Patent Application
20210100911
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 20, 2019, is named 40566-722_301_SL.txt and is 835,387 bytes in size.
The gut epithelium has thwarted efforts to orally administer large therapeutic molecules such as proteins because proteins cannot diffuse across the intact epithelial barrier or cross the barrier through the tight junctions. Once taken up by an epithelial cell, a therapeutic protein either enters the destructive lysosomal trafficking pathway or is released back into the intestinal lumen. This inability to be readily transported across the intestinal epithelium continues to be a limiting factor in developing commercially viable oral formulations, particularly for polypeptide-based therapeutics. A common solution is to use parenteral administration such as intravenous or subcutaneous administration, but these administration routes can often create considerable side effects, lower the therapeutic efficacy, and reduce patient convenience that can negatively affect compliance. There is a need for improved compositions and methods for transporting therapeutics into or across an epithelium, e.g., a gut epithelium.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
In one aspect, the present disclosure provides a carrier-payload complex comprising a carrier capable of endocytosing into a polarized epithelial cell and accumulating in a region of the cell, wherein the payload is heterologous to the carrier. In some embodiments, the region is an apical compartment, a supranuclear compartment, or a basal compartment. In some embodiments, the carrier is retained in the region for at least 5 mins, 10 mins, or 15 minutes in the region. In some embodiments, the carrier is derived from a Cholix polypeptide. In some embodiments, the carrier is a polypeptide having a Cholix sequence with a C-terminus at any one of positions 150-195. In some embodiments, the carrier is a polypeptide having a Cholix sequence with an N-terminus at any one of positions 1-41. In some embodiments, the carrier is a polypeptide having a Cholix sequence with an N-termination truncation at any one of positions 35-40. In some embodiments, the carrier is a polypeptide having a Cholix sequence with a C-terminus at any one of positions 150-205. In some embodiments, the carrier consists of the amino acid residues from the N-terminal position 40 to any one of the C-terminal positions 150-205 of the sequence set forth in SEQ ID NO: 130. In some embodiments, the carrier has a C-terminus at positions 150 or 187 of the sequence set forth in SEQ ID NO: 130. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NO: 137. In some embodiments, the carrier consists of the amino acid sequence set forth in any one of SEQ ID NOs: 137-139. In some embodiments, position numbering is based on alignment of the Cholix polypeptide to the sequence set forth in SEQ ID NO: 130, wherein positions are numbered from an N-terminus to a C-terminus starting with position 1 at the N-terminus. In some embodiments, the carrier is capable of remaining associated with an apical entry receptor longer following endocytosis of the carrier into the polarized epithelial cell than a carrier capable of transcytosing across the polarized epithelial cell. In some embodiments, the apical entry receptor is a TMEM132 receptor. In some embodiments, the polarized epithelial cell comprises a gastrointestinal polarized epithelial cell.
In one aspect, the present disclosure provides a carrier-payload complex comprising (i) a carrier derived from a Cholix polypeptide having a C-terminus at any of positions 195-347 and capable of transcytosing across a polarized epithelial cell, coupled to (ii) a heterologous payload. In some embodiments, position numbering is based on alignment of the Cholix polypeptide to the sequence set forth in SEQ ID NO: 130, wherein positions are numbered from an N-terminus to a C-terminus starting with position 1 at the N-terminus. In some embodiments, the C-terminus is at any one of positions 195-266. In some embodiments, the C-terminus is at any one of the positions 195-266 of any one of the sequences set forth in SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminus is at any one of positions 206, 245, 251, or 266 a sequence set forth in SEQ ID NO: 130. In some embodiments, the C-terminus is at any one of positions 206, 245, 251, or 266 of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminus is at position 206 of any one of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NOs: 131 or 184. In some embodiments, the C-terminus is at position 245 of any one of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NOs: 132 or 183. In some embodiments, the C-terminus is at position 251 of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NOs: 133 or 182. In some embodiments, the C-terminus is at position 266 of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NOs: 134 or 181. In some embodiments, the carrier consists of the sequence set forth in SEQ ID NOs: 1-2, or 4-78 with truncation at any one of the positions 195-347, and has no more than 5, 4, 3, 2, or 1 amino acid variations at any of positions 1-40, 133-151, 152-187, or 188-206 of SEQ ID NOs: 1-2, or 4-78.
