36 amino acids constitute a signal peptide (UniProtKB—P15586). In some embodiments, the mature human GNS amino acid sequence in a GNS therapeutic protein produced by a device described herein consists essentially of amino acids 37-552 of the precursor sequence shown in UniProtKB—P15586.
“N-sulfoglucosamine sulfohydrolase”, “SGSH”, “Sulfamidase” and “heparan-N-sulfatase” may be used interchangeably herein to refer to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) SGSH gene or any fragment, mutant, variant or derivative thereof that has enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature SGSH protein, as measured by any art-recognized SGSH assay. SGSH catalyzes the hydrolysis of N-sulfo-D-glucosamine into D-glucosamine and sulfate. The wild-type human SGSH gene encodes a 502 amino acid precursor polypeptide, of which the N-terminal 20 amino acids constitute a signal peptide (UniProtKB—P51688). In some embodiments, the mature human SGSH amino acid sequence in an SGSH therapeutic protein produced by a device described herein consists essentially of amino acids 21-502 of the precursor sequence shown in UniProtKB—P51688.
“Peptide”, as used herein, is a polypeptide of less than 50 amino acids, typically, less than 25 amino acids.
“Polymer composition”, as used herein, is a composition (e.g., a solution, mixture) comprising one or more polymers. As a class, “polymers’ includes homopolymers, heteropolymers, co-polymers, block polymers, block co-polymers and can be both natural and synthetic. Homopolymers contain one type of building block, or monomer, whereas co-polymers contain more than one type of monomer.
“Polypeptide”, as used herein, is a polymer comprising amino acid residues linked through peptide bonds and having at least two, and in some embodiments, at least 10, 50, 75, 100, 150 or 200 amino acid residues.
“Prevention,” “prevent,” and “preventing”, as used herein, refer to a treatment that comprises administering or applying a therapy, e.g., administering a composition of devices encapsulating cells (e.g., as described herein), prior to the onset of a disease, disorder, or condition to preclude the physical manifestation of said disease, disorder, or condition. In some embodiments, “prevention,” “prevent,” and “preventing” require that signs or symptoms of the disease, disorder, or condition have not yet developed or have not yet been observed. In some embodiments, treatment comprises prevention and in other embodiments it does not.
“Protein”, as used herein, comprises one or more polypeptide chains of at least 50 amino acids in length. In an embodiment, a protein has two or more polypeptide chains have identical or non-identical amino acid sequences of at least 50 amino acids in length. In an embodiment, the polypeptide chains in a protein are noncovalently associated or covalently joined, e.g., via disulfide bond(s).
“RPE cell”, as used herein, refers to a cell having one or more of the following characteristics: a) it comprises a retinal pigment epithelial cell (RPE) (e.g., cultured using the ARPE-19 cell line (ATCC® CRL-2302™)) or a cell derived therefrom, e.g., by stably transfecting cells cultured from the ARPE-19 cell line with an exogenous sequence that encodes an immunomodulatory protein or otherwise engineering such cultured ARPE-19 cells to express an exogenous protein or other exogenous substance, a cell derived from a primary cell culture of RPE cells, a cell isolated directly (without long term culturing, e.g., less than 5 or 10 passages or rounds of cell division since isolation) from naturally occurring RPE cells, e.g., from a human or other mammal, a cell derived from a transformed, an immortalized, or a long term (e.g., more than 5 or 10 passages or rounds of cell division) RPE cell culture; b) a cell that has been obtained from a less differentiated cell, e.g., a cell developed, programmed, or reprogramed (e.g., in vitro) into an RPE cell or a cell that is, except for any genetic engineering, substantially similar to one or more of a naturally occurring RPE cell or a cell from a primary or long term culture of RPE cells (e.g., the cell can be derived from an IPS cell); or c) a cell that has one or more of the following properties: i) it expresses one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; ii) it does not express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; iii) it is naturally found in the retina and forms a monolayer above the choroidal blood vessels in the Bruch's membrane; iv) it is responsible for epithelial transport, light absorption, secretion, and immune modulation in the retina; or v) it has been created synthetically, or modified from a naturally occurring cell, to have the same or substantially the same genetic content, and optionally the same or substantially the same epigenetic content, as an immortalized RPE cell line (e.g., the ARPE-19 cell line (ATCC® CRL-2302™)). In an embodiment, an RPE cell described herein is genetically modified, e.g., to have a new property, e.g., the cell is genetically modified to express and secrete one or more immunomodulatory proteins or an immunomodulatory agent. In other embodiments, an RPE cell is not genetically modified.
“Sequence identity” or “percent identical”, when used herein to refer to two nucleotide sequences or two amino acid sequences, means the two sequences are the same within a specified region, or have the same nucleotides or amino acids at a specified percentage of nucleotide or amino acid positions within the specified when the two sequences are compared and aligned for maximum correspondence over a comparison window or designated region. Sequence identity may be determined using standard techniques known in the art including, but not limited to, any of the algorithms described in US Patent Application Publication No. 2017/02334455. In an embodiment, the specified percentage of identical nucleotide or amino acid positions is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.
“Spherical” as used herein, means a device (e.g., a hydrogel capsule or other particle) having a curved surface that forms a sphere (e.g., a completely round ball) or sphere-like shape, which may have waves and undulations, e.g., on the surface. Spheres and sphere-like objects can be mathematically defined by rotation of circles, ellipses, or a combination around each of the three perpendicular axes, a, b, and c. For a sphere, the three axes are the same length. Generally, a sphere-like shape is an ellipsoid (for its averaged surface) with semi-principal axes within 10%, or 5%, or 2.5% of each other. The diameter of a sphere or sphere-like shape is the average diameter, such as the average of the semi-principal axes.
“Spheroid”, as that term is used herein to refer to a device (e.g., a hydrogel capsule or other particle), means the device has (i) a perfect or classical oblate spheroid or prolate spheroid shape or (ii) has a surface that roughly forms a spheroid, e.g., may have waves and undulations and/or may be an ellipsoid (for its averaged surface) with semi-principal axes within 100% of each other.
“Subject” as used herein refers to a human or non-human animal. In an embodiment, the subject is a human (i.e., a male or female), e.g., of any age group, a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult). In an embodiment, the subject is a non-human animal, for example, a mammal (e.g., a mouse, a dog, a primate (e.g., a cynomolgus monkey or a rhesus monkey)). In an embodiment, the subject is a commercially relevant mammal (e.g., a cattle, pig, horse, sheep, goat, cat, or dog) or a bird (e.g., a commercially relevant bird such as a chicken, duck, goose, or turkey). In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal.
“Treatment,” “treat,” and “treating” as used herein refers to one or more of reducing, reversing, alleviating, delaying the onset of, or inhibiting the progress of one or more of a symptom, manifestation, or underlying cause, of a disease, disorder, or condition. In an embodiment, treating comprises reducing, reversing, alleviating, delaying the onset of, or inhibiting the progress of a symptom of a disease, disorder, or condition. In an embodiment, treating comprises reducing, reversing, alleviating, delaying the onset of, or inhibiting the progress of a manifestation of a disease, disorder, or condition. In an embodiment, treating comprises reducing, reversing, alleviating, reducing, or delaying the onset of, an underlying cause of a disease, disorder, or condition. In some embodiments, “treatment,” “treat,” and “treating” require that signs or symptoms of the disease, disorder, or condition have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition, e.g., in preventive treatment. For example, a therapy (e.g., a device composition) may be administered to a susceptible individual prior to the onset of symptoms (e.g., considering a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. In some embodiments, treatment comprises prevention and in other embodiments it does not.
Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.
When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, “C1-C6 alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.
As used herein, “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 24 carbon atoms (“C1-C24 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1-C12 alkyl”), 1 to 10 carbon atoms (“C1-C12 alkyl”), 1 to 8 carbon atoms (“C1-C8 alkyl”), 1 to 6 carbon atoms (“C1-C6 alkyl”), 1 to 5 carbon atoms (“C1-C5 alkyl”), 1 to 4 carbon atoms (“C1-C4alkyl”), 1 to 3 carbon atoms (“C1-C3 alkyl”), 1 to 2 carbon atoms (“C1-C2 alkyl”), or 1 carbon atom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-C6alkyl”). Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. Each instance of an alkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents; e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
As used herein, “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C2-C24 alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2-C10 alkenyl”), 2 to 8 carbon atoms (“C2-C5 alkenyl”), 2 to 6 carbon atoms (“C2-C6 alkenyl”), 2 to 5 carbon atoms (“C2-C5 alkenyl”), 2 to 4 carbon atoms (“C2-C4 alkenyl”), 2 to 3 carbon atoms (“C2-C3 alkenyl”), or 2 carbon atoms (“C2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Each instance of an alkenyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
As used herein, the term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon triple bonds (“C2-C24 alkenyl”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2-C10 alkynyl”), 2 to 8 carbon atoms (“C2-C8 alkynyl”), 2 to 6 carbon atoms (“C2-C6 alkynyl”), 2 to 5 carbon atoms (“C2-C5 alkynyl”), 2 to 4 carbon atoms (“C2-C4 alkynyl”), 2 to 3 carbon atoms (“C2-C3 alkynyl”), or 2 carbon atoms (“C2 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-C4 alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Each instance of an alkynyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
As used herein, the term “heteroalkyl,” refers to a non-cyclic stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any position of the heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to: —CH2—CH2—O—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2, —S(O)—CH3, —CH2—CH2—S(O)2—CH3, —CH═CH—O—CH3, —Si(CH3)3, —CH2—CH═N—OCH3, —CH═CH—N(CH3)—CH3, —O—CH3, and —O—CH2—CH3. Up to two or three heteroatoms may be consecutive, such as, for example, —CH2—NH—OCH3 and —CH2—O—Si(CH3)3. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as —CH2O, —NRCRD, or the like, it will be understood that the terms heteroalkyl and —CH2O or —NRCRD are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as —CH2O, —NRCRD, or the like. Each instance of a heteroalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
The terms “alkylene,” “alkenylene,” “alkynylene,” or “heteroalkylene,” alone or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, or heteroalkyl, respectively. An alkylene, alkenylene, alkynylene, or heteroalkylene group may be described as, e.g., a C1-C6-membered alkylene, C2-C6-membered alkenylene, C2-C6-membered alkynylene, or C1-C6-membered heteroalkylene, wherein the term “membered” refers to the non-hydrogen atoms within the moiety. In the case of heteroalkylene groups, heteroatoms can also occupy either or both chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula-C(O)2R′— may represent both —C(O)2R′— and —R′C(O)2—.
As used herein, “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic)4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl). An aryl group may be described as, e.g., a C6-C10-membered aryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
As used herein, “heteroaryl” refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). A heteroaryl group may be described as, e.g., a 6-10-membered heteroaryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety.
In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Each instance of a heteroaryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents.
Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other exemplary heteroaryl groups include heme and heme derivatives.
As used herein, the terms “arylene” and “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.
As used herein, “cycloalkyl” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-C10 cycloalkyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-C8cycloalkyl”), 3 to 6 ring carbon atoms (“C3-C6 cycloalkyl”), or 5 to 10 ring carbon atoms (“C5-C10 cycloalkyl”). A cycloalkyl group may be described as, e.g., a C4-C7-membered cycloalkyl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Exemplary C3-C6 cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-C8 cycloalkyl groups include, without limitation, the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C9), bicyclo[1.1.1]pentanyl (C8), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo[3.1.1]heptanyl (C7), and the like. Exemplary C3-C10 cycloalkyl groups include, without limitation, the aforementioned C3-C8 cycloalkyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the cycloalkyl group is either monocyclic (“monocyclic cycloalkyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic cycloalkyl”) and can be saturated or can be partially unsaturated. “Cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.
“Heterocyclyl” as used herein refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety. Each instance of heterocyclyl may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 membered heterocyclyl.
In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.
Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl or thiomorpholinyl-1,1-dioxide. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as a 5,6-bicyclic heterocyclic ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
“Amino” as used herein refers to the radical —NR70R71, wherein R70 and R71 are each independently hydrogen, C1-C8 alkyl, C3-C10 cycloalkyl, C4-C10 heterocyclyl, C6-C10 aryl, and C5-C10 heteroaryl. In some embodiments, amino refers to NH2.
As used herein, “cyano” refers to the radical-CN.
As used herein, “halo” or “halogen,” independently or as part of another substituent, mean, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.
As used herein, “hydroxy” refers to the radical-OH.
Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” cycloalkyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted”, whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, such as any of the substituents described herein that result in the formation of a stable compound. The present disclosure contemplates any and all such combinations to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocyclyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.
Compounds of Formula (I) described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
As used herein, a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, an “S” form of the compound is substantially free from the “R” form of the compound and is, thus, in enantiomeric excess of the “R” form. The term “enantiomerically pure” or “pure enantiomer” denotes that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight, of the enantiomer. In certain embodiments, the weights are based upon total weight of all enantiomers or stereoisomers of the compound.
Compounds of Formula (I) described herein may also comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D or deuterium), and 3H (T or tritium); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; and the like.
The term “pharmaceutically acceptable salt” is meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of Formula (I) used to prepare devices of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds used in the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galacturonic acids and the like (see, e.g., Berge et al, Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds used in the devices of the present disclosure (e.g., a particle, a hydrogel capsule) contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. These salts may be prepared by methods known to those skilled in the art. Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for use in the present disclosure.
Devices of the present disclosure may contain a compound of Formula (I) in a prodrug form. Prodrugs are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds useful for preparing devices in the present disclosure. Additionally, prodrugs can be converted to useful compounds of Formula (I) by chemical or biochemical methods in an ex vivo environment.
Certain compounds of Formula (I) described herein can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of Formula (I) described herein may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.
The term “solvate” refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, e.g., in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates.
The term “hydrate” refers to a compound which is associated with water. Typically, the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R·x H2O, wherein R is the compound and wherein x is a number greater than 0.
The term “tautomer” as used herein refers to compounds that are interchangeable forms of a compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of π electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.
