Compositions and methods relating to immunoglobulin proteases and fusions thereof

Abstract

Provided herein are compositions and methods relating to compositions comprising Ig protease fusion proteins. Also provided herein are compositions and methods for therapeutic treatment of conditions such as autoimmune diseases, allergies, or other immunological disorders, or for therapeutic treatment with such Ig protease fusion proteins in combination with the administration of another therapeutic agent.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Serial No. 63 / 397,383, filed August 11, 2022, U.S. Provisional Application Serial No. 63 / 406,829, filed September 15, 2022, U.S. Provisional Application Serial No. 63 / 413,005, filed October 4, 2022, U.S. Provisional Application Serial No. 63 / 437,523, filed January 6, 2023, U.S. Provisional Application Serial No. 63 / 443,130, filed February 3, 2023, and U.S. Provisional Application Serial No. 63 / 463,942, filed May 4, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (S168170147WO00-SEQ-JAV.xml; size: 67,396 bytes; and creation date August 11, 2023) are incorporated herein by reference in their entirety.

[0003] FIELD OF THE INVENTION Provided herein are compositions and methods relating to immunoglobulin (Ig) proteases and fusions thereof. The Ig proteases and fusions thereof provided herein can be used to cleave Ig (in some embodiments, IgG) and / or may have improved properties. Such Ig proteases and fusions thereof can be for use in methods of treatment, such as methods of treatment with another therapeutic agent. Such Ig proteases and fusions thereof can also be for use in methods of treatment, such as methods of treatment for autoimmune diseases, immunological disorders, transplants, and graft-versus-host disease (GVHD). [Background technology]

[0004] The present invention also relates, at least in part, to a dose of an Ig protease fusion protein for administration in combination with a dose of a synthetic nanocarrier attached to an immunosuppressant, and related compositions, which provide a reduced immune response. The present invention also relates, at least in part, to the foregoing in combination with a dose of a viral vector, such as for gene therapy, which can provide a reduced immune response and / or increase the expression or durability of a transgene or nucleic acid material. Summary of the Invention

[0005] SUMMARY OF THE INVENTION In one aspect, a composition is provided comprising an Ig protease fusion protein comprising (i) an Ig protease domain and (ii) an Fc domain, such as an Fc domain provided herein, wherein, for example, the N-terminus or C-terminus of the Ig protease domain is fused to the Fc domain, and optionally, wherein the Ig protease fusion protein has similar or increased activity compared to a naturally occurring Ig protease, such as any provided herein, for example, IdeS or IdeSORK (e.g., wild-type enzyme). Such activity can be any one of those described herein. In one embodiment, the Ig protease fusion protein has a longer circulating half-life compared to a naturally occurring Ig protease, such as any provided herein, for example, IdeS or IdeSORK (e.g., wild-type enzyme).

[0006] In another aspect, compositions are provided that include an Ig protease fusion protein comprising (i) an Ig protease domain and (ii) albumin, wherein, for example, the N-terminus or C-terminus of the Ig protease domain is fused to albumin, and optionally has similar or increased activity compared to a naturally occurring Ig protease, such as any provided herein, e.g., IdeS or IdeSORK (e.g., wild-type enzyme). In one embodiment, the Ig protease fusion protein has a longer circulating half-life compared to a naturally occurring Ig protease, such as any provided herein, e.g., IdeS or IdeSORK (e.g., wild-type enzyme).

[0007] In one embodiment of any one of the compositions or methods provided herein, the Ig protease fusion protein binds to a region of a target immunoglobulin (e.g., IgG or IgA), and wherein the Ig protease fusion protein cleaves the target immunoglobulin (e.g., IgG or IgA). In one embodiment of any one of the compositions or methods provided herein, the Ig protease domain cleaves the target immunoglobulin (e.g., IgG or IgA) at the hinge region of the target immunoglobulin (e.g., IgG or IgA).

[0008] In one embodiment of any one of the compositions or methods provided herein, the Ig protease domain is or is derived from an Ig protease from a bacterial strain. In one embodiment of any one of the compositions or methods provided herein, the bacterial strain is a Streptococcus bacterial strain. In one embodiment of any one of the compositions or methods provided herein, the Streptococcus bacterial strain is Streptococcus pyogenes. In one embodiment of any one of the compositions or methods provided herein, the Streptococcus bacterial strain is Streptococcus equii. In one embodiment of any one of the compositions or methods provided herein, the Ig protease domain is or is derived from an Ig protease from Streptococcus kroesus. In one embodiment of any one of the compositions or methods provided herein, the bacterial strain is a Mycoplasma bacterial strain.

[0009] In one embodiment of any one of the compositions or methods provided herein, the Ig protease domain is or is derived from IdeS protease. In one embodiment of any one of the compositions or methods provided herein, the Ig protease domain is or is derived from IdeZ protease. In one embodiment of any one of the compositions or methods provided herein, the Ig protease domain is or is derived from IdeMC protease.

[0010] In one embodiment of any one of the compositions or methods provided herein, the Ig protease domain is or is derived from an IdeSORK protease. In one embodiment of any one of the compositions or methods provided herein, the Ig protease domain comprises any one of the sequences of any one of the Ig proteases provided herein or a fragment thereof. The Ig protease may be wild-type or it may be a mutant version thereof.

[0011] In one embodiment of any one of the compositions or methods provided herein, the Fc domain comprises any one of the sequences of any one of the Fc molecules provided herein or fragments thereof. In one embodiment of any one of the compositions or methods provided herein, the Fc domain may be wild-type, or it may be a mutant version thereof. In one embodiment of any one of the compositions or methods provided herein, the Fc domain is of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain is of human Ig. In one embodiment of any one of the compositions or methods provided herein, the Fc domain is of mouse Ig. In one embodiment of any one of the compositions or methods provided herein, the Fc domain is specific for IgG. In one embodiment of any one of the compositions or methods provided herein, the Fc domain is specific for IgA.

[0012] In one embodiment of any one of the compositions or methods provided herein, the Fc domain (eg, human Fc) comprises, or further comprises, a hinge region and a CH2 domain.

[0013] In one embodiment of any one of the compositions or methods provided herein, the Fc domain is a mutant Fc, e.g., providing improved activity (such as any one of the improved activities provided herein). In one embodiment of any one of the compositions or methods provided herein, the mutant Fc may be resistant to proteolysis by Ig proteases. In one embodiment of any one of the compositions or methods provided herein, the human Fc domain is mutated near the boundary between the hinge region and the CH2 domain. In one embodiment of any one of the compositions or methods provided herein, the Fc may have one or more modifications of the hinge region, which may or may not be accompanied by one or more other mutations as provided herein. In one embodiment of any one of the compositions or methods provided herein, the hinge region is shorter (e.g., 3x repeats) and more stable. In one embodiment of any one of the compositions or methods provided herein, any one of the Fc molecules is not glycosylated. In one embodiment of any one of the compositions or methods provided herein, the Fc domain has one or more of reduced or eliminated Fc effector function, reduced or eliminated complement fixation, and / or enhanced binding to FcRn.

[0014] Any one of the Ig protease fusion proteins provided herein may have reduced aggregation, increased stability, increased expression, extended half-life, shortened half-life, reduced Fc to FcRN binding (e.g., IgG1 Fc to FcRN binding), and / or an ablated Ig protease cleavage site.

[0015] In one embodiment of any one of the compositions or methods provided herein, the Fc comprises one or more mutations selected from the following: a GG-SS mutation in the hinge region (if the Fc comprises a hinge region); C220S (e.g., if the Fc comprises a hinge region); H435R; G236S; G237S; N297G replaced by an L234A, L235A, and / or P329A mutation (e.g., all three in one molecule); M428L and / or N434S mutation (e.g., both in one molecule); and a deletion of a terminal lysine (e.g., at the terminus of an Fc molecule or antibody, or portion thereof, including an Fc molecule). The Fc domain as provided herein can have any combination of mutations provided herein, such as the combinations represented by the exemplary molecules provided herein.

[0016] Any one of the foregoing may be a portion of an antibody, such as a full length antibody, or a portion thereof, such as an antigen-binding portion. In one embodiment of any one of the compositions or methods provided herein, the Ig protease fusion protein is monomeric.

[0017] In one embodiment of any one of the compositions or methods provided herein, the Ig protease fusion protein is a dimer, such as a homodimer.For example, the molecules may be complexed so that they are in the form of a dimer.In one embodiment, the fusion protein may be a covalently bonded homodimer. In one embodiment of any one of the compositions or methods provided herein, the Ig protease fusion protein is in a knob-in-hole configuration.

[0018] Also provided is any one of the specific fusion proteins provided herein, comprising any one of the sequences comprising a heavy chain or a portion thereof provided herein and any one of the sequences comprising a light chain or a portion thereof provided herein. Such specific fusion proteins can comprise any one of the light chain molecules provided herein in combination with any one of the heavy chain molecules provided herein. Compositions comprising the specific fusion proteins are also provided, as are methods for using any one of the specific fusion proteins in any one of the methods provided herein. In one embodiment, the specific fusion protein is any one of the specific combinations of sequences provided herein.

[0019] In another aspect, there are provided herein certain fusion proteins and compositions thereof, including those having the following sequences: Any of the foregoing may be expressed in mammalian or non-mammalian cells and thus may be mammalian- or non-mammalian-expressed molecules, respectively. When the Ig protease fusion protein is a molecule expressed in a non-mammalian, such as expressed from E. coli, in one embodiment, N297 is not mutated, or at least is not mutated to G or A. When the Ig protease fusion protein is a molecule expressed in a mammal, in one embodiment, N297 may also be mutated to G, etc.

[0020] In one aspect, a method for producing any one of the Ig protease fusion proteins provided herein is provided. A method for producing an Ig protease fusion protein in a mammalian cell, such as a CHO cell, is also provided. As an example, it has been found that Xork-Fc fusion can be successfully expressed and purified in such cells. Thus, any one of the Ig protease fusion proteins provided herein can be expressed in a mammal.

[0021] Also provided are methods for producing Ig protease fusion proteins in non-mammalian cells, such as E. coli. As an example, it has been found that Xork-Fc fusions can be successfully expressed in such cells. Thus, any one of the Ig protease fusion proteins provided herein can be expressed in non-mammalian cells.

[0022] In another aspect is a nucleic acid encoding any one of the Ig protease fusion proteins provided herein. In another aspect, provided is a vector comprising any one of the nucleic acids provided herein.

[0023] The compositions and methods may be for in vitro or in vivo purposes, such as cleavage of Ig, such as IgG or IgA. Thus, in one aspect, methods are provided for administering an Ig protease fusion protein or any one of the other compositions provided herein to a subject in need thereof, such as for the treatment of a disease or condition that may benefit from Ig cleavage, reduction, removal, etc.

[0024] In another aspect, provided is a method of administering any one of the Ig protease fusion proteins provided herein to a subject in need thereof (such as a subject receiving or to be administered a therapeutic biologic). The subject may be receiving or to be treated with a viral vector. The subject may be receiving or to be treated with gene therapy.

[0025] In one embodiment of any one of the methods provided herein, the therapeutic biologic is a viral vector, such as an adeno-associated virus (AAV) (e.g., AAV8) viral vector. In one embodiment of any one of the methods provided herein, the subject has or is at risk of developing anti-viral vector antibodies, such as anti-AAV (e.g., AAV8) viral vector antibodies.

[0026] In one embodiment of any one of the methods provided herein, the Ig protease fusion protein and the therapeutic biologic are administered concomitantly. In another aspect, provided is a method of administering any one of the Ig protease fusion proteins provided herein to a subject in need thereof (such as a subject with an autoimmune disease or immunological disorder).

[0027] In another aspect, provided is a method of administering any one of the Ig protease fusion proteins provided herein to a subject in need thereof (such as a subject who has received or is about to receive a transplant). In another aspect, provided is a method of administering any one of the Ig protease fusion proteins provided herein to a subject in need thereof (such as a subject with GVHD).

[0028] In one embodiment of any one of the methods provided herein, the subject is a human. In one embodiment of any one of the methods provided herein, the subject is in need of therapeutic treatment. In one embodiment of any one of the methods provided herein, the subject has been or will be administered a therapeutic biological agent, such as a viral vector (such as an AAV viral vector). In one embodiment of any one of the methods provided herein, the subject has been or will be administered gene therapy. In one embodiment of any one of the methods provided herein, the subject has or is at risk of having an autoimmune disease, immunological disorder, or GVHD. In one embodiment of any one of the methods provided herein, the subject has undergone or will undergo a transplant.

[0029] In one embodiment of any one of the methods or compositions provided herein, the Ig protease fusion protein is any one of the Ig protease fusion proteins provided herein. In another aspect, provided are compositions, such as compositions comprising any one of the Ig proteases or Ig protease domains provided herein, as described in any one of the methods provided herein, or any one of the Examples herein.

[0030] In another aspect, provided is a composition, such as a composition comprising any one of the Fc molecules or Fc domains provided herein, as described in any one of the methods provided herein, or any one of the examples herein. In one embodiment, any one of the compositions provided herein is for administration according to any of the methods provided.

[0031] In another aspect, any one of the compositions provided herein is for use in any of the methods provided. In another aspect, any one of the methods provided herein can further comprise administering a synthetic nanocarrier comprising an immunosuppressant. In one embodiment, the synthetic nanocarrier comprising an immunosuppressant can allow for re-medication with another therapeutic biologic, such as an Ig protease fusion protein and / or a viral vector.

[0032] In one embodiment, the method thus further comprises the further administration of an Ig protease fusion protein and / or another therapeutic biologic, such as a viral vector, hi one embodiment, the administration of an Ig protease fusion protein and another therapeutic biologic, such as a viral vector, is performed in combination.

[0033] In one embodiment, any one of the aforementioned methods can include further administration of a synthetic nanocarrier comprising an immunosuppressant in combination with further administration of another therapeutic biologic, such as an Ig protease fusion protein and / or a viral vector. In one embodiment of any one of the aforementioned methods, the administration and / or further administration of a synthetic nanocarrier comprising an immunosuppressant is performed in conjunction with another therapeutic biologic, such as an Ig protease fusion protein and a viral vector.

[0034] In one embodiment of any one of the methods provided herein, the dosing of the viral vector and the synthetic nanocarrier attached to the immunosuppressant is at or about monthly intervals. In one embodiment of any one of the methods provided herein, the administration of the Ig protease fusion protein precedes another therapeutic agent, such as a therapeutic biologic, such as a viral vector.

[0035] In certain aspects, provided are methods of making any one of the compositions or kits provided herein. In one embodiment, the method of making comprises producing one or more doses or dosage forms of a population of synthetic nanocarriers attached to an Ig protease fusion protein and / or a therapeutic biologic and / or an immunosuppressant. In another embodiment of any one of the provided methods of making, the step of producing one or more doses or dosage forms of a population of synthetic nanocarriers attached to an immunosuppressant comprises attaching the immunosuppressant to the synthetic nanocarrier. In another embodiment of any one of the provided methods of making, the method further comprises combining one or more doses or dosage forms of a population of Ig protease fusion protein and / or a therapeutic biologic and / or a synthetic nanocarrier attached to an immunosuppressant in a kit. In another aspect, any one of the compositions or kits provided herein is provided for use in any one of the methods provided herein. [Brief explanation of the drawings]

[0036] [Figure 1] Figure 1 depicts a schematic representation of human IgG cleaved by IdeS or IdeZ proteases. The IgG F(ab')2 and Fc domains are shown after cleavage by IdeS or IdeZ proteases. [Figure 2] Figure 2 depicts a fusion protein engineered by fusing the C-terminus of IdeS (shown in normal text) to the N-terminus of mouse IgG1 Fc (shown in underlined text). The signal sequence is shown in bold. The sequence corresponds to SEQ ID NO: 51.

[0037] [Figure 3]Figures 3A-3B show analyses of purified IdeS-Fc fusion protein. Figure 3A shows the IdeS-Fc fusion protein as a disulfide-linked homodimer at approximately 120,000 daltons under non-reducing conditions and as a single approximately 60,000 dalton band under reducing conditions with increasing concentrations of IdeS-Fc fusion protein. Figure 3B shows a graph of native SEC-HPLC analysis of purified IdeS-Fc fusion protein.

[0038] [Figure 4A-B] Figures 4A-4C show the in vivo activity of IdeS-Fc fusion protein in rabbits. Figure 4A is a table showing rabbits in four different treatment groups. Group 1 rabbits were untreated on the left; Group 2 rabbits were immunized with 1 x 10 vector genomes / kg AAV8 (adeno-associated virus-8) on day 1 and then untreated; Group 3 rabbits were immunized with 1 x 10 vector genomes / kg AAV8 (adeno-associated virus-8) on day 1 and then treated with 0.5 mg of IdeS-Fc fusion protein on day 29; and Group 4 rabbits were immunized with 1 x 10 vector genomes / kg AAV8 (adeno-associated virus-8) on day 1 and then treated with 5.0 mg of IdeS-Fc fusion protein on day 29. FIG. 4B is a graph depicting total rabbit IgG titers in each treatment group measured on days 1, 29, 31, 33, 36, 43, and 57. [Figure 4C] Figures 4A-4C show the activity of IdeS-Fc fusion proteins in vivo in rabbits. Figure 4C is a graph showing the EC50 of anti-AAV8 IgG on days 1, 29, 31, 33, and 36 after dosing with AAV8 on day 1 and IdeS-Fc on day 29.

[0039] [Figure 5A-1]Figures 5A-5B show amino acid sequences. Figure 5A shows the full-length sequence of IdeSORK, a protease isolated from Streptococcus krosus (SEQ ID NO: 1); an N-terminal fragment of IdeSORK with immunoglobulin protease activity (SEQ ID NO: 2); the IdeSORK2.0 protein of SEQ ID NO: 2 engineered to include an additional N-terminal methionine and a C-terminal protein purification tag, -His6 (SEQ ID NO: 3); SEQ ID NOs: 4-11 are sequences of the hinge / CH2 region of various human and mouse IgG subclasses; full-length IdeS is also disclosed as NCBI reference sequence WP_010922160.1 (SEQ ID NO: 12). [Figure 5A-2] Figures 5A-5B show amino acid sequences. Figure 5A shows the full-length sequence of IdeSORK, a protease isolated from Streptococcus krosus (SEQ ID NO: 1); an N-terminal fragment of IdeSORK with immunoglobulin protease activity (SEQ ID NO: 2); the IdeSORK2.0 protein of SEQ ID NO: 2 engineered to include an additional N-terminal methionine and a C-terminal protein purification tag, -His6 (SEQ ID NO: 3); SEQ ID NOs: 4-11 are sequences of the hinge / CH2 region of various human and mouse IgG subclasses; full-length IdeS is also disclosed as NCBI reference sequence WP_010922160.1 (SEQ ID NO: 12). [Figure 5B-1] 5A-5B represent amino acid sequences. Figure 5B shows mature IdeS, also disclosed under NCBI reference sequence number ADF13949.1 (SEQ ID NO: 13); full-length IdeZ, also disclosed under NCBI reference sequence number WP_014622780.1 (SEQ ID NO: 14); mature IdeZ (SEQ ID NO: 15); an exemplary nucleotide sequence encoding the polypeptide of SEQ ID NO: 1 engineered for expression with an N-terminal histidine and a C-terminal His6 (SEQ ID NO: 16); and an exemplary nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 engineered for expression with an N-terminal histidine and a C-terminal His6 (SEQ ID NO: 17). [Figure 5B-2]Figures 5A-5B represent amino acid sequences. Figure 5B shows mature IdeS, also disclosed as NCBI reference sequence ADF13949.1 (SEQ ID NO: 13); full-length IdeZ, also disclosed as NCBI reference sequence WP_014622780.1 (SEQ ID NO: 14); mature IdeZ (SEQ ID NO: 15); an exemplary nucleotide sequence encoding the polypeptide of SEQ ID NO: 1 engineered for expression with an N-terminal histidine and a C-terminal His6 (SEQ ID NO: 16); and an exemplary nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 engineered for expression with an N-terminal histidine and a C-terminal His6 (SEQ ID NO: 17). SEQ ID NO: 17 encodes SEQ ID NO: 3.

[0040] [Figure 6] Figure 6 shows a schematic diagram, example sequences, and specifications for the design of IdeSORK-Fc fusion homodimers. From top to bottom and left to right, SEQ ID NOS: 52, 47, and 53-57 are shown. [Figure 7] FIG. 7 presents tabular HPLC and SDS-PAGE data from IdeSORK-Fc fusions produced from CHO cells.

[0041] [Figure 8] FIG. 8 shows that the IdeSORK-Fc fusion can be successfully expressed in E. coli with good solubility. [Figure 9] Figure 9 is a schematic diagram depicting strategies for engineering IgG proteases for half-life extension. The half-life of IdeSORK IgG proteases can be extended by creating human serum albumin fusions, monomeric Fc fusions, or homodimeric Fc fusions with IdeSORK IgG proteases.

[0042] [Figure 10]Figure 10 is a graph showing that IgG protease-Fc fusion proteins enable AAV transduction in the presence of neutralizing human anti-AAV antibodies. Immunodeficient mice were injected with human serum on day -3, IgG protease on day -2, and AAV8-secreted alkaline phosphatase (SEAP) on day 0. SEAP expression in relative luminescence units (RLU) was measured from immune serum from mice on day 12. Treatment groups were immune serum from mice receiving IdeS protease, IdeSORK protease, IdeSORK-HSA, or IdeSORK-Fc[KIH]. Mice received a molar equivalent of IgG protease based on 1 mg / kg of native IdeS and IdeSORK.

