Transient sirolimus using FASL microgels

Abstract

Provided herein is a method of inducing immune tolerance to graft cells in a human patient in need thereof, the method comprising administering to the patient: (i) graft cells, (ii) a chimeric FasL protein conjugated to a hydrogel, and (iii) transient sirolimus.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 364,607, filed on May 12, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] Reference to Electronically Submitted Sequence Listing The content of the electronically submitted sequence listing (filename: 4999_003PC01_SequenceListing_ST26; size: 8,373 bytes; and creation date: May 5, 2023), submitted together with this application, is incorporated herein by reference in its entirety.

Background Art

[0003] The transplantation of allogeneic graft cells is limited because chronic immunosuppression against the host's immune attack is required to keep the graft alive. Achieving long - term survival of allogeneic grafts without chronic immunosuppression remains a difficult goal to achieve in clinical transplantation. Lifelong immunosuppression is associated with significant risks of morbidity and mortality, including the risk of cancer, renal dysfunction, and beta - cell toxicity, and requires the use of adjuvant therapies such as antiviral, antifungal, and antibacterial agents for chronic prophylaxis to prevent infections. Removing chronic immunosuppression can eliminate these risks and have an immeasurable beneficial impact on reducing the emotional, psychosocial, quality of life, and healthcare costs of patients receiving graft cell transplantation. Therefore, there is an unmet medical need for methods of transplanting graft cells without using chronic immunosuppression.

Summary of the Invention

[0004] Provided herein is a method of transplanting allogeneic graft cells that achieves long-term survival of the graft without the need for chronic immunosuppression. The method includes administering graft cells and Fas ligand (FasL) conjugated to a hydrogel (e.g., a microgel) together with a transient regimen of sirolimus.

[0005] In some embodiments, a method of inducing immune tolerance to graft cells in a human patient in need thereof comprises administering to the human patient (i) graft cells, (ii) a chimeric FasL protein conjugated to a hydrogel, and (iii) sirolimus, wherein sirolimus is administered for 36 weeks or less, optionally 24 weeks or less.

[0006] In some embodiments, sirolimus is administered at an initial dose that maintains a sirolimus trough blood concentration of about 4 ng / mL to about 16 ng / mL. In some embodiments, sirolimus is administered at an initial dose that maintains a sirolimus trough blood concentration of about 9 ng / mL to about 16 ng / mL. In some embodiments, sirolimus is administered at an initial dose that maintains a sirolimus trough blood concentration of about 4 ng / mL to about 15 ng / mL. In some embodiments, sirolimus is administered at an initial dose that maintains a sirolimus trough blood concentration of about 9 ng / mL to about 15 ng / mL. In some embodiments, sirolimus is administered at an initial dose that maintains a sirolimus trough blood concentration of about 9 ng / mL to about 13 ng / mL. In some embodiments, the initial dose is administered over an initial period of about 12 weeks or less. In some embodiments, the initial dose is administered over an initial period of about 1 week to about 12 weeks. In some embodiments, the initial dose is administered over an initial period of about 6 weeks to about 12 weeks.

[0007] In some embodiments, sirolimus is administered in a tapered regimen during a taper period. In some embodiments, the tapered regimen is administered after an initial period of an initial dose. In some embodiments, the tapered regimen occurs after an initial period of an initial dose and includes a first tapered dose administered over a first taper period and a second tapered dose administered over a second taper period. In some embodiments, the first tapered dose maintains a sirolimus trough blood concentration of about 4 ng / mL to about 11 ng / mL. In some embodiments, the first tapered dose maintains a sirolimus trough blood concentration of about 4 ng / mL to about 11 ng / mL. In some embodiments, the first taper period is about 2 weeks or less. In some embodiments, the first taper period is about 1 week to about 2 weeks.

[0008] In some embodiments, the second tapered dose maintains a sirolimus trough blood concentration of about 4 ng / mL to about 9 ng / mL. In some embodiments, the second tapered dose maintains a sirolimus trough blood concentration of about 5 ng / mL to about 9 ng / mL. In some embodiments, the second taper period is about 2 weeks or less. In some embodiments, the second taper period is about 1 week to about 2 weeks.

[0009] In some embodiments, the tapered regimen further includes a third tapered dose administered over a third taper period. In some embodiments, the third tapered dose maintains a sirolimus trough blood concentration of about 3 ng / mL to about 7 ng / mL. In some embodiments, the third taper period is about 2 weeks or less. In some embodiments, the third taper period is about 1 week to about 2 weeks.

[0010] In some embodiments, the tapered regimen further includes a fourth tapered dose administered over a fourth taper period. In some embodiments, the fourth tapered dose maintains a sirolimus trough blood concentration of about 1 ng / mL to about 5 ng / mL. In some embodiments, the fourth taper period is about 2 weeks or less. In some embodiments, the fourth taper period is about 1 week to about 2 weeks.

[0011] In some embodiments, the tapering regimen further comprises a fifth tapering dose administered over a fifth tapering period. In some embodiments, the fifth tapering dose maintains a sirolimus trough blood concentration of from about 0 ng / mL to about 3 ng / mL. In some embodiments, the fifth tapering period is about 2 weeks or less. In some embodiments, the fifth tapering period is from about 1 week to about 2 weeks.

[0012] In some embodiments, the tapering regimen comprises maintaining a sirolimus trough blood concentration of from about 4 ng / mL to about 11 ng / mL for 2 weeks or less, maintaining a sirolimus trough blood concentration of from about 4 ng / mL to about 9 ng / mL for 2 weeks or less, maintaining a sirolimus trough blood concentration of from about 3 ng / mL to about 7 ng / mL for 2 weeks or less, maintaining a sirolimus trough blood concentration of from about 1 ng / mL to about 5 ng / mL for 2 weeks or less, and / or maintaining a sirolimus trough blood concentration of from about 0 ng / mL to about 3 ng / mL for 2 weeks or less.

[0013] In some embodiments, the tapering regimen comprises maintaining a sirolimus trough blood concentration of from about 7 ng / mL to about 11 ng / mL for 2 weeks or less, maintaining a sirolimus trough blood concentration of from about 5 ng / mL to about 9 ng / mL for 2 weeks or less, maintaining a sirolimus trough blood concentration of from about 3 ng / mL to about 7 ng / mL for 2 weeks or less, maintaining a sirolimus trough blood concentration of from about 1 ng / mL to about 5 ng / mL for 2 weeks or less, and / or maintaining a sirolimus trough blood concentration of from about 0 ng / mL to about 3 ng / mL for 2 weeks or less.

[0014] In some embodiments, the tapering period is (i) about 12 weeks or less or (ii) from about 6 weeks to about 12 weeks.

[0015] In some embodiments, the methods provided herein further comprise administering to the subject mesenchymal stem cells, a CD20 agent, and / or a CD47 agent. In some embodiments, the CD20 agent is rituximab.

[0016] In some embodiments, the graft cells are selected from peripheral blood mononuclear cells (PBMCs), bone marrow cells, hematopoietic stem cells, stem cells, stem cell-derived cells, mesenchymal stem cells, dendritic cells, dendritic cells pulsed with self-antigen, human beta cell products, pancreatic islet cells, allogeneic islet cells, hepatocytes, and splenocytes. In some embodiments, the graft cells are pancreatic islet cells or pancreatic islet cells derived from insulin-producing stem cells. In some embodiments, the graft cells are hepatocytes. In some embodiments, the graft cells are stem cells or stem cell-derived cells. In some embodiments, the graft cells are derived from a deceased donor. In some embodiments, the graft cells are allogeneic.

[0017] In some embodiments, the chimeric FasL protein comprises a FasL portion and a streptavidin portion or an avidin portion. In some embodiments, the chimeric FasL chimeric protein further comprises a linker between the FasL portion and the streptavidin portion or the avidin portion. In some embodiments, the chimeric FasL protein comprises a FasL portion and a streptavidin portion. In some embodiments, the FasL portion is a matrix metalloprotease-resistant FasL protein. In some embodiments, the chimeric FasL protein comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the chimeric FasL protein is conjugated to a hydrogel via biotin.

[0018] In some embodiments, the hydrogel is a microgel. In some embodiments, the microgel is from about 125 micrometers to about 175 micrometers. In some embodiments, the microgel is about 150 micrometers. In some embodiments, the hydrogel is a polyethylene glycol (PEG) microgel. In some embodiments, the hydrogel is engineered to present biotin moieties.

[0019] In some embodiments, the graft cells are not encapsulated in the hydrogel.

[0020] In some embodiments, a hydrogel:transplanted cell ratio of 2:1 is administered.

[0021] In some embodiments, immune tolerance to graft cells is induced to treat type 1 diabetes. In some embodiments, at least 5,000 islet equivalents per kilogram of human patient are administered.

[0022] In some embodiments, immune tolerance to graft cells is induced to treat liver failure.

[0023] In some embodiments, a chimeric FasL protein conjugated to graft cells and hydrogel is administered to the reticulum.

[0024] In some embodiments, sirolimus is administered orally. In some embodiments, sirolimus is administered as an oral solution. In some embodiments, sirolimus is administered as an oral tablet. In some embodiments, sirolimus is administered once daily. In some embodiments, sirolimus administration is initiated on the same day or up to 5 days before the day on which a chimeric FasL protein conjugated to graft cells and hydrogel is administered to a human subject.

[0025] In some embodiments, the method further comprises administering prophylactic antiviral and antibacterial agents during sirolimus administration. In some embodiments, the prophylactic antiviral agent is famciclovir. In some embodiments, the antibacterial agent comprises sulfamethoxazole and / or trimethoprim.