The symbol “” as used herein refers to a connection to an entity, e.g., a polymer (e.g., hydrogel-forming polymer such as alginate) or surface of an implantable device, e.g., a particle, a hydrogel capsule. The connection represented by “
” may refer to direct attachment to the entity, e.g., a polymer or an implantable element, may refer to linkage to the entity through an attachment group. An “attachment group,” as described herein, refers to a moiety for linkage of a compound of Formula (I) to an entity (e.g., a polymer or an implantable element (e.g., a device) as described herein), and may comprise any attachment chemistry known in the art. A listing of exemplary attachment groups is outlined in Bioconjugate Techniques (3rd ed, Greg T. Hermanson, Waltham, MA: Elsevier, Inc, 2013), which is incorporated herein by reference in its entirety. In some embodiments, an attachment group comprises alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, —C(O)—, —OC(O)—, —N(RC)—, —N(RC)C(O)—, —C(O)N(RC)—, —N(RC)N(RD), —NCN—, —C(═N(RC)(RD))O—, —S—, —S(O)x—, —OS(O)x—, —N(RC)S(O)—, —S(O)xN(RC)—, —P(RF)y—, —Si(ORA)2—, —Si(RG)(ORA)—, —B(ORA)—, or a metal, wherein each of RA, RC, RD, RF, RG, x and y is independently as described herein. In some embodiments, an attachment group comprises an amine, ketone, ester, amide, alkyl, alkenyl, alkynyl, or thiol. In some embodiments, an attachment group is a cross-linker. In some embodiments, the attachment group is —C(O)(C1-C6-alkylene)-, wherein alkylene is substituted with R1, and R1 is as described herein. In some embodiments, the attachment group is —C(O)(C1-C6-alkylene)-, wherein alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the attachment group is —C(O)C(CH3)2—. In some embodiments, the attachment group is —C(O)(methylene)-, wherein alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the attachment group is —C(O)CH(CH3)—. In some embodiments, the attachment group is —C(O)C(CH3)—.
Devices of the present disclosure contain (e.g., encapsulate) cells genetically modified to express and secrete at least one antigen. In an embodiment, the antigen(s) to be expressed and secreted is chosen based on an established or probable etiology of triggering an autoimmune or allergic disease or condition. In an embodiment, the antigen is a protein, polypeptide or peptide (e.g., as described in Table 5 below). In an embodiment, the device also contains cells genetically modified to express and secrete an immunomodulatory protein, e.g., an immunosuppressive cytokine. The cells may be in the form of single cells, or provided in another form, e.g., disposed on a microcarrier (e.g., a bead or matrix) or as one or more three-dimensional aggregates of cells (e.g., one or more cell clusters).
The genetically modified cell(s) may be derived from a variety of different cell types (e.g., human cells), including adipose cells, epidermal cells, epithelial cells, endothelial cells, fibroblast cells, islet cells, mesenchymal stem cells, keratinocyte cells, pericytes, subtypes of any of the foregoing, cells derived from any of the foregoing, cells derived from induced pluripotent stem cells and mixtures of any of the foregoing. Exemplary cell types include the cell types recited in WO 2017/075631. In some embodiments, the cells are derived from a cell-line shown in Table 2 below.
In some embodiments, the device does not comprise any islet cells or any cells capable of producing insulin in a glucose-responsive manner. In an embodiment, cells contained in a device of the disclosure, e.g., genetically modified RPE cells, have one or more of the following characteristics: (i) are not capable of producing insulin (e.g., insulin A-chain, insulin B-chain, or proinsulin) in an amount effective to treat diabetes or another disease or condition that may be treated with insulin; (ii) not capable of producing insulin in a glucose-responsive manner; or (iii) not derived from an induced pluripotent stem cell that was engineered or differentiated into insulin-producing pancreatic beta cells.
Cells may be genetically modified to express and secrete antigens and any immunomodulatory proteins using any of a variety of genetic engineering techniques are known in the art. For example, a cell may be transfected with an expression vector comprising an exogenous nucleotide sequence(s) encoding the desired protein(s) operably linked to control elements necessary or useful for gene expression, promoters, ribosomal binding sites, enhancers, polyA signal. Typically, the exogenous sequence encodes a precursor form of an antigen polypeptide or immunomodulatory protein, e.g., includes a secretory signal sequence. In some embodiments, the signal sequence consists essentially of an amino acid sequence shown in Table 3 below. In an embodiment, the signal sequence is MGWRAAGALLLALLLHGRLLA (SEQ ID NO: 20).
When engineering cells to co-express two or more antigen polypeptides or immunomodulatory proteins, a multicistronic vector may be employed. In some embodiments, a genetically modified cell comprises an exogenous nucleotide sequence, which encodes an antigen or an immunomodulatory protein described herein, that is stably inserted into one or more genomic locations (e.g., open chromatin region(s)).
In some embodiments, the cells are genetically modified with a regulatable expression system, to allow controlled expression of the antigens and any immunomodulatory proteins. A variety of such systems are known in the art and include, for example: kill switches (see, e.g., Wu, C. et al., Mol. Ther. Methods Clin Dev. 2014; 1:14053); On/Off systems (see, e.g., Gossen, M and Gujar, H., Proc. Natl. Acad. Sci. USA, Vol 89, pp. 5547-5551 (1992); Liberles, S., et al., Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 7825-7830 (1997); feed forward and negative feedback systems (Lillacci, G. et al., Nuc. Acids Res, Vol 46, Issue 18 (12 Oct. 2018) pp. 9855-9863); temperature inducible systems (Miller, I. et al., ACS Synth Biol. 2018; 7 (4): 1167-1173).
In some embodiments, a genetically modified cell described herein is derived from an ARPE-19 cell, and may express and secrete any of the antigen polypeptides and immunomodulatory proteins described herein (or combinations of such molecules).
In an embodiment, a genetically modified cell described herein (e.g., derived from an ARPE-19 cell) comprises any of the coding sequences described herein, e.g., any of the coding sequences shown in
A device of the present disclosure comprises at least one barrier that prevents immune cells from contacting cells contained inside the device. At least a portion of the barrier needs to be sufficiently porous to allow proteins (e.g., antigens and any immunomodulatory protein(s)) expressed and secreted by the cells to exit the device. A variety of device configurations known in the art are suitable.
The device (e.g., particle) can have any configuration and shape appropriate for supporting the viability and productivity of the contained cells after implant into the intended target location. As non-limiting examples, device shapes may be cylinders, rectangles, disks, ovoids, stellates, or spherical. The device can be comprised of a mesh-like or nested structure. In some embodiments, a device is capable of preventing materials over a certain size from passing through a pore or opening. In some embodiments, a device (e.g., particle) is capable of preventing materials greater than 50 kD, 75 kD, 100 kD, 125 kD, 150 kD, 175 kD, 200 kD, 250 kD, 300 kD, 400 kD, 500 kD, 750 kD, or 1,000 kD from passing through.
In an embodiment, the device is a macroencapsulation device. Nonlimiting examples of macrodevices are described in: WO 2019/068059, WO 2019/169089, U.S. Pat. Nos. 9,526,880, 9,724,430 and 8,278,106; European Patent No. EP742818B1, and Sang, S. and Roy, S., Biotechnol. Bioeng. 113 (7): 1381-1402 (2016).
In an embodiment, the device is a macrodevice having one or more cell-containing compartments. A device with two or more cell-containing compartments may be configured to produce two or more antigens and/or immunomodulatory proteins, e.g., cells expressing a first antigen or immunomodulatory protein would be placed in one compartment and cells expressing a second antigen or immunomodulatory protein would be placed in a separate compartment. WO 2018/232027 describes a device with multiple cell-containing compartments formed in a micro-fabricated body and covered by a porous membrane.
In an embodiment, the device is configured as a thin, flexible strand as described in U.S. Pat. No. 10,493,107. This strand comprises a substrate, an inner polymeric coating surrounding the substrate and an outer hydrogel coating surrounding the inner polymeric coating. The protein-expressing cells are positioned in the outer coating.
In some embodiments, a device (e.g., particle) has a largest linear dimension (LLD), e.g., mean diameter, or size that is at least about 0.5 millimeter (mm), preferably about 1.0 mm, about 1.5 mm or greater. In some embodiments, a device can be as large as 10 mm in diameter or size. For example, a device or particle described herein is in a size range of 0.5 mm to 10 mm, 1 mm to 10 mm, 1 mm to 8 mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm, 1 mm to 1.5 mm, 1.5 mm to 8 mm, 1.5 mm to 6 mm, 1.5 mm to 5 mm, 1.5 mm to 4 mm, 1.5 mm to 3 mm, 1.5 mm to 2 mm, 2 mm to 8 mm, 2 mm to 7 mm, 2 mm to 6 mm, 2 mm to 5 mm, 2 mm to 4 mm, 2 mm to 3 mm, 2.5 mm to 8 mm, 2.5 mm to 7 mm, 2.5 mm to 6 mm, 2.5 mm to 5 mm, 2.5 mm to 4 mm, 2.5 mm to 3 mm, 3 mm to 8 mm, 3 mm to 7 mm, 3 mm to 6 mm, 3 mm to 5 mm, 3 mm to 4 mm, 3.5 mm to 8 mm, 3.5 mm to 7 mm, 3.5 mm to 6 mm, 3.5 mm to 5 mm, 3.5 mm to 4 mm, 4 mm to 8 mm, 4 mm to 7 mm, 4 mm to 6 mm, 4 mm to 5 mm, 4.5 mm to 8 mm, 4.5 mm to 7 mm, 4.5 mm to 6 mm, 4.5 mm to 5 mm, 5 mm to 8 mm, 5 mm to 7 mm, 5 mm to 6 mm, 5.5 mm to 8 mm, 5.5 mm to 7 mm, 5.5 mm to 6 mm, 6 mm to 8 mm, 6 mm to 7 mm, 6.5 mm to 8 mm, 6.5 mm to 7 mm, 7 mm to 8 mm, or 7.5 mm to 8 mm.
In some embodiments, a device of the disclosure (e.g., particle, capsule) comprises at least one pore or opening, e.g., to allow for the free flow of materials. In some embodiments, the mean pore size of a device is between about 0.1 μm to about 10 μm. For example, the mean pore size may be between 0.1 μm to 10 μm, 0.1 μm to 5 μm, 0.1 μm to 2 μm, 0.15 μm to 10 μm, 0.15 μm to 5 μm, 0.15 μm to 2 μm, 0.2 μm to 10 μm, 0.2 μm to 5 μm, 0.25 μm to 10 μm, 0.25 μm to 5 μm, 0.5 μm to 10 μm, 0.75 μm to 10 μm, 1 μm to 10 μm, 1 μm to 5 μm, 1 μm to 2 μm, 2 μm to 10 μm, 2 μm to 5 μm, or 5 μm to 10 μm. In some embodiments, the mean pore size of a device is between about 0.1 μm to 10 μm. In some embodiments, the mean pore size of a device is between about 0.1 μm to 5 μm. In some embodiments, the mean pore size of a device is between about 0.1 μm to 1 μm.
In some embodiments, the device comprises a semi-permeable, biocompatible membrane surrounding the genetically modified cells that are encapsulated in a polymer composition (e.g., an alginate hydrogel). The membrane pore size is selected to allow oxygen and other molecules important to cell survival and function to move through the semi-permeable membrane while preventing immune cells from traversing through the pores. In an embodiment, the semi-permeable membrane has a molecular weight cutoff of less than 1000 kD or between 50-700 kD, 70-300 kD, or between 70-150 kD, or between 70 and 130 kD.
In an embodiment, the device may contain a cell-containing compartment that is surrounded with a barrier compartment formed from a cell-free biocompatible material, such as the core-shell microcapsules described in Ma, M et al., Adv. Healthc Mater., 2 (5): 667-672 (2012). Such a barrier compartment could be used with or without the semi-permeable membrane.
Cells in the cell-containing compartment(s) of a device of the disclosure may be encapsulated in a polymer composition. The polymer composition may comprise one or more hydrogel-forming polymers. In some embodiments, the polymer composition in the cell-containing compartment(s) may comprise a cell-binding substance, e.g., a cell-binding peptide described in WO 202/069429. In some embodiments the polymer composition comprises an alginate covalently modified with a peptide of less than 10 amino acids or less comprising RGD, e.g., GRGDSP (SEQ ID NO:34). In addition to the polymer composition in the cell-containing compartment(s), the device (e.g., macrodevice, particle, hydrogel capsule) may comprise or be formed from materials such as metals, metallic alloys, ceramics, polymers, fibers, inert materials, and combinations thereof. A device may be completely made up of one type of material, or may comprise other materials within the cell-containing compartment and any other compartments.
In some embodiments, the device comprises a metal or a metallic alloy. In an embodiment, one or more of the compartments in the device (e.g., the first compartment, the second compartment, or all compartments) comprises a metal or a metallic alloy. Exemplary metallic or metallic alloys include comprising titanium and titanium group alloys (e.g., nitinol, nickel titanium alloys, thermo-memory alloy materials), platinum, platinum group alloys, stainless steel, tantalum, palladium, zirconium, niobium, molybdenum, nickel-chrome, chromium molybdenum alloys, or certain cobalt alloys (e.g., cobalt-chromium and cobalt-chromium-nickel alloys, e.g., ELGILOY® and PHYNOX®). For example, a metallic material may be stainless steel grade 316 (SS 316L) (comprised of Fe, <0.3% C, 16-18.5% Cr, 10-14% Ni, 2-3% Mo, <2% Mn, <1% Si, <0.45% P, and <0.03% S). In metal-containing devices, the amount of metal (e.g., by % weight, actual weight) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w/w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.
In some embodiments, the device comprises a ceramic. In an embodiment, one or more of the compartments in the device (e.g., the first compartment, the second compartment, or all compartments) comprises a ceramic. Exemplary ceramic materials include oxides, carbides, or nitrides of the transition elements, such as titanium oxides, hafnium oxides, iridium oxides, chromium oxides, aluminum oxides, and zirconium oxides. Silicon based materials, such as silica, may also be used. In ceramic-containing devices, the amount of ceramic (e.g., by % weight, actual weight) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w/w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.