In one aspect, the present disclosure provides a carrier-payload complex comprising (i) a carrier derived from a Cholix polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of endocytosis into a polarized epithelial cell or transcytosis of a polarized epithelial cell, coupled to (ii) a heterologous payload. In some embodiments, the carrier comprises a glutamic acid at position 3 and an alanine at position 4. In some embodiments, the carrier is non-toxic. In some embodiments, the carrier is capable of transcytosing the heterologous payload across a polarized epithelial cell. In some embodiments, the carrier comprises a fragment capable of transcytosis of a polarized epithelial cell, wherein the carrier has a C-terminus at any one of the positions 195 to a C-terminal residue of the sequence set forth in any one of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminus is at any one of the positions 195-386 of the sequence set forth in any one of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminus is at position 386 of any one of the sequences set forth in SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminus is at position 386 of any one of the sequences set forth in SEQ ID NOs: 1-2. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NO: 135.
In one aspect, the present disclosure provides a carrier-payload complex comprising (i) a carrier derived from a Cholix polypeptide that does not comprise SEQ ID NO: 179, and does not consist of SEQ ID NO: 126, complexed with (ii) a heterologous payload, wherein the carrier is capable of (a) transcytosing the heterologous payload across a polarized epithelial cell; or (b) transporting the heterologous payload into the polarized epithelial cell. In some embodiments, the carrier comprises at least 75% sequence identity to a sequence set forth in SEQ ID NO: 130, or fragment thereof. In some embodiments, the carrier comprises at least 90% sequence identity to a Cholix variant set forth in SEQ ID NO: 130, or a fragment thereof. In some embodiments, the Cholix polypeptide is a sequence set forth in SEQ ID NO: 130, or fragment thereof. In some embodiments, the carrier comprises a glutamic acid at position 3 and an alanine at position 4. In some embodiments, the carrier is non-toxic. In some embodiments, the carrier is capable of transcytosing the heterologous payload across the polarized epithelial cell. In some embodiments, the carrier has a C-terminal truncation at any one of the positions 195-633 of the sequence set forth in SEQ ID NO: 130. In some embodiments, the C-terminal truncation is at any one of the positions 195-386 of the sequence set forth in SEQ ID NO: 130. In some embodiments, the carrier has a C-terminal truncation at any one of the positions 195-386 of any one of the sequences set forth in SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminal truncation is at position 386 of any one of the sequences set forth in SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminal truncation is at position 386 of any one of the sequences set forth in SEQ ID NOs: 1-2. In some embodiments, the carrier-payload complex comprises an N-terminal methionine. In some embodiments, the carrier is synthetically conjugated to the heterologous payload. In some embodiments, the carrier is genetically fused to the heterologous payload. In some embodiments, the heterologous payload is a therapeutic payload. In some embodiments, the therapeutic payload is a cytokine, an antibody, a hormone, or a nucleic acid. In some embodiments, the therapeutic payload is a cytokine. In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is an IL-10. In some embodiments, the IL-10 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 145. In some embodiments, the interleukin is an IL-22. In some embodiments, the IL-22 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 142. In some embodiments, the therapeutic payload is an antibody. In some embodiments, the antibody is an anti-TNF antibody. In some embodiments, the therapeutic payload is a hormone. In some embodiments, the therapeutic payload is a human growth hormone. In some embodiments, the heterologous payload is covalently coupled to the carrier. In some embodiments, the heterologous payload is coupled to a C-terminus of the carrier. In some embodiments, the heterologous payload is coupled to an N-terminus of the carrier. In some embodiments, the carrier is coupled to the heterologous payload via a spacer. In some embodiments, the spacer is a non-cleavable spacer. In some embodiments, the spacer comprises between 1 and 100 amino acid residues. In some embodiments, the spacer comprises up to 15 repeats of GS (SEQ ID NO: 169), GGS (SEQ ID NO: 170), GGGS (SEQ ID NO: 171), GGGGS (SEQ ID NO: 172), GGGGGS (SEQ ID NO: 173), or a combination thereof. In some embodiments, the spacer comprises an amino acid sequence set forth in SEQ ID NO: 175. In some embodiments, the spacer consists of the amino acid sequence set forth in SEQ ID NO: 176. In some embodiments, the heterologous payload is non-covalently coupled to the carrier. In some embodiments, the heterologous payload is complexed to the carrier via a nanoparticle.