[0043] [Figure 11] Figure 11 is a graph showing IgG protease-Fc with superior activity compared to IdeS at higher human serum doses. SEAP expression in relative brightness units (RLU) was measured from human immune serum using specific proteases. Treatment groups were human serum with IdeS, human immune serum with IdeSORK, and human immune serum with IdeSORK-Fc KIH. Treatment groups received molar equivalents of IgG protease based on 1 mg / kg of native IdeS and IdeSORK.

[0044] [Figure 12]Figure 12 is a graph showing that both IgG protease-Fc fusion and monomer-Fc fusion proteins enable AAV transduction in the presence of neutralizing human anti-AAV antibodies. Immune-deficient mice were injected with human serum on day -3, IgG protease on day -2, and AAV-secreted alkaline phosphatase (SEAP) on day 0. SEAP expression in relative luminance units (RLU) was measured from immune serum from mice on day 12. Treatment groups were immune serum from mice receiving IdeSORK, IdeSORK-Fc monomer, and IdeSORK-Fc homodimer. Mice were administered the molar equivalent of IgG protease based on 1 mg / kg of native IdeSORK.

[0045] [Figure 13] Figure 13 shows a general scheme of the in vivo test of IdeSORK protease activity against human IgG. The numbers shown correspond to the number of days since the time of AAV-SEAP inoculation, designated as day 0. [Figure 14] Figure 14 shows that the Xork1.1 molecule exhibits superior human IgG cleavage in vivo compared to Xork1.0 and Xork1.2, and that Xork1.1-hIgGFc-KIH restores AAV transduction efficiency to the same level as IdeS in passively immunized mice. SEAP activity in groups treated as indicated is shown.

[0046] [Figure 15A] Figures 15A-15B show total IgG levels in passively immunized mice before (day -2) and 48 hours after protease administration (day 0). Grouped (Figure 15A) and individual (Figure 15B) values are shown. The extent of reduction in human IgG levels in the groups immunized with 5% human serum and those treated with either Xork1.1 or IdeS is shown in Figure 15A. [Figure 15B]Figures 15A-15B show total IgG levels in passively immunized mice before (day -2) and 48 hours after protease administration (day 0). Grouped (Figure 15A) and individual (Figure 15B) values are shown. The extent of reduction in human IgG levels in the groups immunized with 5% human serum and those treated with either Xork1.1 or IdeS is shown in Figure 15A.

[0047] [Figure 16] Figure 16 shows that multiple Xork1.1 and Xork1.3 molecules cleave human IgG in vivo. Xork1.1-Fc-HD, Xork1.1-Fc-HD-H435R, and Xork1.3-Fc-HD enable efficient AAV transduction in passively immunized mice at standard and low (0.5x) doses, with Xork1.3-Fc-HD-H435R being slightly less efficient at the low dose. SEAP activity is shown in groups treated as indicated.

[0048] [Figure 17] Figure 17 shows that the Xork1.1-hIgGFc-GGSS molecule produced in E. coli and CHO cells is equally effective over a wide dose range and may be superior to Xork1.1-IgGFc-H435R and Xork1.1-IgG3Fc. SEAP activity in groups treated as indicated is shown. Numbers in parentheses indicate the SEAP activity level in each group, excluding outliers.

[0049] [Figure 18] Figure 18 shows that the Xork1.3-hIgGFc-GGSS dimer produced in E. coli is active over a wide dose range and its activity is not inferior to either the same molecule produced in CHO cells or the Xork1.3 H435R Fc mutant. SEAP activity in groups treated as indicated is shown. Numbers in parentheses indicate the SEAP activity level in each group, excluding outliers.

[0050] [Figure 19] FIG. 19 shows the prevalence of anti-AAV IgG antibodies in humans. [Figure 20] FIG. 20 shows the cleavage of human IgG and pre-existing antibody levels between IdeS and Xork.

[0051] [Figure 21] Figures 21A-21C show: A) Overlay of the Xork sequence on the ldeS crystal structure. The Xork lgG protease has low sequence identity to ldeS but high structural similarity. B) Xork cleaves human lgG with the same specificity and mechanism as IdeS. C) Xork shows very low cross-reactivity with antibodies in normal human serum compared to IdeS. Cross-reactivity of Xork and IdeS with serum from 20 random healthy donors was determined by ELISA.

[0052] [Figure 22A-C] Figures 22A-22E show in vivo activity in a serum passive transfer model of gene therapy. A) Depiction of native Xork, monomeric Xork-Fc, and homodimeric Xork-Fc. B) Passive transfer model. C) Xork 1.1-Fc has more potent in vivo activity than native Xork when equimolar amounts of enzyme are administered. [Figure 22D-E] Figures 22A-22E show in vivo activity in a serum passive transfer model of gene therapy. D) High serum transfer. Xork 1.1 Fc monomer has more potent activity than IdeS in rescuing AAV transduction at equimolar enzyme doses and in the presence of high doses of human immune serum. E) Xork 1.3-Fc shows potent activity at 0.12 mg / kg.

[0053] [Figure 23]Figures 23A-23B show the pharmacodynamics of Xork-Fc: A) Xork 1.1-Fc homodimer produced in CHO cells was administered 14 days, 7 days, 7 hours, or 15 minutes before, or 1 day after passive transfer of human serum (day 0). Animals were treated with AAV-SEAP 3 days after passive transfer of serum, and serum SEAP activity was assessed 12 days later. B) Xork-Fc administered 7 days before passive transfer of human serum shows similar activity as when administered immediately before serum transfer. DETAILED DESCRIPTION OF THE INVENTION

[0054] Detailed Description of the Invention Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified materials or process parameters, as these may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to limit the use of alternative terminology to describe the present invention.

[0055] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a reference to "an Ig protease." As another example, a reference to "a polymer" includes a mixture of two or more such molecules or a mixture of a single polymer species of different molecular weights; a reference to "a synthetic nanocarrier" includes a mixture of two or more such synthetic nanocarriers or a plurality of such synthetic nanocarriers; a reference to "an immunosuppressant" includes a mixture of two or more such materials or a plurality of immunosuppressant molecules, etc.

[0056] As used herein, the term "comprise" or variations thereof, such as "comprises" or "comprising," should be read to indicate the inclusion of any listed integer (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., feature, element, feature, property, method / process step, or limitation), but not the exclusion of any other integer or group of integers. Thus, as used herein, the term "comprising" is inclusive and does not exclude additional, unrecited integers or method / process steps.

[0057] In any one embodiment of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of." The phrase "consisting essentially of" is used herein to require the specified integer(s) or steps, as well as those that do not materially affect the characteristics or functionality of the claimed invention. As used herein, the term "consisting" is used to refer to the sole presence of a listed integer (e.g., a feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., a feature, element, characteristic, property, method / process step, or limitation).

[0058] A. Introduction Autoimmune diseases and other immunological disorders are serious medical conditions that can be chronic and debilitating in nature, resulting in high medical costs and a reduced quality of life. There are over 80 known autoimmune diseases, including, but not limited to, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. Autoimmune diseases or other immunological disorders generally involve a subject's immune system attacking itself and damaging its own tissues.

[0059] Immunoglobulins produced by B cells play an important role in antigen-specific host defense, but they can also play a pathogenic role in autoimmune diseases and other immunological disorders. In normal host defense, secretory immunoglobulins of different isotypes have different roles. For example, IgG antibodies are the most abundant antibody isotype in human blood and play a major role in host defense in peripheral tissues. Autoreactive IgG antibodies can contribute to the pathogenesis of autoimmune diseases. Another example of an undesirable IgG antibody is an IgG antibody that reacts with donor graft tissue, which can cause acute rejection of a transplanted organ. For another example, IgM antibodies are typically the first secretory isotype to be produced and are involved in the early host defense response. For another example, IgA plays an important role in host defense against mucosal pathogens. For yet another example, IgE is thought to be involved in the immune response to parasites. In terms of pathogenic roles, various isotypes play a central role in many autoimmune diseases, such as myasthenia gravis, Graves' disease, and neuromyelitis optica. For example, IgA antibodies are thought to be involved in IgA nephropathy, IgA pemphigus, and linear IgA dermatopathy. As another example, IgE antibodies are thought to be involved in allergies.

[0060] Immunoglobulins, such as IgG isotypes, are also associated with anti-drug antibody (ADA) responses to biologic therapies, which can compromise efficacy or safety. Due to the central role of immunoglobulins in host defense, various microbial pathogens have evolved proteases that selectively cleave specific immunoglobulin isotypes, or in some cases, specific subclasses of certain immunoglobulin isotypes, to evade the host's immune response. For example, various strains of streptococci produce proteases that specifically cleave human IgG (e.g., IdeS from Streptococcus pyogenes and IdeZ from Streptococcus equii). As another example, some strains of mycoplasma bacteria that infect dogs have evolved to produce proteases that specifically cleave canine IgG (e.g., U.S. Patent Application No. 20190262434 A1), and some strains of streptococci that infect pigs have evolved to produce proteases that specifically cleave porcine IgM (but not human IgM). In addition, several types of bacteria produce IgA-specific proteases, including Haemophilus influenzae, Neisseria gonorrhoeae, Neisseria meningitidis, Clostridium ramosum, and Streptococcal pneumoniae. Finally, the parasite Schistosoma mansoni has been reported to produce an IgE-specific protease.

[0061] Ig proteases have therapeutic potential. For example, IgG proteases prevented antibody-mediated acute rejection of kidney allografts in a phase 2 clinical trial (Jordan SC, et al. IgG Endopeptidase in Highly Sensitized Patients Undergoing Transplantation. N Engl J Med. 2017 Aug 3;377(5):442-453. doi: 10.1056 / NEJMoa1612567). For another example, IgG proteases have been applied to animal models of Guillain-Barré syndrome and IgG nephropathy, enabling the administration of AAV gene therapy vectors to non-human primates with pre-existing neutralizing antibodies against the AAV capsid. However, the clinical use of microbial-derived Ig proteases is currently limited by their immunogenicity and short circulating half-life. In addition, many people have pre-existing antibodies to Ig proteases, which may compromise efficacy or safety.

[0062] Provided herein are compositions of Ig protease fusion proteins, methods for their production, and methods for their use.The Ig protease fusion proteins provided herein can be used for any of the purposes provided herein, such as treating any one of the diseases, disorders, or conditions provided herein.Ig protease fusion proteins can also be used in combination with the delivery of biological treatments, including viral vector therapy.Ig protease fusion proteins can also be used in combination with the delivery of synthetic nanocarriers, which in some embodiments include immunosuppressants.

[0063] Ig protease fusion proteins can have improved circulating half-lives in the blood, such as by inclusion of an Fc (Fc domain) or albumin, hi some embodiments, the Ig protease fusion proteins can have extended half-lives compared to naturally occurring or wild-type proteases, such as IdeS or IdeSORK.

[0064] Any one of the compositions described herein can be useful for treating subjects as provided herein. Any one of the compositions described herein can be useful for treating diseases or disorders that Ig cleavage (for example, IgG or IgA cleavage) can benefit from. It is also contemplated that the compositions described herein can be effective when administered in combination with other treatments. It is also contemplated that the compositions described herein can be useful for complementing other treatments, such as gene therapy or other biological treatments. The invention will now be described in more detail below.

[0065] B. Definition "Administering" or "administration" or "administer" means giving a material to a subject in a manner such that there is a pharmacological result in the subject. This may be direct administration or indirect administration, for example, by instructing or directing another subject, including another clinician or the subject themselves, to administer the material.

[0066] A "dosing schedule" refers to the administration of one or more agent doses according to a determined schedule. The schedule may include the number of doses as well as the frequency of such doses or the interval between doses. Such dosing schedules may include a number of parameters that are varied to achieve a particular goal, such as reducing an undesired immune response to an Ig protease and / or a therapeutic biological (e.g., viral vector) antigen and / or increasing or durable expression of a transgene or nucleic acid material. In embodiments, the dosing schedule is any of the dosing schedules provided in the examples below. In some embodiments, a dosing schedule in accordance with the present invention may be used to administer the doses to one or more test subjects. The immune responses in these test subjects can then be evaluated to determine whether they were effective in reducing an undesired humoral immune response and / or increasing or durable expression of a transgene or nucleic acid material. Whether a schedule had the desired effect can be determined using any of the methods provided herein or otherwise known in the art. For example, a sample may be obtained from a subject to which the dosage provided herein has been administered according to a specific administration schedule to determine whether specific immune cells, cytokines, antibodies, etc. have been reduced, produced, activated, etc., and / or whether specific proteins or expression products have been increased, reduced, or produced, etc. Useful methods for detecting the presence and / or number of immune cells include, but are not limited to, flow cytometry (e.g., FACS), ELISpot, proliferative responses, cytokine production, and immunohistochemistry. Useful methods for determining the production levels of proteins such as antibodies are well known in the art and include the assays provided herein. Such assays include ELISA assays.

[0067] "Effective amount" in the context of a composition or dose for administration to a subject refers to the amount of the composition or dose that produces one or more desired responses in the subject. Thus, in some embodiments, an effective amount is any amount of the compositions or doses provided herein that produces one or more of the desired therapeutic effects and / or immune responses provided herein. This amount can be for in vitro or in vivo purposes. For in vivo purposes, the amount can be what a clinician would consider to have clinical benefit for a subject in need thereof. Any one of the compositions or doses (including labeled doses) provided herein can be in an effective amount.

[0068] An effective amount includes reducing the level of an undesired response, but in some embodiments, it includes preventing an undesired response altogether. An effective amount also includes delaying the onset of an undesired response. An effective amount can also be an amount that produces a desired therapeutic endpoint or a desired therapeutic effect. In other embodiments, an effective amount can involve enhancing the level of a desired response, such as a therapeutic endpoint or outcome. An effective amount can produce a therapeutic outcome or endpoint in any of the subjects provided herein. Achievement of any of the foregoing can be monitored by routine methods.

[0069] The effective amount will, of course, depend on the specific subject being treated; the severity of the condition, disease, or disorder; individual patient parameters, including age, physical condition, size, and weight; the duration of treatment; the nature of concurrent treatment (if any); the specific route of administration, and similar factors within the knowledge and expertise of a healthcare practitioner. These factors are well known to those skilled in the art and can be addressed only by routine experimentation. It is generally preferable to use the maximum dose, i.e., the highest safe dose according to sound medical judgment. However, it will be understood by those skilled in the art that a patient may require a lower dose or tolerated dose for medical reasons, psychological reasons, or virtually any other reason.

[0070] "Anti-viral vector immune response" or "immune response to a viral vector" and the like refer to any unwanted immune response to a viral vector. In some embodiments, the unwanted immune response is an antigen-specific immune response to the viral vector or an antigen thereof. In some embodiments, the immune response is specific to a viral antigen of the viral vector. The immune response may be an anti-viral vector antibody response, an anti-viral vector T cell immune response, e.g., a CD4+ T cell or CD8+ T cell immune response, or an anti-viral vector B cell immune response. Similarly, such immune responses may also occur in response to other therapeutic biologics.

[0071] "Antigen" refers to a molecule that can be bound by an immunoglobulin, a B cell antigen receptor, and / or a T cell receptor. Non-limiting examples of antigens include proteins, peptides, polysaccharides, and lipopolysaccharides. Antigens can be classified as exogenous (e.g., molecules foreign to the subject) or endogenous (intracellular, produced within the subject, etc.). Any and all types of antigens known in the art are contemplated in the present disclosure. A subject of any one of the methods provided herein can have an undesired immune response or an undesired level of an immune response to any one of the antigens provided herein.

[0072] "Antigen-specific" refers to any immune response that occurs as a result of the presence of an antigen or a portion thereof, or that generates a molecule that specifically recognizes or binds to the antigen. In some embodiments, when the antigen is that of a viral vector, antigen-specific may mean viral vector-specific. For example, when an immune response is the production of antigen-specific antibodies, antibodies that specifically bind to the antigen are produced. As another example, when an immune response is the proliferation and / or activity of antigen-specific B cells or CD4+ T cells, the proliferation and / or activity occurs as a result of recognition of the antigen or a portion thereof, alone or in complex with an MHC molecule, B cell, etc.

[0073] "Assessing a therapeutic response" refers to any measurement or determination of the level, presence or absence, reduction, increase, etc. of a therapeutic response in vitro or in vivo. Such measurement or determination may be performed on one or more samples obtained from a subject. Such evaluation can be performed as a step in any one of the methods provided herein. The evaluation may be evaluating any one or more of the biomarkers provided herein or known in the art.

[0074] "Attach" or "attached" or "couple" or "coupled" (and the like) refer to the chemical association of one entity (e.g., moiety) with another. In some embodiments, the attachment is covalent, meaning that the attachment occurs in the context of a covalent bond being present between the two entities. In non-covalent embodiments, the non-covalent attachment is mediated by non-covalent interactions, including, but not limited to, charge interactions, affinity interactions, metal coordination, physical adsorption, host-object interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and / or combinations thereof. In embodiments, encapsulation is a form of attachment. In embodiments, the therapeutic biologic, the synthetic nanocarrier attached to the immunosuppressant, and the Ig protease fusion protein are not attached to one another, meaning that the therapeutic biologic, the synthetic nanocarrier attached to the immunosuppressant, and the Ig protease fusion protein have not been subjected to a process specifically intended to chemically associate with one another.

[0075] As used herein, "average" refers to the arithmetic mean unless otherwise specified. As used herein, the term "combination therapy" is intended to define a therapy that includes the use of a combination of two or more materials / agents. Thus, references in this application to "combination therapy," "combination," and the use of materials / agents "in combination" may refer to materials / agents that are administered as part of the same overall treatment regimen. As such, the posology of each of the two or more materials / agents may differ; each may be administered simultaneously or at different times. It will therefore be understood that materials / agents of a combination may be administered sequentially (e.g., before or after) or simultaneously, in the same pharmaceutical formulation (i.e., together) or in different pharmaceutical formulations (i.e., separately). Simultaneously in the same formulation means as a single unit formulation, whereas simultaneously in different formulations means not as a single unit. The posology of each of the two or more materials / agents in a combination therapy may also differ with respect to the route of administration. In one embodiment of any one of the methods provided herein, the materials / agents are administered in combination.

[0076] "In combination" means that two or more materials / agents are administered to a subject in a temporally correlated manner, preferably in a manner sufficiently correlated in time that a first composition has an effect on a second composition (e.g., augmenting the efficacy of the second composition); preferably, two or more materials / agents are administered in combination to provide modulation in a physiological or immunological response, and even more preferably, two or more materials / agents are administered in combination. In embodiments, the combined administration may encompass administration of two or more compositions within a specified period of time. In embodiments, two or more materials / agents are administered sequentially. In embodiments, the materials / agents may be administered in combination repeatedly; i.e., co-administered on more than one occasion. In any one of the embodiments of the methods or compositions provided herein, the Ig protease fusion protein and / or synthetic nanocarrier may be administered in combination or repeatedly co-administered. In some embodiments, two or more compositions are administered within one month, one week, one day, or one hour. In some embodiments, the combined administration includes simultaneous administration of two or more compositions.

[0077] "Determining" or "determining" means ascertaining a fact. Determining may be accomplished in a number of ways, including, but not limited to, conducting an experiment or making a prediction. For example, a dose of an immunosuppressant and / or therapeutic biologic and / or Ig protease fusion protein can be determined by starting with a test dose and determining a dose for administration using known scaling techniques (such as allometric scaling or isometric scaling). Such may also be used to determine a protocol or administration schedule as provided herein. In another embodiment, a dose may be determined by testing various doses in subjects, i.e., through direct experimentation based on experience and guiding data. In an embodiment, "determining" or "determining" includes "causing something to be determined." "Causing something to be determined" means causing, prompting, encouraging, assisting, guiding, instructing, or acting in concert with an entity to ascertain a fact about that entity; this includes direct or indirect, explicit or implicit.

[0078] "Dosage form" means pharmacologically and / or immunologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. Any one of the compositions or doses provided herein may be in a dosage form. A "dose" refers to a specific quantity of a pharmacologically and / or immunologically active material for administration to a subject over a given period of time.

[0079] "Administering" refers to the administration of a pharmacologically and / or immunologically active material or a combination of pharmacologically and / or immunologically active materials to a subject. The materials in the administration may be administered in combination in any one of the methods provided herein. The materials in the administration may be administered individually in separate compositions in any one of the methods provided herein.

[0080] "Encapsulating" means enclosing at least a portion of a substance within a synthetic nanocarrier. In some embodiments, the substance is completely encapsulated within the synthetic nanocarrier. In other embodiments, most or all of the encapsulated substance is not exposed to the local environment external to the synthetic nanocarrier. In other embodiments, no more than 50%, 40%, 30%, 20%, 10%, or 5% (weight / weight) is exposed to the local environment. Encapsulation is distinct from absorption, which places most or all of the substance on the surface of the synthetic nanocarrier, leaving the substance exposed to the local environment external to the synthetic nanocarrier. In any one of the methods or compositions provided herein, the immunosuppressant may be encapsulated in the synthetic nanocarrier.