In some embodiments, the device has two hydrogel compartments, in which the inner, cell-containing compartment is completely surrounded by the second, outer (e.g., barrier) compartment. In an embodiment, the inner boundary of the second compartment forms an interface with the outer boundary of the first compartment. In such embodiments, the thickness of the second (outer) compartment means the average distance between the outer boundary of the second compartment and the interface between the two compartments, e.g., the average of the distances measured at each of the thinnest and thickest points visually observed in the outer compartment. In some embodiments (e.g., the device is about 1.5 mm in diameter), the thinnest and thickest distances for the outer compartment are between 25 and 110 micrometers (μm) and between 270 and 480 μm, respectively. In some embodiments, the thickness of the outer compartment is greater than about 10 nanometers (nm), preferably 100 nm or greater and can be as large as 1 millimeter (mm). For example, the thickness (e.g., average distance) of the outer compartment in a hydrogel capsule device described herein may be 10 nm to 1 mm, 100 nm to 1 mm, 500 nm to 1 millimeter, 1 micrometer (μm) to 1 mm, 1 μm to 1 mm, 1 μm to 500 μm, 1 μm to 250 μm, 1 μm to 1 mm, 5 μm to 500 μm, 5 μm to 250 μm, 10 μm to 1 mm, 10 μm to 500 μm, or 10 μm to 250 μm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is 100 nm to 1 mm, between 1 μm and 1 mm, between 1 μm and 500 μm or between 5 μm and 1 mm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is between about 50 μm and about 100 μm. In some embodiments (e.g., the device is about 1.5 mm in diameter), the thickness of the outer compartment (e.g., average distance) is between about 180 μm and 260 μm or between about 310 μm and 440 μm.
In some embodiments of a two-compartment hydrogel capsule device, the mean pore size of the cell-containing inner compartment and the outer compartment is substantially the same. In some embodiments, the mean pore size of the inner compartment and the second compartment differ by about 1.5%, 2%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some embodiments, the mean pore size of the device (e.g., mean pore size of the first compartment and/or mean pore size of the second compartment) is dependent on a number of factors, such as the material(s) within each compartment and the presence and density of a compound of Formula (I).
The device may form part of a plurality of substantially the same devices in a preparation (e.g., composition). In some embodiments, the devices (e.g., particles, hydrogel capsules) in the preparation have a mean diameter or size between about 0.5 mm to about 8 mm. In some embodiments, the mean diameter or size of devices in the preparation is between about 0.5 mm to about 4 mm or between about 0.5 mm to about 2 mm. In some embodiments, the devices in the preparation are two-compartment hydrogel capsules and have a mean diameter or size of about 0.7 mm to about 1.3 mm or about 1.2 mm to about 1.8 mm.
In some embodiments, the surface of the device comprises a compound capable of mitigating the FBR, an afibrotic compound as described herein below. For devices comprising a barrier compartment surrounding the cell-containing compartment, the afibrotic compound may covalently modify a polymer disposed throughout the barrier compartment and optionally throughout the cell-containing compartment.
In some embodiments, one or more compartments in a device comprises an afibrotic polymer, e.g., an afibrotic compound of Formula (I) covalently attached to a polymer. In an embodiment, some or all the monomers in the afibrotic polymer are modified with the same compound of Formula (I). In some embodiments, some or all the monomers in the afibrotic polymer are modified with different compounds of Formula (I). In some embodiments in which the device is a 2-compartment hydrogel capsule, the afibrotic polymer is present only in the outer, barrier compartment.
One or more compartments in a device may comprise an unmodified polymer that is the same or different than the polymer in any afibrotic polymer that is present in the device. In an embodiment, the first compartment, second compartment or all compartments in the device comprise the unmodified polymer.
Each of the modified and unmodified polymers in the device may be a linear, branched, or cross-linked polymer, or a polymer of selected molecular weight ranges, degree of polymerization, viscosity or melt flow rate. Branched polymers can include one or more of the following types: star polymers, comb polymers, brush polymers, dendronized polymers, ladders, and dendrimers. A polymer may be a thermoresponsive polymer, e.g., gel (e.g., becomes a solid or liquid upon exposure to heat or a certain temperature) or a photocrosslinkable polymer. Exemplary polymers include polystyrene, polyethylene, polypropylene, polyacetylene, poly (vinyl chloride) (PVC), polyolefin copolymers, poly (urethane) s, polyacrylates and polymethacrylates, polyacrylamides and polymethacrylamides, poly (methyl methacrylate), poly (2-hydroxyethyl methacrylate), polyesters, polysiloxanes, polydimethylsiloxane (PDMS), polyethers, poly (orthoester), poly (carbonates), poly (hydroxyalkanoate) s, polyfluorocarbons, PEEK®, Teflon® (polytetrafluoroethylene, PTFE), PEEK, silicones, epoxy resins, Kevlar®, Dacron® (a condensation polymer obtained from ethylene glycol and terephthalic acid), polyethylene glycol, nylon, polyalkenes, phenolic resins, natural and synthetic elastomers, adhesives and sealants, polyolefins, polysulfones, polyacrylonitrile, biopolymers such as polysaccharides and natural latex, collagen, cellulosic polymers (e.g., alkyl celluloses, etc.), polyethylene glycol and 2-hydroxyethyl methacrylate (HEMA), polysaccharides, poly (glycolic acid), poly (L-lactic acid) (PLLA), poly (lactic glycolic acid) (PLGA), a polydioxanone (PDA), or racemic poly (lactic acid), polycarbonates, (e.g., polyamides (e.g., nylon)), fluoroplastics, carbon fiber, agarose, alginate, chitosan, and blends or copolymers thereof. In polymer-containing devices, the amount of a polymer (e.g., by % weight of the device, actual weight of the polymer) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w/w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.
In some embodiments, one or more of the modified and unmodified polymers in the device comprises a polyethylene. Exemplary polyethylenes include ultra-low-density polyethylene (ULDPE) (e.g., with polymers with densities ranging from 0.890 to 0.905 g/cm3, containing comonomer); very-low-density polyethylene (VLDPE) (e.g., with polymers with densities ranging from 0.905 to 0.915 g/cm3, containing comonomer); linear low-density polyethylene (LLDPE) (e.g., with polymers with densities ranging from 0.915 to 0.935 g/cm3, contains comonomer); low-density polyethylene (LDPE) (e.g., with polymers with densities ranging from about 0.915 to 0.935 g/m3); medium density polyethylene (MDPE) (e.g., with polymers with densities ranging from 0.926 to 0.940 g/cm3, may or may not contain comonomer); high-density polyethylene (HDPE) (e.g., with polymers with densities ranging from 0.940 to 0.970 g/cm3, may or may not contain comonomer) and polyethylene glycol.
In some embodiments, one or more of the modified and unmodified polymers in the device comprises a polypropylene. Exemplary polypropylenes include homopolymers, random copolymers (homophasic copolymers), and impact copolymers (heterophasic copolymers), e.g., as described in McKeen, Handbook of Polymer Applications in Medicine and Medical Devices, 3-Plastics Used in Medical Devices, (2014): 21-53.
In some embodiments, one or more of the modified and unmodified polymers in the device comprises a polypropylene. Exemplary polystyrenes include general purpose or crystal (PS or GPPS), high impact (HIPS), and syndiotactic (SPS) polystyrene.
In some embodiments, one or more of the modified and unmodified polymers comprises a comprises a thermoplastic elastomer (TPE). Exemplary TPEs include (i) TPA—polyamide TPE, comprising a block copolymer of alternating hard and soft segments with amide chemical linkages in the hard blocks and ether and/or ester linkages in the soft blocks; (ii) TPC—co-polyester TPE, consisting of a block copolymer of alternating hard segments and soft segments, the chemical linkages in the main chain being ester and/or ether; (iii) TPO—olefinic TPE, consisting of a blend of a polyolefin and a conventional rubber, the rubber phase in the blend having little or no cross-linking; (iv) TPS—styrenic TPE, consisting of at least a triblock copolymer of styrene and a specific diene, where the two end blocks (hard blocks) are polystyrene and the internal block (soft block or blocks) is a polydiene or hydrogenated polydiene; (v) TPU—urethane TPE, consisting of a block copolymer of alternating hard and soft segments with urethane chemical linkages in the hard blocks and ether, ester or carbonate linkages or mixtures of them in the soft blocks; (vi) TPV—thermoplastic rubber vulcanizate consisting of a blend of a thermoplastic material and a conventional rubber in which the rubber has been cross-linked by the process of dynamic vulcanization during the blending and mixing step; and (vii) TPZ—unclassified TPE comprising any composition or structure other than those grouped in TPA, TPC, TPO, TPS, TPU, and TPV.
In some embodiments, the unmodified polymer is an unmodified alginate. In some embodiments, the alginate is a high guluronic acid (G) alginate, and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more guluronic acid (G). In some embodiments, the alginate is a high mannuronic acid (M) alginate, and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more mannuronic acid (M). In some embodiments, the ratio of M:G is about 1. In some embodiments, the ratio of M:G is less than 1. In some embodiments, the ratio of M:G is greater than 1. In an embodiment, the unmodified alginate has a molecular weight of 150 kDa-250 kDa and a G:M ratio of ≥1.5.
In some embodiments, the afibrotic polymer comprises an alginate chemically modified with a Compound of Formula (I). The alginate in the afibrotic polymer may be the same or different than any unmodified alginate that is present in the device. In an embodiment, the density of the Compound of Formula (I) in the afibrotic alginate (e.g., amount of conjugation) is between about 4.0% and about 8.0%, between about 5.0% and about 7.0%, or between about 6.0% and about 7.0% nitrogen (e.g., as determined by combustion analysis for percent nitrogen). In an embodiment, the amount of Compound 101 produces an increase in % N (as compared with the unmodified alginate) of about 0.5% to 2% 2% to 4% N, about 4% to 6% N, about 6% to 8%, or about 8% to 10% N), where % N is determined by combustion analysis and corresponds to the amount of Compound 101 in the modified alginate.
In other embodiments, the density (e.g., concentration) of the Compound of Formula (I) (e.g., Compound 101) in the afibrotic alginate is defined as the % w/w, e.g., % of weight of amine/weight of afibrotic alginate in solution (e.g., saline) as determined by a suitable quantitative amine conjugation assay (e.g. by an assay described in WO2020069429), and in certain embodiments, the density of a Compound of Formula (I) (e.g., Compound 101) is between about 1.0% w/w and about 3.0% w/w, between about 1.3% w/w and about 2.5% w/w or between about 1.5% w/w and 2.2% w/w.
In alginate-containing devices, the amount of modified and unmodified alginates (e.g., by % weight of the device, actual weight of the alginate) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w/w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.
The alginate in an afibrotic polymer can be chemically modified with a compound of Formula (I) using any suitable method known in the art. For example, the alginate carboxylic acid moiety can be activated for coupling to one or more amine-functionalized compounds to achieve an alginate modified with a compound of Formula (I). The alginate polymer may be dissolved in water (30 mL/gram polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 eq) and N-methylmorpholine (1 eq). To this mixture may be added a solution of the compound of Formula (I) in acetonitrile (0.3M). The reaction may be warmed to 55° C. for 16 h, then cooled to room temperature and gently concentrated via rotary evaporation, then the residue may be dissolved, e.g., in water. The mixture may then be filtered, e.g., through a bed of cyano-modified silica gel (Silicycle) and the filter cake washed with water. The resulting solution may then be dialyzed (10,000 MWCO membrane) against water for 24 hours, e.g., replacing the water twice. The resulting solution can be concentrated, e.g., via lyophilization, to afford the desired chemically modified alginate.
In an embodiment, the device comprises at least one cell-containing compartment, and in some embodiments contains two, three, four or more cell-containing compartments. In an embodiment, each cell-containing compartment comprises a plurality of cells (e.g., live cells) and the cells in at least one of the compartments are capable of expressing and secreting at least one antigen when the device is implanted into a subject. In some embodiments, the cells in a single cell-containing compartment express two or more antigens and optionally one or more immunomodulatory proteins.
In an embodiment, all the cells in a cell-containing compartment are derived from a single parental cell-type or a mixture of at least two different parental cell types. In an embodiment, all of the cells in a cell-containing compartment are derived from the same parental cell type, but a first plurality of the derived cells are genetically modified to express a first antigen, and a second plurality of the derived cells are genetically modified to express a second antigen or an immunomodulatory protein. In devices with two or more cell-containing compartments, the cells and the antigen(s) and any immunomodulatory protein(s) produced thereby may be the same or different in each cell-containing compartment. In some embodiments, all of the cell-containing compartments are surrounded by a single barrier compartment. In some embodiments, the barrier compartment is substantially cell-free.
In an embodiment, cells to be incorporated into a device described herein, e.g., a hydrogel capsule, are prepared in the form of a cell suspension prior to being encapsulated within the device. The cells in the suspension may take the form of single cells (e.g., from a monolayer cell culture), or provided in another form, e.g., disposed on a microcarrier (e.g., a bead or matrix) or as a three-dimensional aggregate of cells (e.g., a cell cluster or spheroid). The cell suspension can comprise multiple cell clusters (e.g., as spheroids) or microcarriers.
In addition to antigen(s) and optional immunomodulatory protein(s) expressed by the encapsulated cells, a device (e.g., capsule, particle) may comprise one or more exogenous agents that are not expressed by the cells, and may include, e.g., a nucleic acid (e.g., an RNA or DNA molecule), a protein (e.g., a hormone, an enzyme (e.g., glucose oxidase, kinase, phosphatase, oxygenase, hydrogenase, reductase) antibody, antibody fragment, antigen, or epitope)), an active or inactive fragment of a protein or polypeptide, a small molecule, or drug. In an embodiment, the device is configured to release such an exogenous agent.