[0026] Also provided herein is the use of a chimeric FasL protein conjugated to a hydrogel for inducing immune tolerance in a human patient according to any method provided herein.

[0027] Also provided herein is a chimeric FasL protein conjugated to a hydrogel for use in inducing immune tolerance according to any method provided herein.

Brief Description of the Drawings

[0028]

Figure 1

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 2G

Figure 3

Figure 4-1

Figure 4-2

Figure 4-3

Figure 5-1

Figure 5-2

Figure 6-1

Figure 6-2

Figure 6-3

Figure 7

[0029] Terminology As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise.

[0030] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. The term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A," and "B." Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0031] When an aspect is described herein using the word "comprising," it is understood that alternative similar aspects described using the expressions "consisting of" and / or "consisting essentially of" are also provided. In this disclosure, "comprise," "comprising," "contain," and "have" etc. may mean "include," "including," etc., and "consisting essentially of" or "consisting of" is open-ended and allows for the presence of more than what is listed, provided that the basic or novel features of what is listed are not changed by the presence of more than what is listed, excluding prior art aspects.

[0032] As used herein, the terms "about" and "approximately" when used to modify a numerical value or numerical range indicate that a deviation of up to 10% above or below that value or range is within the intended meaning of the recited value or range. When an aspect is described herein using the word "about" or "approximately" with respect to a numerical value or range, it is understood that another similar aspect with respect to the specific numerical value or range (without "about") is also provided.

[0033] As used herein, the term "sirolimus" (also known as rapamycin) refers to a macrocyclic lactone compound having the chemical name (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadehydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotriacontine-1,5,11,28,29(4H,6H,31H)-pentone. This term includes, for example, the active compound sold under the trade name Rapamune.

[0034] As used herein, "FasL" refers to "Fas ligand", also known as tumor necrosis factor ligand superfamily member 6, apoptosis antigen ligand (APTL), or CD95 ligand (CD95-L). The sequence of the human FasL protein is It is MQQPFNYPYPQIYWVDSSASSPWAPPGTVLPCPTSVPRRPGQRRPPPPPPPPPLPPPPPPPPLPPLPLPPLKKRGNHSTGLCLLVMFFMVLVALVGLGLGMFQLFHLQKELAELRESTSQMHTASSLEKQIGHPSPPPEKKELRKVAHLTGKSNSRSMPLEWEDTYGIVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGKMMSYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL (SEQ ID NO: 7).

[0035] Amino acids 1 to 80 of SEQ ID NO: 7 are the cytoplasmic domain of human FasL. Amino acids 81 to 102 of SEQ ID NO: 7 are the transmembrane domain of human FasL, and amino acids 103 to 281 of SEQ ID NO: 7 are the extracellular domain of human FasL.

[0036] The "chimeric FasL protein" refers to a protein comprising a fusion of a FasL protein or a fragment thereof with a heterologous protein (e.g., streptavidin or avidin) or a fragment thereof.

[0037] As used herein, "biotin" (hexahydro-2-oxo-1H-thieno(3,4-d)imidazole-4-pentanoic acid) includes biotin-containing moieties capable of binding to surfaces such as cell surfaces, such as HS-biotin and EZ-Link™ sulfo-HS-LC-biotin (Pierce). Biotin and protein-reactive biotins are commercially available.

[0038] As used herein, "hydrogel" refers to a polymer material swollen with water. These include, for example, hydrogel polymer networks having dimensions much larger than cells (e.g., greater than 500 μm). Hydrogels are typically formed by cross-linking organic polymers (natural or synthetic) by covalent, ionic, or hydrogen bonds to form a three-dimensional open lattice structure that takes up water molecules to form a gel. Examples of materials that can be used to form hydrogels include macromer-based materials (including PEG macromers) assembled using different cross-linking methods (e.g., Michael-type addition, thiol-ene, click reactions, etc.), polysaccharides (such as alginic acid), polyphosphazenes, and polyacrylates, or block copolymers, such as Pluronic™ or Tetronic™ polyethylene oxide-polypropylene glycol block copolymers that can be cross-linked by temperature, free radical polymerization, click reaction, or pH, respectively.

[0039] As used herein, "microgel" refers to a hydrogel having smaller dimensions (e.g., on the order of tens or hundreds of μm).

[0040] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers modified by natural or intervening means, such as by any other operation or modification, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. This definition also includes polypeptides containing, for example, one or more analogs of amino acids (including non-natural amino acids, etc.) and other modifications known in the art.

[0041] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those purified to the extent that they are no longer in their natural form. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure. As used herein, "substantially pure" refers to a substance that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0042] "Percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). Percent identity can be determined by aligning the two sequences while introducing gaps to maximize identity between the sequences. The alignment can be generated using programs known in the art. For the purposes herein, alignment of nucleotide sequences can be performed with the blastn program set to default parameters, and alignment of amino acid sequences can be performed with the blastp program set to default parameters (see world wide web ncbi.nlm.nih.gov of the National Center for Biotechnology Information (NCBI)).

[0043] As used herein, amino acids having hydrophobic side chains include alanine (A), isoleucine (I), leucine (L), methionine (M), valine (V), phenylalanine (F), tryptophan (W), and tyrosine (Y). Amino acids having aliphatic hydrophobic side chains include alanine (A), isoleucine (I), leucine (L), methionine (M), and valine (V). Amino acids having aromatic hydrophobic side chains include phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0044] As used herein, amino acids having polar neutral side chains include asparagine (N), cysteine (C), glutamine (Q), serine (S), and threonine (T).

[0045] As used herein, amino acids having charged side chains include aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (K). Amino acids having acidic charged side chains include aspartic acid (D) and glutamic acid (E). Amino acids having basic charged side chains include arginine (R), histidine (H), and lysine (K).

[0046] As used herein, the term "host cell" can be any cell type, e.g., primary cells, cells in culture, or cells derived from a cell line. In some embodiments, the term "host cell" refers to a cell that has been transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parental cell that was transfected with the nucleic acid molecule, e.g., due to mutations that may occur in the progeny or environmental influences, or due to the incorporation of the nucleic acid molecule into the host cell genome.

[0047] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that enables the biological activity of the active ingredient and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. The formulation may be sterile.

[0048] As used herein, terms such as "administer", "administering", and "administration" refer to methods that can be used to enable delivery of an active agent (e.g., graft cells, microgels, and / or sirolimus) to a desired biological site of action.

[0049] As used herein, the terms "subject" and "patient" are used interchangeably. A subject can be an animal. In some embodiments, the subject is a mammal such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate). In some embodiments, the subject is a human.

[0050] The term "therapeutically effective amount" refers to an amount effective to treat a disease or disorder in a subject.

[0051] Terms such as "treating" or "treatment" or "treat" or "alleviating" or "alleviate" refer to therapeutic measures that effect the cure, mitigation, alleviation of symptoms, and / or arrest of the progression of a diagnosed pathological condition or disorder. Thus, those in need of treatment include those already diagnosed with a disorder or suspected of having a disorder.

[0052] As used herein, a "transient" dose or regimen refers to administration for a limited period of time and is not chronic. For example, transient administration can last for 36 weeks or less, 30 weeks or less, or 24 weeks or less.

[0053] As used herein, a "tapering" dose or regimen refers to administration in an amount that decreases over time. When the dose of a regimen refers to a range, "tapering" refers to a range where the upper limit decreases, e.g., a dosing regimen that achieves a trough blood concentration of 9 - 13 ng / mL for a period of time, then a trough blood concentration of 7 - 11 ng / mL for a period of time, then a trough blood concentration of 5 - 9 ng / mL is a "tapering" regimen.

[0054] Alternatively, the pharmacological and / or physiological effect can be a prophylactic effect, i.e., an effect of completely or partially preventing a disease or its symptoms. In this regard, the disclosed methods include administering a "prophylactically effective amount" of a drug (e.g., one or more antibodies or antigen-binding fragments thereof). A "prophylactically effective amount" refers to an amount effective for the dosage and period required to achieve the desired prophylactic result.

[0055] Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

[0056] Transient sirolimus administration Provided herein is a method of inducing immune tolerance to graft cells using a transient dose of sirolimus. In some embodiments, sirolimus is administered for 36 weeks or less. In some embodiments, sirolimus is administered for 30 weeks or less. In some embodiments, sirolimus is administered for 24 weeks or less.

[0057] According to the methods provided herein, sirolimus can be administered for about 1 to about 36 weeks after graft cells are administered to a patient. In some embodiments, sirolimus is administered for about 4 to about 36 weeks after graft cells are administered to a patient. In some embodiments, sirolimus is administered for about 8 to about 36 weeks after graft cells are administered to a patient. In some embodiments, sirolimus is administered for about 10 to about 36 weeks after graft cells are administered to a patient. In some embodiments, sirolimus is administered for about 12 to about 36 weeks after graft cells are administered to a patient.

[0058] According to the method provided herein, sirolimus can be administered for about 1 week to about 30 weeks after the patient has been administered graft cells. In some embodiments, sirolimus is administered for about 4 to about 30 weeks after the patient has been administered graft cells. In some embodiments, sirolimus is administered for about 8 to about 30 weeks after the patient has been administered graft cells. In some embodiments, sirolimus is administered for about 10 to about 30 weeks after the patient has been administered graft cells. In some embodiments, sirolimus is administered for about 12 to about 30 weeks after the patient has been administered graft cells.