In one aspect, the present disclosure provides a method of transcytosing a heterologous payload across a polarized epithelial cell, comprising: (a) contacting an apical membrane of the polarized epithelial cell with a carrier-payload complex; and (b) transcytosing the carrier-payload complex across the polarized epithelial cell, wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the polarized epithelial cell, coupled to the heterologous payload. In some embodiments, contacting the apical membrane of the polarized epithelial cell with the carrier-payload complex comprises interacting of the carrier with the apical entry receptor TMEM132. In some embodiments, interacting of the carrier with the membrane protein TMEM132 results in receptor-mediated endocytosis of the carrier-payload complex. In some embodiments, the carrier that interacts with TMEM132 comprises the amino acid residues 135-151 of SEQ ID NO: 130, or a sequence having at least 90% sequence identity thereto. In some embodiments, the transcytosing of the carrier-payload complex across the polarized epithelial cell comprises interacting of the carrier with any one or more of GRP75, ERGIC-53, and perlecan. In some embodiments, the transcytosing of the carrier-payload complex across the polarized epithelial cell further comprises co-localization of the carrier-payload complex with any one or more of COPI, EEA1, and Rab7 at the apical side, and with Rab11a at the basal side of the epithelial cell. In some embodiments, the carrier that interacts with GRP7 or ERGIC-53 comprises the amino acid residues 1-40 and 152-187 of SEQ ID NO: 130, or a sequence having at least 90% sequence identity thereto. In some embodiments, the carrier that interacts with perlecan comprises the amino acid residues 188-205 of SEQ ID NO: 130, or a sequence having at least 90% sequence identity thereto. In some embodiments, the method can further comprise, subsequent to (b) delivering the carrier-payload complex into the Lamina propria. In some embodiments, the polarized epithelial cell is a polarized gut epithelial cell.
In one aspect, the present disclosure provides a method of orally delivering a heterologous payload to a subject, comprising: orally administering a carrier-payload complex to the subject, wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby delivering the heterologous payload to the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the epithelium, coupled to the heterologous payload.
In one aspect, the present disclosure provides a carrier-payload complex for orally delivering a heterologous payload to a subject by a method comprising: orally administering the carrier-payload complex to the subject, wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby treating a disease the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the epithelium, coupled to the heterologous payload.
In one aspect, the present disclosure provides a use of a carrier-payload complex for orally delivering a heterologous payload to a subject by a method comprising: orally administering the carrier-payload complex to the subject; wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby treating a disease in the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the epithelium, coupled to the heterologous payload.
In one aspect, the present disclosure provides a method of treating a disease in a subject, comprising: orally administering a carrier-payload complex to the subject, wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby treating a disease in the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the epithelium, coupled to the heterologous payload.
In one aspect, the present disclosure provides a carrier-payload complex for use in treating a disease in a subject by a method comprising: orally administering the carrier-payload complex to the subject, wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby treating the disease in the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence e set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the epithelium, coupled to the heterologous payload.