In some embodiments, the devices described herein comprise at least one compound of Formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
In some embodiments, the compound of Formula (I) is a compound of Formula (I-a):
or a pharmaceutically acceptable salt thereof, wherein:
In some embodiments, for Formulas (I) and (I-a), A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, —O—, —C(O)O—, —C(O)—, —OC(O)—, —N(RC)C(O)—, —N(RC)C(O)(C1-C6-alkylene)-, —N(RC)C(O)(C1-C6-alkenylene)-, or —N(RC)—. In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, —O—, —C(O)O—, —C(O)—, —OC(O)—, or —N(RC)—. In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, —O—, —C(O)O—, —C(O)—, —OC(O)—, or —N(RC)—. In some embodiments, A is alkyl, —O—, —C(O)O—, —C(O)—, —OC(O), or —N(RC)—. In some embodiments, A is —N(RC)C(O)—, —N(RC)C(O)(C1-C6-alkylene)-, or —N(RC)C(O)(C1-C6-alkenylene)-. In some embodiments, A is —N(RC)—. In some embodiments, A is —N(RC)—, and RC is hydrogen or alkyl. In some embodiments, A is —NH—. In some embodiments, A is —N(RC)C(O)(C1-C6-alkylene)-, wherein alkylene is substituted with R1. In some embodiments, A is —N(RC)C(O)(C1-C6-alkylene)-, and R1 is alkyl (e.g., methyl). In some embodiments, A is —NHC(O)C(CH3)2—. In some embodiments, A is —N(RC)C(O)(methylene)-, and R1 is alkyl (e.g., methyl). In some embodiments, A is —NHC(O)CH(CH3)—. In some embodiments, A is —NHC(O)C(CH3)—.
In some embodiments, for Formulas (I) and (I-a), L1 is a bond, alkyl, or heteroalkyl. In some embodiments, L1 is a bond or alkyl. In some embodiments, L′ is a bond. In some embodiments, L1 is alkyl. In some embodiments, L1 is C1-C6 alkyl. In some embodiments, L1 is —CH2—, —CH(CH3)—, —CH2CH2CH2, or —CH2CH2—. In some embodiments, L1 is —CH2— or —CH2CH2—.
In some embodiments, for Formulas (I) and (I-a), L3 is a bond, alkyl, or heteroalkyl. In some embodiments, L3 is a bond. In some embodiments, L3 is alkyl. In some embodiments, L3 is C1-C12 alkyl. In some embodiments, L3 is C1-C6 alkyl. In some embodiments, L3 is —CH2—. In some embodiments, L3 is heteroalkyl. In some embodiments, L3 is C1-C12 heteroalkyl, optionally substituted with one or more R2 (e.g., oxo). In some embodiments, L3 is C1-C6 heteroalkyl, optionally substituted with one or more R2 (e.g., oxo). In some embodiments, L3 is —C(O)OCH2—, —CH2(OCH2CH2)2—, —CH2(OCH2CH2)3—, CH2CH2O—, or —CH2O—. In some embodiments, L3 is —CH2O—.
In some embodiments, for Formulas (I) and (I-a), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, M is heteroalkyl, aryl, or heteroaryl. In some embodiments, M is absent. In some embodiments, M is alkyl (e.g., C1-C6 alkyl). In some embodiments, M is —CH2—. In some embodiments, M is heteroalkyl (e.g., C1-C6 heteroalkyl). In some embodiments, M is (—OCH2CH2—)z, wherein z is an integer selected from 1 to 10. In some embodiments, z is an integer selected from 1 to 5. In some embodiments, M is —OCH2CH2—, (—OCH2CH2—)2, (—OCH2CH2—)3, (—OCH2CH2—)4, or (—OCH2CH2—)5. In some embodiments, M is —OCH2CH2—, (—OCH2CH2—)2, (—OCH2CH2—)3, or (—OCH2CH2—)4. In some embodiments, M is (—OCH2CH2—)3. In some embodiments, M is aryl. In some embodiments, M is phenyl. In some embodiments, M is unsubstituted phenyl. In some embodiments, M is
In some embodiments, M is
In some embodiments, M is phenyl substituted with 1-4 R3 (e.g., 1 R3). In some embodiments, R3 is CF3.
In some embodiments, for Formulas (I) and (I-a), P is absent, heterocyclyl, or heteroaryl. In some embodiments, P is absent. In some embodiments, for Formulas (I) and (I-a), P is a tricyclic, bicyclic, or monocyclic heteroaryl. In some embodiments, P is a monocyclic heteroaryl. In some embodiments, P is a nitrogen-containing heteroaryl. In some embodiments, P is a monocyclic, nitrogen-containing heteroaryl. In some embodiments, P is a 5-membered heteroaryl. In some embodiments, P is a 5-membered nitrogen-containing heteroaryl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, pyrrolyl, oxazolyl, or thiazolyl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, or pyrrolyl. In some embodiments, P is imidazolyl. In some embodiments, P is
In some embodiments, P is triazolyl. In some embodiments, P is 1,2,3-triazolyl. In some embodiments, P is
In some embodiments, P is
In some embodiments, P is heterocyclyl. In some embodiments, P is a 5-membered heterocyclyl or a 6-membered heterocyclyl. In some embodiments, P is imidazolidinonyl. In some embodiments, P is
In some embodiments, P is thiomorpholinyl-1,1-dioxidyl. In some embodiments, P is
In some embodiments, for Formulas (I) and (I-a), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, Z is heterocyclyl. In some embodiments, Z is monocyclic or bicyclic heterocyclyl. In some embodiments, Z is an oxygen-containing heterocyclyl. In some embodiments, Z is a 4-membered heterocyclyl, 5-membered heterocyclyl, or 6-membered heterocyclyl. In some embodiments, Z is a 6-membered heterocyclyl. In some embodiments, Z is a 6-membered oxygen-containing heterocyclyl. In some embodiments, Z is tetrahydropyranyl. In some embodiments, Z is
In some embodiments, Z is a 4-membered oxygen-containing heterocyclyl. In some embodiments, Z is
In some embodiments, Z is a bicyclic oxygen-containing heterocyclyl. In some embodiments, Z is phthalic anhydridyl. In some embodiments, Z is a sulfur-containing heterocyclyl. In some embodiments, Z is a 6-membered sulfur-containing heterocyclyl. In some embodiments, Z is a 6-membered heterocyclyl containing a nitrogen atom and a sulfur atom. In some embodiments, Z is thiomorpholinyl-1,1-dioxidyl. In some embodiments, Z is
In some embodiments, Z is a nitrogen-containing heterocyclyl. In some embodiments, Z is a 6-membered nitrogen-containing heterocyclyl. In some embodiments, Z is
In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic nitrogen-containing heterocyclyl, optionally substituted with one or more R5. In some embodiments, Z is 2-oxa-7-azaspiro[3.5]nonanyl. In some embodiments, Z is
In some embodiments, Z is 1-oxa-3,8-diazaspiro[4.5]decan-2-one. In some embodiments, Z is
In some embodiments, for Formulas (I) and (I-a), Z is aryl. In some embodiments, Z is monocyclic aryl. In some embodiments, Z is phenyl. In some embodiments, Z is monosubstituted phenyl (e.g., with 1 R5). In some embodiments, Z is monosubstituted phenyl, wherein the one R5 is a nitrogen-containing group. In some embodiments, Z is monosubstituted phenyl, wherein the one R5 is NH2. In some embodiments, Z is monosubstituted phenyl, wherein the one R5 is an oxygen-containing group. In some embodiments, Z is monosubstituted phenyl, wherein the one R5 is an oxygen-containing heteroalkyl. In some embodiments, Z is monosubstituted phenyl, wherein the one R5 is OCH3. In some embodiments, Z is monosubstituted phenyl, wherein the one R5 is in the ortho position. In some embodiments, Z is monosubstituted phenyl, wherein the one R5 is in the meta position. In some embodiments, Z is monosubstituted phenyl, wherein the one R5 is in the para position.
In some embodiments, for Formulas (I) and (I-a), Z is alkyl. In some embodiments, Z is C1-C12 alkyl. In some embodiments, Z is C1-C10 alkyl. In some embodiments, Z is C1-C8 alkyl. In some embodiments, Z is C1-C8 alkyl substituted with 1-5 R5. In some embodiments, Z is C1-C8 alkyl substituted with 1 R5. In some embodiments, Z is C1-C8 alkyl substituted with 1 R5, wherein R5 is alkyl, heteroalkyl, halogen, oxo, —ORA1, —C(O)ORA1, —C(O)RB1, —OC(O)RB1, or —N(RC1)(RD1). In some embodiments, Z is C1-C8 alkyl substituted with 1 R5, wherein R5 is —ORA1 or —C(O)ORA1. In some embodiments, Z is C1-C8 alkyl substituted with 1 R5, wherein R5 is ORA1 or —C(O)OH. In some embodiments, Z is —CH3.
In some embodiments, for Formulas (I) and (I-a), Z is heteroalkyl. In some embodiments, Z is C1-C12 heteroalkyl. In some embodiments, Z is C1-C10 heteroalkyl. In some embodiments, Z is C1-C8 heteroalkyl. In some embodiments, Z is C1-C6 heteroalkyl. In some embodiments, Z is a nitrogen-containing heteroalkyl optionally substituted with one or more R5. In some embodiments, Z is a nitrogen and sulfur-containing heteroalkyl substituted with 1-5 R5. In some embodiments, Z is N-methyl-2-(methylsulfonyl) ethan-1-aminyl.
In some embodiments, Z is —ORA or —C(O)ORA. In some embodiments, Z is —ORA (e.g., —OH or —OCH3). In some embodiments, Z is —OCH3. In some embodiments, Z is —C(O)ORA (e.g., —C(O)OH).
In some embodiments, Z is hydrogen.
In some embodiments, L2 is a bond and P and L3 are independently absent. In some embodiments, L2 is a bond, P is heteroaryl, L3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L3 is heteroalkyl, and Z is alkyl.
In some embodiments, the compound of Formula (I) is a compound of Formula (I-b):
In some embodiments, the compound of Formula (I-b) is a compound of Formula (I-b-i):
In some embodiments, the compound of Formula (I-b-i) is a compound of Formula (I-b-ii):
In some embodiments, the compound of Formula (I) is a compound of Formula (I-c):
In some embodiments, the compound of Formula (I) is a compound of Formula (I-d):
In some embodiments, the compound of Formula (I) is a compound of Formula (I-e):
In some embodiments, the compound of Formula (I) is a compound of Formula (I-f):
In some embodiments, the compound of Formula (I) is a compound of Formula (II):
In some embodiments, the compound of Formula (II) is a compound of Formula (II-a):
In some embodiments, the compound of Formula (I) is a compound of Formula (III):
In some embodiments, the compound of Formula (III) is a compound of Formula (III-a):
In some embodiments, the compound of Formula (III-a) is a compound of Formula (III-b):
In some embodiments, the compound of Formula (III-a) is a compound of Formula (III-c):
In some embodiments, the compound of Formula (III-c) is a compound of Formula (III-d):
In some embodiments, the compound of Formula (I) is a compound of Formula (III-e):
In some embodiments, the compound of Formula (I) is a compound of Formula (III-f):
In some embodiments, the compound of Formula (I) is a compound of Formula (III-g):
In some embodiments, the compound of Formula (I) is a compound of Formula (III-h):
In some embodiments, the compound of Formula (I) is a compound of Formula (III-i):
In some embodiments, X is S(O)x. In some embodiments, x is 2. In some embodiments, X is S(O)2.
In some embodiments, each of R2a, R2b, R2c, and R2d is independently hydrogen.
In some embodiments, RC is hydrogen, —C(O)(C1-C6-alkyl), or —C(O)(C1-C6-alkenyl). In some embodiments, each of alkyl and alkenyl is substituted with 1 R6 (e.g., —CH3). In some embodiments, RC is hydrogen.
In some embodiments, n is 1. In some embodiments, q is 2, 3, 4, or 5. In some embodiments, q is 3. In some embodiments, m is 1. In some embodiments, p is 0. In some embodiments, R12 is halo (e.g., Cl).
In some embodiments, the compound is a compound of Formula (I). In some embodiments, L2 is a bond and P and L3 are independently absent.
In some embodiments, the compound is a compound of Formula (I-a). In some embodiments of Formula (II-a), L2 is a bond, P is heteroaryl, L3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L3 is heteroalkyl, and Z is alkyl. In some embodiments, L2 is a bond and P and L3 are independently absent. In some embodiments, L2 is a bond, P is heteroaryl, L3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L3 is heteroalkyl, and Z is alkyl.
In some embodiments, the compound is a compound of Formula (I-b). In some embodiments, P is absent, L1 is —NHCH2, L2 is a bond, M is aryl (e.g., phenyl), L3 is —CH2O, and Z is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, e.g., thiomorpholinyl-1,1-dioxide). In some embodiments, the compound of Formula (I-b) is Compound 116.
In some embodiments of Formula (I-b), P is absent, L1 is —NHCH2, L2 is a bond, M is absent, L3 is a bond, and Z is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (I-b) is Compound 105.
In some embodiments, the compound is a compound of Formula (I-b-i). In some embodiments of Formula (I-b-i), each of R2a and R2b is independently hydrogen or CH3, each of R2c and R2d is independently hydrogen, m is 1 or 2, n is 1, X is O, p is 0, M2 is phenyl optionally substituted with one or more R3, R3 is —CF3, and Z2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (I-b-i) is Compound 100, Compound 106, Compound 107, Compound 108, Compound 109, or Compound 111.
In some embodiments, the compound is a compound of Formula (I-b-ii). In some embodiments of Formula (I-b-ii), each of R2a, R2b, R2c, and R2d is independently hydrogen, q is 0, p is 0, m is 1, and Z2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl). In some embodiments, the compound of Formula (I-b-ii) is Compound 100.
In some embodiments, the compound is a compound of Formula (I-c). In some embodiments of Formula (I-c), each of R2c and R2d is independently hydrogen, m is 1, p is 1, q is 0, R5 is —CH3, and Z is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, e.g., piperazinyl). In some embodiments, the compound of Formula (I-c) is Compound 113.
In some embodiments, the compound is a compound of Formula (I-d). In some embodiments of Formula (I-d), each of R2a, R2b, R2c, and R2d is independently hydrogen, m is 1, n is 3, X is O, p is 0, and Z is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (I-d) is Compound 110 or Compound 114.
In some embodiments, the compound is a compound of Formula (I-f). In some embodiments of Formula (I-f), each of R2a and R2b is independently hydrogen, n is 1, M is —CH2—, P is a nitrogen-containing heteroaryl (e.g., imidazolyl), L3 is —C(O)OCH2—, and Z is CH3. In some embodiments, the compound of Formula (I-f) is Compound 115.
In some embodiments, the compound is a compound of Formula (II-a). In some embodiments of Formula (II-a), each of R2a and R2b is independently hydrogen, n is 1, q is 0, L3 is —CH2(OCH2CH2)2, and Z is —OCH3. In some embodiments, the compound of Formula (II-a) is Compound 112.