[0059] According to the method provided herein, sirolimus can be administered for about 1 week to about 24 weeks after the patient has been administered graft cells. In some embodiments, sirolimus is administered for about 4 to about 24 weeks after the patient has been administered graft cells. In some embodiments, sirolimus is administered for about 8 to about 24 weeks after the patient has been administered graft cells. In some embodiments, sirolimus is administered for about 10 to about 24 weeks after the patient has been administered graft cells. In some embodiments, sirolimus is administered for about 12 to about 24 weeks after the patient has been administered graft cells.

[0060] In some embodiments, sirolimus is administered at a dose that maintains a sirolimus trough blood concentration of about 4 ng / mL to about 16 ng / mL. The trough blood concentration of about 4 ng / mL to about 16 ng / mL is maintained for an initial period of about 12 weeks or less, for example, about 1 week to about 12 weeks, about 2 weeks to about 12 weeks, about 3 weeks to about 12 weeks, about 4 weeks to about 12 weeks, about 5 weeks to about 12 weeks, about 6 weeks to about 12 weeks, about 7 weeks to about 12 weeks, about 8 weeks to about 12 weeks, about 9 weeks to about 12 weeks, about 10 weeks to about 12 weeks, or about 11 weeks to about 12 weeks.

[0061] In some embodiments, sirolimus is administered at a dose that maintains a sirolimus trough blood concentration of about 4 ng / mL to about 15 ng / mL. The trough blood concentration of about 4 ng / mL to about 15 ng / mL is maintained for an initial period of about 12 weeks or less, for example, about 1 week to about 12 weeks, about 2 weeks to about 12 weeks, about 3 weeks to about 12 weeks, about 4 weeks to about 12 weeks, about 5 weeks to about 12 weeks, about 6 weeks to about 12 weeks, about 7 weeks to about 12 weeks, about 8 weeks to about 12 weeks, about 9 weeks to about 12 weeks, about 10 weeks to about 12 weeks, or about 11 weeks to about 12 weeks.

[0062] In some embodiments, sirolimus is administered at a dose that maintains a sirolimus trough blood concentration of about 9 ng / mL to about 16 ng / mL. The trough blood concentration of about 9 ng / mL to about 16 ng / mL is maintained for an initial period of about 12 weeks or less, for example, about 1 week to about 12 weeks, about 2 weeks to about 12 weeks, about 3 weeks to about 12 weeks, about 9 weeks to about 12 weeks, about 5 weeks to about 12 weeks, about 6 weeks to about 12 weeks, about 7 weeks to about 12 weeks, about 8 weeks to about 12 weeks, about 9 weeks to about 12 weeks, about 10 weeks to about 12 weeks, or about 11 weeks to about 12 weeks.

[0063] In some embodiments, sirolimus is administered at a dose that maintains a sirolimus trough blood concentration of about 9 ng / mL to about 15 ng / mL. The trough blood concentration of about 9 ng / mL to about 15 ng / mL is maintained for an initial period of about 12 weeks or less, for example, about 1 week to about 12 weeks, about 2 weeks to about 12 weeks, about 3 weeks to about 12 weeks, about 9 weeks to about 12 weeks, about 5 weeks to about 12 weeks, about 6 weeks to about 12 weeks, about 7 weeks to about 12 weeks, about 8 weeks to about 12 weeks, about 9 weeks to about 12 weeks, about 10 weeks to about 12 weeks, or about 11 weeks to about 12 weeks.

[0064] In some embodiments, sirolimus is administered at a dose that maintains a sirolimus trough blood concentration of about 9 ng / mL to about 13 ng / mL. The trough blood concentration of about 9 ng / mL to about 13 ng / mL is maintained for an initial period of about 12 weeks or less, such as about 1 week to about 12 weeks, about 2 weeks to about 12 weeks, about 3 weeks to about 12 weeks, about 9 weeks to about 12 weeks, about 5 weeks to about 12 weeks, about 6 weeks to about 12 weeks, about 7 weeks to about 12 weeks, about 8 weeks to about 12 weeks, about 9 weeks to about 12 weeks, about 10 weeks to about 12 weeks, or about 11 weeks to about 12 weeks.

[0065] Sirolimus can be administered in a tapering regimen in which multiple doses are administered and each dose decreases compared to the previous dose. Sirolimus can be administered in a tapering regimen in which multiple doses are administered, each dose targeting a blood trough range, and the upper limit of each range decreases compared to the previous dose. For example, sirolimus can be administered followed by one or more tapering doses after an initial dose. In some embodiments, the method includes an initial dose and at least one tapering dose (e.g., 1 to 10 tapering doses or 1 to 5 tapering doses). In some embodiments, the method includes an initial dose and at least two tapering doses (e.g., 2 to 10 tapering doses or 2 to 5 tapering doses). In some embodiments, the method includes an initial dose and at least three tapering doses (e.g., 3 to 10 tapering doses or 3 to 5 tapering doses).

[0066] The initial dose can maintain a sirolimus trough blood concentration of about 4 ng / mL to about 16 ng / mL. The initial dose can maintain a sirolimus trough blood concentration of about 9 ng / mL to about 16 ng / mL. The initial dose can maintain a sirolimus trough blood concentration of about 4 ng / mL to about 15 ng / mL. The initial dose can maintain a sirolimus trough blood concentration of about 9 ng / mL to about 15 ng / mL. The initial dose can maintain a sirolimus trough blood concentration of about 9 ng / mL to about 13 ng / mL. The trough blood concentration is the initial dose and can be maintained for an initial period of about 12 weeks or less, such as about 1 week to about 12 weeks, about 2 weeks to about 12 weeks, about 3 weeks to about 12 weeks, about 4 weeks to about 12 weeks, about 5 weeks to about 12 weeks, about 6 weeks to about 12 weeks, about 7 weeks to about 12 weeks, about 8 weeks to about 12 weeks, about 9 weeks to about 12 weeks, about 10 weeks to about 12 weeks, or about 11 weeks to about 12 weeks.

[0067] Subsequently, for example, after the initial dose, sirolimus can be administered in one or more tapered doses over one or more tapering periods. For example, in some embodiments, the first tapered dose maintains a sirolimus trough blood concentration of about 4 ng / mL to about 11 ng / mL, such as about 7 ng / mL to about 11 ng / mL. The first tapering period can be about 2 weeks or less, such as about 1 week to about 2 weeks. In some embodiments, the second tapered dose maintains a sirolimus trough blood concentration of about 4 ng / mL to about 9 ng / mL, such as about 5 ng / mL to about 9 ng / mL. The second tapering period can be about 2 weeks or less, such as about 1 week to about 2 weeks. In some embodiments, the third tapered dose maintains a sirolimus trough blood concentration of about 3 ng / mL to about 7 ng / mL. The third tapering period can be about 2 weeks or less, such as about 1 week to about 2 weeks. In some embodiments, the fourth tapered dose maintains a sirolimus trough blood concentration of about 1 ng / mL to about 5 ng / mL. The fourth tapering period can be about 2 weeks or less, such as about 1 week to about 2 weeks. In some embodiments, the fifth tapered dose maintains a sirolimus trough blood concentration of about 0 ng / mL to about 3 ng / mL. The fifth tapering period can be about 2 weeks or less, such as about 1 week to about 2 weeks.

[0068] In some embodiments, the taper regimen comprises maintaining a sirolimus trough blood concentration of from about 4 ng / mL to about 11 ng / mL for no more than 2 weeks, maintaining a sirolimus trough blood concentration of from about 4 ng / mL to about 9 ng / mL for no more than 2 weeks, maintaining a sirolimus trough blood concentration of from about 3 ng / mL to about 7 ng / mL for no more than 2 weeks, maintaining a sirolimus trough blood concentration of from about 1 ng / mL to about 5 ng / mL for no more than 2 weeks, and / or maintaining a sirolimus trough blood concentration of from about 0 ng / mL to about 3 ng / mL for no more than 2 weeks.

[0069] In some embodiments, the taper regimen comprises maintaining a sirolimus trough blood concentration of from about 7 ng / mL to about 11 ng / mL for no more than 2 weeks, maintaining a sirolimus trough blood concentration of from about 5 ng / mL to about 9 ng / mL for no more than 2 weeks, maintaining a sirolimus trough blood concentration of from about 3 ng / mL to about 7 ng / mL for no more than 2 weeks, maintaining a sirolimus trough blood concentration of from about 1 ng / mL to about 5 ng / mL for no more than 2 weeks, and / or maintaining a sirolimus trough blood concentration of from about 0 ng / mL to about 3 ng / mL for no more than 2 weeks.

[0070] In some embodiments, the taper period is no more than about 12 weeks. For example, the taper period can be from about 1 week to about 12 weeks, from about 2 weeks to about 12 weeks, from about 3 weeks to about 12 weeks, from about 4 weeks to about 12 weeks, from about 5 weeks to about 12 weeks, from about 6 weeks to about 12 weeks, from about 7 weeks to about 12 weeks, from about 8 weeks to about 12 weeks, from about 9 weeks to about 12 weeks, from about 10 weeks to about 12 weeks, or from about 11 weeks to about 12 weeks.

[0071] Sirolimus can be administered orally. In some embodiments, sirolimus is administered as an oral solution. In some embodiments, sirolimus is administered as an oral tablet.

[0072] In some embodiments, sirolimus is administered once daily.