[0081] An "expression control sequence" is any sequence that can affect expression and can include promoters, enhancers, and operators. In one embodiment of any one of the methods or compositions provided, the expression control sequence is a promoter. In one embodiment of any one of the methods or compositions provided, the expression control sequence is a liver-specific promoter or a constitutive promoter. A "liver-specific promoter" is one that confers expression exclusively or preferentially in cells of the liver. A "constitutive promoter" is one that is generally active and is not considered exclusive or preferential for a particular cell. In any one of the nucleic acids or viral vectors provided herein, the promoter can be any one of the promoters provided herein.

[0082] "Fc domain" refers to a portion of an antibody that interacts with an Fc receptor, or a portion of an Ig protease fusion protein that includes a portion thereof that interacts with an Fc receptor. In some embodiments of the present disclosure, the Fc domain is of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain is of a mouse Ig. In some embodiments, the Fc domain is of a human Ig. As used herein, the term refers to an entire Fc molecule or portion thereof that interacts with an Fc receptor and / or provides the required activity and desired result consistent with the disclosure provided herein.

[0083] By "generating" is meant causing, directly or indirectly, the production of an effect such as an immune response or a physiological response (eg, a tolerogenic immune response). "Graft-versus-host disease" (GVHD) is a complication that can occur after transplantation of pluripotent cells (e.g., stem cells) or bone marrow, in which the newly transplanted material attacks the transplant recipient's body. In some cases, GVHD occurs after a blood transfusion. GVHD can be divided into acute and chronic forms. The acute or fulminant form of the disease (aGVHD) is usually observed within the first 100 days after transplantation, while the chronic form of graft-versus-host disease (cGVHD) usually occurs after 100 days. It represents a major challenge to transplantation due to the associated morbidity and mortality. The occurrence of moderate to severe cGVHD cases can have a detrimental effect on long-term survival.

[0084] A "homodimeric Ig protease fusion protein" or "homodimeric Ig protease fusion protein" refers to the presence of two identical Ig protease domains in an Ig protease fusion protein as provided herein, which may be coupled or conjugated to a molecule (such as an Fc domain) that can extend the half-life of the Ig protease domain. The two Ig protease domains may be coupled or conjugated to each other by any means, or may be coupled (e.g., covalently) or conjugated to each other by their respective attachment to an Fc domain. The Fc domain may be composed of units that are coupled or conjugated together to form the Fc domain. An Ig protease fusion protein is referred to as a "dimer" or "dimeric" if the Ig protease domains are not identical. Examples of monomeric or homodimeric Ig protease fusion proteins are provided herein.

[0085] "Identifying a subject" refers to any act or series of acts that allows a clinician to recognize a subject as one who may benefit from the methods or compositions provided herein, or any other indicators as provided. Preferably, the identified subject is one who needs therapeutic treatment as provided herein. In some embodiments, the subject is identified based on symptoms (and / or lack thereof), a pattern of behavior (e.g., that places the subject at risk), and / or based on one or more tests (e.g., biomarker assays) described herein. In some embodiments of any one of the methods provided herein, the subject is one who will benefit from or is in need of a treatment provided herein. In one embodiment of any one of the methods provided herein, the method further comprises identifying a subject in need of a composition or method as provided herein. The act or series of acts may be direct, by oneself, or indirect, such as (but not limited to) by an unrelated third party acting through reliance on one's words or actions.

[0086] "Immunoglobulin" or "Ig" refers to a glycoprotein molecule that recognizes and binds to an antigen. Immunoglobulins can be classified by class, or by subclass, or by immunoglobulin isotype. In some embodiments, an immunoglobulin of a particular class or isotype may differ in structure and / or biological function compared to another immunoglobulin of a different class or isotype.

[0087] "Immunoglobulin isotype" refers to the classification of an immunoglobulin by the heavy chains it contains (e.g., IgA contains alpha heavy chains, IgD contains delta heavy chains, IgE contains epsilon heavy chains, IgG contains gamma heavy chains, and IgM contains mu heavy chains). In some embodiments, immunoglobulin isotypes differ in function and / or antigen response compared to different immunoglobulin isotypes. In some embodiments, immunoglobulin isotypes are further classified by subclass (e.g., IgA1, IgA2, IgD, IgE, IgG2, IgG2a, IgG2b, IgG3, IgG4; or IgM).

[0088] "Immunoglobulin (Ig) protease" refers to an enzyme that cleaves / hydrolyzes one or more peptide bonds in an immunoglobulin. In some embodiments, the protease can be selected from naturally occurring or wild-type or endogenous Ig proteases or variants thereof. In some embodiments, the Ig protease can be selected from Ig proteases from bacterial strains. In some embodiments, the bacterial strain is a Streptococcus bacterial strain. In some embodiments, the bacterial strain is a Mycoplasma bacterial strain. In some embodiments, the Ig protease is an IdeS protease. In some embodiments, the Ig protease is an IdeZ protease. In some embodiments, the Ig protease is an IdeMC protease. In some embodiments, the Ig protease is an IdeSORK protease. In some embodiments, each of the proteases herein can be wild-type, or a mutant or truncated version thereof. In embodiments, the Ig protease cleaves and / or hydrolyzes one or more target immunoglobulins, such as IgG or IgA molecules.

[0089] In some embodiments, the Ig protease may be of human origin. In some embodiments, the Ig protease may include any portion of a human protease capable of cleaving the hinge region of human Ig, including, for example, cathepsin G and numerous matrix metalloproteinases (Ryan MH, et al. Proteolysis of purified IgGs by human and bacterial enzymes in vitro and the detection of specific proteolytic fragments of endogenous IgG in rheumatoid synovial fluid. Mol Immunol. 2008 Apr;45(7):1837-46. doi: 10.1016 / j.molimm.2007.10.043. Epub 2007 Dec 21. PMID: 18157932). In some embodiments, structure-based protein design can aid in the generation of protease domains that can be evaluated and optimized for specificity and / or activity.

[0090] In some embodiments, the Ig protease is a mutant version of any one of the sequences provided herein.In some embodiments, the Ig protease is a fragment of a full-length protease, for example, a fragment of any one of the sequences provided herein, which has the catalytic or hydrolytic activity of the full-length enzyme.The mutant version may comprise one or more amino acid substitutions compared to wild-type.In any one of the methods or compositions provided herein, a wide variety of Ig proteases or domains thereof can be used according to the present invention.

[0091] "Immunoglobulin A (IgA) protease" refers to an enzyme that cleaves and / or hydrolyzes one or more peptide bonds in immunoglobulin A. In some embodiments, the IgA protease is from a Streptococcus bacterial strain. In some embodiments, the IgA protease is from a Neisseria bacterial strain. In some embodiments, the IgA protease is from a Clostridium bacterial strain. In some embodiments, the IgA protease is from a Capnocytophaga bacterial strain. In some embodiments, the IgA protease is from a Bacteroides bacterial strain. In some embodiments, the IgA protease is from a Gemella bacterial strain. In some embodiments, the IgA protease is from a Prevotella bacterial strain.

[0092] "Immunoglobulin G (IgG) protease" refers to an enzyme that cleaves and / or hydrolyzes one or more peptide bonds of immunoglobulin G. In some embodiments, the IgG protease is from a Streptococcus bacterial strain. In one embodiment, the Streptococcus bacterium is Streptococcus pyogenes. In one embodiment, the Streptococcus bacterial strain is Streptococcus equii. In one embodiment, the Streptococcus bacterial strain is Streptococcus kroesus. In some embodiments, the IgG protease is from a Mycoplasma bacterial strain. In one embodiment, the Mycoplasma bacterial strain is Mycoplasma canis. In one embodiment, the IgG protease is based on any one of the IgG proteases of U.S. Publication No. 2019-0262434 A1, which IgG protease of U.S. Publication No. 2019-0262434 A1 is incorporated herein by reference. In one embodiment, the IgG protease is based on the protease described in WO2022 / 223818, the disclosure of which is incorporated herein by reference, including the Ig proteases described herein. In some embodiments, the IgG protease is an IdeSORK protease. IdeSORK may also be referred to herein as "Xork."

[0093] An "Ig protease domain" refers to a portion of an Ig protease fusion protein, or a fragment or portion thereof, that cleaves / hydrolyzes one or more peptide bonds in an immunoglobulin (e.g., in the hinge region of an immunoglobulin). In one embodiment of any one of the compositions or methods provided herein, the Ig protease domain of the Ig protease fusion protein comprises any fragment having the protease activity of any one of the Ig proteases provided herein. In some embodiments, the Ig protease domain comprises the active site of an Ig protease enzyme. In some embodiments, the Ig protease domain is or is from a Streptococcus bacterial strain. In some embodiments, the Ig protease domain is or is from an IgA protease. In some embodiments, the Ig protease domain is or is from an IgG protease. In some embodiments, the Ig protease domain is or is from an IdeSORK. In some embodiments of any one of the compositions or methods provided herein, the Ig protease domain of the Ig protease fusion protein is a full-length Ig protease.In some embodiments of any one of the compositions or methods provided herein, the Ig protease domain of the Ig protease fusion protein is a fragment of a full-length Ig protease.In some embodiments of any one of the compositions or methods provided herein, the Ig protease domain of the Ig protease fusion protein is a mutant version of a wild-type Ig protease or a fragment thereof.

[0094] As used herein, the term "immunosuppressant" refers to a compound capable of inducing a tolerogenic immune response specific to an antigen, and is also referred to herein as an "immunosuppressive effect." The immunosuppressive effect generally refers to the production or expression of cytokines or other factors by antigen-presenting cells (APCs) that reduce, inhibit, or prevent an undesired immune response, or promote a desirable immune response, such as a regulatory immune response, to a specific antigen. When an APC acquires (under the immunosuppressive effect) an immunosuppressive function on immune cells that recognize the antigen presented by the APC, the immunosuppressive effect is said to be specific to the presented antigen.

[0095] Immunosuppressants include, but are not limited to, statins; mTOR inhibitors such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors such as trichostatin A; corticosteroids; inhibitors of mitochondrial function such as rotenone; P38 inhibitors; 6Bio, dexamethasone, TCPA-1, IKK These include NF-κB inhibitors such as NF-κB VII; adenosine receptor agonists; prostaglandin E2 agonists (PGE2) such as misoprostol; phosphodiesterase inhibitors such as phosphodiesterase 4 (PDE4) inhibitors such as rolipram; histone deacetylase (HDAC) inhibitors, proteasome inhibitors; kinase inhibitors; G protein-coupled receptor agonists; G protein-coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3KB inhibitors such as TGX-221; autophagy inhibitors such as 3-methyladenine; aryl hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and oxidized ATP such as P2X receptor blockers. Immunosuppressants also include methotrexate, IDO, vitamin D3, cyclosporines such as cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine (AZA), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol, and triptolide. In embodiments, the immunosuppressant may include any of the agents provided herein.

[0096] An immunosuppressant may be a compound that provides an immunosuppressive effect directly on APCs, or it may be a compound that provides an immunosuppressive effect indirectly (i.e., after being processed in some manner after administration). Immunosuppressants therefore include prodrug forms of any of the compounds provided herein.

[0097] In one embodiment of any of the methods or compositions provided herein, the immunosuppressant provided herein is formulated with a synthetic nanocarrier. In a preferred embodiment, the immunosuppressant is an element added to the material that constitutes the structure of the synthetic nanocarrier. For example, in one embodiment, when the synthetic nanocarrier is composed of one or more polymers, the immunosuppressant is a compound added to and attached to the one or more polymers. As another example, in one embodiment, when the synthetic nanocarrier is composed of one or more lipids, the immunosuppressant is also a compound added to and attached to the one or more lipids. In an embodiment, for example, when the material of the synthetic nanocarrier also provides an immunosuppressive effect, the immunosuppressant is an element present in addition to the material of the synthetic nanocarrier that provides the immunosuppressive effect.

[0098] "Loading," when included in a composition comprising (e.g., coupled to) a synthetic nanocarrier, is the amount of immunosuppressant in the composition, based on the total dry recipe weight (wt / wt) of material in the entire synthetic nanocarrier. Generally, such loading is calculated as an average across the entire population of synthetic nanocarriers. In one embodiment, the average loading across the synthetic nanocarriers is between 0.1% and 25%, 30%, 35%, 40%, 45%, or 50%. In another embodiment, the average loading across the synthetic nanocarriers is between 1% and 25%, 30%, 35%, 40%, 45%, or 50%. In a further embodiment, the loading is between 1% and 15%. In another embodiment, the loading is between 0.1% and 10%. In a further embodiment, the loading is between 5% and 15%. In yet a further embodiment, the loading is between 7% and 12%. In yet a further embodiment, the loading is between 8% and 12%. In yet another embodiment, the loading amount is between 7% and 10%. In yet another embodiment, the loading amount is between 8% and 10%. In further embodiments, the loading amount is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, averaged across the population of synthetic nanocarriers. In any one of the methods, compositions, or kits provided herein, the loading amount of the immunosuppressant, such as rapamycin, can be any one of the loading amounts provided herein.

[0099] The immunosuppressant (e.g., rapamycin) loading of nanocarriers in suspension can be calculated by dividing the immunosuppressant content of the nanocarriers, as determined by HPLC analysis of the test article, by the mass of the nanocarriers. The total polymer content can be determined by gravimetric yield of the mass of the dried nanocarriers or by measuring the total organic content of the nanocarrier solution according to pharmacy procedures, and can be corrected for PVA content.

[0100] "Maximum dimension of a synthetic nanocarrier" refers to the largest dimension of the nanocarrier measured along any axis of the synthetic nanocarrier. "Minimum dimension of a synthetic nanocarrier" refers to the smallest dimension of the synthetic nanocarrier measured along any axis of the synthetic nanocarrier. For example, for a spherical synthetic nanocarrier, the largest and smallest dimensions will be substantially the same and will be the size of its diameter. Similarly, for a cuboidal synthetic nanocarrier, the smallest dimension of the synthetic nanocarrier will be the smallest of its height, width, or length, while the largest dimension of the synthetic nanocarrier will be the largest of its height, width, or length. In one embodiment, the smallest dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 100 nm. In one embodiment, the largest dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or less than 5 μM. Preferably, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is greater than 110 nm, more preferably greater than 120 nm, more preferably greater than 130 nm, and more preferably even greater than 150 nm. Preferably, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is less than 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 500 nm, 450 nm, 400 nm, 350 nm, or 300 nm. The aspect ratio of the maximum and minimum dimensions of synthetic nanocarriers may vary depending on the embodiment.Illustratively, the aspect ratio of the largest dimension to the smallest dimension of a synthetic nanocarrier may vary from 1:1 to 1,000,000:1, preferably from 1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferably from 1:1 to 1000:1, even more preferably from 1:1 to 100:1, and even more preferably from 1:1 to 10:1.

[0101] Preferably, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or less than 3 μm, more preferably equal to or less than 2 μm, more preferably equal to or less than 1 μm, more preferably equal to or less than 800 nm, more preferably equal to or less than 600 nm, and more preferably still equal to or less than 500 nm. In a preferred embodiment, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 100 nm, more preferably equal to or greater than 120 nm, more preferably equal to or greater than 130 nm, more preferably equal to or greater than 140 nm, and more preferably still equal to or greater than 150 nm. Measurements of synthetic nanocarrier dimensions (e.g., effective diameter) may be obtained in some embodiments by suspending synthetic nanocarriers in a liquid (usually aqueous) medium and using dynamic light scattering (DLS) (e.g., using a Brookhaven ZetaPALS instrument). For example, a suspension of synthetic nanocarriers can be diluted from an aqueous buffer into purified water to achieve a final synthetic nanocarrier suspension concentration of approximately 0.01-0.1 mg / mL. The diluted suspension may be prepared directly in or transferred to a cuvette suitable for DLS analysis. The cuvette may then be placed in the DLS, equilibrated to a controlled temperature, and then scanned for a sufficient time to obtain a stable and reproducible distribution based on appropriate inputs for the viscosity of the medium and the refractive index of the sample. The effective diameter or mean value of the distribution is then reported. Determining the effective size of high-aspect-ratio or non-spherical synthetic nanocarriers may require magnification techniques, such as electron microscopy, to obtain more accurate measurements."Dimension" or "size" or "diameter" of a synthetic nanocarrier refers to the mean value of the particle size distribution, e.g., as obtained using dynamic light scattering.

[0102] A "monomeric Ig protease fusion protein" or "monomeric Ig protease fusion protein" refers to the presence of one Ig protease domain in an Ig protease fusion protein as provided herein, which domain may be coupled or conjugated to a molecule (such as an Fc domain) that can extend the half-life of the Ig protease domain. The Ig protease domain may be coupled or conjugated to an Fc domain. The Fc domain may be composed of units that are coupled or conjugated together to form the Fc domain. In embodiments, the coupling may be by a covalent bond. Examples of monomeric Ig protease fusion proteins are provided herein.

[0103] "Non-naturally occurring" or "non-natural" refers to any aspect of the present disclosure, including, but not limited to, polynucleotides, peptides, protein domains, proteases, and / or Ig protease fusion proteins that have been modified, synthetic, and / or engineered and are not found in nature. In one embodiment of any one of the compositions or methods provided herein, the Ig protease fusion protein is non-naturally occurring.

[0104] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a pharmacologically inactive material that is used together with a pharmacologically active material to formulate a composition. Pharmaceutically acceptable excipients include various materials known in the art, including but not limited to sugars (e.g., glucose, lactose, etc.), preservatives such as antibacterial agents, reconstitution aids, coloring agents, saline (e.g., phosphate-buffered saline), and buffers. Any one of the compositions provided herein may include a pharmaceutically acceptable excipient or carrier.

[0105] "Providing" means an act or series of acts performed by an individual that supplies an item or series of items or methods needed for the practice of the invention. The act or series of acts may be performed directly or indirectly by the individual.

[0106] "Providing a subject" refers to any act or series of acts that allows a clinician to contact a subject and administer a composition provided herein to the subject or perform a method provided herein on the subject. Preferably, the subject is in need of a composition provided herein. The act or series of acts may be performed directly or indirectly by itself. In one embodiment of any one of the methods provided herein, the method further comprises providing a subject.

[0107] "Rapalog" refers to rapamycin and molecules structurally related to (analogs of) rapamycin (sirolimus). Examples of rapalogs include, but are not limited to, temsirolimus (CCI-779), deforolimus, everolimus (RAD001), ridaforolimus (AP-23573), and zotarolimus (ABT-578). Additional examples of rapalogs can be found, for example, in WO Publication WO 1998 / 002441 and U.S. Patent No. 8,455,510, the disclosures of which are incorporated herein by reference in their entireties. In any one of the methods, compositions, or kits provided herein, the immunosuppressant may be a rapalog, such as rapamycin.

[0108] "Reducing an immune response," as used herein, refers to lowering or eliminating an unwanted immune response to an Ig protease or other therapeutic agent that would be expected to occur, for example, following administration of the Ig protease or other therapeutic agent (e.g., without treatment with an immunosuppressant, which can be included in a synthetic nanocarrier). In some embodiments, a reduced immune response may be measured by measuring antibody titers. In some embodiments, a reduced immune response is a persistently reduced antibody titer for at least 1 week, 2 weeks, 1 month, 2 months, 2 months, 3 months, 4 months, or 5 months, etc. In some embodiments, a subject of any one of the methods provided herein is one in need of persistent antibody reduction or inhibition for at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, or 5 months.

[0109] " Subject " refers to animals, including warm-blooded mammals such as humans and primates; birds; domestic animals or livestock animals such as cats, dogs, sheep, goats, cows, horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish; reptiles; zoo animals and wild animals. In any one of the methods or compositions provided herein, the subject is human. In any one of the methods, compositions and kits provided herein, the subject is any one of the subjects provided herein, for example, has any of the conditions defined herein or requires any one of the treatments provided herein.

[0110] "Synthetic nanocarrier(s)" means a discrete object not found in nature and possessing at least one dimension less than or equal to 5 microns in size. Synthetic nanocarriers may be in a variety of different shapes, including, but not limited to, spherical, cubic, conical, rectangular, cylindrical, toroidal, etc. Synthetic nanocarriers comprise one or more surfaces.

[0111] Synthetic nanocarriers can be, but are not limited to, one or more lipid-based nanoparticles (also referred to herein as lipid nanoparticles, i.e., nanoparticles whose structural material is predominantly lipid), polymeric nanoparticles, metal nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles (i.e., particles composed primarily of viral structural proteins but which are not or have low infectivity), peptide- or protein-based particles (also referred to herein as protein particles, i.e., particles whose structural material is predominantly peptides or proteins) (e.g., albumin nanoparticles), and / or nanoparticles developed using a combination of nanomaterials, such as lipid-polymer nanoparticles. Synthetic nanocarriers can be in a variety of different shapes, including, but not limited to, spherical, cubic, conical, rectangular, cylindrical, toroidal, etc. Examples of synthetic nanocarriers include: (1) biodegradable nanoparticles disclosed in U.S. Patent 5,543,158 to Gref et al., (2) polymeric nanoparticles in published U.S. Patent Application 20060002852 to Saltzman et al., (3) lithographically constructed nanoparticles in published U.S. Patent Application 20090028910 to DeSimone et al., (4) WO 2009 / 051837 to von Andrian et al., (5) nanoparticles disclosed in published U.S. Patent Application 2008 / 0145441 to Penades et al., (6) nanoprecipitated nanoparticles disclosed in P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles," Nanomedicine. 5(6):843-853 (2010), and (7) Look et al., Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus erythematosus in mice” J.Clinical Investigation 123(4):1741-1749 (2013), (8) nucleic acid-attached virus-like particles disclosed in published U.S. patent application 20060251677 to Bachmann et al., (9) virus-like particles disclosed in WO2010047839A1 or WO2009106999A2, (10) nanoprecipitated nanoparticles disclosed in P. Paolicelli et al., “Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853 (2010), (11) apoptotic cells, apoptotic bodies, or synthetic or semi-synthetic mimetics disclosed in U.S. Publication No. 2002 / 0086049, or (12) Look et al., Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus erythematosus in mice” J. Clinical Investigation 123(4):1741-1749(2013).