In some embodiments of Formula (II-a), each of R2a and R2b is independently hydrogen, n is 1, L3 is a bond or —CH2, and Z is hydrogen or —OH. In some embodiments, the compound of Formula (II-a) is Compound 103 or Compound 104.
In some embodiments, the compound is a compound of Formula (III). In some embodiments of Formula (III), each of R2a, R2b, R2c, and R2d is independently hydrogen, m is 1, n is 2, q is 3, p is 0, RC is hydrogen, and Z1 is heteroalkyl optionally substituted with R5 (e.g., —N(CH3)(CH2CH2)S(O)2CH3). In some embodiments, the compound of Formula (III) is Compound 120.
In some embodiments, the compound is a compound of Formula (III-b). In some embodiments of Formula (III-b), each of R2a, R2b, R2c, and R2d is independently hydrogen, m is 0, n is 2, q is 3, p is 0, and Z2 is aryl (e.g., phenyl) substituted with 1 R5 (e.g., —NH2). In some embodiments, the compound of Formula (III-b) is Compound 102.
In some embodiments, the compound is a compound of Formula (III-b). In some embodiments of Formula (III-b), each of R2a, R2b, R2c, and R2d is independently hydrogen, m is 1, n is 2, q is 3, p is 0, RC is hydrogen, and Z2 is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, e.g., a nitrogen-containing spiro heterocyclyl, e.g., 2-oxa-7-azaspiro[3.5]nonanyl). In some embodiments, the compound of Formula (III-b) is Compound 121.
In some embodiments, the compound is a compound of Formula (III-d). In some embodiments of Formula (III-d), each of R2a, R2b, R2c, and R2d is independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O)2. In some embodiments of Formula (III-d), each of R2a and R2b is independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O)2. In some embodiments, the compound of Formula (III-d) is Compound 101, Compound 117, Compound 118, or Compound 119.
In some embodiments, the compound is a compound of Formula (I-b), (I-d), or (I-e). In some embodiments, the compound is a compound of Formula (I-b), (I-d), or (II). In some embodiments, the compound is a compound of Formula (I-b), (I-d), or (I-f). In some embodiments, the compound is a compound of Formula (I-b), (I-d), or (III).
In some embodiments, the compound of Formula (I) is not a compound disclosed in WO2012/112982, WO2012/167223, WO2014/153126, WO2016/019391, WO 2017/075630, US2012-0213708, US 2016-0030359 or US 2016-0030360.
In some embodiments, the compound of Formula (I) comprises a compound shown in Table 4, or a pharmaceutically acceptable salt thereof. In some embodiments, the exterior surface and/or one or more compartments within a device described herein comprises a small molecule compound shown in Table 4, or a pharmaceutically acceptable salt thereof.
Conjugation of any of the compounds in Table 4 to a polymer (e.g., an alginate) may be performed as described in Example 2 of WO 2019/195055 or any other suitable chemical reaction.
In some embodiments, the compound is a compound of Formula (I) (e.g., Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (II), (II-a), (III), (III-a), (III-b), (III-c), or (III-d)), or a pharmaceutically acceptable salt thereof and is selected from:
or a pharmaceutically acceptable salt thereof.
In some embodiments, the device described herein comprises the compound of
or a pharmaceutically acceptable salt of either compound.
In some embodiments, a compound of Formula (I) (e.g., Compound 101 in Table 4) is covalently attached to an alginate (e.g., an alginate with approximate MW<75 kDa, G:M ratio ≥1.5) at a conjugation density of at least 2.0% and less than 9.0%, or 3.0% to 8.0%, 4.0-7.0, 5.0 to 7.0, or 6.0 to 7.0 or about 6.8 as determined by combustion analysis for percent nitrogen as described in WO 2020/069429.
In some embodiments, the device is configured to locally release an immunosuppressant compound (e.g., as defined herein) during a desired release period after the device is implanted into a subject. In an embodiment, the desired release period is at least 5 days, 10 days, 15 days or 30 days. In an embodiment, the immunosuppressant is continuously released throughout the release period by an extended release formulation present in the device.
The extended release formulation can comprise a solid, semi-solid, gel, or liquid form of the immunosuppressant. In addition, the extended release formulation may comprise the immunosuppressant in conjunction with another component, such as a polymer, solvent, or other excipient. The extended release formulations described herein may provide control over immunosuppressant release from the device; for example, the extended release formulation may liberate a small amount of immunosuppressant into or out of the device over time, e.g., to provide a substantially constant concentration of the immunosuppressant in or around the device and or subject over an extended period of time. In an embodiment, the extended release formulation provides a dosage of the immunosuppressant for longer than 1 day (e.g., greater than 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months or more), e.g., upon administration to a subject.
In an embodiment, the extended release formulation of the immunosuppressant is a solid (e.g., a crystal or a granule). In an embodiment, the extended release formulation of the immunosuppressant is a semi-solid or a gel. In an embodiment, the extended release formulation of the immunosuppressant is a liquid (e.g., a solution, e.g., an aqueous solution).
The extended release formulation of the immunosuppressant may comprise another component, such as a polymer. Exemplary polymers may be biodegradable or non-biodegradable. In an embodiment, the polymer is a synthetic biodegradable polymer, e.g., a polymer that is not naturally occurring. In an embodiment, the polymer is a naturally occurring biodegradable polymer, e.g., a polymer that is found in nature, e.g., a polypeptide or polysaccharide. Exemplary polymers include polyesters, polyethers, polycarbonates, polyvinyl alcohols, polyurethanes, polypropylenes, polyphosphazenes, polyanhydrides, alginate, dextran, hyaluronic acid, cellulose, xanthan gum, scleoroglucan, and the like.
The polymer may be linear or branched. A branched polymer may be a star polymer, comb polymer, brush polymer, dendronized polymer, ladder, or dendrimer. In an embodiment, the polymer may be cross-linked. Further, the polymer may comprise selected molecular weight ranges, degrees of polymerization, viscosities or melt flow rates. The polymer may be thermoresponsive (e.g., a gel, e.g., which becomes a solid or liquid upon exposure to heat or a certain temperature) or can also be photocrosslinkable (e.g., a gel, e.g., which becomes a solid upon photocrosslinking).
In an embodiment, the polymer in an extended release formulation of the immunosuppressant has a molecular weight (Mw) greater than about 1,000 Da (e.g., greater than about 2,500 Da, 5,000 Da, 7,500 Da, 10,000 Da, 12,500 Da, 15,000 Da, 20,000 Da, 25,000 Da, 50,000 Da, or more). In an embodiment, the polymer has a Mw of less than about 100,000 Da (e.g., less than about 90,000 Da, 80,000 Da, 75,000 Da, 50,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, or less). In an embodiment, the polymer has a Mw of between 1,000 Da and 100,000 Da. For example, the polymer may have a Mw between 1,000 Da and 50,000 Da, 2,500 Da and 40,000 Da, 5,000 Da and 30,000 Da, 5,000 Da and 20,000 Da, or 10,000 Da and 20,000. In an embodiment, the polymer has a Mw between 1,000 Da and 50,000 Da. In an embodiment, the polymer has a Mw between 5,000 Da and 20,000 Da.
The polymer may comprise any type of end group on its termini. For example, the polymer may comprise an amine, ester, acid, hydroxyl, acyl, amide, or ether end group on one or more of its termini. In an embodiment, the polymer comprises the same end group on each termini. In an embodiment, the polymer comprises a plurality of end groups on its termini (e.g., at least 2, 3, 4, 5 different end groups on its termini). In an embodiment, the polymer comprises an acid group on at least one of its termini. In an embodiment, the polymer comprises an ester group on at least one of its termini.
In an embodiment, the polymer in an extended release formulation of the immunosuppressant is a polyester. Polyesters degrade through the nonspecific hydrolysis of their ester bonds. Exemplary polyesters include poly (lactic acid) (PLA), poly (L-lactic acid) (PLLA), poly (D-lactic acid) (PDLA), poly (glycolic acid) (PGA), poly (lactic glycolic acid) (PLGA), poly (caprolactone) (PCL), and poly (lactic caprolactone) (PLCL). In an embodiment, the polyester polymer comprises lactic acid (e.g., a lactide). In an embodiment, the polyester polymer comprises a glycolic acid (e.g., a glycolide). In an embodiment, the polyester polymer comprises poly (lactic glycolic acid) (PLGA) (e.g., poly (D,L-lactic glycolic acid), poly (D-lactic glycolic acid), or poly (L-lactic glycolic acid)). The PLGA may be obtained from any commercial supplier or may be prepared de novo. In an embodiment, the PLGA is a Resomer R PLGA.
The polymer may be a homogeneous polymer or a blended polymer (e.g., a co-polymer of a block co-polymer). In the case of blended polymers, the polymer may comprise a plurality of monomer units (e.g., at least 2, 3, 4, 5, or 6 types of monomer units). In an embodiment, for blended polymers comprising 2 monomer units (Unit A and Unit B), the ratio of Unit A to Unit B is between 0.1:99.1 to 99.1:0.1. For example, the ratio of Unit A to Unit B may be 0.1:99.1, 0.5:99.5, 1:99, 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, 99:1, 99.5:0.5, or 99.1:0.1. Similarly, a blended polymer may comprise 3 monomer units (Unit A, Unit B, and Unit C) with exemplary ratios of Unit A to Unit B to Unit C between 1:1:99 to 99:1:1 (including varying amounts of Unit B).
In an embodiment, the polymer in an extended release formulation of the immunosuppressant has a viscosity (e.g., inherent viscosity) of between 0.01 dL/g and 2.5 dL/g. For example, the polymer may have a viscosity of between 0.05 dL/g and 2.0 dL/g, 0.1 dL/g to 0.5 dL/g, 0.1 dL/g to 0.3 dL/g, or 1.0 dL/g and 1.5 dL/g. In an embodiment, the polymer is free of a contaminant, such as a metal, catalyst, free radical, oxygen, microbe, or endotoxin. In an embodiment, the polymer is sterile. In an embodiment, the polymer is provided as a powder, granules, pellets, crystals, a gel, or a liquid.
The extended release formulation of the immunosuppressant may comprise another component, such as a solvent. The solvent may be an organic solvent that is miscible with water, or an organic solvent that is not miscible with water. In an embodiment, the solvent is an organic solvent that aids in the solubility and stability of the immunosuppressant over time. In an embodiment, the organic solvent is any organic solvent approved by the Food and Drug Administration (FDA) for use in humans. Exemplary organic solvents include dimethylamide (DMA), dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dimethylsulfoxide (DMSO), formamide, tetrahydrofuran (THF), ethanol, isopropanol, acetone, ethyl acetate, 1,4-dioxane, pyridine, and triethylamine (TEA). In an embodiment, the organic solvent comprises an amine (e.g., a primary amine, secondary amine, or tertiary amine). In an embodiment, the organic solvent comprises a ketone. In an embodiment, the organic solvent has a boiling point greater than about 100° C., e.g., greater than about 150° C., 200° C., 250° C., 300° C. or higher.
Exemplary extended release formulations described herein may comprise a both a polymer and an organic solvent, e.g., at a fixed ratio of polymer to organic solvent. Factors such as solubility, stability, toxicity, or volume of the final composition are often taken into consideration when determining the ratio of the polymer with the organic solvent. In one embodiment, the ratio of the polymer to the organic solvent is between 50:50 and 90:10 (w/w) (e.g., 50:50, 60:40, 70:30, 80:20, or 90:10). In another embodiment, the ratio of the polymer to the organic solvent is between 10:90 and 50:50 (w/w) (e.g., 10:90, 20:80, 30:70, 40:60, or 50:50).
The concentration of immunosuppressant in the extended release formulation may be within the range of 0.01% to 50% w/w (e.g., about 0.1% to 40% w/w or 1% to 25% w/w). In some embodiments, the concentration of immunosuppressant in the extended release formulation is about 0.1% to 25% w/w, e.g., between about 0.1% and 20%, about 0.1% and 15%, about 0.1% and 10%, about 0.5% and 25%, about 0.5% and 20%, about 0.5% and 15%, about 0.5% and 10%, about 1% and 25%, about 1% and 20%, about 1% and 15%, or about 1% and 10% w/w). In some embodiments, the concentration of immunosuppressant in the extended release formulation is about 0.05%, about 0.1%, about 0.5%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, about 13%, about 13.5%, about 14%, about 14.5%, about 15%, about 17.5%, about 20%, or about 25% w/w.
The concentration of immunosuppressant in the extended release formulation may be within the range of 0.01% to 50% w/v (e.g., about 0.1% to 40% w/v or 1% to 25% w/v). In some embodiments, the concentration of immunosuppressant in the extended release formulation is about 0.1% to 25% w/v, e.g., between about 0.1% and 20%, about 0.1% and 15%, about 0.1% and 10%, about 0.5% and 25%, about 0.5% and 20%, about 0.5% and 15%, about 0.5% and 10%, about 1% and 25%, about 1% and 20%, about 1% and 15%, or about 1% and 10% w/v). In some embodiments, the concentration of immunosuppressant in the extended release formulation is about 0.05%, about 0.1%, about 0.5%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, about 13%, about 13.5%, about 14%, about 14.5%, about 15%, about 17.5%, about 20%, or about 25% w/v.
The extended release formulations compositions provided are formulated for sustained or delayed release of an immunosuppressant described herein (e.g., a compound of Formula (I)). Sustained release, as described herein, refers to the property of a composition by which release of an immunosuppressant from the extended release formulation occurs over an extended period of time as compared to the release from an isotonic saline solution. Such release profile may result in prolonged delivery (over, say 1 to about 2,000 hours, or alternatively about 2 to about 800 hours) of effective amounts (e.g., about 0.0001 mg/kg/hour to about 10 mg/kg/hour, e.g., 0.001 mg/kg/hour, 0.01 mg/kg/hour, 0.1 mg/kg/hour, 1.0 mg/kg/hour) of the immunosuppressant or any other material associated with the sustained release formulation. To illustrate further, a wide range of degradation rates or release rates may be obtained by adjusting the features of the extended release formulation, such as the identity of the polymer or any related feature of the polymer (including the molecular weight, viscosity, end group, etc), the identity of the organic solvent, the concentration of any one of the polymer, organic solvent, or immunosuppressant, or ratios thereof.