[0073] The administration of sirolimus can be initiated on the same day as the administration of the chimeric FasL protein conjugated to the graft cells and the hydrogel. Alternatively, the administration of sirolimus can be initiated prior to the day of administration of the chimeric FasL protein conjugated to the graft cells and the hydrogel. For example, the administration of sirolimus can be initiated about 5 days prior to the day of administration of the chimeric FasL protein conjugated to the graft cells and the hydrogel. The administration of sirolimus can be initiated about 4 days prior to the day of administration of the chimeric FasL protein conjugated to the graft cells and the hydrogel. The administration of sirolimus can be initiated about 3 days prior to the day of administration of the chimeric FasL protein conjugated to the graft cells and the hydrogel. The administration of sirolimus can be initiated about 2 days prior to the day of administration of the chimeric FasL protein conjugated to the graft cells and the hydrogel. The administration of sirolimus can be initiated about 1 day prior to the day of administration of the chimeric FasL protein conjugated to the graft cells and the hydrogel.

[0074] FasL hydrogel FasL hydrogels (e.g., microgels) are known in the art. Exemplary FasL hydrogels (e.g., microgels) are provided in WO2018 / 165547, which is hereby incorporated by reference in its entirety.

[0075] As provided herein, the chimeric FasL protein can comprise a FasL portion and a heterologous portion.

[0076] In some embodiments, the FasL portion in the chimeric FasL protein comprises a fragment of FasL (e.g., human FasL) that binds to TNFRSF6 / FAS. TNFRSF6 / FAS is a receptor that binds FasL and transmits an apoptosis signal to the cell. In some embodiments, the FasL portion comprises a fragment of FasL (e.g., human FasL) that binds to TNFRSF6B / DcR3. TNFRSF6B / DcR3 is a soluble decoy receptor for FasL. In some embodiments, the FasL portion comprises an apoptosis-inducing fragment of FasL. In some embodiments, the FasL portion comprises the extracellular domain of FasL.

[0077] In some embodiments, the FasL portion does not include a transmembrane domain. In some embodiments, the FasL portion does not include a cytoplasmic domain. In some embodiments, the FasL portion includes neither a transmembrane domain nor a cytoplasmic domain.

[0078] In some embodiments, the FasL portion in the chimeric FasL protein is resistant to matrix metalloprotease (MMP). In some embodiments, the FasL portion lacks an MMP-sensitive site. See Yolcu et al, Immunity 17:795-808 (2002). By way of example, the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:5 are FasL portions that do not have an MMP-sensitive site.

[0079] In some embodiments, the chimeric FasL protein comprises a human FasL portion comprising an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acids 131-281 of SEQ ID NO: 7. In some embodiments, the human FasL portion comprising an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acids 131-281 of SEQ ID NO: 7 is capable of binding to TNFRSF6 / FA. In some embodiments, the human FasL portion comprising an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acids 131-281 of SEQ ID NO: 7 is capable of inducing apoptosis in FasL-sensitive cells such as effector T cells (T eff ) and / or A20 lymphoma cells. In some embodiments, the human FasL portion comprising an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acids 131-281 of SEQ ID NO: 7 is capable of binding to TNFRSF6 / FA and is capable of inducing apoptosis in FasL-sensitive cells such as T eff and / or A20 lymphoma cells.

[0080] In some embodiments, the chimeric FasL protein comprises a human FasL portion comprising the following amino acid sequence: IGHPSPPPEKKELRKVAHLTGKSNSRSMPLEWEDTYGIVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGKMMSYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL (SEQ ID NO: 1).

[0081] In some embodiments, the chimeric FasL protein comprises a rat FasL portion having the following amino acid sequence:

[0082] IANPSTPSETKKPRSVAHLTGNPRSRSIPLEWEDTYGTALISGVKYKKGGLVINEAGLYFVYSKVYFRGQSCNSQPLSHKVYMRNFKYPGDLVLMEEKKLNYCTTGQIWAHSSYLGAVFNLTVADHLYVNISQLSLINFEESKTFFGLYKL (SEQ ID NO: 5).

[0083] In some embodiments, the heterologous portion in the chimeric FasL protein comprises a protein capable of binding to biotin. In some embodiments, the heterologous portion in the chimeric FasL protein comprises streptavidin or a fragment thereof. As used herein, "streptavidin or a fragment thereof" refers to full-length streptavidin or a fragment of full-length streptavidin capable of binding to biotin. In some embodiments, the heterologous portion in the chimeric FasL protein comprises avidin or a fragment thereof. As used herein, "avidin or a fragment thereof" refers to full-length avidin or a fragment of full-length avidin capable of binding to biotin. In some embodiments, the heterologous portion in the chimeric FasL protein comprises streptavidin or a fragment thereof or avidin or a fragment thereof.

[0084] The streptavidin or avidin fragments that may be included in the chimeric FasL proteins provided herein include "core streptavidin" ("CSA"), which may include streptavidin residues 13-138, 14-138, 13-139 or 14-139, which are cleavage forms of the full-length streptavidin polypeptide. See, for example, Pahler et al., J. Biol. Chem., 262:13933-37 (1987). Other cleavage forms of streptavidin and avidin that retain strong binding to biotin can also be used. See, for example, Sano et al, J Biol Chem. 270(47):28204-09 (1995) (describing core streptavidin variants 16-133 and 14-138) (U.S. Patent No. 6,022,951). Variants of streptavidin and core forms of streptavidin that retain substantial or increased biotin binding activity can also be used. See, for example, Chilcoti et al, Proc Natl Acad Sci, 92(5):1754-58 (1995); Reznik et al, Nat Biotechnol, 14(8):1007-11 (1996). For example, variants with reduced immunogenicity, such as those engineered to remove potential T cell or lymphocyte epitopes by site-directed mutagenesis, can also be used. See Meyer et al, Protein Sci., 10:491-503 (2001). Similarly, variants of avidin and core forms of avidin that retain substantial or increased biotin binding activity may be used. See Hiller et al, J Biochem, 278:573-85 (1991); and Livnah et al, Proc Natl Acad Sci USA 90:5076-80 (1993). For convenience, in the description herein, the terms "avidin" and "streptavidin" (or "SA") include fragments, variants, and core forms of these molecules.

[0085] Avidin and streptavidin can be obtained from commercial suppliers. Furthermore, the nucleic acid sequences encoding streptavidin and avidin, as well as the amino acid sequences of streptavidin and avidin, are also known. See, for example, GenBank accession numbers X65082; X03591; NM_205320; X05343; Z21611; and Z21554.

[0086] In some embodiments, the chimeric FasL protein comprises a streptavidin portion having the following amino acid sequence: ITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGATEANAWKSTLVGHDTFTKVKPSAASS (SEQ ID NO: 2)

[0087] In some embodiments, the chimeric FasL protein comprises a streptavidin portion having an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0088] In some embodiments, the chimeric FasL protein comprises a FasL portion on the C-terminal side of a heterologous moiety (e.g., streptavidin or a fragment thereof or avidin or a fragment thereof). The FasL portion located on the C-terminal side of the heterologous moiety (e.g., streptavidin or a fragment thereof or avidin or a fragment thereof) may be directly at the C-terminus of the heterologous moiety or may be separated from the heterologous moiety by a linker (e.g., an amino acid linker). In some embodiments, the chimeric FasL protein comprises a FasL portion on the N-terminal side of a heterologous moiety (e.g., streptavidin or a fragment thereof or avidin or a fragment thereof). The FasL portion located on the N-terminal side of the heterologous moiety (e.g., streptavidin or a fragment thereof or avidin or a fragment thereof) may be directly at the N-terminus of the heterologous moiety or may be separated from the heterologous moiety by a linker (e.g., an amino acid linker).

[0089] In some embodiments, the FasL portion and the heterologous moiety in the chimeric FasL protein are fused via an amino acid linker. In some embodiments, the linker is a glycine-serine linker. The glycine-serine linker may comprise, for example, the amino acid sequence GGGGSGGGGSG (SEQ ID NO: 3).

[0090] In some embodiments, a chimeric FasL protein comprising a human FasL portion comprises the following amino acid sequence: ITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGATEANAWKSTLVGHDTFTKVKPSAASSGGGGSGGGGSGEFIGHPSPPPEKKELRKVAHLTGKSNSRSMPLEWEDTYGIVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGKMMSYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL (SEQ ID NO: 4).

[0091] In some embodiments, the chimeric FasL protein comprising the rat FasL moiety comprises the following amino acid sequence: ITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGATEANAWKSTLVGHDTFTKVKPSAASSGGGGSGGGGSGEFIANPSTPSETKKPRSVAHLTGNPRSRSIPLEWEDTYGTALISGVKYKKGGLVINEAGLYFVYSKVYFRGQSCNSQPLSHKVYMRNFKYPGDLVLMEEKKLNYCTTGQIWAHSSYLGAVFNLTVADHLYVNISQLSLINFEESKTFFGLYKL (SEQ ID NO: 6).

[0092] In the amino acid sequence of SEQ ID NO: 4, amino acids 1 to 124 correspond to the streptavidin moiety, amino acids 125 to 135 correspond to the glycine - serine linker, and amino acids 138 to 188 correspond to the FasL moiety.

[0093] As provided herein, a hydrogel (e.g., a microgel) is engineered to present the chimeric FasL protein. For example, the hydrogel (e.g., a microgel) can be biotinylated and bound to a chimeric FasL protein comprising streptavidin or a fragment thereof or avidin or a fragment thereof. Thus, in some embodiments, the biotinylated hydrogel (e.g., a microgel) is bound to the chimeric FasL protein via a non - covalent bond between biotin and streptavidin or a fragment thereof or avidin or a fragment thereof.