[0112] Synthetic nanocarriers may have a minimum dimension equal to or smaller than about 100 nm, preferably equal to or smaller than 100 nm, and do not include a surface with complement-activating hydroxyl groups, or alternatively, include a surface consisting essentially of moieties that are not complement-activating hydroxyl groups. In one embodiment, synthetic nanocarriers having a minimum dimension equal to or smaller than about 100 nm, preferably equal to or smaller than 100 nm, do not include a surface that substantially activates complement, or include a surface consisting essentially of moieties that do not substantially activate complement. In a more preferred embodiment, synthetic nanocarriers according to the invention having a minimum dimension equal to or smaller than about 100 nm, preferably equal to or smaller than 100 nm, do not include a surface that activates complement, or alternatively, include a surface consisting essentially of moieties that do not activate complement. In an embodiment, synthetic nanocarriers exclude virus-like particles. In embodiments, synthetic nanocarriers may possess an aspect ratio greater than or equal to 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

[0113] "Target immunoglobulin" refers to one or more immunoglobulins cleaved by an Ig protease. In some embodiments, the target immunoglobulin may be all immunoglobulins in a particular isotype subclass (e.g., all IgG isotype subclasses, all IgA isotype subclasses, all IgA isotype subclasses, IgE, or IgD). In some embodiments, the target immunoglobulin may be a particular immunoglobulin isotype (e.g., IgA1, IgA2, IgD, IgE, IgG2, IgG2, IgG2a, IgG2b, IgG3, IgG4; or IgM).

[0114] A "therapeutic biologic" refers to any protein, carbohydrate, lipid, or nucleic acid that can be administered to a subject and have a therapeutic effect. In some embodiments of any one of the methods or compositions provided herein, the therapeutic biologic can be a therapeutic polynucleotide or a therapeutic protein.

[0115] "Therapeutic polynucleotide" refers to any polynucleotide or polynucleotide-based therapy that can be administered to a subject and have a therapeutic effect. Such therapy includes gene therapy, gene silencing, etc. Examples of such therapy are known in the art and include, but are not limited to, naked RNA (including messenger RNA, modified messenger RNA, and forms of RNAi). In one embodiment of any one of the compositions or methods provided herein, the therapeutic polynucleotide is a viral vector.

[0116] "Therapeutic protein" refers to any protein or protein-based therapy that can be administered to a subject and have a therapeutic effect. Such therapies include protein replacement and protein supplementation therapies. Such therapies also include the administration of exogenous or foreign proteins, antibody therapy, and the like. Therapeutic proteins include, but are not limited to, enzymes, enzyme cofactors, hormones, blood clotting factors, cytokines, growth factors, monoclonal antibodies, antibody-drug conjugates, and polyclonal antibodies.

[0117] "Expression of a transgene or nucleic acid material" refers to the level of expression product of a transgene or nucleic acid material from a viral vector in a subject when the transgene or nucleic acid material is delivered by the viral vector. In some embodiments, the level of expression may be determined by measuring transgene protein concentration in various tissues or systems of interest in the subject. Alternatively, if the expression product is a nucleic acid, the level of expression may be measured by the nucleic acid product. Increased expression can be determined, for example, by measuring the amount of expression product in a sample obtained from the subject and comparing it to a previous sample. Durability of expression may be measured by similar or other methods that will be apparent to one of skill in the art. The sample may be a tissue sample. In some embodiments, expression products can be measured using flow cytometry.

[0118] "Transplant" refers to biological materials such as cells, tissues, and organs (in whole or in part) that can be administered to a subject. Transplants can be autografts, allografts, or xenografts of biological materials such as organs, tissues, skin, bone, nerves, tendons, neurons, blood vessels, fat, cornea, pluripotent cells, and differentiated cells (obtained or induced in vivo or in vitro). In some embodiments, the graft is formed from, for example, cartilage, bone, extracellular matrix, or collagen matrix. Transplants can also be single cells, cell suspensions, and cells in tissues and organs that can be transplanted. Transplantable cells typically have a therapeutic function, e.g., a function that is missing or diminished in the recipient subject. Non-limiting examples of transplantable cells are beta cells, hepatocytes, hematopoietic stem cells, neural stem cells, neurons, glial cells, or myelinating cells. Transplantable cells can be unmodified cells, e.g., cells obtained from a donor subject and usable for transplantation without any genetic or epigenetic modifications. In other embodiments, the transplantable cells can be modified cells, e.g., cells obtained from a subject with a genetic defect, where the genetic defect has been corrected, or cells derived from reprogrammed cells, e.g., differentiated cells derived from cells obtained from the subject.

[0119] "Transplantation" refers to the process of transferring (transferring) a graft to a recipient subject (e.g., from a donor subject, from an in vitro source (e.g., differentiated autologous or heterologous native or induced pluripotent cells)), and / or from one bodily location to another in the same subject.

[0120] "Treating" refers to administering one or more therapeutic agents with the expectation that the subject may benefit from such administration. In some embodiments, the subject is one who is suspected of having an undesired presence of Ig, such as IgG or IgA, and / or in whom cleavage of Ig, such as IgG or IgA, is desired. In some embodiments, a subject who is suspected of having a disease, disorder, or condition is one who a clinician believes may have the disease, disorder, or condition. Treating can be direct or indirect, for example, by inducing or instructing another subject, including another clinician or the subject themselves, to treat the subject.

[0121] An "undesirable immune response" refers to any undesirable immune response that results from exposure to an antigen and promotes or worsens a disease, disorder, or condition (or a symptom thereof) provided herein, or is a symptom of a disease, disorder, or condition provided herein. Such an immune response may generally have a negative impact on the subject's health or may be a symptom of a negative impact on the subject's health. The undesirable immune response may be an undesirable humoral immune response, which may include antigen-specific antibody production, antigen-specific B cell proliferation and / or activity, or antigen-specific CD4+ T cell proliferation and / or activity. Generally, as used herein, these undesirable immune responses are specific to therapeutic biologics, such as Ig proteases or viral vectors, and counteract the desired beneficial effects of administration with the agent, respectively.

[0122] "Viral vector" refers to a vector construct having viral components, such as capsid and / or coat proteins, adapted to contain and deliver a transgene or nucleic acid material (e.g., encoding a therapeutic agent such as a therapeutic protein), which can be expressed as provided herein. Viral vectors can be based on, without limitation, retroviruses (e.g., murine retroviruses, avian retroviruses, Moloney murine leukemia virus (MomULV), Harvey murine sarcoma virus (HamUSV), mouse mammary tumor virus (MumTV), gibbon ape leukemia virus (GalV), and Rous sarcoma virus (RSV)), lentiviruses, herpesviruses, adenoviruses, adeno-associated viruses, alphaviruses, and the like. Other examples are described elsewhere herein or known in the art. Viral vectors can be based on naturally occurring variants, strains, or serotypes of viruses, such as any one of those provided herein. Viral vectors can also be based on viruses selected through molecular evolution. Viral vectors can be engineered vectors, recombinant vectors, mutant vectors, or hybrid vectors.In some embodiments, viral vectors are "chimeric viral vectors".In such embodiments, this means that viral vectors are composed of viral components derived from more than one virus or viral vector.The AAV vectors provided herein are based on AAV, such as AAV8, and have viral components such as capsid and / or coat protein, which can package transgenes or nucleic acid materials for delivery.

[0123] "Viral vector antigen" refers to an antigen associated with a viral vector (i.e., the viral vector, or a fragment thereof that can generate an immune response to the viral vector (e.g., production of anti-viral vector-specific antibodies)). A viral vector antigen may be presented for recognition by the immune system (e.g., presented by a cell of the immune system, such as an antigen-presenting cell (including, but not limited to, a dendritic cell, B cell, or macrophage)). A viral vector antigen may be presented for recognition by, for example, a T cell. Such an antigen may be recognized by a T cell through presentation of an epitope of the antigen bound to a class I or class II major histocompatibility complex molecule (MHC), initiating an immune response in the T cell. Viral vector antigens generally include proteins, polypeptides, peptides, polynucleotides, etc., or are contained or expressed in, on, or by a cell. In some embodiments, a viral vector antigen comprises an MHC class I-restricted epitope and / or an MHC class II-restricted epitope and / or a B-cell epitope. In some embodiments, one or more tolerogenic immune responses specific to the viral vector are generated as a result of using the methods, compositions, or kits provided herein.

[0124] "% by weight" or "% by weight" is the ratio of one weight to another weight multiplied by 100. For example, % by weight can be the ratio of the weight of one component to the weight of another component multiplied by 100, or the ratio of the weight of one component to the total weight of more than one component multiplied by 100. Generally, with respect to synthetic nanocarriers, % by weight is measured average over the entire population of synthetic nanocarriers or average over all synthetic nanocarriers in a composition or suspension.

[0125] C. Compositions and Related Methods Provided herein are compositions and related methods of Ig protease fusion proteins useful for treating diseases or disorders, for example, by neutralizing pathogenic immunoglobulins associated with the disease or disorder. The Ig protease fusion protein of any one of the methods and compositions provided herein can have enhanced activity, such as an extended half-life and / or optimized protease activity. As described herein, any one of the compositions and methods provided herein may reduce the level of an important biomarker for a disease or disorder, or a marker for monitoring treatment with a therapeutic agent. The Ig protease fusion protein of any one of the methods and compositions provided herein can be used to reduce anti-drug antibodies or antibodies that neutralize viral vectors (e.g., AAV vectors). Any one of the compositions and methods provided herein may reduce the level of an important biomarker for an immune response. The Ig protease fusion protein of any one of the methods and compositions provided herein may be administered in combination with a synthetic nanocarrier comprising an immunosuppressant. The Ig protease fusion protein of any one of the methods and compositions provided herein may be re-administered in combination with a synthetic nanocarrier comprising, for example, an immunosuppressant.

[0126] Immunoglobulin (Ig) proteases and their Ig protease domains A wide variety of Ig proteases can be used in accordance with the present invention, any protease domain of which can be comprised in an Ig protease fusion protein, including mutants and truncated forms, as provided herein. In some embodiments of the present disclosure, the protease domain can be selected from a naturally occurring or endogenous Ig protease or variant thereof. In some embodiments of the present disclosure, the Ig protease can be from an Ig protease from a bacterial strain. In some embodiments, the bacterial strain is a Streptococcus bacterial strain. In some embodiments, the bacterial strain is a Mycoplasma bacterial strain. In some embodiments, the bacterial strain is a Neisseria bacterial strain. In some embodiments, the bacterial strain is a Clostridium bacterial strain. In some embodiments, the bacterial strain is a Capnocytophaga bacterial strain. In some embodiments, the bacterial strain is a Bacteroides bacterial strain. In some embodiments, the bacterial strain is a Gemella bacterial strain. In some embodiments, the bacterial strain is a Prevotella bacterial strain.

[0127] In some embodiments, the Ig protease may have specificity for one or more target immunoglobulins, such as IgG or IgA immunoglobulins. In some embodiments, the target IgA may be all IgA isotype subclasses. In some embodiments, the target IgG may be all IgG isotype subclasses. In some embodiments, the target IgG may be a specific subset of IgG isotype subclasses (e.g., IgG1, IgG2, IgG2A, IgG2B, IgG3, or IgG4). In some embodiments, the target IgG is specific for multiple (i.e., more than one) IgG subclasses or all IgG subclasses. In some embodiments, the target IgG is specific for all IgG subclasses containing lambda light chains or all IgG subclasses containing kappa light chains. In some embodiments, the target IgG may be all IgG isotype subclasses.

[0128] In some embodiments, the Ig protease may be of human origin, e.g., to minimize immunogenicity. In some embodiments, the Ig protease domain can include any portion of a human protease capable of cleaving the hinge region of human Ig, including, for example, cathepsin G and numerous matrix metalloproteinases (Ryan MH, et al. Proteolysis of purified IgGs by human and bacterial enzymes in vitro and the detection of specific proteolytic fragments of endogenous IgG in rheumatoid synovial fluid. Mol Immunol. 2008 Apr;45(7):1837-46. doi: 10.1016 / j.molimm.2007.10.043. Epub 2007 Dec 21. PMID: 18157932). In some embodiments, structure-based protein design can aid in the generation of protease domains that can be evaluated and optimized for specificity and / or activity. In some embodiments of any one of the compositions or methods provided herein, the Ig protease domain can be replaced with a homologous or structurally related domain with similar or novel specificity, such as a sequence based on a human protein to reduce immunogenicity.

[0129] In some embodiments, the Ig protease is an IdeS protease. In some embodiments, the Ig protease is an IdeZ protease. In some embodiments, the Ig protease is an IdeMC protease. In one embodiment, the Ig protease is any one of the IgG proteases in U.S. Publication No. 2019-0262434 A1, which IgG proteases are incorporated herein by reference. In some embodiments, the Ig protease is an IdeSORK protease. Exemplary sequences of such proteases are provided below.

[0130] In one embodiment of any one of the compositions or methods provided herein, the Ig protease of the invention comprises a sequence at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical (including all values in between) to any one of the sequences provided herein. Such non-identical or mutated versions include truncated or other mutant versions of any one of the Ig proteases provided herein. In one embodiment of any one of the compositions or methods provided herein, the Ig protease domain is of any one of the truncated or mutant versions of the Ig proteases provided herein. Preferably, any one of the Ig proteases provided herein also exhibits an activity such that the cleavage activity against Ig is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more of the activity of the Ig proteases provided herein.

[0131] In some embodiments, the Ig protease contains one or more amino acid substitutions relative to wild-type IdeSORK (SEQ ID NO: 1) and / or is a truncated version thereof. In one embodiment, the Ig protease comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1; the amino acid sequence of SEQ ID NO: 2; an amino acid sequence that is an N-terminal fragment of the sequence of SEQ ID NO: 1; or an amino acid sequence at least 50% identical to the amino acid sequence of any one of the foregoing. In one embodiment, the Ig protease comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0132] In one embodiment, the Ig protease comprises a truncated form (N-terminally truncated) of a polypeptide having the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 19. For example, the sequence of SEQ ID NO: 19 is as follows: TLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQ PSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEET but does not have the N-terminal 25 amino acids and the His tag of SEQ ID NO:3.

[0133] Thus, an Ig protease may comprise the sequence of SEQ ID NO: 18 or SEQ ID NO: 20, but without at least one of amino acids 1-25 at its N-terminus. In one embodiment, an Ig protease comprises the sequence of SEQ ID NO: 18 or SEQ ID NO: 20, but without amino acids 1 and 2 at its N-terminus. In another embodiment, an Ig protease comprises the sequence of SEQ ID NO: 18 or SEQ ID NO: 20, but without amino acids 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-23, 1-24, or 1-25 at its N-terminus. In one embodiment, an Ig protease comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 19.

[0134] SEQ ID NO: 18 SKRKLLKKIEKKDTSSVLTQKKQTKTLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGN KFLQPERLLNMYINGYGYLTSQDKAKTSQPSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTI KLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEETEKERIRLEEEADKAKLEQDRIQKEAEEKLALEKAEKERIRLEEEADKAKLEQDRIQKEAEEKLALEKAEKERIRLEEEADKAKLEQDRIQKEAEEKLALEKAEKE RIRLEEEADKAKLEQDRIQKEAEEKLALEKAEKERIRLEEEAAKLEQEKQIATAPQPDKKQENTTSEQEKPAPTELPPLVNKADETETPRETAPDQTPSATNTFRKILPKMNAVSQFFSQLMGTIQIVFAFILKIFK

[0135] SEQ ID NO: 19 TLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQ PSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEET

[0136] SEQ ID NO: 20 SKRKLLKKIEKKDTSSVLTQKKQTKTLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGYG YLTSQDKAKTSQPSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEET

[0137] The Ig protease or any one of its domains can further comprise an N-terminal methionine and / or a His tag or other tag at the C-terminus. In one embodiment, the His tag or other tag can be coupled to the remainder of the polypeptide by a linker. In one embodiment, the Ig protease or domain thereof is engineered to include an additional N-terminal methionine and / or a protein purification or other tag at the C-terminus, which can be joined to the C-terminus by a linker.

[0138] In one embodiment, the Ig protease has protease activity against any immunoglobulin molecule as set forth in any one of SEQ ID NOs: 4-8, wherein the Ig protease cleaves the CH2 / hinge sequence between positions corresponding to positions 249 and 250 of human IgG according to the Kabat numbering system (positions 236 and 237 according to the EU numbering system).

[0139] Fc domain and its modified versions In some embodiments of any one of the compositions or methods provided, any Ig protease domain provided herein can be combined with any one of the Ig Fc domains or albumin proteins provided herein, and can be included as part of any one of the Ig protease fusion proteins provided herein. In one embodiment, the Ig protease domain is engineered to be fused to an Fc molecule as provided herein. In some embodiments of any one of the methods or compositions provided, the Ig protease fusion proteins of the invention have an extended circulating half-life.

[0140] Any Fc domain can be used in any one of the compositions or methods provided, and can be combined with any one of the Ig protease domains provided herein, or can be included as part of any of the Ig protease fusion proteins provided herein. In some embodiments of the present disclosure, the Ig Fc domain can be selected from a full-length Ig Fc or a fragment thereof. In some embodiments of the present disclosure, the Fc is from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc is from a human Ig. In one embodiment, the Fc further comprises a hinge region and / or a CH2 domain.

[0141] An exemplary sequence for human IgG1 is as follows, with the constant domains underlined:

number

[0142] An exemplary sequence of a human IgG1 constant domain is as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22)

[0143] An exemplary sequence of a human IgG2 constant domain is as follows: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23)

[0144] An exemplary sequence of a human IgG3 constant domain is as follows: ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 24)

[0145] An exemplary sequence of a human IgG4 constant domain is as follows: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 25)

[0146] In one embodiment of any one of the compositions or methods provided herein, Fc molecule or domain is mutated compared to wild type.In one embodiment of any one of the compositions or methods provided herein, Fc molecule or domain (including its part) comprises one or more or all or any combination of the following mutations: GG-SS mutation in hinge region (when Fc comprises hinge region); C220S (for example, when Fc comprises hinge region); H435R; G236S; G237S; N297G (for example, all three in one molecule), replaced by L234A, L235A and / or P329A mutation; M428L and / or N434S mutation (for example, both in one molecule); And terminal lysine deletion (for example, at the end of Fc molecule or domain or antibody, or its part (including Fc molecule or domain)).In some embodiments, Fc can have one or more modifications of hinge region, with or without any one or more of the above mutations. In one embodiment, the hinge region is shorter (e.g., 3x repeats) and more stable. In one embodiment, the Fc molecule or any one of the domains is aglycosylated.

[0147] In one embodiment of any one of the compositions or methods provided herein, the Fc molecule or domain (including portions thereof) comprises one or more, or all, or any combination of the mutations in Table 1 below. In one embodiment of any one of the compositions or methods provided herein, the Fc molecule or domain (including portions thereof) comprises one or more, or all, or any combination of the sequences in Table 1 below.

[0148] In one embodiment of any one of the compositions or methods provided herein, the Fc molecule or domain comprises at least two (any combination of two) of the mutations provided herein. In one embodiment of any one of the compositions or methods provided herein, the Fc molecule or domain comprises at least three (any combination of three) of the mutations provided herein. In one embodiment of any one of the compositions or methods provided herein, the Fc molecule or domain comprises at least four (any combination of four) of the mutations provided herein. In one embodiment of any one of the compositions or methods provided herein, the Fc molecule or domain comprises at least five (any combination of five) of the mutations provided herein. In one embodiment of any one of the compositions or methods provided herein, the Fc molecule or domain comprises any mutation of a particular Fc molecule or domain of a particular example thereof or example of an Ig protease fusion protein provided herein.

[0149] Table 1 [Table 1]

[0150] The Fc molecule or domain, or portion thereof, can have any combination of the aforementioned mutations, such as the combinations represented by the exemplary molecules provided herein. Thus, in one aspect, there is any one of the mutant Fc molecules or domains provided herein, or compositions thereof. In one embodiment of any one of the compositions or methods provided herein, the Fc molecule or domain is any one of the Fc molecules provided in the examples. Any one of the foregoing may be a portion of an antibody, such as a full-length antibody, or a portion thereof, such as an antigen-binding portion.

[0151] Any one of the Fc molecules provided herein can have reduced aggregation, increased stability, increased expression, extended half-life, shortened half-life, reduced Fc to FcRn binding (e.g., IgG1 Fc to FcRn binding), and / or removed Ig protease cleavage sites. Any one of the Fc molecules provided herein can have any one or more, or all, or a combination of the activities of the same provided herein.