In one aspect, the present disclosure provides a use of a carrier-payload complex in the manufacture of a medicament for treating a disease in a subject by a method comprising: orally administering the carrier-payload complex to the subject, wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby treating the disease in the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the epithelium, coupled to the heterologous payload. In some embodiments, the method further comprises binding of the heterologous payload to a receptor in the Lamina propria. In some embodiments, the method further comprises delivering the heterologous payload into systemic circulation. In some embodiments, the carrier is a Cholix derived polypeptide. In some embodiments, the carrier comprises amino acid residues 1-206, 1-245, 1-251, 1-266, or 1-386 of the sequence set forth in SEQ ID NO: 130. In some embodiments, the carrier comprises amino acid residues 1-206, 1-245, 1-251, 1-266, or 1-386 of the sequence set forth in SEQ ID NOs: 1-2. In some embodiments, the carrier comprises any one of the amino acid sequences set forth in SEQ ID NOs: 131-135 or 180-184. In some embodiments, the polarized epithelium is a polarized gut epithelium. In some embodiments, the disease is ulcerative colitis, pouchitis, proctitis, Crohn's disease, Multiple sclerosis (MS), Systemic lupus erythematosus (SLE), Graft versus host disease (GVHD), Rheumatoid arthritis, or Psoriasis. In some embodiments, the disease is ulcerative colitis. In some embodiments, the ulcerative colitis is mild-to-moderate or moderate to severe. In some embodiments, the disease is Crohn's disease. In some embodiments, the Crohn's disease is Fistulizing Crohn's disease. In some embodiments, the payload is an interleukin. In some embodiments, the interleukin comprises the amino acid sequence of SEQ ID NOs: 142 or 145.
In one aspect, the present disclosure provides a method of transporting a heterologous payload into a polarized epithelial cell, comprising: (a) contacting the apical membrane of the polarized epithelial cell with a carrier-payload complex; and (b) transporting the carrier-payload complex into the polarized epithelial cell, wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transporting the carrier-payload complex into the epithelial cell, coupled to the heterologous payload. In some embodiments, contacting the apical membrane of the polarized epithelial cell with the carrier-payload complex comprises interacting of the carrier with the apical entry receptor TMEM132. In some embodiments, interacting of the carrier with the apical entry receptor TMEM132 results in receptor-mediated endocytosis of the carrier-payload complex. In some embodiments, the method further comprises transporting the heterologous payload to an apical compartment or a basal compartment. In some embodiments, the carrier of the carrier-payload complex remains associated with TMEM132 after endocytosis. In some embodiments, the carrier that interacts with TMEM132 comprises amino acid residues 135-151 of SEQ ID NO: 130, or a sequence having at least 90% sequence identity thereto. In some embodiments, the carrier is a Cholix derived polypeptide. In some embodiments, the carrier consists of amino acid residues 1-151, 1-187, 41-187, or 40-205 of a sequence set forth in SEQ ID NO: 130. In some embodiments, the carrier consists of amino acid residues 1-151, 1-187, 41-187, or 40-205 of the sequence set forth in SEQ ID NOs: 1-2. In some embodiments, the carrier consists of any one of the amino acid sequences set forth in SEQ ID NOs: 136-139. In some embodiments, the carrier is non-covalently coupled to the heterologous payload via a nanoparticle. In some embodiments, a ratio of the heterologous payload to the carrier on the nanoparticle is at least 15,000:1. In some embodiments, the heterologous payload is a glucose-lowering agent. In some embodiments, the glucose-lowering agent is non-covalently associated with the nanoparticle. In some embodiments, the heterologous payload is an siRNA. In some embodiments, the siRNA is non-covalently associated with the nanoparticle. In some embodiments, the carrier is covalently-linked to the nanoparticle or is spray-dried on the nanoparticle.
In one aspect, the present disclosure provides a method comprising transporting a heterologous payload into a polarized epithelial cell, comprising: (a) contacting the apical membrane of the polarized epithelial cell with a carrier-payload complex; and (b) transporting the carrier-payload complex into the polarized epithelial cell, wherein the carrier-payload complex is the carrier-payload complex comprising any of the endocytosing carriers herein.
In one aspect, the disclosure includes a carrier-payload complex comprising a carrier capable of accumulating in an apical compartment of a polarized epithelial cell at least 5 minutes after endocytosis of the carrier, coupled to a heterologous payload.