[0094] In some embodiments provided herein, the hydrogel (e.g., microgel) comprises polyethylene glycol (PEG). In some embodiments, the hydrogel (e.g., microgel) comprises maleimide-terminated 4-arm poly(ethylene) glycol (PEG-4MAL) macromer. Such hydrogels (e.g., microgels) can be synthesized by microfluidic crosslinking. See Headen et al, Advanced Materials, 26:3003-3008 (2014). The PEG-4MAL platform enables the stoichiometric covalent incorporation of thiol-containing molecules and results in improved crosslinking efficiency for forming structurally defined hydrogels. See Phelps et al, Advanced Materials, 24:64-70, 62 (2012).

[0095] In some embodiments, the hydrogel (e.g., microgel) is about 125 micrometers to about 175 micrometers in size. In some embodiments, the hydrogel (e.g., microgel) is about 150 micrometers.

[0096] Biotinylated hydrogels (e.g., microgels) can be prepared by reacting biotin-PEG-thiol with PEG-4MAL macromer and crosslinking with dithiothreitol (DTT) via microfluidic crosslinking to produce 150 μm diameter microgels. The resulting microgels can present covalently bound biotin capable of capturing streptavidin (SA) with high affinity.

[0097] In some embodiments, FasL in the chimeric FasL protein conjugated to the hydrogel (e.g., microgel) can form FasL trimers.

[0098] In some embodiments, the chimeric FasL protein conjugated to the hydrogel (e.g., microgel) has apoptotic activity.

[0099] In some embodiments, a hydrogel (e.g., a microgel) containing a chimeric FasL protein encapsulates graft cells. In some embodiments, a hydrogel (e.g., a microgel) containing a chimeric FasL protein does not encapsulate graft cells.

[0100] In some embodiments, the hydrogel (e.g., a microgel) contains multiple sites for crosslinking so that additional compounds can be conjugated to the hydrogel (e.g., a microgel).

[0101] Grafted cells As used herein, "grafted cells" refers to cells (or tissues or organs containing cells) that are administered to a subject in need thereof. According to the methods provided herein, sirolimus and a chimeric FasL protein conjugated to a hydrogel can induce specific immune tolerance to graft cells. Graft cells can be cells derived from a donor, e.g., a deceased donor. Graft cells may be stem cells or stem cell-derived cells.

[0102] Types of graft cells include PBMCs, bone marrow cells, hematopoietic stem cells, stem cells, stem cell-derived cells, mesenchymal stem cells, dendritic cells, dendritic cells incubated with self-antigens, human beta cell products, pancreatic islet cells, allogeneic islet cells, hepatocytes, and splenocytes.

[0103] In some embodiments, the graft cells are pancreatic islet cells or pancreatic islet cells derived from insulin-producing stem cells. In some embodiments, the graft cells are pancreatic islet cells. In some embodiments, the graft cells are pancreatic islet cells derived from insulin-producing stem cells.

[0104] In some embodiments, the graft cells are hepatocytes.

[0105] The types of graft cells include islet cells (e.g., pancreatic islet cells), splenocytes, PBMCs, bone marrow cells, mesenchymal stem cells, hematopoietic stem cells, stem cells, induced pluripotent stem cells, human beta cell products, hepatocytes, dendritic cells, macrophages, endothelial cells, cardiomyocytes, and vascular cells, as well as immune cells including T cells. In any of the embodiments provided herein, the graft cells can be administered as a preparation of isolated cells or as part of a tissue or organ.

[0106] In some embodiments provided herein, the graft cells are allogeneic. In some embodiments, the graft cells are xenogeneic. In some embodiments, the graft cells are of human origin. In some embodiments, the graft cells are derived from non-human primates, dogs, cats, cows, sheep, horses, rabbits, mice, or rats.

[0107] The graft cells for use provided herein can be graft cells obtained from a deceased donor.

[0108] In some embodiments provided herein, the graft cells are autologous or syngeneic (derived from the subject being treated). For example, autologous graft cells can be derived from autologous tissue by the pluripotency of induced pluripotent cells and differentiation into the desired autologous graft cells. In some embodiments, cells derived from the subject are used to induce immune tolerance to self that has been disrupted in autoimmune diseases.

[0109] In some embodiments, the graft cells are embedded in a hydrogel (e.g., microgel) containing a chimeric FasL protein. In some embodiments, the graft cells are not embedded in a hydrogel (e.g., microgel) containing a chimeric FasL protein.

[0110] For example, pancreatic islet cells can be administered to treat diabetes together with a tapering and / or transient sirolimus regimen and a chimeric FasL protein conjugated to a hydrogel.

[0111] In another example, hepatocytes can be administered to treat acute liver failure or liver system metabolic disorders, together with sirolimus and a chimeric FasL protein conjugated to a hydrogel.

[0112] Use and Indications Provided herein is a method for inducing immune tolerance to graft cells. In some embodiments, the method is for the prevention or risk reduction of rejection of a cell graft or tissue graft and / or for the treatment of type I diabetes. The method can include administering a graft cell, a chimeric FasL protein conjugated to a hydrogel, and sirolimus.

[0113] In some embodiments, a hydrogel (e.g., a microgel) containing the chimeric FasL protein and the graft cells are administered simultaneously. In some embodiments, the graft cells are not encapsulated in the hydrogel (e.g., a microgel). In such embodiments, the graft cells and the hydrogel (e.g., a microgel) may be administered in the same pharmaceutical composition or in separate pharmaceutical compositions.

[0114] In some embodiments, the graft cells are encapsulated in a hydrogel (e.g., a microgel).

[0115] In some embodiments, the hydrogel and the graft cells are administered at a ratio of hydrogel (e.g., a microgel) approximately 2: cell 1.

[0116] In some embodiments, the methods of inducing immune tolerance provided herein are for the treatment of type I diabetes in a human patient. Thus, in some embodiments, at least 3,000 islet equivalents per kilogram of patient are administered. In some embodiments, at least 5,000 islet equivalents per kilogram of patient are administered. In some embodiments, at least 10,000 islet equivalents per kilogram of patient are administered. In some embodiments, at least 11,000 islet equivalents per kilogram of patient are administered. In some embodiments, from about 3,000 to about 20,000 islet equivalents per kilogram of patient are administered. In some embodiments, from about 5,000 to about 20,000 islet equivalents per kilogram of patient are administered. In some embodiments, from about 10,000 to about 20,000 islet equivalents per kilogram of patient are administered. In some embodiments, from about 11,000 to about 20,000 islet equivalents per kilogram of patient are administered.

[0117] In some embodiments, the hydrogel and graft cells are administered at a ratio of hydrogel (e.g., microgel) to allogeneic islet 1 of about 2:1.

[0118] In some embodiments, the methods of inducing immune tolerance provided herein are for the treatment of liver failure, for example, by administering a therapeutically effective amount of hepatocytes, a chimeric FasL protein conjugated to a hydrogel, and transient sirolimus.

[0119] In some embodiments of the methods provided herein, the graft cells and the chimeric FasL protein conjugated to the hydrogel are administered into the omentum.

[0120] In some embodiments, the hydrogel (e.g., microgel), graft cells, and sirolimus are administered or are for administration in combination with mesenchymal stem cells (MSCs), an anti-CD20 agent (such as rituximab), or an anti-CD46 agent.

[0121] In some embodiments, sirolimus is administered in combination with a prophylactic antiviral agent (e.g., famciclovir). In some embodiments, sirolimus is administered in combination with an antibacterial agent (e.g., sulfamethoxazole and / or trimethoprim). In some embodiments, sirolimus is administered in combination with a prophylactic antiviral agent (e.g., famciclovir) and an antibacterial agent (e.g., sulfamethoxazole and / or trimethoprim).

[0122] As used herein, "combination" administration can refer to concurrent or sequential administration. Concurrent administration can include administration in the same pharmaceutical composition or in separate pharmaceutical compositions.

Example

[0123] Example 1 Biotinylated PEG microgels (150 μm in diameter) were synthesized by microfluidic crosslinking and functionalized with SA-FasL as described previously (Headon, D.M., et al., Nat. Mater. 17:732-739 (2018)). Four diabetic non-human primates (NHPs) (Figure 1B; Tables 1 and 2) were transplanted with allogeneic islets (14,800 - 18,700 islet equivalent quantity (IEQ) / kg) mixed with SA-FasL-presenting microgels (150,000 - 200,000 microgels delivering 0.2 mg of SA-FasL) onto the surface of the omentum. A 3-month course of sirolimus (rapamycin) monotherapy was used with a target sirolimus trough blood concentration of 40 ng / ml for the first 2 weeks and then 20 ng / ml until 3 months post-transplantation, at which point sirolimus was discontinued without tapering. Sirolimus concentration became undetectable 2 weeks after discontinuation. Three diabetic NHPs (Figure 1B; Tables 1 and 2) were co-transplanted with unmodified PEG microgels lacking SA-FasL protein and allogeneic islets onto the omentum under a similar sirolimus course.

Table 1

Table 2

[0124] After islet transplantation, rapid glycemic control was achieved in all NHPs administered the SA-FasL-presenting microgels and was maintained over observation periods exceeding 134, 170, 177, and 188 days (Figures 2A and 2B). The animals ended with functioning grafts. In contrast, control NHPs that received the same sirolimus regimen and had equivalent sirolimus plasma concentrations (P = 0.63; Figures 2D and 2E), but did not receive SA-FasL, maintained glycemic control for only 21, 27, and 35 days and had a mean survival time of 27.7 days (Figures 2A and 2E, P = 0.010).