[0152] In one embodiment of any of the compositions or methods provided herein, the Fc domain of the invention comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, or at least 99% (including all values in between) identical to any one of the Fc sequences provided herein.

[0153] Ig protease fusion protein In another aspect, there are provided herein certain Ig protease fusion proteins, including those having any one of the Ig protease domains provided herein and / or any one of the Fc domains provided herein. In another aspect, there are provided herein certain Ig protease fusion proteins comprising the sequences provided herein, such as:

[0154] Protease-IgGFc (CS, GG-SS) MTLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQPSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEETEPKSSDKTHTCPPCPAPELLSSPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 39)

[0155] Protease-IgGFc (C-S, GG-SS, H435R) MTLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQPSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEETEPKSSDKTHTCPPCPAPELLSSPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPG(SEQ ID NO:40)

[0156] Protease-IgGFc (C-S, GG-SS) MSKRKLLKKIEKKDTSSVLTQKKQTKTLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQPSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEETEPKSSDKTHTCPPCPAPELLSSPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 41)

[0157] Protease-IgGFc (C-S, GG-SS, H435R) (SEQ ID NO: 42)

[0158] Protease-hIgG3Fc (short hinge, CS, GGSS) MTLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGY GYLTSQDKAKTSQPSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKN SGGSLRHLYSLSTGEQIWKKYFEETEPKSSDTPPPCPRCPAPELLSSPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 43)

[0159] Protease-hIgG3Fc (short hinge, CS, GGSS) MSKRKLLKKIEKKDTSSVLTQKKQTKTLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQPSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEETEPKSSDTPPPCPRCPAPELLSSPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG(SEQ ID NO:44)

[0160] Protease (Xork1.3)-IgGFc (C-S, GG-SS) MTLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQPSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEETEPKSSDKTHTCPPCPAPELLSSPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 45)

[0161] Protease (Xork1.3)-IgGFc (C-S, GG-SS, H435R) MTLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQPSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEETEPKSSDKTHTCPPCPAPELLSSPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPG (SEQ ID NO: 46)

[0162] Protease (Xork1.1)-IgGFc (C-S, GG-SS) MSKRKLLKKIEKKDTSSVLTQKKQTKTLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQPSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEETEPKSSDKTHTCPPCPAPELLSSPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:47)

[0163] Protease (Xork1.1)-IgGFc (C-S, GG-SS, H435R) (SEQ ID NO: 48)

[0164] Protease (Xork1.3)-hIgG3Fc (short hinge, CS, GGSS) MTLWADGVQVDDKDFKPSTENFGTNYLAAEYGIGKGYYDINKKFDGTDDLCSGVVAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNTDIFELNKLHDEDQSNFFDFIKKSFGNKFLQPERLLNMYINGY GYLTSQDKAKTSQPSPSKLNFFQKVFKNNLLTDKTPINDIDEFSSQTKNALQNHKVLAVSFASIKNRGLGHVVTVWGADFDENGKVVALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDSSTIKLTGFEDKN SGGSLRHLYSLSTGEQIWKKYFEETEPKSSDTPPPCPRCPAPELLSSPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 49)

[0165] Protease (Xork1.1)-hIgG3Fc (short hinge, CS, GGSS) (SEQ ID NO: 50)

[0166] In one embodiment of any one of the compositions or methods provided herein, the Ig protease fusion protein of the invention comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical (including all values in between) to any one of the Ig protease fusion protein sequences provided herein.

[0167] In one embodiment of any one of the Ig protease fusion proteins provided herein, the fusion protein is in the form of a monomer.In another embodiment, the fusion protein may be complexed with another fusion protein to form a dimer.In one embodiment, the fusion protein dimer is a homodimer.In the case of a homodimer, the disulfide bond in the hinge region of Fc may dimerize the fusion protein.In one embodiment, the dimerization may be the result of a covalent bond.

[0168] The units that form the Fc domain are coupled or conjugated via the knobs-in-holes (KIH) mechanism. "Knobs-in-holes" or "KIH" refers to an antibody engineering strategy used for dimerization in antibody production. In Ig, e.g., IgG, amino acids that form the interface of a domain can be mutated at positions that affect domain interactions to promote dimer formation. Knobs are represented by amino acids with large flank changes (e.g., tyrosine), and holes are represented by amino acids with small side chains (e.g., threonine). An amino acid with a large side chain (knob) can be introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a small side chain (hole) can be introduced into the heavy chain of an antibody that specifically binds to a second antigen. Coexpression of the two with knob and hole mutations can result in dimer formation due to the interaction of the heavy chain hole and heavy chain knob. Either one of the Ig protease fusion proteins may be in a KIH format.

[0169] Any of the foregoing may be expressed in mammalian or non-mammalian cells and thus may be a mammalian- or non-mammalian-expressed molecule, respectively. In one embodiment, when the Fc fusion is a molecule expressed in a non-mammal (such as one expressed from E. coli), in one embodiment, N297 is not mutated, or at least is not mutated to G or A. In one embodiment, when the Fc fusion is a molecule expressed in a mammal, in one embodiment, N297 may also be mutated (e.g., to G).

[0170] Also provided is a method for producing an Fc molecule or a fusion thereof in mammalian cells, such as CHO cells. As an example, it has been found that an Ig protease (Xork)-Fc fusion can be expressed in such cells and successfully purified. Thus, any one of the Fc molecules or fusions thereof provided herein can be an Fc molecule or a fusion thereof expressed in a mammal. Also provided is a method for producing an Fc molecule or a fusion thereof in non-mammalian cells, such as E. coli cells. Methods for such production are known in the art (see, e.g., U.S. Patent No. 6,835,809; the method is incorporated herein by reference). As an example, it has been found that an Ig protease (Xork)-Fc fusion can be expressed in such cells and successfully purified. Thus, any one of the Fc molecules or fusions thereof provided herein can be an Fc molecule or a fusion thereof expressed in a non-mammalian.

[0171] Albumin protein Any albumin protein can be used in some embodiments of any one of the provided compositions or methods, and can be combined with any one of the Ig protease domains provided herein, or can be included as part of any one of the Ig protease fusion proteins provided herein.In some embodiments, albumin protein can be mutated, truncated, or engineered to improve binding to FcRn.In some embodiments, albumin protein is human albumin protein.

[0172] Transgenes and viral vectors The transgene or nucleic acid material provided herein, for example, the transgene or nucleic acid material of a viral vector, may encode any protein or portion thereof, or nucleic acid or portion thereof, that is beneficial to a subject, for example, a subject with a disease or disorder. In embodiments, the subject has or is suspected of having a disease or disorder in which the subject's endogenous version of the protein is deficient or is produced in limited amounts or not at all. The subject may have any one of the diseases or disorders provided herein, and the transgene or nucleic acid material encodes any one of the therapeutic proteins or portions thereof provided herein. The transgene or nucleic acid material provided herein may encode a functional version of any protein that causes a disease or disorder in the subject through some defect in its endogenous version in the subject (including a defect in the expression of the endogenous version).

[0173] The sequence of the transgene or nucleic acid material may also include expression control sequences. Expression control sequences include promoters, enhancers, and operators and are generally selected based on the expression system in which the expression construct is to be utilized. In some embodiments, promoter and enhancer sequences are selected for their ability to increase gene expression, while operator sequences may be selected for their ability to regulate gene expression. The transgene may also include sequences that facilitate, and preferably promote, homologous recombination in the host cell. The transgene may also include sequences necessary for replication in the host cell.

[0174] Exemplary expression control sequences include liver-specific promoter sequences and constitutive promoter sequences, such as any one of the promoters provided herein. Generally, the promoter is operatively linked upstream (i.e., 5') of the sequence encoding the desired expression product. The transgene may also include a suitable polyadenylation sequence operatively linked downstream (i.e., 3') of the coding sequence.

[0175] Viruses have evolved specialized mechanisms for transporting their genomes inside the cells they infect; viral vectors based on these viruses can transduce cells for specific applications.The examples of viral vectors that can be used as provided herein are known in the art or are described herein.Suitable viral vectors include, for example, the vectors based on adeno-associated virus (AAV).

[0176] The viral vector provided herein can be based on adeno-associated virus (AAV).AAV vectors are particularly noteworthy for use in therapeutic applications, such as those described herein.AAV is a DNA virus that is not known to cause human diseases.Generally, AAV requires co-infection with helper viruses (such as adenoviruses or herpesviruses) or the expression of helper genes for efficient replication.For the description of AAV-based vectors, see, for example, U.S. Patent Nos. 8,679,837, 8,637,255, 8,409,842, 7,803,622, and 7,790,449, and U.S. Publication Nos. 20150065562, 20140155469, 20140037585, 20130096182, 20120100606, and 20070036757. The AAV vector may be a recombinant AAV vector. The AAV vector may be a self-complementary (sc) AAV vector, for example, as described in U.S. Patent Publication Nos. 2007 / 01110724 and 2004 / 0029106, and U.S. Patent Nos. 7,465,583 and 7,186,699.

[0177] The adeno-associated virus on which the viral vector is based may be of a particular serotype, such as AAV8 or AAV2. In some embodiments of any one of the methods or compositions provided herein, the AAV vector is therefore an AAV8 vector or an AAV2 vector. Viral vectors can be produced using methods known to those skilled in the art or as otherwise described herein. For example, viral vectors can be constructed and / or purified using the methods described in, for example, U.S. Patent No. 4,797,368 and Laughlin et al., Gene, 23, 65-73 (1983).

[0178] Viral vectors, such as AAV vectors, may be produced using recombinant methods. For example, the methods may involve culturing host cells that contain a nucleic acid sequence encoding an AAV capsid protein or a fragment thereof, a functional rep gene, a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene, and sufficient helper functions to allow packaging of the recombinant AAV vector into the AAV capsid protein.

[0179] The components to be cultured in a host cell to package a viral vector into a capsid may be provided in trans to the host cell. Alternatively, any one or more of the required components (e.g., recombinant viral vector, rep sequence, cap sequence, and / or helper functions) may be provided by a stable host cell engineered to contain one or more of the required components using methods known to those of skill in the art. Most preferably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may also be under the control of a constitutive promoter. The recombinant viral vector, rep sequence, cap sequence, and helper functions to produce the viral vector may be delivered to the packaging host cell using any suitable genetic elements. The selected genetic elements may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of the present invention are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are well known, and the selection of a suitable method is not a limiting factor for the present invention. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993), and U.S. Patent No. 5,478,745.

[0180] In some embodiments, recombinant AAV vectors may be produced using a triple transfection method (e.g., as described in detail in U.S. Pat. No. 6,001,650, the contents of which regarding the triple transfection method are incorporated herein by reference). Typically, recombinant AAV is produced by transfecting a host cell with a recombinant AAV vector (e.g., containing a transgene) to be packaged into an AAV particle, an AAV helper function vector, and an accessory function vector. Generally, the AAV helper function vector encodes AAV helper function sequences (rep and cap) that function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). The accessory function vector may encode nucleotide sequences for non-AAV-derived viral and / or cellular functions on which AAV depends for replication. Accessory functions include those functions required for AAV replication, including, but not limited to, moieties involved in AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and activation of AAV capsid assembly. Viral-based accessory functions can be derived from any known helper virus, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus. Other methods for producing viral vectors are known in the art. Moreover, viral vectors are commercially available.

[0181] Use of Ig protease fusion proteins The present disclosure contemplates that the Ig protease fusion protein may be administered to a subject. In some embodiments of the present disclosure, a method of administering an Ig protease fusion protein to a subject is provided, wherein the Ig protease fusion protein cleaves immunoglobulins, such as IgG or IgA, in the subject. In some embodiments, a method of administering an Ig protease fusion protein to a subject is provided.

[0182] It is contemplated that any of the Ig protease fusion proteins contemplated by the present disclosure can be used for therapeutic treatment of autoimmune diseases, allergies, or other immunological disorders, etc. Any of the Ig protease fusion proteins can be used in conjunction with biological therapies, such as therapeutic protein therapies or therapeutic polynucleotide therapies, such as viral vector therapies.

[0183] Administration according to the present invention can be by various routes, including but not limited to subcutaneous, intravenous and intraperitoneal routes.For example, the administration mode for any one of the compositions of the treatment methods provided can be by intravenous administration.Compositions referred to herein can be prepared and administered by conventional methods.

[0184] The compositions of the invention can be administered in an effective amount, such as those described herein. In some embodiments of any one of the methods or compositions provided, repeated cycles of administration of the Ig protease fusion protein are contemplated.

[0185] Aspects of the present invention relate to determining a protocol for the methods of administration as provided herein. The protocol can be determined by varying the frequency and dosage of at least the Ig protease fusion protein and / or synthetic nanocarrier and / or other treatment, and then evaluating a desired or undesired therapeutic or immune response. The protocol can include the frequency and dosage of administration of at least the Ig protease fusion protein and / or synthetic nanocarrier and / or other treatment. Any one of the methods provided herein can include a step of determining a protocol, or the administering step is carried out according to a protocol determined to achieve one or more desired results as described herein.

[0186] Synthetic Nanocarriers A wide variety of synthetic nanocarriers can be used in accordance with the present invention. In some embodiments, synthetic nanocarriers are spheres or spherical. In some embodiments, synthetic nanocarriers are flat or tabular. In some embodiments, synthetic nanocarriers are cubic or cuboid. In some embodiments, synthetic nanocarriers are oval or ellipsoid. In some embodiments, synthetic nanocarriers are cylindrical, conical, or pyramidal.

[0187] In some embodiments, it is desirable to use a population of synthetic nanocarriers that are relatively homogeneous with respect to size or shape, so that each synthetic nanocarrier has similar properties. In other embodiments, the synthetic nanocarriers may comprise metal particles, quantum dots, ceramic particles, etc. For example, based on the total number of synthetic nanocarriers, at least 80%, at least 90%, or at least 95% of the synthetic nanocarriers may have a minimum or maximum dimension that falls within 5%, 10%, or 20% of the average diameter or average dimension of the synthetic nanocarriers.

[0188] Synthetic nanocarriers can be solid or hollow and can comprise one or more layers. In some embodiments, each layer has a unique composition and unique properties relative to the other layer(s). By way of example only, synthetic nanocarriers may have a core / shell structure, where the core is one layer (e.g., a polymeric core) and the shell is a second layer (e.g., a lipid bilayer or monolayer). Synthetic nanocarriers may comprise multiple distinct layers.

[0189] In some embodiments, synthetic nanocarriers may optionally comprise one or more lipids. In some embodiments, synthetic nanocarriers may comprise liposomes. In some embodiments, synthetic nanocarriers may comprise lipid bilayers. In some embodiments, synthetic nanocarriers may comprise lipid monolayers. In some embodiments, synthetic nanocarriers may comprise micelles. In some embodiments, synthetic nanocarriers may comprise a core comprising a polymer matrix surrounded by a lipid layer (e.g., a lipid bilayer, a lipid monolayer, etc.). In some embodiments, synthetic nanocarriers may comprise a non-polymeric core (e.g., a metal particle, a quantum dot, a ceramic particle, a bone particle, a virus particle, a protein, a nucleic acid, a carbohydrate, etc.) surrounded by a lipid layer (e.g., a lipid bilayer, a lipid monolayer, etc.).

[0190] In some embodiments, synthetic nanocarriers may comprise metal particles, quantum dots, ceramic particles, etc. In some embodiments, non-polymeric synthetic nanocarriers are aggregates of non-polymeric components, such as aggregates of metal atoms (e.g., gold atoms).

[0191] In some embodiments, synthetic nanocarriers may optionally comprise one or more amphiphilic entities. In some embodiments, amphiphilic entities can facilitate the production of synthetic nanocarriers with increased stability, improved uniformity, or increased viscosity. In some embodiments, amphiphilic entities can associate with the interior surface of a lipid membrane (e.g., a lipid bilayer, a lipid monolayer, etc.). Many amphiphilic entities known in the art are suitable for use in making synthetic nanocarriers in accordance with the present invention.Such amphiphilic entities include, but are not limited to, phosphoglycerides; phosphatidylcholine; dipalmitoylphosphatidylcholine (DPPC); dioleylphosphatidylethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol esters; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; surfactant fatty acids such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides; sorbitan trioleate (Span® 85) glycocholate; sorbitan monolaurate (Span® 20); polysorbate 20 (Tween® 20); polysorbate 60 (Tween® 60); polysorbate 65 (Tween® 65). (Trademark) 65); Polysorbate 80 (Tween® 80); Polysorbate 85 (Tween® 85); Polyoxyethylene monostearate; Surfactin; Poloxamer; Sorbitan fatty acid esters such as sorbitan trioleate; Lecithin; Lysolecithin; Phosphatidylserine; Phosphatidylinositol; Sphingomyelin; Phosphatidylethanolamine (cephalin); Cardiolipin; Phosphatidic acid; Cerebrosides; Dicetyl phosphate; Dipalmitoyl phosphate Examples of amphiphilic entities include phatidylglycerol, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, tyloxapol, poly(ethylene glycol) 5000-phosphatidylethanolamine, poly(ethylene glycol) 400-monostearate, phospholipids, synthetic and / or natural detergents with high surfactant properties, deoxycholate, cyclodextrins, chaotropic salts, ion pairing agents, and combinations thereof. The amphiphilic entity component may be a mixture of various amphiphilic entities.Those skilled in the art will recognize that this is an exemplary, but not exhaustive, list of substances with surface activity. Any amphiphilic entity can be used in the production of synthetic nanocarriers to be used in accordance with the present invention.

[0192] In some embodiments, synthetic nanocarriers may optionally contain one or more carbohydrates. The carbohydrates may be natural or synthetic. The carbohydrates may be derivatized natural carbohydrates. In some embodiments, the carbohydrates comprise monosaccharides or disaccharides, including, but not limited to, glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellobiose, mannose, xylose, arabinose, glucuronic acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid. In some embodiments, the carbohydrate is a polysaccharide, including, but not limited to, pullulan, cellulose, microcrystalline cellulose, hydroxypropylmethylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, hydroxyethyl starch, carrageenan, glycon, amylose, chitosan, N,O-carboxymethylchitosan, alginic acid and alginic acid, starch, chitin, inulin, konjac, glucomannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan. In some embodiments, the synthetic nanocarrier does not include (or specifically excludes) carbohydrates such as polysaccharides. In some embodiments, the carbohydrate may include a carbohydrate derivative, such as a sugar alcohol, including, but not limited to, mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.

[0193] In some embodiments, synthetic nanocarriers may comprise one or more polymers. In some embodiments, synthetic nanocarriers comprise one or more polymers that are non-methoxy-terminated Pluronic® polymers. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight / weight) of the polymers comprising the synthetic nanocarrier are non-methoxy-terminated Pluronic® polymers. In some embodiments, all of the polymers comprising the synthetic nanocarrier are non-methoxy-terminated Pluronic® polymers. In some embodiments, synthetic nanocarriers comprise one or more polymers that are non-methoxy-terminated polymers. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight / weight) of the polymers comprising the synthetic nanocarrier are non-methoxy-terminated polymers. In some embodiments, all of the polymers comprising the synthetic nanocarrier are non-methoxy-terminated polymers. In some embodiments, the synthetic nanocarrier comprises one or more polymers that do not include a Pluronic® polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight / weight) of the polymers comprising the synthetic nanocarrier do not comprise Pluronic® polymers. In some embodiments, none of the polymers comprising the synthetic nanocarrier comprise Pluronic® polymers. In some embodiments, such polymers can be surrounded by a coating layer (e.g., liposomes, lipid monolayers, micelles, etc.). In some embodiments, various elements of the synthetic nanocarrier can be attached to polymers.

[0194] Immunosuppressants can be attached to synthetic nanocarriers in any of a number of ways. Generally, attachment can be the result of binding between the immunosuppressant and the synthetic nanocarrier. This binding can result in the immunosuppressant being attached to the surface of the synthetic nanocarrier and / or being contained (encapsulated) within the synthetic nanocarrier. However, in some embodiments, the immunosuppressant is encapsulated by the synthetic nanocarrier as a result of the structure of the synthetic nanocarrier, rather than being bound to the synthetic nanocarrier. In preferred embodiments, the synthetic nanocarrier comprises a polymer as provided herein, and the immunosuppressant is attached to the polymer.

[0195] When attachment occurs as a result of binding between the immunosuppressant and the synthetic nanocarrier, attachment may occur via a coupling moiety. The coupling moiety can be any moiety through which the immunosuppressant is bound to the synthetic nanocarrier. Such moieties include covalent bonds, such as amide or ester bonds, as well as other molecules that bind (covalently or non-covalently) the immunosuppressant to the synthetic nanocarrier. Such molecules include linkers or polymers, or units thereof. For example, the coupling moiety can include a charged polymer to which the immunosuppressant is electrostatically bound. As another example, the coupling moiety can include a polymer or unit thereof to which it is covalently bound.

[0196] In preferred embodiments, synthetic nanocarriers comprise polymers as provided herein. These synthetic nanocarriers may be entirely polymeric, or they may be mixtures of polymers and other materials.

[0197] In some embodiments, the polymers of the synthetic nanocarrier associate to form a polymeric matrix. In some of these embodiments, a component, such as an immunosuppressant, can be covalently associated with one or more polymers of the polymeric matrix. In some embodiments, the covalent association is mediated by a linker. In some embodiments, a component can be non-covalently associated with one or more polymers of the polymeric matrix. For example, in some embodiments, a component can be encapsulated within, surrounded by, and / or dispersed throughout the polymeric matrix. Alternatively or additionally, a component can be associated with one or more polymers of the polymeric matrix through hydrophobic interactions, charge interactions, van der Waals forces, etc. A wide variety of polymers and methods for forming polymeric matrices therefrom are known in the art.