In some embodiments, the carrier is capable of accumulating in the apical compartment of the polarized epithelial cell at least 10 minutes after endocytosis of the carrier. In some embodiments, the carrier is capable of accumulating in the apical compartment of the polarized epithelial cell at least 15 minutes after endocytosis of the carrier.
In some embodiments, the polarized epithelial cell comprises a gastrointestinal polarized epithelial cell. In some embodiments, the polarized epithelial cell comprises a rat gastrointestinal polarized epithelial cell.
In another aspect, the disclosure includes a carrier-payload complex comprising a carrier capable of accumulating in an apical compartment of a polarized epithelial cell at least 5 minutes after intraluminal application of the carrier to a gastrointestinal tract of a mammal, coupled to a heterologous payload.
In some embodiments, the carrier is capable of accumulating in the apical compartment of the polarized epithelial cell at least 10 minutes after intraluminal application of the carrier to the gastrointestinal tract of the mammal. In some embodiments, the carrier is capable of accumulating in the apical compartment of polarized epithelial cell at least 15 minutes after intraluminal application of the carrier to the gastrointestinal tract carrier of the mammal.
In some embodiments, the intraluminal application comprises intraluminal injection into a rat jejunum.
In another aspect, the disclosure includes a carrier-payload complex comprising a carrier capable of accumulating in a supranuclear compartment of a polarized epithelial cell at least 5 minutes after endocytosis of the carrier, coupled to a heterologous payload.
In some embodiments, the carrier is capable of accumulating in the supranuclear compartment of the polarized epithelial cell at least 10 minutes after endocytosis of the carrier. In some embodiments, the carrier is capable of accumulating in the supranuclear compartment of the polarized epithelial cell at least 15 minutes after endocytosis of the carrier.
In some embodiments, the polarized epithelial cell comprises a gastrointestinal polarized epithelial cell. In some embodiments, the polarized epithelial cell comprises a rat gastrointestinal polarized epithelial cell.
In another aspect, the disclosure includes a carrier-payload complex comprising a carrier capable of accumulating in a supranuclear compartment of a polarized epithelial cell at least 5 minutes after intraluminal application of the carrier to a gastrointestinal tract of a mammal, coupled to a heterologous payload.
In some embodiments, the carrier is capable of accumulating in the supranuclear compartment of the polarized epithelial cell at least 10 minutes after intraluminal application of the carrier to the gastrointestinal tract of the mammal. In some embodiments, the carrier is capable of accumulating in the supranuclear compartment of polarized epithelial cell at least 15 minutes after intraluminal application of the carrier to the gastrointestinal tract carrier of the mammal.
In some embodiments, the intraluminal application comprises intraluminal injection into a rat jejunum.
In another aspect, the disclosure includes a carrier-payload complex comprising a carrier capable of accumulating in a basal compartment of a polarized epithelial cell at least 5 minutes after endocytosis of the carrier, coupled to a heterologous payload.
In some embodiments, the carrier is capable of accumulating in the basal compartment of the polarized epithelial cell at least 10 minutes after endocytosis of the carrier. In some embodiments, the carrier is capable of accumulating in the basal compartment of the polarized epithelial cell at least 15 minutes after endocytosis of the carrier.
In some embodiments, the polarized epithelial cell comprises a gastrointestinal polarized epithelial cell. In some embodiments, the polarized epithelial cell comprises a rat gastrointestinal polarized epithelial cell.
In another aspect, the disclosure includes a carrier-payload complex comprising a carrier capable of accumulating in a basal compartment of a polarized epithelial cell at least 5 minutes after intraluminal application of the carrier to a gastrointestinal tract of a mammal, coupled to a heterologous payload.
In some embodiments, carrier is capable of accumulating in the basal compartment of the polarized epithelial cell at least 10 minutes after intraluminal application of the carrier to the gastrointestinal tract of the mammal. In some embodiments, the carrier is capable of accumulating in the basal compartment of polarized epithelial cell at least 15 minutes after intraluminal application of the carrier to the gastrointestinal tract carrier of the mammal.
In some embodiments, the intraluminal application comprises intraluminal injection into a rat jejunum.