[0125] Figure 2B shows the mean non-fasting blood glucose levels (left axis) and the total exogenous insulin requirements (EIR; right axis) administered, averaged across all SA-FasL-presenting microgel recipients. Prior to islet transplantation, NHPs in the SA-FasL-microgel group had mean non-fasting blood glucose levels around 400 mg / dL and required 16 - 22 units of exogenous insulin per day. After transplantation, fasting blood glucose levels (using the time 0 readings of the intravenous glucose tolerance test (IVGTT) when NHPs were fasted overnight (Figure 2C)) were within the normal range of naïve NHPs (e.g., 37 - 92 mg / dL in M9118), but random non-fasting blood glucose levels in some NHPs fluctuated above normal and sometimes exceeded 300 mg / dL. Nevertheless, SA-FasL microgel NHPs maintained good glycemic control within the normal range during the study period (STZ post / transplantation pre vs. post, P = 0.0035) (Figure 2B). Three SA-FasL microgel NHPs required exogenous insulin after transplantation, but only 10 - 20% of the pre-transplantation amount was needed to maintain postprandial blood glucose levels below 250 mg / dL. Subject 4 (M9118) initially required 1 - 2 units of insulin per day, but by 4 months after transplantation, the graft became fully functional and exogenous insulin was only needed intermittently. In SA-FasL microgel NHPs, the EIR after transplantation was significantly lower than that post-STZ / transplantation pre (P = 0.0092). All NHPs experienced an initial weight loss after STZ induction and after transplantation, but gradually recovered, indicating that post-transplantation normoglycemia was not due to malnutrition. Both STZ induction and transplantation surgery were expected to immediately cause weight loss. The continued initial mild weight loss after transplantation is highly likely related to the rapamycin therapy. The results of metabolic markers showed normal liver and kidney function in SA-FasL microgel NHPs (Figure 6, dashed squares). When the islet grafts were surgically removed at the end of the study, SA-FasL microgel NHPs rapidly returned to a diabetic state (blood glucose levels: post-transplantation vs. post-graft removal, P < 0.0001; EIR: post-transplantation vs. post-graft, P = 0.0042) (Figure 2B), indicating that glycemic control was due to the graft.

[0126] At a specific time point, an intravenous glucose tolerance test (IVGTT) was performed to evaluate glucose handling kinetics and insulin and C-peptide concentrations. After a glucose bolus stimulation, SA-FasL microgel NHPs returned to normoglycemia within 90 minutes and had a profile equivalent to that of naive NHPs before diabetes induction [area under the curve (AUC) analysis: P < 0.0001 for naive before STZ vs. after STZ (after STZ induction but before transplantation); P < 0.0001 for 3 months after transplantation vs. after STZ; P = 0.0002 for 6 months after transplantation vs. after STZ] (Figure 2C). After graft removal, blood glucose levels continued to rise and reached levels equivalent to pre-transplant diabetes levels (AUC: P = 0.0760 for after graft removal vs. after STZ), indicating that blood glucose control was due to the graft. Consistent with these measures of graft function, the background levels of serum insulin and C-peptide in SA-FasL microgel NHPs were measured before islet transplantation after STZ administration and were clearly low after graft removal as well (insulin: P < 0.0001 for before STZ vs. after STZ; P = 0.9998 for after STZ vs. after graft removal (PGR); C-peptide: P = 0.0060 for before STZ vs. after STZ; P = 0.4032 for after STZ vs. after graft removal) (Figure 2D). SA-FasL microgel NHPs restored insulin and C-peptide expression (insulin: P = 0.0022 for 3 months after transplantation vs. after STZ; P = 0.0007 for 6 months after transplantation vs. after STZ; C-peptide: P = 0.0248 for 3 months after transplantation vs. after STZ; P = 0.0230 for 6 months after transplantation vs. after STZ) and were at levels similar to the pre-diabetic state. Notably, the stimulated insulin and C-peptide concentrations were higher than the corresponding fasting concentrations indicating glucose responsiveness (insulin: P = 0.0274, C-peptide: P = 0.0149). Glycated hemoglobin (HbA1c) values in SA-FasL microgel NHPs were well controlled after transplantation, and at the end of the study, A1C values were below 6% in almost all (3 / 4) of the NHPs. SA-FasL protein was detected in the serum of all NHPs as early as 1 day after transplantation in the range of 4 - 56 ng / mL and decreased to background levels on days 7 - 14.This result is consistent with the in vivo measurement in mouse SA-FasL microgels, showing a local half-life of 3.0 days.

[0127] Figure 2E shows the mean random non-fasting blood glucose levels (left axis) and total daily EIR227 (right axis) averaged across all recipients who received the control microgels (simply referred to as "microgels" herein). Prior to islet transplantation, the microgel NHPs had an average non-fasting blood glucose level of around 400 mg / dL and required 15 - 20 units of exogenous insulin per day. After transplantation, the microgel NHPs recovered uneventfully, and the average blood glucose level returned to the normal range. However, around 30 days after transplantation, the blood glucose level and the required external insulin dose increased and remained elevated at a level equivalent to the pre-transplant diabetic state, indicating graft rejection (blood glucose level: P = 0.0050 for post-transplant vs post-rejection; EIR: P = 0.0441 for post-transplant vs post-graft rejection). After transplantation, the NHPs initially lost weight but began to gain weight around day 25. The IVGTT showed that the blood glucose level remained elevated even after a glucose bolus injection, indicating poor graft function (AUC: P = 0.0013 for pre-STZ vs post-STZ; P = 0.1036 for 1 month post-transplant vs post-STZ) (Figure 2F). Similarly, the insulin and C-peptide concentrations 1 month after transplantation remained at the pre-transplant diabetic levels (insulin: P < 0.0001 for pre-STZ vs post-STZ; P = 0.9504 for 1 month post-transplant vs post-STZ; C-peptide: P < 0.0134 for pre-STZ vs post-STZ; P > 0.9999 for 1 month post-transplant vs post-STZ) (Figure 2G).

[0128] In autopsy pathology, the liver, heart, lung, kidney, and intestinal tissues of SA-FasL microgel NHPs showed normal H&E staining. The recipient pancreas contained only a few islet-like clusters of endocrine gland cells, and no insulin-positive β cells were observed, indicating that host β cells were effectively eliminated by STZ treatment, supporting that post-transplant blood glucose control is attributed to the graft. Histological analysis of the omental transplantation site of SA-FasL microgel NHPs revealed a number of well-granulated cell clusters resembling islets, with minimal or no infiltrating lymphocytes, suggesting no rejection reaction. Histology of the omentum of microgel NHPs showed islet-like clusters infiltrated by lymphocytes, consistent with graft rejection. Immunostaining of the graft site showed well-preserved insulin + structures corresponding to the transplanted islets in long-term (day 177) SA-FasL microgel NHPs, while sections of microgel control NHPs at the time of rejection (day 21) showed structures with loss of insulin staining peripherally. Importantly, immunostaining analysis of the graft site showed that FoxP3+ (T reg marker) cells were present at a higher frequency (cell number, intensity) in SA-FasL microgel NHPs compared to microgel NHPs (FoxP3 + cell number: P = 0.0143; FoxP3 + intensity: P = 0.043, Figures 3A - B). This finding is consistent with tests in diabetic mice, indicating that T reg plays an important role in the establishment of immune tolerance induced by SA-FasL-microgels (Headon, DM, et al., Nat. Mater. 17:732 - 739 (2018)).

[0129] Peripheral blood mononuclear cells (PBMCs) were collected at specific time points, immunostained, and evaluated by flow cytometry using a validated gating strategy. Figure 4 shows the longitudinal profile of immune cell populations. CD20 + B cells as well as CD3 + , CD4 + and CD8 +The levels of T cells remained stable over time among individual NHPs in both groups, with minor fluctuations (CD20 + : P = 0.5069, P = 0.2531; CD3 + : P = 0.3405, P = 0.4959; CD4 + : P = 0.5079, P = 0.5142; CD8 + : P = 0.2287, P = 0.5277). Similarly, no differences over time were observed in CD28 + CD95 - CD4 + or CD8 + naive, CD28 + CD95 + CD4 + or CD8 + central memory, CD28 - CD95 + CD4 + or CD8 + effector memory T cells either (CD4 + naive: P = 0.4621, P = 0.5102; CD8 + naive: P = 0.2524, P = 0.5626; CD4 + central memory: 283P = 0.5977, P = 0.4785; CD8 + central memory: P = 0.4420, P = 0.4790; CD4 + effector memory: 284P = 0.4815, P = 0.4829; CD8 + effector memory: P = 0.2442, P = 0.0844). In SA-FasL microgel NHPs, the lack of differences over time in these immune cell populations in the systemic circulation may be due to the local immunosuppressive effect of SA-FasL on the graft (Yolcu, ES., at al., J. Immunol. 187:5901 - 5909 (2011) and Headon, DM, et al., Nat. Mater. 17:732 - 739 (2018)).