[0198] The polymer may be a natural polymer or a non-natural (synthetic) polymer. The polymer may be a homopolymer or a copolymer comprising two or more monomers. With respect to sequence, the copolymer may be random, block, or may contain a combination of random and block sequences. Typically, the polymer according to the present invention is an organic polymer.

[0199] In some embodiments, the polymer comprises a polyester, polycarbonate, polyamide, or polyether, or units thereof. In other embodiments, the polymer comprises poly(ethylene glycol) (PEG), polypropylene glycol, poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) copolymer, or polycaprolactone, or units thereof. In some embodiments, the polymer is preferably biodegradable. Thus, in these embodiments, when the polymer comprises a polyether, such as poly(ethylene glycol) or polypropylene glycol, or units thereof, the polymer preferably comprises a block copolymer of the polyether and a biodegradable polymer, such that the polymer is biodegradable. In other embodiments, the polymer does not solely comprise a polyether, such as poly(ethylene glycol) or polypropylene glycol, or units thereof.

[0200] Other examples of polymers suitable for use in the present invention include, but are not limited to, polyethylene, polycarbonate (e.g., poly(1,3-dioxan-2-one)), polyanhydrides (e.g., poly(sebacic anhydride)), polypropyl fumarate, polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactide, polyglycolide, polylactide-co-glycolide, polycaprolactone, polyhydroxy acids (e.g., poly(β-hydroxyalkanoates))), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine, polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

[0201] In some embodiments, polymers in accordance with the present invention include polymers approved for human use by the U.S. Food and Drug Administration (FDA) under 21 C.F.R. § 177.2600, including, but not limited to, polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2-one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.

[0202] In some embodiments, the polymer can be hydrophilic. For example, the polymer may contain anionic groups (e.g., phosphate groups, sulfate groups, carboxylate groups); cationic groups (e.g., quaternary amine groups); or polar groups (e.g., hydroxyl groups, thiol groups, amine groups). In some embodiments, synthetic nanocarriers comprising a hydrophilic polymer matrix create a hydrophilic environment within the synthetic nanocarrier. In some embodiments, the polymer can be hydrophobic. In some embodiments, synthetic nanocarriers comprising a hydrophobic polymer matrix create a hydrophobic environment within the synthetic nanocarrier. The selection of the hydrophilic or hydrophobic nature of the polymer can affect the properties of materials incorporated (e.g., attached) within the synthetic nanocarrier.

[0203] In some embodiments, the polymer may be modified with one or more moieties and / or functional groups. A variety of moieties or functional groups can be used in accordance with the present invention. In some embodiments, the polymer may be modified with polyethylene glycol (PEG), carbohydrates, and / or acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Some embodiments may be made using the general teachings of U.S. Patent No. 5,543,158 to Gref et al. or WO Publication No. WO 2009 / 051837 by Von Andrian et al.

[0204] In some embodiments, the polymer may be modified with a lipid or fatty acid group. In some embodiments, the fatty acid group may be one or more of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, or lignoceric acid. In some embodiments, the fatty acid group may be one or more of palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, alpha-linoleic acid, gamma-linoleic acid, arachidonic acid, gadoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, or erucic acid.

[0205] In some embodiments, the polymer may be a polyester, including copolymers containing lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide) (collectively referred to herein as "PLGA"); and homopolymers containing glycolic acid units (collectively referred to herein as "PGA"), and homopolymers containing lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide (collectively referred to herein as "PLA"). In some embodiments, exemplary polyesters include, for example, polyhydroxy acids; PEG copolymers, and copolymers of lactide and glycolide (e.g., PLA-PEG copolymer, PGA-PEG copolymer, PLGA-PEG copolymer), and derivatives thereof. In some embodiments, polyesters include, for example, poly(caprolactone), poly(caprolactone)-PEG copolymer, poly(L-lactide-L-lysine) copolymer, poly(serine ester), poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

[0206] In some embodiments, the polymer may be PLGA. PLGA is a biocompatible and biodegradable copolymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lactic acid. The degradation rate of PLGA can be adjusted by changing the ratio of lactic acid:glycolic acid. In some embodiments, PLGA to be used in accordance with the present invention is characterized by a lactic acid:glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.

[0207] In some embodiments, the polymer may be one or more acrylic acid polymers. In some embodiments, acrylic acid polymers include, for example, copolymers of acrylic acid and methacrylic acid, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymers, poly(methyl methacrylate), poly(methacrylic anhydride), methyl methacrylate, polymethacrylate, poly(methyl methacrylate) copolymers, polyacrylamide, aminoalkyl methacrylate copolymers, glycidyl methacrylate copolymers, polycyanoacrylate, and combinations comprising one or more of the foregoing polymers. The acrylic acid polymer may also include fully polymerized copolymers of acrylic acid esters and methacrylic acid esters with a low content of quaternary ammonium groups.

[0208] In some embodiments, the polymer can be a cationic polymer. Generally, cationic polymers can condense and / or protect negatively charged strands of nucleic acids. Amine-containing polymers such as poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethyleneimine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993, Bioconjugate Chem., 4:372) are positively charged at physiological pH and form ion pairs with nucleic acids. In embodiments, synthetic nanocarriers may not include (or may exclude) cationic polymers.

[0209] In some embodiments, the polymer can be a degradable polyester with cationic side chains (Putnam et al., 1999, Macromolecules, 32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633; and Zhou et al., 1990, Macromolecules, 23:3399). Examples of these polyesters are poly(L-lactide-L-lysine) copolymers (Barrera et al., 1993, J. Am. Chem. Soc., 115:11010), poly(serine esters) (Zhou et al., 1990, Macromolecules, 23:3399), poly(4-hydroxy-L-proline esters) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633), and poly(4-hydroxy-L-proline esters) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633). 121:5633).

[0210] The properties of these and other polymers and methods for preparing them are well known in the art (e.g., U.S. Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181). More generally, various methods for synthesizing certain suitable polymers are described in Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, edited by Goethals, Pergamon Press, 1980; Principles of Polymerization by Odian, John Wiley & Sons, 4th ed., 2004; Contemporary Polymer Chemistry by Allcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

[0211] In some embodiments, the polymers may be linear or branched polymers. In some embodiments, the polymers may be dendrimers. In some embodiments, the polymers may be substantially crosslinked to one another. In some embodiments, the polymers may be substantially free of crosslinks. In some embodiments, the polymers may be used in accordance with the present invention without undergoing a crosslinking step. It should further be understood that synthetic nanocarriers may comprise block copolymers, graft copolymers, blends, mixtures, and / or adducts of any of the foregoing polymers with other polymers. Those skilled in the art will recognize that the polymers listed herein represent an exemplary, but not exhaustive, list of polymers that may be used in accordance with the present invention.

[0212] In some embodiments, synthetic nanocarriers do not include polymeric components. In some embodiments, synthetic nanocarriers may include metal particles, quantum dots, ceramic particles, etc. In some embodiments, non-polymeric synthetic nanocarriers are aggregates of non-polymeric components, such as aggregates of metal atoms (e.g., gold atoms).

[0213] The compositions of the present invention can include components such as immunosuppressants in combination with pharmaceutically acceptable excipients such as preservatives, buffers, saline, or phosphate-buffered saline. The compositions can be prepared using conventional pharmaceutical manufacturing and compounding techniques to achieve a useful dosage form. In one embodiment, the composition (such as one containing an immunosuppressant) is suspended in a sterile saline solution for injection together with a preservative.

[0214] In embodiments, when preparing synthetic nanocarriers as carriers, methods for attaching components to synthetic nanocarriers can be useful. When the components are small molecules, it can be advantageous to attach the components to a polymer prior to assembly of the synthetic nanocarrier. In embodiments, rather than attaching the components to a polymer, it can also be advantageous to prepare synthetic nanocarriers with surface groups that are used to attach the components to the synthetic nanocarrier through the use of these surface groups, and then use this polymer conjugate in the construction of the synthetic nanocarrier.

[0215] In certain embodiments, the attachment can be a covalent linker. In embodiments, the immunosuppressant according to the present invention can be covalently attached to the outer surface via a 1,2,3-triazole linker formed by a 1,3-dipolar cycloaddition reaction between an azide group on the surface of the nanocarrier and an immunosuppressant containing an alkyne group, or between an alkyne on the surface of the nanocarrier and an immunosuppressant containing an azide group. Such cycloaddition reactions are preferably carried out in the presence of a Cu(I) catalyst, together with a suitable Cu(I)-ligand and a reducing agent to reduce the Cu(II) compound to a catalytically active Cu(I) compound. This Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be referred to as a click reaction.

[0216] Additionally, covalent coupling may involve covalent linkers, including amide linkers, disulfide linkers, thioether linkers, hydrazone linkers, hydrazide linkers, imine or oxime linkers, urea or thiourea linkers, amidine linkers, amine linkers, and sulfonamide linkers.

[0217] Amide linkers are formed via an amide bond between an amine on one component, such as an immunosuppressant, and a carboxylic acid group on a second component, such as a nanocarrier. The amide bond in the linker can be made using any of the conventional amide bond-forming reactions with a suitably protected amino acid, such as an N-hydroxysuccinimide-activated ester.

[0218] Disulfide linkers are created through the formation of a disulfide (SS) bond between two sulfur atoms, for example, in the form R1-SS-R2. Disulfide bonds can be formed by thiol exchange of a component containing a thiol / mercaptan group (-SH) with another activated thiol group on a polymer or nanocarrier, or by thiol exchange of a nanocarrier containing a thiol / mercaptan group with a component containing an activated thiol group.

[0219] Triazole linkers, particularly 1,2,3-triazoles of the form: [ka] Here, R1 and R2 can be any chemical entity and are produced by a 1,3-dipolar cycloaddition reaction between an azide attached to a first component, such as a nanocarrier, and a terminal alkyne attached to a second component, such as an immunosuppressant. The 1,3-dipolar cycloaddition reaction is carried out with or without a catalyst, preferably using a Cu(I) catalyst to link the two components through a 1,2,3-triazole functional group. This chemistry is described in detail by Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) and Meldal, et al., Chem. Rev., 2008, 108(8), 2952-3015, and is often referred to as the "click" reaction or CuAAC.

[0220] In some embodiments, a polymer containing an azide group or an alkyne group at the end of the polymer chain is prepared.This polymer is then used to prepare a synthetic nanocarrier, so that a plurality of alkyne groups or azide groups are located on the surface of the nanocarrier.Alternatively, a synthetic nanocarrier can be prepared by another route, and then functionalized with an alkyne group or an azide group.The component is prepared in the presence of either an alkyne group (when the polymer contains an azide) or an azide group (when the polymer contains an alkyne) group.The component is then reacted with the nanocarrier through a 1,3-dipolar cycloaddition reaction, with or without a catalyst, which covalently attaches the component to the particle through a 1,4-disubstituted 1,2,3-triazole linker.

[0221] Thioether linkers are made by the formation of a sulfur-carbon (thioether) bond, for example, in the form R1-S-R2. Thioethers can be made by either alkylation of a thiol / mercaptan (—SH) group on one component with an alkylating group, such as a halide or epoxide, on a second component. Thioether linkers can also be formed by Michael addition of a thiol / mercaptan group on one component to an electron-deficient alkene group on a second component containing a maleimide or vinyl sulfone group as a Michael acceptor. Alternatively, thioether linkers can be prepared by radical thiol-ene reaction of a thiol / mercaptan group on one component with an alkene group on a second component.

[0222] Hydrazone linkers are made by reaction of a hydrazide group on one component with an aldehyde / ketone group on a second component. Hydrazide linkers are formed by the reaction of a hydrazine group on one component with a carboxylic acid group on a second component. Such reactions are generally carried out using chemistry similar to the formation of amide bonds, in which the carboxylic acid is activated with an activating reagent.

[0223] Imine or oxime linkers are formed by the reaction of an amine or N-alkoxyamine (or aminooxy) group on one component with an aldehyde or ketone group on a second component. Urea or thiourea linkers are prepared by reaction of an amine group on one component with an isocyanate or thioisocyanate group on a second component.

[0224] Amidine linkers are prepared by reaction of an amine group on one component with an imidoester group on a second component. Amine linkers are prepared by the alkylation reaction of an amine group on one component with an alkylating group such as a halide, epoxide, or sulfonate ester group on a second component. Alternatively, amine linkers can also be prepared by reductive amination of an amine group on one component with an aldehyde or ketone group on a second component using a suitable reducing reagent such as sodium cyanoborohydride or sodium triacetoxyborohydride.

[0225] Sulfonamide linkers are made by reaction of an amine group on one component with a sulfonyl halide (such as sulfonyl chloride) group on a second component. The sulfone linker is made by Michael addition of a nucleophile to a vinyl sulfone. Either the vinyl sulfone or the nucleophile can be on the surface of the nanocarrier or attached to a component.

[0226] Components can also be conjugated to nanocarriers via non-covalent conjugation methods. For example, negatively charged immunosuppressants can be conjugated to positively charged nanocarriers through electrostatic adsorption. Components containing metal ligands can also be conjugated to nanocarriers containing metal complexes via metal-ligand complexes.

[0227] In embodiments, components can be attached to a polymer, such as polylactic acid-block-polyethylene glycol, prior to assembly of the synthetic nanocarrier, or the synthetic nanocarrier can be formed with reactive or activatable groups on its surface. In the latter case, components can be prepared with groups that are compatible with the attachment chemistry presented by the surface of the synthetic nanocarrier. In other embodiments, peptide components can be attached to VLPs or liposomes using a suitable linker. A linker is a compound or reagent that can couple two molecules together. In one embodiment, the linker can be a homobifunctional or heterobifunctional reagent as described in Hermanson 2008. For example, a VLP or liposomal synthetic nanocarrier containing carboxylic acid groups on its surface can be treated with the homobifunctional linker adipic acid dihydrazide (ADH) in the presence of EDC to form the corresponding synthetic nanocarrier with an ADH linker. The resulting ADH-linked synthetic nanocarrier is then conjugated with an acid group-containing peptide component via the other end of the ADH linker on the nanocarrier to produce the corresponding VLP or liposome-peptide conjugate.

[0228] For a detailed description of available conjugation methods, see Hermanson GT, Bioconjugate Techniques, 2nd ed., Academic Press, Inc. (2008). In addition to covalent attachment, components can be attached by adsorption to preformed synthetic nanocarriers, or they can be attached by encapsulation during the formation of the synthetic nanocarriers.

[0229] By way of example, synthetic nanocarriers comprising rapamycin can be produced or made available by any one of the following methods: 1) PLA with an inherent viscosity of 0.41 dL / g is purchased from Evonik Industries (Rellinghauser Strasse 1-11, 45128 Essen, Germany) (product code: Resomer Select 100 DL 4A). PLA-PEG-OMe block copolymer with methyl ether-terminated PEG blocks of approximately 5,000 Da and a total inherent viscosity of 0.50 DL / g is purchased from Evonik Industries (Rellinghauser Strasse 1-11, 45128 Essen, Germany) (product code: Resomer Select 100 DL mPEG 5000 (15 wt% PEG)). Rapamycin is purchased from Concord Biotech Limited (1482-1486 Trasad Road, Dholka 382225, Ahmedabad, India) (product code: SIROLIMUS). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa·s) is purchased from MilliporeSigma (EMD Millipore, 290 Concord Road, Billerica, Massachusetts 01821) (product code 1.41350). Dulbecco's Phosphate Buffered Saline 1× (DPBS) is purchased from Lonza (Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland) (product code 17-512Q). Sorbitan monopalmitate is purchased from Croda International (300-A Columbus Circle, Edison, NJ 08837) (product code SPAN 40). Solutions are prepared as follows: Solution 1 is prepared by dissolving PLA at 150 mg / mL and PLA-PEG-Ome at 50 mg / mL in dichloromethane. Solution 2 is prepared by dissolving rapamycin at 100 mg / mL in dichloromethane. Solution 3 is prepared by dissolving SPAN 40 at 50 mg / mL in dichloromethane.Solution 4 is prepared by dissolving PVA at 75 mg / mL in 100 mM phosphate buffer (pH 8). An O / W emulsion is prepared by adding Solution 1 (0.50 mL), Solution 2 (0.12 mL), Solution 3 (0.10 mL), and dichloromethane (0.28 mL) into a thick-walled glass pressure tube. The combined organic phase solutions are then mixed by repeated pipetting. To this mixture, Solution 4 (3 mL) is added. The pressure tube is then vortex mixed for 10 seconds. The crude emulsion is then homogenized by sonicating it at 30% amplitude for 1 minute using a Branson Digital Sonifier 250 and a 1 / 8" tapered tip, with the pressure tube immersed in an ice-water bath. The emulsion is then added to a 50 mL beaker containing DPBS (30 mL). This is stirred at room temperature for 2 hours to evaporate the dichloromethane and form nanocarriers. The nanocarrier portion is washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging it at 75,600 × g for 50 minutes at 4 ° C, removing the supernatant, and resuspending the pellet in DPBS containing 0.25% w / v PVA. The washing procedure is repeated, and the pellet is resuspended in DPBS containing 0.25% w / v PVA to reach a nanocarrier suspension with a nominal concentration of 10 mg / mL based on the polymer. The nanocarrier suspension is then filtered using a 0.22 μm PES membrane syringe filter from MilliporeSigma (EMD Millipore, 290 Concord Rd. Billerica MA, product code SLGP033RB). The filtered nanocarrier suspension is stored at -20 ° C. 2) PLA with an inherent viscosity of 0.41 dL / g is purchased from Evonik Industries (Rellinghauser Strasse 1-11, 45128 Essen, Germany) (product code: Resomer Select 100 DL 4A). PLA-PEG-OMe block copolymer with methyl ether-terminated PEG blocks of approximately 5,000 Da and a total inherent viscosity of 0.50 DL / g is purchased from Evonik Industries (Rellinghauser Strasse 1-11, 45128 Essen, Germany) (product code: Resomer Select 100 DL mPEG 5000 (15 wt% PEG)). Rapamycin is purchased from Concord Biotech Limited (1482-1486 Trasad Road, Dholka 382225, Ahmedabad, India) (product code: SIROLIMUS). Sorbitan monopalmitate was purchased from Sigma-Aldrich (3050 Spruce St., St. Louis, MO 63103) (product code 388920). EMPROVE® polyvinyl alcohol (PVA) 4-88, USP (85-89% hydrolyzed, 3.4-4.6 mPa·s viscosity) was purchased from MilliporeSigma (EMD Millipore, 290 Concord Road, Billerica, Massachusetts 01821) (product code 1.41350). Dulbecco's phosphate-buffered saline 1× (DPBS) was purchased from Lonza (Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland) (product code 17-512Q). Solutions are prepared as follows: Solution 1: A mixture of polymer, rapamycin, and sorbitan monopalmitate is prepared by dissolving PLA at 37.5 mg / mL, PLA-PEG-Ome at 12.5 mg / mL, rapamycin at 8 mg / mL, and sorbitan monopalmitate at 2.5 in dichloromethane. Solution 2: Polyvinyl alcohol is prepared at 50 mg / mL in 100 mM pH 8 phosphate buffer.An O / W emulsion is prepared by combining Solution 1 (1.0 mL) and Solution 2 (3 mL) in a small glass pressure tube and vortex mixing for 10 seconds. The formulation is then homogenized by sonicating at 30% amplitude for 1 minute using a Branson Digital Sonifier 250 and a 1 / 8" tapered tip, with the pressure tube immersed in an ice-water bath. The emulsion is then added to a 50 mL beaker containing DPBS (15 mL) and covered with aluminum foil. A second O / W emulsion is prepared using the same materials and methods as above, and then added to the same beaker with a fresh aliquot of DPBS (15 mL). The combined emulsion is then left uncovered and stirred at room temperature for 2 hours to evaporate the dichloromethane and form nanocarriers. The nanocarrier portion is washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 75,600 × g for 50 minutes at 4 ° C, removing the supernatant, and resuspending the pellet in DPBS containing 0.25% w / v PVA. The washing procedure is repeated, and the pellet is then resuspended in DPBS containing 0.25% w / v PVA to reach a nanocarrier suspension with a nominal concentration of 10 mg / mL based on the polymer. The nanocarrier suspension is then filtered using a 0.22 μm PES membrane syringe filter from MilliporeSigma (EMD Millipore, 290 Concord Rd. Billerica MA, product code SLGP033RB). The filtered nanocarrier suspension is then stored at -20 ° C.

[0230] immunosuppressants Any immunosuppressant provided herein can be used in the provided methods or compositions, and in some aspects can be attached to or included in synthetic nanocarriers. Immunosuppressants include, but are not limited to, statins; mTOR inhibitors such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase (HDAC) inhibitors; corticosteroids; inhibitors of mitochondrial function such as rotenone; P38 inhibitors; NF-κβ inhibitors; adenosine receptor agonists; prostaglandin E2 agonists; phosphodiesterase inhibitors such as phosphodiesterase 4 inhibitors; proteasome inhibitors; kinase inhibitors; G protein-coupled receptor agonists; G protein-coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors and oxidized ATP. Immunosuppressants also include IDO, vitamin D3, cyclosporin A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine, 6-mercaptopurine, aspirin, niflumic acid, estriol, tripolide, interleukins (e.g., IL-1, IL-10), cyclosporin A, siRNAs targeting cytokines or cytokine receptors, and the like.