One protocol generally accepted in the field that may be used to determine the release rate of the immunosuppressant from an extended release formulation described herein entails disposing the extended release formulation into a physiological environment and removing samples of the mixture and various time points for analysis, e.g., by HPLC.
The release rate of the immunosuppressant from the extended release formulation may also be characterized by the amount of immunosuppressant released per day per quantity of polymer present in the sustained release formulation. For example, in some embodiments, the release rate may vary from about 1 ng or less of the immunosuppressant per day per quantity of polymer to about 500 or more ng/day/quantity of polymer. Alternatively, the release rate may be about 0.05, 0.5, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 ng/day/quantity of polymer. In still other embodiments, the release rate of the immunosuppressant may be 10,000 ng/day/ng/day/quantity of polymer, or even higher.
In an embodiment, the release of immunosuppressant from an extended release formulation described herein may be described as the half-life (i.e., T1/2) of such material in the formulation.
In an embodiment, an extended release formulation described herein provides a dosage of the immunosuppressant for greater than 1 day (e.g., greater than 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months or more), e.g., upon administration of the device to a subject. In an embodiment, the extended release formulation provides a dosage of the immunosuppressant for greater than 1 week (e.g., greater than 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months or more), e.g., upon administration to a subject. In an embodiment, the extended release formulation provides a dosage of the immunosuppressant between 1 week and 10 weeks (e.g., 1 week and 8 weeks, 1 week and 6 weeks, 1 week and 4 weeks, 2 weeks and 8 weeks, 3 weeks and 6 weeks), e.g., upon administration to a subject. In an embodiment, the extended release formulation provides a dosage of the immunosuppressant between 0.5 ug and 500 ug per day (e.g., between 1 ug and 500 ug, 1 ug and 250 ug, 1 ug and 100 ug, 1 ug and 50 ug, 5 ug and 500 ug, 5 ug and 250 ug, 5 ug and 100 ug, 5 ug and 50 ug, 10 ug and 250 ug, 10 ug and 100 ug, 10 ug and 50 ug, 50 ug and 500 ug, 50 ug and 250 ug, 50 ug and 100 ug).
In an embodiment, the extended release formulation has the property that it forms a drug depot within a device described herein. A drug depot refers to a localized mass of a particular immunosuppressant (e.g., solid particles of a rapamycin compound described herein), wherein the immunosuppressant is gradually released from the device, e.g., by diffusion. A drug depot may comprise at least one region which provides a bolus of the immunosuppressant, and a remaining region which provides a sustained release of the immunosuppressant.
In an embodiment, the extended release formulation comprises a plurality of particles of the immunosuppressant suspended in a hydrogel in the device, e.g., within a hydrogel that forms the outer compartment or the inner, cell-containing compartment of a two-compartment device described herein. In an embodiment, the particles comprise the immunosuppressant in crystalline form.
In an embodiment, the immunosuppressant particles are prepared by adding a desired quantity of an amorphous powder of the immunosuppressant to a desired volume of a solution comprising a hydrogel-forming polymer (e.g., an alginate or alginate mixture described herein), sonicating the resulting powder-polymer mixture until a substantially homogenous suspension is formed, and contacting droplets of the resulting suspension with a cross-linking solution. In an embodiment, the quantity of the immunosuppressant powder and the volume of the polymer solution are selected to achieve a mixture of 2.5 mg to 5.0 mg powder per mL polymer solution. In an embodiment, the genetically-modified cells are added to the homogenous suspension and the resulting cell-containing suspension is used to form the inner compartment of a two-compartment hydrogel capsule as described herein. In an embodiment, the immunosuppressant particles comprise a rapamycin compound, e.g., rapamycin.
In some embodiments, the immunosuppressant in an extended release formulation is rapamycin, an analogue or derivative of rapamycin, or a pharmaceutically acceptable salt of rapamycin, analogue or derivative. The structure of rapamycin is:
The term rapamycin compound as used herein refers to rapamycin and compounds with a structural similarity to rapamycin, e.g., compounds with a similar macrocyclic structure which have been modified to enhance therapeutic benefit. In an embodiment, the mTOR inhibitor is rapamycin or the rapamycin derivative everolimus. In an embodiment, the mTOR inhibitor is the rapamycin ester cell cycle inhibitor-779 (CCI-779). Additional rapamycin compounds which may be used in the devices described herein include but are not limited to the rapamycin analogues and derivatives described in any of the following U.S. Pat. Nos. 6,015,809; 6,004,973; 5,985,890; 5,955,457; 5,922,730; 5,912,253; 5,780,462; 5,665,772; 5,637,590; 5,567,709; 5,563,145; 5,559,122; 5,559,120; 5,559,119; 5,559,112; 5,550,133; 5,541,192; 5,541,191; 5,532,355; 5,530,121; 5,530,007; 5,525,610; 5,521,194; 5,519,031; 5,516,780; 5,508,399; 5,508,290; 5,508,286; 5,508,285; 5,504,291; 5,504,204; 5,491,231; 5,489,680; 5,489,595; 5,488,054; 5,486,524; 5,486,523; 5,486,522; 5,484,791; 5,484,790; 5,480,989; 5,480,988; 5,463,048; 5,446,048; 5,434,260; 5,411,967; 5,391,730; 5,389,639; 5,385,910; 5,385,909; 5,385,908; 5,378,836; 5,378,696; 5,373,014; 5,362,718; 5,358,944; 5,346,893; 5,344,833; 5,302,584; 5,262,424; 5,262,423; 5,260,300; 5,260,299; 5,233,036; 5,221,740; 5,221,670; 5,202,332; 5,194,447; 5,177,203; 5,169,851; 5,164,399; 5,162,333; 5,151,413; 5,138,051; 5,130,307; 5,120,842; 5,120,727; 5,120,726; 5,120,725; 5,118,678; 5,118,677; 5,100,883; 5,023,264; 5,023,263; and 5,023,262, each of which is incorporated herein by reference in its entirety.
Any of the rapamycin compounds described herein may be used to prepare rapamycin particles for encapsulation in hydrogel capsules. In an embodiment, rapamycin particles are prepared using rapamycin.
In an embodiment, the rapamycin particles are biodegradable nanoparticles described in WO2021183781, WO2016073799.
Each compartment of a device described herein may comprise an unmodified polymer, a polymer modified with a compound of Formula (I), or a blend thereof. Briefly, to prepare a device configured as a two-compartment hydrogel capsule, a volume of a first polymer solution (e.g., comprising an unmodified polymer, a polymer modified with a cell-binding peptide, or a blend thereof, and genetically modified cells) is loaded into a first syringe connected to the inner lumen of a coaxial needle. The first syringe may then be connected to a syringe pump oriented vertically above a vessel containing an aqueous cross-linking solution which comprises a cross-linking agent, a buffer, and an osmolarity-adjusting agent. A volume of the second polymer solution (e.g., comprising an unmodified polymer, a polymer modified with a compound of Formula (I), or a blend thereof) is loaded into a second syringe connected to the outer lumen of the coaxial needle. The second syringe may then be connected to a syringe pump oriented horizontally with respect to the vessel containing the cross-linking solution. A high voltage power generator may then be connected to the top and bottom of the needle. The syringe pumps and power generator can then be used to extrude the first and second polymer solutions through the syringes with settings determined to achieve a desired droplet rate of polymer solution into the cross-linking solution. The skilled artisan may readily determine various combinations of needle lumen sizes, voltage range, flow rates, droplet rate and drop distance to create 2-compartment hydrogel capsule compositions in which the majority (e.g., at least 80%, 85%, 90% or more) of the capsules are within 10% of the target size and desired shape. After exhausting the first and second volumes of polymer solution, the droplets may be allowed to cross-link in the cross-linking solution for certain amount of time, e.g., about five minutes.
An exemplary process for preparing a composition of millicapsules (e.g., 1.5 mm diameter) is described in WO 2019/195055.
A device described herein may be provided as a preparation or composition for implantation or administration to a subject, e.g., a subject with an autoimmune disease or an allergic condition. In some embodiments, a device preparation or composition comprises at least 2, 4, 8, 16, 32, 64 or more devices, and at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the devices in the preparation or composition have a characteristic as described herein, e.g., mean diameter or mean pore size or cell density.
A device composition may be configured for implantation, or implanted or disposed into or onto any site of the body. In some embodiments, a device, preparation or composition is configured for implantation, implanted or disposed into the subcutaneous fat of a subject, or into the muscle tissue of a subject. In some embodiments, the device, device preparation or device composition is configured for implantation or implanted or disposed into the peritoneal cavity (e.g., the omentum). In some embodiments, the device is configured for implantation or implanted or disposed into or onto the lesser sac, also known as the omental bursa or bursalis omentum. The lesser sac refers to a cavity located in the abdomen formed by the omentum, and is in close proximity to, for example, the greater omentum, lesser omentum, stomach, small intestine, large intestine, liver, spleen, gastrosplenic ligament, adrenal glands, and pancreas. Typically, the lesser sac is connected to the greater sac via the omental foramen (i.e., the Foramen of Winslow). In some embodiments, the lesser sac comprises a high concentration of adipose tissue. A device, device preparation or device composition may be implanted in the peritoneal cavity (e.g., the omentum, e.g., the lesser sac) or disposed on a surface within the peritoneal cavity (e.g., omentum, e.g., lesser sac) via injection or catheter. Additional considerations for implantation or disposition of a device, preparation or composition into the omentum (e.g., the lesser sac) are provided in M. Pellicciaro et al. (2017) CellR4 5 (3): e2410.
In some embodiments, a device or preparation is easily retrievable from a subject, e.g., without causing injury to the subject or without causing significant disruption of the surrounding tissue. In an embodiment, the device or preparation can be retrieved with minimal or no surgical separation of the device(s) from surrounding tissue, e.g., via minimally invasive surgical approach, extraction, or resection.
A device, composition or preparation can be configured to provide continuous delivery of an antigen and optionally an immunomodulatory protein for a variety of time periods after implant into a mammalian recipient (e.g., a human patient), including: a short continuous delivery (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) or prolonged delivery (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 or longer).
In some embodiments, the device composition or preparation does not contain any capsule, device, implant or other object disclosed in any of WO2012/112982, WO2012/167223, WO2014/153126, WO2016/019391, WO2016/187225, WO 2018/232027, WO 2019/068059, WO 2019/169089, US2012-0213708, US 2016-0030359, and US 2016-0030360.
Devices described herein, and compositions or preparations thereof, may be useful in preventing or treating undesired antigen-specific immune responses, such as those involved in autoimmune diseases, immune reactions to therapeutic proteins, graft rejection and allergies. Antigens that may be continuously delivered by a device described herein include autoantigens (self-antigens), alloantigens, therapeutic proteins and allergens.
Autoantigens are endogenous proteins or fragments thereof that elicit a pathogenic immune response. Of particular interest are autoantigens that induce a T cell mediated inflammatory pathogenic response. Administration of a device described herein to continuously deliver the relevant target autoantigen in combination with an immunomodulatory agent are of potential use in the treatment of autoimmune diseases characterized by the involvement of pro-inflammatory T cells, such as multiple sclerosis, experimental autoimmune encephalitis, rheumatoid arthritis and insulin dependent diabetes mellitus.
The genetically modified cells may express and secrete one or more autoantigens associated with an autoimmune disease such as multiple sclerosis (MS), rheumatoid arthritis (RA), Type 1 diabetes, autoimmune uveitis (AU), primary biliary cirrhosis (PBC), myasthenia gravis (MG), neuromyelitis optica, Sjogren's syndrome, pemphigus vulgaris (PV), scleroderma, pernicious anemia, systemic lupus erythematosus (SLE) and Grave's disease. Table 5 provides a list of autoimmune diseases and associated autoantigens that may be delivered by a device described herein.
A device described herein can be administered to individuals who are receiving therapy with a therapeutic protein and may or may not have previously exhibited ADA to the therapeutic protein. It can also be administered to individuals who are planning to begin a therapeutic regimen with a particular therapeutic protein or peptide.
In an embodiment, the antigen expressed and secreted by genetically modified cells in the device comprises an amino acid sequence of any of the peptide antigens described in International Application WO 2018/127828 A1.
Selection of antigen(s) and any immunomodulatory protein(s) to be produced by a device for treating a particular autoimmune disease or allergic condition would typically include consideration of the antigens known to trigger the autoimmune or allergic response. Table 5 below lists exemplary diseases and conditions paired with exemplary antigens that may be useful as antigen molecules that are expressed and secreted by the genetically modified cells described herein, or from which such molecules could be derived.
Devices described herein are useful to prevent or reduce ADA to a therapeutic protein or peptide produced by cells contained in the device following administration of the device to a subject, e.g., a human. In some embodiments, the therapeutic substance is for the prevention or treatment of a disease, disorder, or condition, e.g., those described in international application publications WO 2017/075631 A1, WO 2019/195055 A1 and WO 2020/069429 A1.
In some embodiments, the therapeutic agent is a peptide or polypeptide (e.g., a protein), such as a hormone, enzyme, cytokine (e.g., a pro-inflammatory cytokine or an anti-inflammatory cytokine), growth factor, clotting factor, or lipoprotein. A therapeutic peptide or therapeutic protein (e.g., a hormone, growth factor, clotting factor or coagulation factor, antibody molecule, enzyme, cytokine, cytokine receptor, or a chimeric protein including cytokines or a cytokine receptor) produced by a genetically modified cell in a device described herein can have a naturally occurring amino acid sequence, or may contain a variant of the naturally occurring sequence. The variant can be a naturally occurring or non-naturally occurring amino acid substitution, mutation, deletion or addition relative to the reference sequence, e.g., a naturally occurring sequence. The naturally occurring amino acid sequence may be a polymorphic variant. The naturally occurring amino acid sequence can be a human or a non-human amino acid sequence. In some embodiments, the naturally occurring amino acid sequence or naturally occurring variant thereof is a human sequence.