[0130] The production of IgG antibodies against donor MHC antigens was evaluated by flow cytometry at multiple time points before and after transplantation. In SA-FasL microgel NHPs, positive donor-specific antibodies against MHC class I (P = 0.2904) or MHC class II (P = 0.0758) were not detected over time (Figures 5A and 5B). In contrast, in the sera of control microgel NHPs 1 month after transplantation, there was a significant increase in the titer of antibodies against MHC class II (P = 0.0326), but no significant increase in the titer of antibodies against class I (P = 0.3292) (Figures 5A and 5B). Donor and third-party CD4 + and CD8 + The mixed lymphocyte reaction against T cells also showed no change between pre- and post-transplantation conditions in SA-FasL microgel NHPs (CD4 + Donor: P = 0.4200; CD4 + Third party: P = 0.6949; CD8 + Donor: P = 0.4145; CD8 + Third party: P = 0.7858) (Figures 5C - 5F). Microgel NHPs showed no change in the CD4 + T cell response to donor (P = 0.6082) and third-party (P = 0.0555) stimulants, but CD8 +The T cell compartment showed increased responses to both donor (P = 0.0057) and third - party (P = 0.0216) antigens (Figs. 5C - 5F). In the enzyme - linked immunosorbent spot (ELISpot) assay, no differences in IFN - γ secretion were shown at each time point before and after transplantation in SA - FasL microgel NHP (donor: P = 0.2485; third - party: P = 0.1445) or microgel NHP (donor: P = 0.0824; third - party: P = 0.1413) (Figs. 5G - 5H). In the multiplexed immunobead assay, no significant differences were shown in the pre - and post - transplantation levels of pro - inflammatory and anti - inflammatory cytokines and chemokines in the sera of SA - FasL microgel NHP and microgel NHP. Finally, antibodies against the SA - FasL protein were not detected in the sera of SA - FasL microgel NHP at the early time points but were expressed approximately 2 weeks after transplantation. Most of the antibodies were against the SA domain of the SA - FasL protein. These results showed that antibodies against SA - FasL were expressed approximately 2 weeks after transplantation when SA - FasL was no longer detected systemically and were mainly against the SA part of the chimeric protein. Importantly, the presence of such antibodies did not adversely affect the effectiveness of the protocol for maintaining the survival of allogeneic islet grafts.

[0131] These results indicate that SA - FasL - presenting microgels co - transplanted with allogeneic islets are effective in the long - term (>6 months) survival and maintenance of excellent glycemic control in diabetic NHPs without chronic immunosuppression.

[0132] Example 2 To demonstrate the safety, tolerability, and activity of short - term administration of iTOL - 101 and sirolimus in poorly controlled diabetic adults despite intensive diabetes management, a Phase 1 / 2 trial is conducted. A schematic of the study design is shown in Fig. 7.

[0133] iTOL - 101 iTOL - 101 consists of two components: 1) the iTOL - 100 agent (SA - FasL microgel) and 2) the allogeneic cadaveric islet agent.

[0134] The dosage of islets of the same species varies among subjects, but at least 5,000 islet equivalents (IEQ) / kg of functional islets are delivered to each subject. The evaluation of the total IEQ and the minimum dosage required for transplantation (5,000 IEQ / kg) is determined according to the standard operating procedure (SOP) used in the National Institutes of Health (NIH) CIT Phase 3 clinical trial (ClinicalTrials.gov identifier: NCT00434811).

[0135] Mix the iTOL-100 dosage of 0.7 mg of SA-FasL in approximately 11 mL of Dulbecco's phosphate-buffered saline with 350,000 islets (5,000 IEQ / kg) to obtain the final delivery dosage. The volume of iTOL-100 is adjusted volume:volume according to any increase in the total amount of islets to be implanted.

[0136] Participants Participants in the study meet the following inclusion criteria: · Adult men and women aged 18 to 65 years; · Have a medical history consistent with type 1 diabetes (T1D), onset before the age of 40, and insulin-dependent status for more than 5 years at the time of enrollment; · Disappearance of stimulated C-peptide (<0.3 ng / mL) in the mixed meal tolerance test (MMTT) measured at 60 and 90 minutes after meal initiation; · In the 12 months prior to study enrollment, under the direction of an endocrinologist or diabetologist, active intensive diabetes management involving continuous blood glucose monitoring, insulin injection administration more than 3 times per day, or insulin pump therapy, and more than 2 clinical evaluations; and · Hypoglycemia unawareness measured by a Clarke score ≥4 and at least one of the following criteria: o At least 2 episodes of severe hypoglycemia defined by the American Diabetes Association (ADA) criteria in the 12 months prior to enrollment despite intensive diabetes management interventions; o HbA1c >8.0 and glycemic variability >36% (cv); Time when oHbA1c > 7.0 and less than the range (blood glucose level < 70 mg / dL) > 8%

[0137] The test participants do not meet any of the following exclusion criteria: · Body mass index (BMI) > 30 kg / m2 or body weight ≤ 50 kg; · Insulin requirement > 1.0 IU / kg / day or < 15 U / day; · HbA1c > 10%; · Untreated proliferative diabetic retinopathy; · Blood pressure: systolic > 160 mmHg or diastolic > 100 mmHg; · Estimated glomerular filtration rate (eGFR) calculated using the subject's serum creatinine measurement and the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula or the Modification of Diet in Renal Disease [MDRD] study formula < 80 mL / min / 1.73 m 2 。eGFR < 70 mL / min / 1.73 m 2 Strict vegetarians (vegans) were also excluded. The complete (raw) GFR value is used for subjects with a body surface area > 1.73 m 2 ; or · Presence or history of macroalbuminuria (> 300 mg / g creatinine).

[0138] Administration of iTOL-101 and sirolimus Mix the two components of iTOL-101 in a clinical facility and administer iTOL-101 to the surface of the greater omentum by laparoscopic surgery using a 12 French gastrostomy catheter under sedation before transplantation and general anesthesia. To promote adhesion to the surface of the greater omentum of the island and avoid cell pelletization, human recombinant thrombin is added as needed and an amount approximately equal to the volume of the implanted graft is added to cover the surface of the graft.

[0139] After placing iTOL-101 on the greater omentum, a peritoneal sac (greater omentum flap) is created by folding the greater omentum over the iTOL-101 graft. The entire procedure, including the preparation and delivery of iTOL-101 and the formation of the greater omentum flap, can be completed in less than 30 minutes.

[0140] Sirolimus (mTOR inhibitor) is initiated at the time of the subject's hospital admission, 1 to 3 days prior to surgery, for a total of 24 weeks. The subject is administered sirolimus adjusted to maintain a trough blood concentration of 9 - 13 ng / mL for the first 12 weeks, followed by a 12-week tapering regimen that decreases every 2 weeks (+ / - 7 days) based on the trough blood concentration. Prophylactic antiviral agents (famciclovir) and antibacterial agents (sulfamethoxazole and trimethoprim) are also administered to the subject for 24 weeks according to the sirolimus labeling. The sirolimus regimen is as follows: · 9 - 13 ng / mL for the first 12 weeks; · 7 - 11 ng / mL until week 14; · 5 - 9 ng / mL until week 16; · 3 - 7 ng / mL until week 18; · 1 - 5 ng / mL until week 20; · 0 - 3 ng / mL until week 22; · 0 at week 24.

[0141] Safety and Efficacy The following safety endpoints are examined: · The occurrence of fever, nausea, vomiting, and abdominal pain; · Evaluation of the laparoscopic port site at the time of and after implantation (e.g., bleeding, erythema, pain, tenderness, induration, pus); · The incidence of all adverse events (AEs) reported during the study (including any immune reactions where a relationship is suspected); · The incidence of clinically significant grade 3 and 4 laboratory finding abnormalities; · Evaluation of retinopathy at 3, 6, and 12 months; · Evaluation of eGFR at 3, 6, and 12 months; · Allosensitization at 12 months.

[0142] To show that the administration of iTOL-101 and a temporary course of sirolimus are safe, a review of safety endpoints is conducted.

[0143] The following efficacy endpoints are considered. · The number of insulin-independent subjects at 6 and 12 months; · The number of subjects with a ≥50% decrease in insulin requirement compared to baseline at 6 and 12 months; · The number of subjects with peak C-peptide ≥100 pmol / L during MMTT at 3, 6, and 12 months; · The number of subjects with >70% of the time within the range (blood glucose 70 - 180 mg / dL) at 3, 6, and 12 months; · The number of subjects with blood glucose variability <36% (cv) at 3, 6, and 12 months; · The number of subjects with HbA1c <6.5 or a ≥2% decrease in HbA1c from baseline at 3, 6, and 12 months; · The number of subjects with at least one severe hypoglycemic episode at 12 months; and · The number of subjects with a >50% decrease or disappearance of severe hypoglycemic events at 3, 6, and 12 months.

[0144] To show that the administration of iTOL-101 and a temporary course of sirolimus are effective in the treatment of diabetes, a review of efficacy endpoints is conducted. * * *

Claims

1. A pharmaceutical composition comprising a hydrogel-conjugated chimeric FasL protein for use in a method for inducing immune tolerance to graft cells in a human patient in need thereof, the method comprising administering to the human patient (i) the graft cells, (ii) the hydrogel-conjugated chimeric FasL protein, and (iii) sirolimus, wherein the sirolimus is administered for 36 weeks or less (e.g., 24 weeks or less), the pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the method comprises administering sirolimus in an initial dose that maintains a sirolimus trough blood concentration of about 4 ng / mL to about 16 ng / mL or about 9 ng / mL to about 16 ng / mL.

3. The pharmaceutical composition according to claim 1, wherein the method comprises administering sirolimus in an initial dose that maintains a sirolimus trough blood concentration of about 4 ng / mL to about 15 ng / mL or about 9 ng / mL to about 15 ng / mL.

4. The pharmaceutical composition according to claim 1, wherein the method comprises administering sirolimus in an initial dose that maintains a sirolimus trough blood concentration of about 9 ng / mL to about 13 ng / mL.

5. The pharmaceutical composition according to any one of claims 2 to 4, wherein the initial dose is administered over an initial period of approximately 12 weeks or less.