In another aspect, the disclosure includes a carrier-payload complex comprising. a carrier derived from a Cholix polypeptide having a C-terminus at any of positions 195-347, coupled to a heterologous payload. In some embodiments, the position numbering is based on alignment of the Cholix polypeptide to the sequence set forth in SEQ ID NO: 130, wherein positions are numbered from an N-terminus to a C-terminus starting with position 1 at the N-terminus.
In some embodiments, the C-terminal truncation is at any one of the positions 195-266 of a sequence set forth in SEQ ID NO: 130. In some embodiments, the C-terminal truncation is at any one of the positions 195-266 of any one of the sequences set forth in SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminal truncation is at any one of positions 206, 245, 251, or 266 a sequence set forth in SEQ ID NO: 130. In some embodiments, the C-terminal truncation is at any one of positions 206, 245, 251, or 266 of SEQ ID NOs: 1-2, or 4-78.
In some embodiments, the C-terminal truncation is at position 206 of any one of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NOs: 131 or 184. In some embodiments, the C-terminal truncation is at position 245 of any one of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NOs: 132 or 183. In some embodiments, the C-terminal truncation is at position 251 of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NOs: 133 or 182. In some embodiments, the C-terminal truncation is at position 266 of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NOs: 134 or 181. In some embodiments, the carrier consists of the sequence set forth in SEQ ID NOs: 1-2, or 4-78 with truncation at any one of the positions 195-347, and has no more than 5, 4, 3, 2, or 1 amino acid variations at any of positions 1-40, 133-151, 152-187, or 188-206 of SEQ ID NOs: 1-2, or 4-78.
In another aspect, the disclosure includes a carrier-payload complex comprising a carrier derived from a Cholix polypeptide having an N-terminus at positions 1-41 and a C-terminus at positions 150-195, or consisting of the amino acid residues from any one of the N-terminal positions 35-40 to any one of the C-terminal positions 150-205 of the sequence set forth in SEQ ID NO: 130, coupled to a heterologous payload. In some embodiments, the position numbering is based on alignment of the Cholix polypeptide to the sequence set forth in SEQ ID NO: 130, wherein positions are numbered from an N-terminus to a C-terminus starting with position 1 at the N-terminus.
In some embodiments, the carrier is capable of remaining associated with an apical entry receptor following endocytosis of the carrier into a polarized epithelial cell. In some embodiments, the apical entry receptor is a TMEM132 receptor (e.g., TMEM132A).
In some embodiments, the carrier consists of the amino acid residues from the N-terminal position 40 to any one of the C-terminal positions 150-205 of the sequence set forth in SEQ ID NO: 130. In some embodiments, the carrier has a C-terminal truncation at positions 150 or 186 of the sequence set forth in SEQ ID NO: 130.
In some embodiments, the carrier is capable of transporting the payload to a basal compartment or a supranuclear compartment in a polarized epithelial cell. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the carrier is capable of transporting the payload to an apical compartment in a polarized epithelial cell.
In some embodiments, the carrier consists of the amino acid sequence set forth in any one of SEQ ID NOs: 137-139.
In another aspect, the disclosure includes a carrier-payload complex comprising a derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of endocytosis into a polarized epithelial cell or transcytosis of a polarized epithelial cell, coupled to a heterologous payload.
In some embodiments, the carrier comprises a glutamic acid at position 3 and an alanine at position 4.
In some embodiments, the carrier is non-toxic.
In some embodiments, the carrier is capable of transcytosing the heterologous payload across a polarized epithelial cell.
In some embodiments, the carrier comprises a fragment capable of transcytosis of a polarized epithelial cell, wherein the carrier has a C-terminal truncation at any one of the positions 195 to a C-terminal residue of the sequence set forth in any one of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminal truncation is at any one of the positions 195-386 of the sequence set forth in any one of SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminal truncation is at position 386 of any one of the sequences set forth in SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminal truncation is at position 386 of any one of the sequences set forth in SEQ ID NOs: 1-2. In some embodiments, the carrier consists of the amino acid sequence set forth in SEQ ID NO: 135.