The therapeutic peptide or polypeptide may be expressed as part of a fusion protein, which comprises a first amino acid sequence which forms a therapeutic domain (e.g., has substantially the same activity as the therapeutic peptide or polypeptide) and a second amino acid sequence with a desired property. In an embodiment, the second amino acid sequence facilitates secretion of the fusion protein by the encapsulated cells, e.g., a signal peptide sequence from a heterologous protein. In an embodiment, the second amino acid sequence forms a tissue-targeting moiety, e.g., binds to a tissue-specific protein of interest, e.g., a blood-brain barrier transporter, liver, a tumor. In an embodiment, the second amino acid sequence forms a half-life-extending domain, e.g., an immunoglobulin Fc, albumin, a serum albumin binding domain. In an embodiment, the fusion protein comprises amino acid sequences for both a tissue-targeting domain and a half-life extending domain in addition to the amino acid sequence for the therapeutic domain.
In some embodiments, the therapeutic protein is a clotting factor or a coagulation factor, e.g., a blood clotting factor or a blood coagulation factor. In an embodiment, the blood clotting protein is a human FVII protein, a human FVIII protein, or a human FIX protein. Non-limiting examples of amino acid sequences for these blood clotting factors, and exogenous coding sequences, are described in WO 2019/067766, WO 2020/198695 and WO 2021/10227.
In some embodiments, the therapeutic protein is a cytokine or a cytokine receptor, or a chimeric protein including cytokines or their receptors.
In some embodiments, the therapeutic protein is an enzyme, e.g., an enzyme that is deficient or missing in a lysosomal storage disorder (LSD). In an embodiment, the enzyme is an alpha-galactosidase A (GLA) protein. Exemplary amino acid and exogenous coding sequences for a human GLA protein are described in WO 2020/198685. In an embodiment, the enzyme is an acid alpha-glucosidase (GAA) protein, an alpha-L-iduronidase (IDUA) protein, an alpha-N-acetyl-glucosaminidase (NAGLU) protein, a beta-glucoronidase (GUSB) protein, a glucocerebrosidase (GBA) protein, a heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) protein, an iduronate-2-sulfatase (IDS) protein, an N-acetylgalactosamine-6-sulfatase (GNS) protein or an N-sulfoglucosamine sulfohydrolase (SGSH) protein.
1. An implantable device comprising a first plurality of mammalian cells genetically modified to express and secrete one or more antigens, wherein the device is configured to deliver at least one immunomodulatory agent that facilitates immune tolerance induction to the secreted antigen(s) and wherein the device exhibits the following properties when implanted into a subject:
2. An implantable device comprising a first plurality of mammalian cells genetically modified to express and secrete one or more antigens, wherein the device is configured to:
3. The implantable device of embodiment 2, which comprises at least one of the following features:
4. The implantable device of any one of embodiments 1 to 3, wherein the immunomodulatory agent promotes an antigen-specific regulatory T cell (Treg) response or inhibits an antigen-specific effector T cell response (Teff) response.
5. The implantable device of any one or embodiments 1 to 4, which comprises feature (iv), wherein the signal peptide sequence consists essentially of MGWRAAGALLLALLLHGRLLA (SEQ ID NO:20).
6. The implantable device of any one of embodiments 4 to 6, which comprises one or both of feature (ii) and feature (iii).
7. The implantable device of embodiment 6, which does not comprise feature (ii).
8. The implantable device of embodiment 6, which does not comprise feature (iii).
9. The implantable device of any one of embodiments 1 to 8, which comprises feature (i).
10. The implantable device of any one of embodiments 1 to 9, which comprises feature (v).
11. The implantable device of any one of embodiments 1 to 10, which comprises feature (vi).
12. The implantable device of any one of embodiments 1 to 10, which comprises feature (vii).
13. The implantable device of any one of embodiments 1 to 12, wherein each plurality of genetically modified cells is contained in a cell-containing compartment surrounded by a barrier compartment.
14. The implantable device of embodiment 13, wherein the cell-containing compartment comprises a first hydrogel-forming polymer and the barrier compartment comprises a second hydrogel-forming polymer.
15. The implantable device of embodiment 14, wherein one or both of the first hydrogel-forming polymer and the second hydrogel forming polymer is an alginate.
16. The implantable device of any one of embodiments 1 to 13, which comprises two or more cell-containing compartments.
17. The implantable device of any one of embodiments 1 to 16, wherein the antigen secreted by the first plurality of genetically modified mammalian cells is a therapeutic protein or therapeutic peptide, optionally wherein the therapeutic protein is a blood clotting factor, a coagulation factor, a hormone, an enzyme, an antibody, a cytokine or a soluble cytokine receptor.
18. The implantable device of any one of embodiments 1 to 16, wherein the antigen secreted by the first plurality of genetically modified mammalian cells is an antigen listed in Table 5 for an autoimmune disease.
19. The implantable device of any one of embodiments 1 to 16, wherein the antigen secreted by the first plurality of genetically modified mammalian cells is an enzyme.
20. The implantable device of any one of embodiments 1 to 16, wherein the antigen secreted by the first plurality of genetically modified mammalian cells is an enzyme deficient in an LSD.
21. The implantable device of any one of embodiments 1 to 16, wherein the antigen secreted by the first plurality of genetically modified mammalian cells is a GLA protein.
22. The implantable device of any one of the above embodiments, wherein the device is configured to deliver an immunosuppressant compound to the subject for at least 20 days or at least 30 days.
23. A hydrogel capsule comprising:
24. The hydrogel capsule of embodiment 23, wherein the cell-containing compartment further comprises living cells genetically modified to express and secrete an immunomodulatory protein.
25. The hydrogel capsule of embodiment 24, wherein the antigen-secreting cells are also genetically modified to express and secrete an immunomodulatory protein.
26. The hydrogel capsule of any one of embodiments 23 to 25, wherein the extended release formulation is present in one or both of the cell-containing compartment and the barrier compartment.
27. The hydrogel capsule of embodiment 26, wherein the first polymer composition comprises a hydrogel-forming polymer and the extended release formulation of the immunosuppressant compound is prepared by a process which comprises adding a desired quantity of an amorphous powder of the immunosuppressant compound to a desired volume of a solution comprising the hydrogel-forming polymer, sonicating the resulting mixture until a substantially homogenous suspension is formed, adding the living cells to the suspension and contacting droplets of the polymer, immunosuppressant compound and cell suspension with a cross-linking solution, optionally wherein the hydrogel-forming polymer is an alginate.
28. The hydrogel capsule of any one of embodiments 23 to 27, wherein at least a portion of the living cells are genetically modified to continuously express and secrete a second antigen, wherein the first and second antigens are expressed and secreted by the same cells or by different cells.
29. A device composition comprising a preparation of hydrogel capsules and a pharmaceutically acceptable excipient, wherein each hydrogel capsule in the preparation is a hydrogel capsule as defined in any of embodiments 23 to 28, and optionally wherein the composition has a volume of less than 10 milliliters, less than 8 ml, or less than 5 ml.
30. An implantable device comprising mammalian cells genetically modified to express and secrete a therapeutic substance, wherein the device is configured to:
31. The implantable device of embodiment 30, wherein the immunosuppressant compound is delivered from an extended release formulation.
32. The implantable device of embodiment 30 or 31, wherein the device is a hydrogel capsule which comprises:
33. The implantable device of embodiment 32, wherein the first hydrogel-forming polymer is an alginate and the second hydrogel-forming polymer is an alginate, optionally wherein the barrier compartment further comprises an unmodified alginate.
34. The implantable device of embodiment 32 or 33, wherein the cell-binding peptide consists of GRGDSP.
35. The implantable device of any one of embodiments 30 to 34, which induces a lower ADA response following implant into a subject than the ADA response induced by a reference device.
36. The implantable device of any one of embodiments 30 to 35, wherein the therapeutic substance is a blood clotting factor, a coagulation factor, a hormone, an enzyme, an antibody, a cytokine or a soluble cytokine receptor.
37. The implantable device of any one of embodiments 30 to 36, wherein the therapeutic substance is an enzyme.
38. The implantable device of any one of embodiments 30 to 37, wherein the therapeutic substance is an enzyme that is deficient or missing in a lysosomal storage disorder (LSD).
39. The implantable device of any one of embodiments 32 to 38, wherein the therapeutic substance is an arylsulfatase B (ARSB) protein, a GLA protein, a GAA protein, an IDUA protein, a NAGLU protein, a GUSB protein, a GBA protein, an HGSNAT protein, an IDS protein, a GNS protein or an SGSH protein.
40. The implantable device of any one of embodiments 32 to 39, wherein the therapeutic substance is a human IDUA protein.
41. The implantable device of any one of embodiments 32 to 39, wherein the therapeutic substance is a human GLA protein.
42. The implantable device of any one of embodiments 32 to 39, wherein the therapeutic substance is a human ARSB protein.
43. The implantable device of any one of embodiments 32 to 36, wherein the therapeutic substance is a FVIII protein, a FIX protein or a FVII protein.
44. A device composition comprising a preparation of devices and a pharmaceutically acceptable excipient, wherein each device in the preparation is a device as defined in any of embodiments 30 to 43, optionally wherein the composition has a volume of less than 10 milliliters, less than 8 ml, or less than 5 ml.
45. The device composition of embodiment 44, wherein each device in the preparation is a device as defined in any of embodiments 30 to 43.
46. A method of providing a therapeutic substance to a subject, comprising administering to the subject the device of any one of embodiments 30 to 43 or the device composition of embodiment 44 or 45.
47. The method of embodiment 46, further comprising measuring the level of antibodies to the therapeutic substance in a serum sample from the subject collected at two or more time points following the administration, optionally wherein the time points are selected from the group consisting of day 7, day 14, day 21, day 28, day 35, day 42 and day 48.
48. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein the immunosuppressant compound is an mTorr inhibitor.
49. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein the immunosuppressant compound is cyclosporine or a rapamycin compound.
50. The implantable device, hydrogel capsule, or method of any one of the above embodiments, wherein the immunosuppressant compound is a rapamycin compound.
51. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein the immunosuppressant compound is rapamycin.
52. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein the extended release formulation of the immunosuppressant is a sustained release formulation.
53. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein the extended release formulation of the immunosuppressant is a controlled release formulation.
54. The implantable device, hydrogel capsule, or method of any one of the above embodiments, wherein the immunomodulatory protein is an IL-10 protein, an IL-22 protein, or a soluble CTLA-4 protein (sCTLA-4).
55. The implantable device, hydrogel capsule or method of embodiment 54, wherein the immunomodulatory protein is an IL-10 protein, optionally wherein the IL-10 protein is encoded by an exogenous coding sequence shown in
56. The implantable device, hydrogel capsule or method of embodiment 54, wherein the immunomodulatory protein is an IL-22 protein, optionally wherein the IL-22 protein is encoded by an exogenous coding sequence shown in
57. The implantable device, hydrogel capsule or method of embodiment 54, wherein the immunomodulatory protein is an sCTLA-4 protein, optionally wherein the sCTLA-4 protein is encoded by an exogenous coding sequence shown in
58. The implantable device, hydrogel capsule or method of embodiment 54, which comprises an extended release formulation of rapamycin and a soluble FLT3-Ligand protein (sFLT3-L), optionally wherein the sFLT3-L protein is encoded by an exogenous coding sequence shown in
59. The implantable device, hydrogel capsule, or method of any one of the above embodiments, wherein the mammalian cells are also genetically modified to express and secrete the immunomodulatory protein.
60. The implantable device, hydrogel capsule, or method of any one of the above embodiments, wherein all of the genetically modified cells contained in the device or hydrogel capsule are derived from RPE cells or induced pluripotent stem cells.
61. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein all of the genetically modified cells contained in the device or hydrogel capsule are derived from ARPE-19 cells.
62. The implantable device, hydrogel capsule, or method of any one of the above embodiments, wherein the FBR-mitigating compound is a compound of Formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
63. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein the FBR-mitigating compound is selected from the compounds shown in Table 4 above, or a pharmaceutically acceptable salt of the selected compound.
64. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein the FBR-mitigating compound is selected from the group consisting of Compounds 100, 101, 102, 114, 122 and 123 shown in Table 4 above, or a pharmaceutically acceptable salt of the selected compound.
65. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein the FBR-mitigating compound is Compound 100 or a pharmaceutically acceptable salt thereof.
66. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein the FBR-mitigating compound is Compound 101 or a pharmaceutically acceptable salt thereof.
67. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein the FBR-mitigating compound is Compound 114 or a pharmaceutically acceptable salt thereof.
68. The implantable device, hydrogel capsule or method of any one of the above embodiments, wherein the FBR-mitigating compound is Compound 122 or a pharmaceutically acceptable salt thereof.
69. The implantable device or hydrogel capsule or method of any one of the above embodiments, wherein the FBR-mitigating compound is Compound 123 or a pharmaceutically acceptable salt thereof.
70. A genetically modified cell comprising an exogenous nucleotide coding sequence shown in
71. The genetically modified cell of embodiment 70, which is derived from ARPE-19 cells.
In order that the disclosure described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the implantable devices, and compositions and methods provided herein and are not to be construed in any way as limiting their scope.
Genetically modified ARPE-19 cells expressing one or more immunomodulatory proteins described herein may be cultured to produce a composition of cells suitable for encapsulation in two compartment hydrogel capsules. The genetically-modified cells are grown in complete growth medium (DMEM: F12 with 10% FBS) in 150 cm2 cell culture flasks or CellSTACK® Culture Chambers (Corning Inc., Corning, NY). To passage cells, the medium in the culture flask are aspirated, and the cell layer is briefly rinsed with phosphate buffered saline (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, Gibco). 5-10 mL of 0.05% (w/v) trypsin/0.53 mM EDTA solution (“TrypsinEDTA”) is added to the flask, and the cells are observed under an inverted microscope until the cell layer is dispersed, usually between 3-5 minutes. To avoid clumping, cells are handled with care and hitting or shaking the flask during the dispersion period is minimized. If the cells do not detach, the flasks are placed at 37° C. to facilitate dispersal. Once the cells disperse, 10 mL complete growth medium is added and the cells are aspirated by gentle pipetting. The cell suspension is transferred to a centrifuge tube and spun down at approximately 125× g for 5-10 minutes to remove TrypsinEDTA. The supernatant is discarded, and the cells are resuspended in fresh growth medium. Appropriate aliquots of cell suspension are added to new culture vessels, which are incubated at 37° C. The medium is renewed weekly.