6. The pharmaceutical composition according to claim 5, wherein the method comprises administering the sirolimus in a tapering regimen over a tapering period, and optionally the tapering regimen may be administered after the initial period of the initial dose.

7. The pharmaceutical composition according to claim 6, wherein the tapering regime is performed after the initial period of the initial dose and includes a first tapering dose administered over a first tapering period and a second tapering dose administered over a second tapering period.

8. The pharmaceutical composition according to claim 7, wherein the first tapering dose is a dose that maintains a sirolimus trough blood concentration of approximately 4 ng / mL to approximately 11 ng / mL.

9. The pharmaceutical composition according to claim 7, wherein the first tapering dose is a dose that maintains a sirolimus trough blood concentration of approximately 4 ng / mL to approximately 11 ng / mL.

10. The pharmaceutical composition according to claim 7, wherein the first gradual reduction period is (i) about two weeks or less or (ii) about one week to about two weeks.

11. The pharmaceutical composition according to claim 7, wherein the second tapering dose is a dose that maintains a sirolimus trough blood concentration of approximately 4 ng / mL to approximately 9 ng / mL.

12. The pharmaceutical composition according to claim 7, wherein the second tapering dose is a dose that maintains a sirolimus trough blood concentration of approximately 5 ng / mL to approximately 9 ng / mL.

13. The pharmaceutical composition according to claim 7, wherein the second gradual reduction period is (i) about two weeks or less or (ii) about one week to about two weeks.

14. The pharmaceutical composition according to claim 7, wherein the tapering regime further comprises a third tapering dose administered over a third tapering period.

15. The pharmaceutical composition according to claim 14, wherein the third tapering dose is a dose that maintains a sirolimus trough blood concentration of approximately 3 ng / mL to approximately 7 ng / mL.

16. The pharmaceutical composition according to claim 14, wherein the third gradual reduction period is (i) about two weeks or less or (ii) about one week to about two weeks.

17. The pharmaceutical composition according to claim 14, wherein the tapering regime further comprises a fourth tapering dose administered over a fourth tapering period.

18. The pharmaceutical composition according to claim 17, wherein the fourth dose reduction is a dose that maintains a sirolimus trough blood concentration of approximately 1 ng / mL to approximately 5 ng / mL.

19. The pharmaceutical composition according to claim 17, wherein the fourth tapering period is (i) about two weeks or less or (ii) about one week to about two weeks.

20. The pharmaceutical composition according to claim 17, wherein the tapering regime further comprises a fifth tapering dose administered over a fifth tapering period.

21. The pharmaceutical composition according to claim 20, wherein the fifth dose of the gradual reduction is a dose that maintains a trough sirolimus blood concentration of about 0 ng / mL to about 3 ng / mL.

22. The pharmaceutical composition according to claim 20, wherein the fifth gradual reduction period is (i) about two weeks or less or (ii) about one week to about two weeks.

23. The pharmaceutical composition according to claim 6, wherein the tapering regime includes maintaining a sirolimus trough blood concentration of approximately 4 ng / mL to approximately 11 ng / mL for two weeks or less, maintaining a sirolimus trough blood concentration of approximately 4 ng / mL to approximately 9 ng / mL for two weeks or less, maintaining a sirolimus trough blood concentration of approximately 3 ng / mL to approximately 7 ng / mL for two weeks or less, maintaining a sirolimus trough blood concentration of approximately 1 ng / mL to approximately 5 ng / mL for two weeks or less, and / or maintaining a sirolimus trough blood concentration of approximately 0 ng / mL to approximately 3 ng / mL for two weeks or less.

24. The pharmaceutical composition according to claim 6, wherein the tapering regime includes maintaining a sirolimus trough blood concentration of approximately 7 ng / mL to approximately 11 ng / mL for two weeks or less, maintaining a sirolimus trough blood concentration of approximately 5 ng / mL to approximately 9 ng / mL for two weeks or less, maintaining a sirolimus trough blood concentration of approximately 3 ng / mL to approximately 7 ng / mL for two weeks or less, maintaining a sirolimus trough blood concentration of approximately 1 ng / mL to approximately 5 ng / mL for two weeks or less, and / or maintaining a sirolimus trough blood concentration of approximately 0 ng / mL to approximately 3 ng / mL for two weeks or less.

25. The pharmaceutical composition according to claim 6, wherein the gradual reduction period is (i) about 12 weeks or less or (ii) about 6 weeks to about 12 weeks.

26. The pharmaceutical composition according to any one of claims 1 to 4, wherein the method further comprises administering mesenchymal stem cells, an anti-CD20 agent, and / or an anti-CD47 agent to the subject.

27. The pharmaceutical composition according to claim 26, wherein the anti-CD20 agent is rituximab.

28. The pharmaceutical composition according to any one of claims 1 to 4, wherein the graft cells are selected from PBMCs, bone marrow cells, hematopoietic stem cells, stem cells, stem cell-derived cells, mesenchymal stem cells, dendritic cells, dendritic cells pulsed with autoantigens, human beta cell products, pancreatic islet cells, allogeneic islet cells, hepatocytes, and spleen cells.

29. The pharmaceutical composition according to claim 28, wherein the graft cells are islet cells or islet cells derived from insulin-producing stem cells.

30. The pharmaceutical composition according to claim 28, wherein the graft cells are hepatocytes.

31. The pharmaceutical composition according to claim 28, wherein the graft cells are stem cells or stem cell-derived cells.

32. The pharmaceutical composition according to any one of claims 1 to 4, wherein the graft cells are derived from a deceased donor.

33. The pharmaceutical composition according to any one of claims 1 to 4, wherein the graft cells are allogeneic.

34. The pharmaceutical composition according to any one of claims 1 to 4, wherein the chimeric FasL protein comprises a FasL portion and a streptavidin portion or avidin portion, and optionally the chimeric FasL chimeric protein further comprises a linker between the FasL portion and the streptavidin portion or avidin portion.

35. The pharmaceutical composition according to claim 34, wherein the chimeric FasL protein comprises a FasL portion and a streptavidin portion.

36. The pharmaceutical composition according to claim 34, wherein the FasL portion is a matrix metalloproteinase-resistant FasL protein.

37. The pharmaceutical composition according to any one of claims 1 to 4, wherein the chimeric FasL protein comprises the amino acid sequence of SEQ ID NO:

4.

38. The pharmaceutical composition according to any one of claims 1 to 4, wherein the chimeric FasL protein is conjugated to the hydrogel via biotin.

39. The pharmaceutical composition according to any one of claims 1 to 4, wherein the hydrogel is a microgel.

40. The pharmaceutical composition according to claim 39, wherein the microgel is approximately 125 micrometers to approximately 175 micrometers.

41. The pharmaceutical composition according to claim 40, wherein the microgel is approximately 150 micrometers in size.

42. The pharmaceutical composition according to any one of claims 1 to 4, wherein the hydrogel is a polyethylene glycol (PEG) microgel.

43. The pharmaceutical composition according to any one of claims 1 to 4, wherein the hydrogel is operated to present a biotin moiety.

44. The pharmaceutical composition according to any one of claims 1 to 4, wherein the method comprises simultaneously administering the chimeric FasL protein conjugated to the hydrogel and the graft cells, wherein the graft cells are not embedded in the hydrogel.

45. The pharmaceutical composition according to any one of claims 1 to 4, wherein the method comprises administering a hydrogel to transplanted cells in a ratio of 2:

1.

46. The pharmaceutical composition according to any one of claims 1 to 4, wherein the method induces immune tolerance to the graft cells for the treatment of type 1 diabetes.

47. The pharmaceutical composition according to claim 46, wherein the method comprises administering an amount equivalent to at least 5,000 islands per kilogram of the human patient.

48. The pharmaceutical composition according to any one of claims 1 to 4, wherein the method induces immune tolerance to the graft cells for the treatment of liver failure.

49. The pharmaceutical composition according to any one of claims 1 to 4, wherein the method comprises administering the graft cells and the chimeric FasL protein conjugated to the hydrogel to a net.

50. The pharmaceutical composition according to any one of claims 1 to 4, wherein the method comprises orally administering the sirolimus.

51. The pharmaceutical composition according to claim 50, wherein the method comprises administering the sirolimus as an oral liquid.

52. The pharmaceutical composition according to claim 50, wherein the method comprises administering the sirolimus as an oral tablet.

53. The pharmaceutical composition according to any one of claims 1 to 4, wherein the method comprises administering the sirolimus once a day.

54. The pharmaceutical composition according to any one of claims 1 to 4, wherein the sirolimus administration is initiated on the same day as or up to five days before the day on which the chimeric FasL protein conjugated to the graft cells and the hydrogel is administered to the human subject.

55. The pharmaceutical composition according to any one of claims 1 to 4, wherein the method further comprises administering a prophylactic antiviral agent and an antibacterial agent during the administration of sirolimus.

56. The pharmaceutical composition according to claim 55, wherein the aforementioned preventive antiviral agent is famciclovir.

57. The pharmaceutical composition according to claim 56, wherein the antibacterial agent comprises sulfamethoxazole and / or trimethoprim.

58. Use of a chimeric FasL protein conjugated to a hydrogel in the manufacture of a pharmaceutical for use in a method of inducing immune tolerance to graft cells in a human patient in need thereof, The method comprises administering to the human patient (i) the graft cells, (ii) a chimeric FasL protein conjugated to a hydrogel, and (iii) sirolimus, wherein the sirolimus is administered for 36 weeks or less (for example, 24 weeks or less).

Images