[0231] Examples of statins include atorvastatin (LIPITOR®, TORVAST®), cerivastatin, fluvastatin (LESCOL®, LESCOL® XL), lovastatin (MEVACOR®, ALTOCOR®, ALTOPREV®), mevastatin (COMPACTIN®), pitavastatin (LIVALO®, PIAVA®), rosuvastatin (PRAVACHOL®, SELEKTINE®, LIPOSTAT®), rosuvastatin (CRESTOR®), and simvastatin (ZOCOR®, LIPEX®).

[0232] Examples of mTOR inhibitors include rapamycin and its analogs (e.g., CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap), C16-(S)-butylsulfonamidorapamycin (C16-BSrap), C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry & Biology 2006, 13:99-107), AZD8055, BEZ235 (NVP-BEZ235), chrysophanic acid (chrysophanol), deforolimus (MK-8669), everolimus (RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (available from Selleck, Houston, TX, USA).

[0233] Examples of TGF-β signaling agents include TGF-β ligands (e.g., activin A, GDF1, GDF11, bone morphogenetic protein, nodal, TGF-β) and their receptors (e.g., ACVR1B, ACVR1C, ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B, TGFβRI, TGFβRII), R-SMADS / CO-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD8), and ligand inhibitors (e.g., follistatin, noggin, chordin, DAN, lefty, LTBP1, THBS1, decorin).

[0234] Examples of inhibitors of mitochondrial function include atractyloside (dipotassium salt), bongkrekic acid (triammonium salt), carbonyl cyanide m-chlorophenylhydrazone, carboxyatractyloside (e.g., from Atractylis gummifera), CGP-37157, (-)-deguelin (e.g., from Mundulea sericea), F16, hexokinase II VDAC binding domain peptide, oligomycin, rotenone, Ru360, SFK1, and valinomycin (e.g., from Streptomyces fulvissimus) (EMD4Biosciences, USA).

[0235] Examples of P38 inhibitors include SB-203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) 1H-imidazole), SB-239063 (trans-1-(4-hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-yl)imidazole), SB-220025 (5-(2-amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole)), and ARRY-797.

[0236] Examples of NF (e.g., NK-κβ) inhibitors include IFRD1, 2-(1,8-naphthyridin-2-yl)-phenol, 5-aminosalicylic acid, BAY 11-7082, BAY 11-7085, CAPE (caffeic acid phenethyl ester), diethyl maleate, IKK-2 inhibitor IV, IMD 0354, lactacystin, MG-132 [Z-Leu-Leu-Leu-CHO], NFκB activation inhibitor III, NF-κB activation inhibitor II, JSH-23, parthenolide, phenylarsine oxide (PAO), PPM-18, pyrrolidinedithiocarbamic acid ammonium salt, QNZ, RO 106-9920, rocaglamide, rocaglamide AL, rocaglamide C, rocaglamide I, rocaglamide J, rocaglaol, (R)-MG-132, sodium salicylate, triptolide (PG490), and wedelolactone.

[0237] Examples of adenosine receptor agonists include CGS-21680 and ATL-146e. Examples of prostaglandin E2 agonists include E-prostanoid 2 and E-prostanoid 4. Examples of phosphodiesterase inhibitors (non-selective and selective inhibitors) are caffeine, aminophylline, IBMX (3-isobutyl-1-methylxanthine), paraxanthine, pentoxifylline, theobromine, theophylline, methylated xanthines, vinpocetine, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone (PERFAN®), milrinone, levosimendan, mesembrine, ibudilast, picra, cyclosporine ... These include milast, luteolin, drotaverine, roflumilast (DAXAS™, DALIRESP™), sildenafil (REVATION®, VIAGRA®), tadalafil (ADCIRCA®, CIALIS®), vardenafil (LEVITRA®, STAXYN®), udenafil, avanafil, icariin, 4-methylpiperazine, and pyrazolopyrimidine-7-one.

[0238] Examples of proteasome inhibitors include bortezomib, disulfiram, epigallocatechin-3-gallate, and salinosporamide A. Examples of kinase inhibitors include bevacizumab, BIBW 2992, cetuximab (ERBITUX®), imatinib (GLEEVEC®), trastuzumab (HERCEPTIN®), gefitinib (IRESSA®), ranibizumab (LUCENTIS®), pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib, E7080, pazopanib, and mubritinib.

[0239] Examples of glucocorticoids include hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (DOCA), and aldosterone.

[0240] Examples of retinoids include retinol, retinal, tretinoin (retinoic acid, RETIN-A®), isotretinoin (ACCUTANE®, AMNESTEEM®, CLARAVIS®, SOTRET®), alitretinoin (PANRETIN®), etretinate (TEGISON™) and its metabolite acitretin (SORIATANE®), tazarotene (TAZORAC®, AVAGE®, ZORAC®), bexarotene (TARGRETIN®), and adapalene (DIFFERIN®).

[0241] Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonist, IGFBP, TNF-BF, uromodulin, alpha-2-macroglobulin, cyclosporin A, pentamidine, and pentoxifylline (PENTOPAK®, PENTOXIL®, TRENTAL®).

[0242] Examples of peroxisome proliferator-activated receptor antagonists include GW9662, PPARγ antagonist III, G335, and T0070907 (EMD4Biosciences, USA). Examples of peroxisome proliferator-activated receptor agonists include pioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY 171883, PPARγ activators, Fmoc-Leu, troglitazone, and WY-14643 (EMD4Biosciences, USA).

[0243] Examples of histone deacetylase inhibitors include hydroxamic acids (or hydroxamates) such as trichostatin A, cyclic tetrapeptides (such as trapoxin B) and depsipeptides, aliphatic acid compounds such as benzamides, electrophilic ketones, phenylbutyrate, and valproic acid, hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589), benzamides such as entinostat (MS-275), CI994, and mocetinostat (MGCD0103), nicotinamide, derivatives of NAD, dihydrocoumarins, naphthopyranones, and 2-hydroxynaphthaldehydes.

[0244] Examples of calcineurin inhibitors include cyclosporine, pimecrolimus, voclosporin, and tacrolimus. Examples of phosphatase inhibitors include BN82002 hydrochloride, CP-91149, calyculin A, cantharidic acid, cantharidin, cypermethrin, ethyl-3,4-defostatin, fostriecin sodium salt, MAZ51, methyl-3,4-defostatin, NSC 95397, norcantharidin, okadaic acid ammonium salt from Prorocentrum concavum, okadaic acid, okadaic acid potassium salt, okadaic acid sodium salt, phenylarsine oxide, various phosphatase inhibitor cocktails, protein phosphatase 1C, protein phosphatase 2A inhibitors, protein phosphatase 2A1, protein phosphatase 2A2, and sodium orthovanadate.

[0245] Preferably, in any one embodiment of the methods, compositions, or kits provided herein, the immunosuppressant is rapamycin. In some such embodiments, the rapamycin is preferably encapsulated in a synthetic nanocarrier. Rapamycin is the active ingredient of Rapamune, an immunosuppressant that has been widely used in humans and is currently FDA-approved for the prevention of organ rejection in kidney transplant patients aged 13 years or older.

[0246] If coupled to a synthetic nanocarrier, the amount of immunosuppressant coupled to the synthetic nanocarrier (wt / wt), based on the total dry recipe weight of the material in the entire synthetic nanocarrier, is as described elsewhere herein. Preferably, in some embodiments of any one of the methods, compositions, or kits provided herein, the loading amount of immunosuppressant, such as rapamycin or a rapalog, is between 7% and 12% by weight, or between 8% and 12% by weight.

[0247] Compositions and Kits The compositions provided herein may contain inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonic acid, acetic acid, or citric acid), and pH adjusters (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citric acid or acetic acid, amino acids and their salts), antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium deoxycholate), solutions and / or cryoprotectants. / lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmolality adjusters (e.g., salts or sugars), antimicrobial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsiloxane, preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity adjusters (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose), and cosolvents (e.g., glycerol, polyethylene glycol, ethanol).

[0248] The compositions according to the present invention may contain pharmaceutically acceptable excipients. The compositions may be prepared using conventional pharmaceutical manufacturing and compounding techniques to achieve useful dosage forms. Suitable techniques for use in the practice of the present invention may be found in Handbook of Industrial Mixing: Science and Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of Dosage Form Design, 2nd Edition, Edited by M.E. Auten, 2001, Churchill Livingstone. In one embodiment, the composition is suspended in sterile saline solution for injection together with a preservative.

[0249] It should be understood that the compositions of the present invention can be made in any suitable manner, and that the invention is in no way limited to compositions that can be produced using the methods described herein. Selection of an appropriate method of manufacture may require attention to the properties of the particular components involved.

[0250] In some embodiments, the composition is produced under aseptic conditions or initially or finally sterilized.This can ensure that the resulting composition is sterile and non-infectious, thus improving safety compared to non-sterile compositions.This provides a valuable safety measure, especially when the subject receiving the composition has an immune deficiency, is suffering from an infectious disease, and / or is prone to infectious diseases.In some embodiments, the composition can be lyophilized and stored in a suspension or as a lyophilized powder, depending on the formulation strategy to prevent loss of activity over a long period of time.The compositions referred to herein can be produced and prepared for administration using conventional methods.

[0251] The compositions of the present invention can be administered in an effective amount, such as the effective amount described elsewhere herein. The dose of the compositions provided herein can contain various amounts of the Ig protease fusion protein and / or synthetic nanocarrier containing the immunosuppressant and / or other therapeutic agent according to the present invention. The amount of elements present in the composition for administration can vary depending on their properties, the therapeutic benefit to be achieved, and other such parameters. In some embodiments of any one of the methods or compositions provided herein, the dose of the Ig protease fusion protein and / or synthetic nanocarrier containing the immunosuppressant and / or other therapeutic agent is any one of the doses provided herein.

[0252] Another aspect of the present disclosure relates to kits. In some embodiments, the kit comprises any one or more of the compositions provided herein. In some embodiments of any one of the kits provided, the kit comprises any one or more of the compositions comprising an Ig protease fusion protein as provided herein. Preferably, the composition(s) comprising the Ig protease fusion protein are in an effective amount. The composition(s) comprising the Ig protease fusion protein may be in one container or more than one container in the kit. In some embodiments of any one of the kits provided, the kit further comprises any one or more of the synthetic nanocarrier compositions and / or other therapeutic agents provided herein. Preferably, in some embodiments, the synthetic nanocarrier composition(s) are in an amount that provides one or more doses of the immunosuppressant provided herein. The Ig protease fusion protein and / or synthetic nanocarrier and / or other therapeutic agent may be in one container or more containers in the kit. In some embodiments of any one of the kits provided, the container is a vial or an ampoule. In some embodiments of any one of the kits provided, the composition(s) are in lyophilized form, each in separate containers or in the same container, so that they may be reconstituted at a later time. In some embodiments of any one of the kits, the lyophilized compositions further comprise a sugar, such as mannitol. In some embodiments of any one of the kits provided, the composition(s) are in the form of a frozen suspension, each in separate containers or in the same container, so that they may be reconstituted at a later time. In some embodiments of any one of the kits, the frozen suspension further comprises PBS. In some embodiments of any one of the kits, the kit further comprises PBS and / or 0.9% sodium chloride (USP). In some embodiments of any one of the kits provided, the kit further comprises instructions for reconstitution, mixing, administration, etc.In some embodiments of any one of the kits provided herein, the instruction manual comprises the description of any one of the methods described herein.The instruction manual can be in any suitable form, for example, as a printed insert or label.In some embodiments of any one of the kits provided herein, the kit further comprises one or more syringes or other devices that can deliver the composition(s) to the subject in vivo.

[0253] example Example 1: Synthesis of engineered IdeS-mouse Fc fusion protein An IdeS-mouse Fc fusion protein was engineered to improve the circulating half-life of Ig proteases for therapeutic potential (Figure 2). IdeS, derived from Streptococcus pyogenes, cleaves IgG from humans, non-human primates, and rabbits, but not mouse IgG. The C-terminus of IdeS (Figure 2, normal text) was fused to the N-terminus of mouse IgG1 Fc (Figure 2, underlined text) to design a fusion protein. A signal sequence was also included in the fusion protein (Figure 2, bold text).

[0254] The IdeS-Fc fusion protein was cloned into a mammalian expression plasmid and transiently transfected into 293 cells. The cell supernatant was purified on a HiTrap MabSelect SuRe column. The column was washed with 1.5 M NaCl, 1.5 M glycine (pH 8.5), and eluted with 50 mM Tris, 25 mM arginine (pH 10). The IdeS-Fc fusion protein eluted as a disulfide-linked homodimer of approximately 120,000 daltons, which collapsed to a single band of approximately 60,000 daltons on a reducing SDS-PAGE gel (Figure 3A). Native SEC-HPLC analysis of the purified IdeS-Fc fusion protein was also performed (Figure 3B).

[0255] Example 2: In vivo activity of engineered IdeS-mouse Fc fusion proteins The IdeS-mouse Fc fusion protein was evaluated in vivo in rabbits. Rabbits were left untreated (group 1) or injected with 1 x 10 12 Animals were immunized with 10 vector genomes / kg of AAV8 (adeno-associated virus-8) and then either untreated (group 2) or treated with 0.5 mg (group 3) or 5 mg (group 4) of IdeS-mouse Fc fusion protein on day 29. Control animals were left untreated (group 1) (Figure 4A). After dosing with IdeS-Fc on day 29, total IgG was assessed (Figure 4B). In rabbits treated with 5 mg of IdeS-Fc, total IgG declined to below detectable, quantifiable levels by day 31 and recovered by day 36 (Figure 4B). Anti-AAV8-specific IgG showed a similar pattern (Figure 4C).

[0256] Example 3: Synthesis of an engineered IdeS-human Fc fusion protein (hypothetical) It is contemplated that for therapeutic use in humans, it would be advantageous to use human Fc rather than mouse Fc. However, because IdeS cleaves human IgG, the IdeS-Fc fusion protein would have to be mutated to be resistant to autoproteolysis. IdeS cleaves human IgG near the boundary between the hinge region and the CH2 domain. The P1, P2, P1', and P2' residues of the IdeS cleavage site are L, G, G, and P, respectively, in human IgG1, IgG3, and IgG4, and V, A, G, and P in human IgG2 (Wenig K, et al. Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG. Proc Natl Acad Sci US A. 2004 Dec 14;101(50):17371-6. doi: 10.1073 / pnas.0407965101). However, IdeS does not cleave short synthetic peptides spanning the cleavage site, indicating a secondary docking on IgG that provides specificity (Vincents B, et al. Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding. Biochemistry. 2004 Dec 14;43(49):15540-9. doi: 10.1021 / bi048284d).However, human CH2 fusion proteins are cleaved by IdeS, indicating that binding exists within the CH2 domain (Novarra S, et al. A hingeless Fc fusion system for site-specific cleavage by IdeS. MAbs. 2016 Aug-Sep;8(6):1118-25. doi: 10.1080 / 19420862.2016.1186321). Thus, the present invention contemplates mutations that block IdeS-human Fc fusion protein autoproteolysis, which may be made at the cleavage site near the interface between the hinge region and the CH2 domain or within the putative docking site in CH2.

[0257] Analysis of the crystal structure of IdeS overlaid on the homologous structure of papain shows that the carbonyl oxygen of the P1 residue (Gly-236) of IgG forms a hydrogen bond with the amide of the peptide bond between the side chain nitrogens of Cys-94 and Lys-84 of IdeS (Wening PNAS 2004). This orientation orients the amide nitrogen of the P1' residue (Gly-237) of IdeS near the ND1 of the imidazole of His-262. The putative S1' subsite in IdeS is narrower than that of papain, suggesting that P1' residues larger than glycine may cause steric hindrance (Wening et al. PNAS 2004).

[0258] In some embodiments, the IdeS protease domain may be replaced with a mutated, truncated, or engineered version that has one or more desired activities and / or functions. In some embodiments, the IdeS protease domain may be replaced with a homologous protease domain from another strain of streptococcal bacteria, such as the IdeZ protease domain or a mutated, truncated, or engineered version thereof. In some embodiments, the IdeS protease domain may be replaced with the IdeMC protease domain (U.S. Patent Application No. 20190262434 A2), an IgG protease domain produced by canine-specific mycoplasma strains, or a mutated, truncated, or engineered version of the IdeMC protease domain.

[0259] In some embodiments, the Fc domain may be derived from human IgG1, IgG2, IgG3, or IgG4, or versions that have been mutated or engineered to reduce or eliminate Fc effector functions or complement fixation, for example, to enhance binding to FcRn. In some embodiments, the hinge region of the Fc domain may be mutated, truncated, or deleted.

[0260] Example 4: Synthesis of an Engineered Ig Protease-Albumin Fusion Protein (Hypothetical) In some embodiments, the non-naturally occurring Ig protease may be engineered to contain albumin, a plasma protein that also has a long circulating half-life mediated through FcRn binding. In some embodiments, the albumin of the non-naturally occurring Ig protease may be a human albumin protein. In some embodiments, the non-naturally occurring Ig protease may comprise albumin and the Ig protease domain of IdeS from Streptococcus pyogenes.

[0261] In some embodiments, the IdeS protease domain may be replaced with a mutated, truncated, or engineered version that has one or more desired activities and / or functions. In some embodiments, the IdeS protease domain may be replaced with a homologous protease domain from another strain of streptococcal bacteria, such as IdeZ, or a mutated, truncated, or engineered version thereof. In some embodiments, the IdeS protease domain may be replaced with the protease domain of IdeMC, an IgG protease domain produced by a canine-specific mycoplasma strain (e.g., U.S. Patent Application No. 20190262434 A2), or a mutated, truncated, or engineered version of the IdeMC protease domain.

[0262] Example 5: Administration of Engineered Ig Protease Fusion Proteins (Hypothetical) The Ig protease fusion protein can be administered to a subject according to any one of the methods known to those skilled in the art. In some embodiments, the route of administration includes oral or intravenous administration. In some embodiments, the route of administration may be localized. In other embodiments, the route of administration may be systemic. It is contemplated that the subject may have any one of the diseases, disorders, or conditions provided herein.

[0263] Example 6: Xork-Fc fusion homodimers can be expressed in CHO and E. coli An Ig protease (Xork)-Fc fusion was designed as described in Figure 6. As an example, it was found that the Ig protease (Xork)-Fc fusion could be successfully expressed in CHO cells (Figure 7). Furthermore, it was found that an exemplary Ig protease (Xork)-Fc fusion could be prepared from E. coli cells (Figure 8).

[0264] Example 7: Xork-Fc, an engineered IgG protease, exhibits low cross-reactivity to antibodies pre-existing in human serum and enables efficient AAV transduction in an in vivo model of passive transfer of neutralizing human serum Pre-existing neutralizing antibodies against AAV are highly prevalent and a major exclusion factor for enrollment in many gene therapy trials. Strategies are needed to expand access to critical gene therapies to patients with pre-existing anti-AAV antibodies. Recently, bacterial proteases specific for human IgG have been proposed as a way to transiently clear IgG from the circulation, opening a window in which AAV can be administered. A novel IgG-specific protease, IdeSork (Xork), has been developed. In contrast to IdeS, Xork, an IgG protease derived from the common human pathogen Streptococcus pyogenes, is derived from a streptococcal species not known to infect humans. As a result, compared to the moderate to high levels of pre-existing antibodies against IdeS, the levels of pre-existing antibodies against Xork are low or absent in normal human serum. Xork cleaves human IgG in vitro with the same specificity and mechanism as IdeS. The in vivo activity of Xork was optimized by engineering an Fc fusion protein to extend its half-life. Inhibition of AAV transduction in vivo by passive transfer of human serum containing pre-existing anti-AAV antibodies was effectively prevented by treatment with Xork-Fc. Combining Xork-Fc with ImmTOR tolerogenic nanoparticles prevented the de novo formation of anti-Xork antibodies, allowing for rechallenge with Xork.

[0265] Example 8: Administration of an engineered Ig protease-Fc fusion protein in combination with a synthetic nanocarrier containing rapamycin (e.g., ImmTOR) (hypothetical) The Ig protease domain-Fc fusion protein can be administered to a subject in combination with a synthetic nanocarrier containing rapamycin according to any one of the methods provided herein or known to those skilled in the art. In some embodiments, the route of administration includes oral or intravenous administration. In some embodiments, the route of administration may be localized. In other embodiments, the route of administration may be systemic. It is contemplated that the subject may have any one of the diseases, disorders, or conditions provided herein.

[0266] Example 9: Synthesis of synthetic nanocarriers containing immunosuppressants (hypothetical) Synthetic nanocarriers comprising an immunosuppressant, such as rapamycin, can be produced using any method known to those skilled in the art. Preferably, in some embodiments of any one of the methods or compositions provided herein, the synthetic nanocarriers comprising an immunosuppressant are produced by any one of the methods in U.S. Publication Nos. US 2016 / 0128986 A1 and US 2016 / 0128987 A1, and such methods of production and the resulting synthetic nanocarriers described are incorporated herein by reference in their entirety. In any one of the methods or compositions provided herein, the synthetic nanocarriers comprising an immunosuppressant are such incorporated synthetic nanocarriers.