Chemically-modified Polymer. A polymeric material may be chemically modified with a compound of Formula (I) (or pharmaceutically acceptable salt thereof) prior to formation of a device described herein (e.g., a hydrogel capsule). For example, in the case of alginate, the alginate carboxylic acid is activated for coupling to one or more amine-functionalized compounds to achieve an alginate modified with an afibrotic compound, e.g., a compound of Formula (I). The alginate polymer is dissolved in water (30 mL/gram polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 eq) and N-methylmorpholine (1 eq). To this mixture is added a solution of the compound of interest (e.g., Compound 101 shown in Table 4) in acetonitrile (0.3M).
The amounts of the compound and coupling reagent added depends on the desired concentration of the compound bound to the alginate, e.g., conjugation density. A medium conjugation density of Compound 101 typically ranges from 2% to 5% N, while a high conjugation density of Compound 101 typically ranges from 5.1% to 8% N. To prepare a solution of low molecular weight alginate, chemically modified with a medium conjugation density of Compound 101 (CM-LMW-Alg-101-Medium polymer), the dissolved unmodified low molecular weight alginate (approximate MW<75 kDa, G:M ratio ≥1.5) is treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (5.1 mmol/g alginate) and N-methylmorpholine (10.2 mmol/g alginate) and Compound 101 (5.4 mmol/g alginate). To prepare a solution of low molecular weight alginate, chemically modified with a high conjugation density of Compound 101 (CM-LMW-Alg-101-High polymer), the dissolved unmodified low-molecular weight alginate (approximate MW<75 kDa, G:M ratio ≥1.5) is treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (5.1 mmol/g alginate) and N-methylmorpholine (10.2 mmol/g alginate) and Compound 101 (10.5 mmol/g alginate).
The reaction is warmed to 55° C. for 16 h, then cooled to room temperature and gently concentrated via rotary evaporation, then the residue is dissolved in water. The mixture is filtered through a bed of cyano-modified silica gel (Silicycle) and the filter cake is washed with water. The resulting solution is then extensively dialyzed (10,000 MWCO membrane) and the alginate solution is concentrated via lyophilization to provide the desired chemically-modified alginate as a solid or is concentrated using any technique suitable to produce a chemically modified alginate solution with a viscosity of 25 cP to 35 cP.
The conjugation density of a chemically modified alginate is measured by combustion analysis for percent nitrogen. The sample is prepared by dialyzing a solution of the chemically modified alginate against water (10,000 MWCO membrane) for 24 hours, replacing the water twice followed by lyophilization to a constant weight.
CBP-Alginates. A polymeric material may be covalently modified with a cell-binding peptide prior to formation of a device described herein (e.g., a hydrogel capsule described herein) using methods known in the art, see, e.g., Jeon O, et al., Tissue Eng Part A. 16:2915-2925 (2010) and Rowley, J. A. et al., Biomaterials 20:45-53 (1999).
For example, in the case of alginate, an alginate solution (1%, w/v) is prepared with 50 mM of 2-(N-morpholino)-ethanesulfonic acid hydrate buffer solution containing 0.5M NaCl at pH 6.5, and sequentially mixed with N-hydroxysuccinimide and 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide (EDC). The molar ratio of N-hydroxysuccinimide to EDC is 0.5:1.0. The peptide of interest is added to the alginate solution. The amounts of peptide and coupling reagent added depends on the desired concentration of the peptide bound to the alginate, e.g., peptide conjugation density. By increasing the amount of peptide and coupling reagent, higher conjugation density can be obtained. After reacting for 24 h, the reaction is purified by dialysis against ultrapure deionized water (diH2O) (MWCO 3500) for 3 days, treated with activated charcoal for 30 min, filtered (0.22 mm filter), and concentrated to the desired viscosity.
The conjugation density of a peptide-modified alginate is measured by combustion analysis for percent nitrogen. The sample is prepared by dialyzing a solution of the chemically modified alginate against water (10,000 MWCO membrane) for 24 hours, replacing the water twice followed by lyophilization to a constant weight.
70:30 mixture of chemically-modified and unmodified alginate. A low molecular weight alginate (PRONOVA™ VLVG alginate, NovaMatrix, Sandvika, Norway, cat. #4200506, approximate molecular weight <75 kDa; G:M ratio ≥1.5) is chemically modified with Compound 101 to produce chemically modified low molecular weight alginate (CM-LMW-Alg-101) solution with a viscosity of 25 cp to 35 cP and a conjugation density of 5.1% to 8% N, as determined by combustion analysis for percent nitrogen. A solution of high molecular weight unmodified alginate (U-HMW-Alg) is prepared by dissolving unmodified alginate (PRONOVA™ SLG100, NovaMatrix, Sandvika, Norway, cat. #4202106, approximate molecular weight of 150 kDa-250 kDa) at 3% weight to volume in 0.9% saline. The CM-LMW-Alg solution is blended with the U-HMW-Alg solution at a volume ratio of 70% CM-LMW-Alg to 30% U-HMW-Alg (referred to herein as a 70:30 CM-Alg: UM-Alg solution).
Unmodified alginate solution. An unmodified medium molecular weight alginate (SLG20, NovaMatrix, Sandvika, Norway, cat. #4202006, approximate molecular weight of 75-150 kDa), is dissolved at 1.4% weight to volume in 0.9% saline to prepare a U-MMW-Alg solution.
Unmodified alginate solution. An unmodified medium molecular weight alginate (SLG20, NovaMatrix, Sandvika, Norway, cat. #4202006, approximate molecular weight of 75-150 kDa), is dissolved at 1.4% weight to volume in 0.9% saline to prepare a U-MMW-Alg solution.
Alginate Solution Comprising Cell Binding Sites. A solution of SLG20 alginate is modified with a peptide consisting of GRGDSP (SEQ ID NO:34) as described above and concentrated to a viscosity of about 100 cP. The amount of the peptide and coupling reagent used are selected to achieve a target peptide conjugation density of about 0.2 to 0.3, as measured by combustion analysis.
An amount of a solid, amorphous form of the desired immunosuppressant (e.g., a glucocorticoid) is added to an alginate solution (e.g., GRGDSP-medium molecular weight alginate in saline, viscosity of about 100 cP) in a conical tube (e.g., 15 mL to 50 mL) in an amount sufficient to form a suspension with a desired concentration of the solid immunosuppressant in the alginate solution (e.g., 1 mg to 10 mg compound per mL alginate solution). The tube is placed in a 5.7 liter sonication bath (Ultrasonic Cleaner, VWR Catalog No. 97043-940, Input: 117V˜60 Hz, Operating frequency: 35 kHz) at room temperature for 3 minutes to 10 minutes, removed and vortexed for one minute at 3,000 RPM (Thermo Scientific™ LP Vortex Mixer). The sonication and vortex steps are repeated until no visible powder was observed, indicating that a fine, substantially homogenous suspension has been generated. The suspension is kept at 4° C. until use and is used within 6 hours.
Genetically modified cells, and optionally immunosuppressant particles, are encapsulated in two-compartment hydrogel capsules according to the protocol described below.
Prior to fabricating hydrogel capsules, buffers and alginate solutions are sterilized by filtration through a 0.2-μm filter using aseptic processes.
Immediately before encapsulation, a desired volume of a composition comprising the cells (e.g., from a culture of the cells as described in Example 1) is centrifuged at 1,400 r.p.m. for 1 min and washed with calcium-free Krebs-Henseleit (KH) Buffer (4.7 mM KCl, 25 mM HEPES, 1.2 mM KH2PO4, 1.2 mM MgSO4×7H2O, 135 mM NaCl, PH≈7.4, ≈290 mOsm). After washing, the cells ae centrifuged again and all of the supernatant is aspirated. The cell pellet is resuspended in the GRGDSP-modified alginate solution described in Example 3 or in Example 4 at a desired cell density (e.g., about 50 to 150 million suspended single cells per ml alginate solution).
To prepare two-compartment hydrogel millicapsules of about 1.5 mm diameter an electrostatic droplet generator is set up as follows: an ES series 0-100-kV, 20-watt high-voltage power generator (EQ series, Matsusada, NC, USA) is connected to the top and bottom of a coaxial needle. For capsules without an immunosuppressant, a suitable needle has an inner lumen of 22G, outer lumen of 18G, Ramé-Hart Instrument Co., Succasunna, NJ, USA. To prepare capsules that co-encapsulate immunosuppressant particles in the inner compartment, the inner lumen of the coaxial needle may need to have a larger diameter to avoid needle clogging by the immunosuppressant particles, e.g., a useful coaxial needle has an inner lumen of 21G and an outer lumen of 17G, Ramé-Hart Instrument Co., Succasunna, NJ, USA).
The inner lumen is attached to a first 5-ml Luer-lock syringe (BD, NJ, USA), which is connected to a syringe pump (Pump 11 Pico Plus, Harvard Apparatus, Holliston, MA, USA) that is oriented vertically. The outer lumen is connected via a luer coupling to a second 5-ml Luer-lock syringe which is connected to a second syringe pump (Pump 11 Pico Plus) that is oriented horizontally. A first alginate solution containing the genetically modified cells (as single cells) suspended in a GRGDSP-modified alginate solution is placed in the first syringe and a cell-free alginate solution comprising a mixture of a chemically-modified alginate and unmodified alginate is placed in the second syringe. The two syringe pumps move the first and second alginate solutions from the syringes through both lumens of the coaxial needle and single droplets containing both alginate solutions are extruded from the needle into a glass dish containing a cross-linking solution. The settings of each Pico Plus syringe pump are 12.06 mm diameter and the flow rates of each pump are adjusted to achieve a flow rate ratio of 1:1 for the two alginate solutions. Thus, with the total flow rate set at 10 ml/h, the flow rate for each alginate solution was about 5 mL/h. Control (empty) capsules are prepared in the same manner except that the alginate solution used for the inner compartment is a cell-free solution.
After extrusion of the desired volumes of alginate solutions, the alginate droplets are crosslinked for five minutes in a cross-linking solution which contained 25 mM HEPES buffer, 20 mM BaCl2, 0.2M mannitol and 0.01% of poloxamer 188. Capsules that fall to the bottom of the crosslinking vessel are collected by pipetting into a conical tube. After the capsules settle in the tube, the crosslinking buffer is removed, and capsules are washed four times in HEPES buffer, two times in 0.9% saline, and two times in culture media and stored in an incubator at 37° C.
A rapamycin particle suspension is prepared as described in Example 4 using amounts of the rapamycin compound (solid, amorphous form) and alginate solution to achieve a concentration of 2.5 mg of the rapamycin solid/mL alginate solution. Two-compartment hydrogel capsules containing the suspension in the inner compartment are prepared as in Example 5, except that cells are not typically included. Multiple capsules (e.g., five) are placed in each of a desired number of replicate wells (e.g., four) in a multiwell tissue culture plate (e.g., Falcon® 12-well Clear Flat Bottom Not Treated Multiwell Cell Culture Plate). Each of the replicate wells contains 1 mL of a cell culture media (e.g., DMEMF12, Gibco Cat. No. 113 30-032 with rFBS, Gibco Cat. No. 26140-095). The media in each well is replaced with fresh 1 mL of the media at day 1, day 4 and day 7. Pictures of each well are taken using a BZX Fluorescence Microscope at day 1, day 4, day 7 to visually assess the capsules for the presence of encapsulated rapamycin particles.
The quantity of the rapamycin compound diffusing from the capsules into the media over time is estimated by removing an aliquot (e.g., 20 μL) of the media supernatant from each replicate well on multiple days (e.g., day 1, day 4 and day 7) and diluting the removed aliquot 10-fold with Methanol (e.g., 180 μL). The sample is vortexed briefly and centrifuged at 12,500×g for 10 minutes. The supernatant is removed and transferred to an LCMS vial for analysis. The samples are analyzed using a Thermo Fisher Vanquish UPLC interfaced to a Thermo Fisher Q Exactive mass spectrometer. The chromatographic separation is performed on a 50 mm×2.1 mm Waters BEH C18 chromatography column with 1.7 μm particle size. Mobile phases A and B are 0.1-% aqueous formic acid and 0.1% formic acid in acetonitrile, respectively. The column temperature is held at 65° C. and the mobile phase is flowed at 500 μL/min. Injection volume is 5 μL. The separation is accomplished with a gradient started at 10% mobile phase B and increasing to 99% over 2.5 minutes. The column is held at 99% B for 1 minute before returning to initial conditions. The initial conditions (10% B) are held for 1.5 minutes before the next LC injection. The Q Exactive mass spectrometer is configured with an electrospray ionization source operating in positive ion mode. Data are acquired using a full scan method scanning from 400-1250 m/z at a resolution of 70,000. Data are acquired using a full scan method scanning from 400-1250 m/z at a resolution of 70,000. For rapamycin, data are analyzed by extracting a 10 ppm window centered on the rapamycin-sodium adduct+1 ions and the retention time is 1.35 minutes and the m/z is 936.5443.
A suspension of rapamycin particles prepared as in Example 4 is likely to provide the desired extended release of rapamycin from the capsules if less than 10% of the encapsulated amount has been released at day 7, and preferably less than <5%, <2%, or <1% has been released at day 7.
Using the process described in Example 4 we generated empty alginate spheres or alginate spheres containing rapamycin particles. The bright field micrographs show empty alginate spheres (
Using the processes described in Examples 4 and 5, we generated alginate spheres which contained ARPE19 cells genetically modified to express and secrete an hGLA protein (
The rate of rapamycin release from spheres containing hGLA-secreting cells and rapamycin particles was measured using the process described in Example 6. As depicted in
Immunocompetent C57BL/6 mice were implanted with alginate spheres containing hGLA-secreting cells or with alginate spheres containing hGLA-secreting cells and rapamycin particles. Weekly bleeds were performed and hGLA-specific IgG titers were measured in the plasma over 45 days. Recipient mice implanted with spheres containing hGLA-secreting cells uniformly developed high-titers of antibodies specific to hGLA. In contrast, mice implanted with alginate spheres co-encapsulating rapamycin particles and hGLA-secreting cells developed significantly lower titers of hGLA-specific antibodies (
This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference in their entirety. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, Figures, or Examples but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/013943 | 1/26/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63141892 | Jan 2021 | US |