In another aspect, the disclosure includes a carrier-payload complex comprising a carrier derived from a Cholix polypeptide that does not comprise SEQ ID NO: 179, and does not consist of SEQ ID NO: 126, complexed with a heterologous payload, wherein the carrier is capable of transcytosing the heterologous payload across a polarized epithelial cell; or transporting the heterologous payload into the polarized epithelial cell.
In some embodiments, the carrier comprises at least 75% sequence identity to a sequence set forth in SEQ ID NO: 130, or fragment thereof. In some embodiments, the carrier comprises at least 90% sequence identity to a Cholix variant set forth in SEQ ID NO: 130, or a fragment thereof. In some embodiments, the Cholix polypeptide is a sequence set forth in SEQ ID NO: 130, or fragment thereof. In some embodiments, the carrier comprises a glutamic acid at position 3 and an alanine at position 4.
In some embodiments, the carrier is non-toxic.
In some embodiments, the carrier is capable of transcytosing the heterologous payload across the polarized epithelial cell.
In some embodiments, the carrier has a C-terminal truncation at any one of the positions 195-633 of the sequence set forth in SEQ ID NO: 130. In some embodiments, the C-terminal truncation is at any one of the positions 195-386 of the sequence set forth in SEQ ID NO: 130. In some embodiments, the carrier has a C-terminal truncation at any one of the positions 195-386 of any one of the sequences set forth in SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminal truncation is at position 386 of any one of the sequences set forth in SEQ ID NOs: 1-2, or 4-78. In some embodiments, the C-terminal truncation is at position 386 of any one of the sequences set forth in SEQ ID NOs: 1-2. In some embodiments, the carrier-payload complex comprises an N-terminal methionine.
In some embodiments, the carrier is synthetically conjugated to the heterologous payload. In some embodiments, the carrier is genetically fused to the heterologous payload.
In some embodiments, the heterologous payload is a therapeutic payload. In some embodiments, the therapeutic payload is a cytokine, an antibody, a hormone, or a nucleic acid. In some embodiments, the therapeutic payload is a cytokine. In some embodiments, the cytokine is an interleukin.
In some embodiments, the interleukin is an IL-10. In some embodiments, the IL-10 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 145. In some embodiments, the interleukin is an IL-22. In some embodiments, the IL-22 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 142.
In some embodiments, the therapeutic payload is an antibody. In some embodiments, the antibody is an anti-TNF antibody. In some embodiments, the therapeutic payload is a hormone. In some embodiments, the therapeutic payload is a human growth hormone.
In some embodiments, the heterologous payload is covalently coupled to the carrier. In some embodiments, the heterologous payload is coupled to a C-terminus of the carrier. In some embodiments, the heterologous payload is coupled to an N-terminus of the carrier. In some embodiments, the carrier is coupled to the heterologous payload via a spacer.
In some embodiments, the spacer is a non-cleavable spacer. In some embodiments, the spacer comprises between 1 and 100 amino acid residues. In some embodiments, the spacer comprises up to 15 repeats of GS (SEQ ID NO: 169), GGS (SEQ ID NO: 170), GGGS (SEQ ID NO: 171), GGGGS (SEQ ID NO: 172), GGGGGS (SEQ ID NO: 173), or a combination thereof. In some embodiments, the spacer comprises an amino acid sequence set forth in SEQ ID NO: 175. In some embodiments, the spacer consists of the amino acid sequence set forth in SEQ ID NO: 176.
In some embodiments, the heterologous payload is non-covalently coupled to the carrier. In some embodiments, the heterologous payload is complexed to the carrier via a nanoparticle.
In one aspect, the present disclosure includes a polynucleotide encoding any of the carrier-payload complexes (e.g., delivery constructs) described herein, e.g., those comprising, consisting essentially of, or consisting of any one of the amino acid sequences set forth in SEQ ID NOs: 147-150, 152-159 or 188.
In one aspect, the present disclosure includes a vector comprising any of such polynucleotides that encode a carrier-payload complex (e.g., delivery construct) of this disclosure.