[0267] Example 10: In vivo activity testing of Xork IgG protease candidate molecules (reduction of AAV neutralization by human immune serum in passively immunized mice) material and method Experimental Overview. The experiment utilized immunologically naive female C57BL / 6 mice (17 g, 3–4 per group). Pretested AAV8-positive human serum (pooled samples CP16 or P1) or control normal serum was diluted 5–100-fold (thus, the final injectable solution had 1–20% of the original serum). The diluted serum was then heat-inactivated at 56°C for 30 minutes, after which 100 μL per mouse was injected (iv; retroorbital). One control group was left uninjected or injected with PBS only. 24 hours (1 day) after serum injection, mice were either mock-treated or injected with equimolar amounts of various Xork IgG protease molecules, with a typical dose of 2.095E. -08 Two days after IgG protease treatment, all mice in the study were injected with AAV8-SEAP at 5E 11 Mice were injected (iv, ro) at 200 mg / kg. Mice were bled 12 days after AAV inoculation to determine SEAP activity in the serum. A typical study timeline is shown in Figure 13.

[0268] Evaluation of Results. The efficacy of the protease molecule in cleaving human IgG was measured in vivo in mice passively immunized against AAV8. In this setting, mice were administered human serum containing IgG against AAV8 and then injected with AAV8 carrying the reporter gene secreted alkaline phosphatase (SEAP). When protease treatment was not administered, the level of AAV vector transduction, as measured by SEAP expression, was significantly (up to 20-fold) lower than in naive, unimmunized mice. Therefore, the activity of the protease (administered 1 day after serum transfer and 2 days before AAV inoculation) was measured by its ability to increase the activity of the AAV-encoded transgene relative to that in passively immunized, untreated animals. The results were expressed as follows, relative to SEAP activity in mice not injected with human immune serum (naive animals): The mean SEAP activity in groups sham-immunized with PBS or immunized with normal (non-immune) human serum and subsequently left untreated with IgG protease was considered 100%, and the activities in all other experimental groups were expressed as a percentage relative to this group. One group was always injected with immune serum and subsequently treated with protease; the resulting SEAP activity (usually within 5-10% of the non-immune control) represents the maximal level of AAV transduction inhibition seen in this study (i.e., typically in the 90-95% range). SEAP activity in the experimental group that was statistically indistinguishable from that of mock-immunized mice was considered to be indicative of complete cleavage of human anti-AAV IgG by the tested molecule; SEAP that was lower than that in mock-injected controls but higher than that in non-protease-treated immunized controls was considered to be indicative of partial IgG cleavage by the tested molecule; and activity that was significantly different from that in non-protease-treated immunized controls was considered to be indicative of absent or insufficient human IgG cleavage.

[0269] SEAP activity measurement. SEAP expression was determined using the Phospha-Light SEAP Reporter Gene Assay System (Invitrogen, Carlsbad, CA). Samples were diluted 1:10, heat-inactivated (65°C, 30 min), and chilled on ice. Once samples reached room temperature, they were added to an opaque assay plate, followed by the addition of assay buffer (5 min) and substrate (20 min) according to the manufacturer's recommendations. Light emission was read at 477 nm in a SpectraMax L (Molecular Devices, San Jose, CA, USA) and reported in relative luminance units (RLU), which are proportional to the concentration of SEAP in serum.

[0270] AAV IgG ELISA. In one study, the levels of total IgG or IgG to AAV8 at various time points were assayed by ELISA and expressed as maximum (top) OD or by AAV8 neutralization assay (EC 50 (These values are expressed as 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 10.1, 11.2, 12.3, 13.4, 14.5, 15.6, 16.7, 17.8, 18.9, 19.9, 20.1, 21.1, 22.1, 23.2, 24.2, 25.3, 26.3, 27.4, 28.1, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 55.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, 78.2, 79.2, 80.2, 81.2, 82.2, 83.2, 84.2, 85 The observed optical density (OD) was proportional to the amount of anti-AAV8 human IgG antibody in the sample and was reported. 50The EC was determined as follows: A positive control anti-AAV8 IgG antibody (Fitzgerald Industries International, Acton, MA) and samples were diluted 1:40, followed by serial dilutions of 1:3. Plates were then processed as described above, and EC was calculated using a four-parameter logistic curve fit function in the Softmax Pro software program (Molecular Devices, San Jose, CA). 50 The positive control anti-AAV8-IgG antibody was used to calculate the EC 50 was used as a standard curve for determining

[0271] PK assay. ELISA plates were coated with a custom polyclonal goat anti-Xork antibody at 1 μg / mL overnight at 4°C. The polyclonal anti-Xork antibody can capture Xork-containing molecules (Xork1.1 homodimer, Xork1.1 Fc H435R, Xork1.1 E. coli, Xork H435R E. coli, and Xork1.1 IgG3Fc). After washing with PBST, the wells were blocked with 1% casein for 1-2 hours at room temperature. The plate was washed with PBST, and then samples were added. The samples were incubated for 2 hours at room temperature to allow capture of Xork-containing molecules. The plate was then washed with PBST, and then mouse anti-human IgG Fc-HRP was added. A secondary antibody detected the presence of human Fc captured in the wells. A final wash with PBST was then performed. Intact molecules (containing both Xork and human Fc) were visualized using 3,3',5,5'-tetramethylbenzidine (TMB) oxidized with HRP conjugated to a mouse anti-human IgG Fc secondary antibody. The reaction was then stopped with sulfuric acid. Plates were then read at 450 and 570 nm using a spectrophotometer. Sample optical densities (OD) (OD at 570 nm subtracted from OD at 450 nm) were used to interpolate the concentration of intact Xork molecules using appropriate standard curves (Xork1.1 homodimer, Xork1.1 Fc H435R, Xork1.1 E. coli, Xork H435R E. coli, and XORK1.1 IgG3Fc) that accounted for any sample dilutions performed.

[0272] result Experiment 1. The activity of Xork candidate molecules was increased by their fusion to the human IgG Fc domain, resulting in levels of human IgG cleavage indistinguishable from that of IdeS protease. Twelve groups of three mice each were inoculated with 2% or 5% CP16 immune serum pools as described above, and two additional groups were injected with naive (non-immune) serum or sham-injected. Five pairs of experimental groups (each receiving 2% or 5% immune serum) were treated with IgG protease as described above and inoculated with AAV8-SEAP two days later. SEAP activity in serum was measured 12 days after AAV inoculation. One pair of experimental groups was not treated with protease prior to AAV injection to assess the level of inhibition of AAV transduction. The molecules tested were the clinically approved IgG protease from Streptococcus pyogenes (IdeS) and four Xork candidate molecules: Xork1.0, Xork1.2, and two fusion molecules carrying Xork1.0 attached to either human serum albumin (HSA) or the human IgG Fc domain (knob-in-hole structure, KIH); the latter two constructs are designated Xork1.1-HSA and Xork1.1-hIgGFc-KIH. Passive immunization with immune serum resulted in a significant decrease in transduction efficiency, as demonstrated by SEAP activity in these groups being 21% and 9% of that of untreated controls (2% and 5% after treatment with serum, respectively, Figure 14). Treatment with Xork1.0 and Xork1.2 did not result in any increase in SEAP activity compared to untreated controls, and treatment with Xork1.1-HSA was partially effective (Figure 14). Conversely, treatment with Xork1.1-hIgGFc-KIH resulted in a significant increase in transduction activity, as measured by SEAP expression, with the levels achieved being indistinguishable from those seen in mice treated with IdeS protease. Furthermore, levels of human IgG measured before and 2 days after administration of the protease molecule provided a complete complement of the SEAP activity data.This shows the most significant reduction in total human IgG in the groups treated with Xork1.1 and IdeS, with virtually no reduction in IgG in the groups treated with Xork 1.0 or Xork1.2 (Figure 15).

[0273] Experiment 2. Xork1.1 and Xork1.3 candidate molecules enable full AAV transduction activity in passively immunized mice at standard and low doses of protease. Twelve groups of three or four mice each were inoculated with 2% or 5% CP16 immune serum pools as described above, and two additional groups were injected with naive (non-immune) serum or sham-injected. Five pairs of experimental groups (each receiving 2% or 5% immune serum) were treated with Xork1.1 or Xork1.3 IgG protease (a fusion construct consisting of the Xork catalytic domain and the human IgG Fc domain). Two days later, they were inoculated with AAV8-SEAP, and serum SEAP activity was measured 12 days after AAV inoculation. One pair of experimental groups was not treated with protease prior to AAV injection to assess the level of inhibition of AAV transduction. Furthermore, groups 1-2, 7-8, and 9-10 were classified into Xork1.1-Fc (mono-dimer or HD), Xork1.1-Fc-HD-H435R, and Xork1.3-Fc-HD-H435R, respectively, with the standard 2.095E -08 M / kg (the latter two have a single amino acid mutation in the Fc domain as shown), while groups 3-4 and 5-6 were treated with a two-fold lower dose (1.048E, respectively). -08Xork1.1-Fc-HD and Xork1.3-Fc-HD were tested (M / kg Xork1.1-Fc-HD and Xork1.3-Fc-HD). In almost all mouse groups treated with Xork1.1-Fc or Xork-1.3-Fc, the level of SEAP expression was at least equal to or higher than that in the control group that did not receive passive immunization (Figure 16). This demonstrated the high IgG cleavage efficacy of these molecules, as the protease-untreated group administered 5% AAV immune serum showed a 94% reduction in SEAP activity (Figure 16). The only test molecule that did not provide full AAV transduction efficacy was Xork1.3-Fc-HD-H435R, although SEAP levels in mice treated with the construct were only approximately 20% lower than those in non-immunized controls (Figure 16).

[0274] Experiment 3. The in vivo activity of Xork1.1-hIgGFc-GGSS is unaffected by its means of production, demonstrated across a wide dose range, and may be superior to that of Xork1.1-hIgGFc-H435R and Xork1.1-hIgG3-Fc. Nine groups of four mice each were inoculated with a 10% P1 human immune serum pool, as previously described for the CP16 pool; one group received a higher 20% serum dose; two additional groups were injected with naive (non-immune) serum or sham-injected. Eight experimental groups were treated with three different Xork1.1 molecules produced in E. coli (Groups 1-3) or serial dilutions of the Xork1.1-hIgGFc-GGSS dimer produced in eukaryotic CHO cells (Groups 4-8; either undiluted or the standard 2.095E -08 M / kg, and its 2x, 20x, 200x, and 2,000x dilutions, respectively. They were then inoculated with AAV8-SEAP 2 days later, and SEAP activity in serum was measured 12 days after AAV inoculation. Two groups (one inoculated with 10% immune serum and the other with 20% immune serum) were not treated with protease prior to AAV injection to assess the level of inhibition of AAV transduction.

[0275] Although the level of AAV transduction enabled by the Xork1.1-hIgGFc-GGSS dimer produced in CHO cells appears slightly superior, the activity of this molecule when diluted 2-fold is very similar to that of its E. coli-produced counterpart, the Xork1.1-IgGFc fusion, indicating that these two molecules are of similar potency (Figure 17). It is also possible that the activity of these molecules is significantly superior to that of Xork1.1-IgGFc-H435R and Xork1.1-IgG3Fc, since a 20-fold lower dose of Xork1.1-hIgGFc in Group 6 resulted in SEAP activity similar to that in Groups 2 and 3. In addition, the reduction in activity by 2- and 20-fold diluted Xork1.1-hIgGFc-GGSS was not dramatic, and significant reduction in transduction was observed at >20-fold dilutions (i.e., doses of 1.048E). -09 Xork1.1-hIgGFc-GGSS was diluted 200-2,000 times (Group 7, Group 8) or 1.048E -10 , 1.048E -11 Even when diluted to 1 M / kg, the resulting SEAP activity is higher than that in the non-protease treated control (Group 9).

[0276] Experiment 4. Evaluation of the in vivo activity of Xork1.3-hIgGFc-GGSS homodimer produced in E. coli and CHO cells relative to Xork1.3-hIgGFc-GGSS-H435R produced in E. coli. Seven groups of four mice each were inoculated with a 10% P1 immune serum pool, and one group received naive (non-immune) serum. Six experimental groups were treated with either Xork1.3-hIgGFc-GGSS-homodimer molecules produced in E. coli (Groups 1-2) or CHO cells (Groups 5-6), or with Xork1.3-hIgGFc-GGSS-H435R produced in E. coli (Groups 3-4). Each molecule was administered at two doses: 1.048E -08 M / kg and 1.048E -09 M / kg (0.5x and 0.05x the standard 2.095E, respectively) -08 Mice were then inoculated with AAV8-SEAP 2 days later, and serum SEAP activity was measured 12 days after AAV inoculation. One group inoculated with 10% immune serum was not treated with protease prior to AAV injection to assess the level of inhibition of AAV transduction.

[0277] Similar to its Xork1.1 counterpart, the Xork1.3-hIgGFc-GGSS homodimer enabled approximately 100% AAV transduction in passively immunized mice, with SEAP activity levels in treated animals indistinguishable from those injected with naive, non-immune serum (Figure 18). This activity was observed over a wide concentration range, exceeding the standard 2.095E -08 Even at a 20-fold dilution, this low 1.048E -09At a dose of 100 M / kg, E. coli-derived Xork1.3-hIgGFc-GGSS-HD enabled higher transduction levels than those achieved with the other constructs tested in this study, namely, E. coli-derived Xork1.3-hIgGFc-H435R and CHO-produced Xork1.3-hIgGFc-GGSS-HD. In any case, CHO-made Xork1.3-hIgGFc-GGSS-HD showed no advantage over its E. coli-produced counterpart in human IgG cleavage in an in vivo passive immunization model.

[0278] Example 11: Xork-Fc, an Engineered IgG Protease, Exhibits Low Cross-Reactivity to Antibodies Pre-Existing in Human Serum and Enables Efficient AAV Transduction in an In Vivo Model of Passive Transfer of Neutralizing Human Serum Pre-existing neutralizing antibodies to AAV are prevalent and a major exclusion factor for enrollment in many gene therapy clinical trials. Individuals may not be able to be treated with viral vectors, such as AAV-mediated gene therapy, due to prior exposure to neutralizing antibodies. New strategies will help expand access to important gene therapies to patients with pre-existing anti-AAV antibodies.

[0279] Bacterial proteases specific for human IgG could be a way to transiently remove IgG from the circulation, opening a window during which AAV can be administered. For example, a novel IgG-specific protease, IdeSork (Xork), has been developed. In contrast to IdeS, Xork, an IgG protease derived from the common human pathogen Streptococcus pyogenes, is derived from a species of streptococcus not known to infect humans. As a result, the levels of pre-existing antibodies against Xork are low or absent in normal human serum, compared with the moderate to high levels of pre-existing antibodies against IdeS.

[0280] The protease specifically and efficiently cleaves human IgG and exhibits low cross-reactivity with human serum. Xork has been shown in vitro to cleave human IgG with the same specificity and mechanism as IdeS. The in vivo activity of Xork was optimized by engineering an Fc fusion protein to extend its half-life. The nearly complete (up to 97%) inhibition of AAV transduction in vivo by passive transfer of human serum containing pre-existing anti-AAV antibodies was efficiently prevented by treatment with Xork-Fc. Combining Xork-Fc with ImmTOR tolerogenic nanoparticles prevented the de novo formation of anti-Xork antibodies, allowing for rechallenge with Xork.

[0281] Thus, as provided herein, synthetic nanocarriers comprising an Ig protease fusion protein (e.g., Xork-Fc) plus an immunosuppressant (e.g., ImmTOR) can address two major challenges in gene therapy: 1) increasing the number of patients eligible for gene therapy by reducing pre-existing anti-AAV antibodies, and 2) reducing the de novo formation of anti-AAV antibodies, thereby enabling re-medication. Thus, provided herein is a method of administering both a synthetic nanocarrier comprising an immunosuppressant as provided herein and an Ig protease (e.g., IgG protease) fusion protein to a subject who is also receiving viral vector therapy. Related compositions are also provided.

Claims

1. below: (i) an Ig protease domain, and (ii) an Fc domain; 1. A composition comprising an Ig protease fusion protein comprising: Optionally, wherein the Ig protease fusion protein has a longer circulating half-life compared to a naturally occurring Ig protease. The composition.

2. below: (i) an Ig protease domain, and (ii) albumin proteins; 1. A composition comprising an Ig protease fusion protein comprising: Optionally, wherein the Ig protease fusion protein has a longer circulating half-life compared to a naturally occurring Ig protease. The composition.

3. 3. The composition of claim 1 or 2, wherein the Ig protease fusion protein binds to a region of a target immunoglobulin and wherein the Ig protease fusion protein cleaves the target immunoglobulin.

4. 4. The composition of claim 3, wherein the Ig protease domain cleaves the target immunoglobulin at the hinge region of the target immunoglobulin.

5. The composition of claim 3 or 4, wherein the target immunoglobulin is IgG or IgA.

6. The composition of any one of claims 1 and 3 to 5, wherein the Ig protease domain is from an Ig protease from a bacterial strain.

7. The composition of claim 6 , wherein the bacterial strain is a Streptococcus bacterial strain.

8. The composition of claim 7, wherein the streptococcal bacterial strain is Streptococcus pyogenes.

9. 9. The composition of claim 8, wherein the streptococcal bacterial strain is Streptococcus equii.

10. The composition of claim 6 , wherein the bacterial strain is a Mycoplasma strain.

11. The composition of any one of claims 1 and 3 to 5, wherein the Ig protease domain is from an Ig protease from Streptococcus kroesus.

12. The composition of any one of claims 1 and 3 to 5, wherein the Ig protease domain is that of the IdeS protease.

13. The composition of any one of claims 1 and 3 to 5, wherein the Ig protease domain is that of the IdeZ protease.

14. The composition of any one of claims 1 and 3 to 5, wherein the Ig protease domain is that of the IdeMC protease.

15. The composition of any one of claims 1 and 3 to 5, wherein the Ig protease domain is that of IdeSORK.

16. The composition of any one of claims 1 and 3 to 15, wherein the Fc domain is a mouse Fc domain.

17. The composition of any one of claims 1 and 3 to 15, wherein the Fc domain is a human Fc domain.

18. The composition of any one of claims 1 and 3 to 17, wherein the Fc domain is an IgG Fc domain.

19. 19. The composition of claim 18, wherein the Fc domain is selected from IgG1, IgG2, IgG3, or IgG4.

20. The composition of any one of claims 1 to 19, wherein the Fc domain further comprises a hinge region and a CH2 domain.

21. 21. The composition of any one of claims 1 to 20, wherein the Fc domain is mutated to be resistant to proteolysis by Ig proteases.

22. 22. The composition of any one of claims 1 to 21, wherein the Fc domain is mutated near the boundary between the hinge region and the CH2 domain.

23. 23. The composition of any one of claims 1 to 22, wherein the hinge domain is mutated to render it resistant to proteolysis by Ig proteases.

24. 24. The composition of any one of claims 1 to 23, wherein the Fc domain has one or more of reduced or ablated Fc effector function, reduced or ablated complement fixation, and / or enhanced binding to FcRn.

25. 25. The composition of any one of claims 1 to 24, wherein the Ig protease domain comprises any one of the Ig protease sequences provided herein or a fragment thereof.

26. 26. The composition of any one of claims 1 and 3-25, wherein the Fc domain is any one of the Fc domains provided herein or a fragment thereof that interacts with an Fc receptor.

27. 27. The composition of any one of claims 1 and 3 to 26, wherein the Ig protease fusion is in monomeric form.

28. 27. The composition of any one of claims 1 and 3 to 26, wherein the Ig protease fusion is in a dimeric form, such as a homodimeric form.

29. 27. The composition of any one of claims 1 and 3 to 26, wherein the Ig protease fusion is in the KIH form.

30. An Ig protease fusion protein comprising any one of the sequences provided herein.

31. A method for producing an Ig protease fusion protein according to any one of claims 1 to 30.

32. 31. A method of administering any one of the Ig protease fusion proteins of any one of claims 1 to 30 to a subject in need thereof, such as a subject having an autoimmune disease, an immunological disorder, GVHD, or who has undergone or is about to undergo a transplant.

33. 31. A method of administering any one of the Ig protease fusion proteins of any one of claims 1 to 30 to a subject in need thereof, such as a subject receiving or to be administered a therapeutic biological.

34. 34. The method of claim 33, wherein the Ig protease fusion protein and the therapeutic biologic are administered concomitantly.

35. 35. The method of claim 33 or 34, wherein the therapeutic biological is a therapeutic polynucleotide or a therapeutic protein.

36. 36. The method of claim 35, wherein the therapeutic polynucleotide is a viral vector.

37. 37. The method of claim 36, wherein the viral vector is an AAV viral vector.

38. The method of any one of claims 33 to 37, wherein a synthetic nanocarrier comprising an immunosuppressant is also administered to the subject.

39. 39. The method of claim 38, wherein a synthetic nanocarrier comprising an immunosuppressant is administered in combination with the Ig protease fusion protein and / or therapeutic biologic.

40. 40. The method of any one of claims 33-39, wherein the administration of the Ig protease fusion protein and / or therapeutic biological is repeated.

41. The method of claim 40, wherein the administration of the synthetic nanocarrier comprising the immunosuppressant is repeated.

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