In one aspect, the present disclosure includes a method of transcytosing a heterologous payload across a polarized epithelial cell, comprising: (a) contacting an apical membrane of the polarized epithelial cell with a carrier-payload complex; and (b) transcytosing the carrier-payload complex across the polarized epithelial cell, wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the polarized epithelial cell, coupled to the heterologous payload. In some embodiments, contacting the apical membrane of the polarized epithelial cell with the carrier-payload complex comprises interacting of the carrier with the apical entry receptor TMEM132. In some embodiments, interacting of the carrier with the membrane protein TMEM132 results in receptor-mediated endocytosis of the carrier-payload complex. In some embodiments, the carrier that interacts with TMEM132 comprises the amino acid residues 135-151 of SEQ ID NO: 130, or a sequence having at least 90% sequence identity thereto. In some embodiments, the transcytosing of the carrier-payload complex across the polarized epithelial cell comprises interacting of the carrier with any one or more of GRP75 (e.g., GRP75B), ERGIC-53, and perlecan. In some embodiments, the transcytosing of the carrier-payload complex across the polarized epithelial cell further comprises co-localization of the carrier-payload complex with any one or more of COPI, EEA1, and Rab7 at the apical side, and with Rab11a at the basal side of the epithelial cell. In some embodiments, the carrier that interacts with GRP7 or ERGIC-53 comprises the amino acid residues 1-40 and 152-187 of SEQ ID NO: 130, or a sequence having at least 90% sequence identity thereto. In some embodiments, the carrier that interacts with perlecan comprises the amino acid residues 188-205 of SEQ ID NO: 130, or a sequence having at least 90% sequence identity thereto. In some embodiments, the method further comprises, subsequent to (b) delivering the carrier-payload complex into the Lamina propria. In some embodiments, the polarized epithelial cell is a polarized gut epithelial cell.
In one aspect, the present disclosure includes a method of orally delivering a heterologous payload to a subject, comprising: orally administering a carrier-payload complex to the subject, wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby delivering the heterologous payload to the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the polarized epithelium, coupled to the heterologous payload.
In one aspect, the present disclosure includes a carrier-payload complex for orally delivering a heterologous payload to a subject by a method comprising: orally administering the carrier-payload complex to the subject, wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby treating a disease the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the polarized epithelium, coupled to the heterologous payload.
In one aspect, the present disclosure includes a use of a carrier-payload complex for orally delivering a heterologous payload to a subject by a method comprising: orally administering the carrier-payload complex to the subject; wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby treating a disease in the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the polarized epithelium, coupled to the heterologous payload.
In one aspect, the present disclosure includes a method of treating a disease in a subject, comprising: orally administering a carrier-payload complex to the subject, wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby treating a disease in the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the polarized epithelium, coupled to the heterologous payload.
In one aspect, the present disclosure includes a carrier-payload complex for use in treating a disease in a subject by a method comprising: orally administering the carrier-payload complex to the subject, wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby treating the disease in the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the polarized epithelium, coupled to the heterologous payload.
In one aspect, the present disclosure includes a use of a carrier-payload complex in the manufacture of a medicament for treating a disease in a subject by a method comprising: orally administering the carrier-payload complex to the subject, wherein the carrier is capable of transcytosing the carrier-payload complex across a polarized epithelium, thereby treating the disease in the subject, and wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transcytosing the carrier-payload complex across the polarized epithelium, coupled to the heterologous payload. In some embodiments, the method further comprises binding of the heterologous payload to a receptor in the Lamina propria. In some embodiments, the method further comprises delivering the heterologous payload into systemic circulation. In some embodiments, the carrier is a Cholix derived polypeptide. In some embodiments, the carrier comprises amino acid residues 1-206, 1-245, 1-251, 1-266, or 1-386 of a sequence set forth in SEQ ID NO: 130. In some embodiments, the carrier comprises amino acid residues 1-206, 1-245, 1-251, 1-266, or 1-386 of the sequence set forth in any one of SEQ ID NOs: 1-2. In some embodiments, the carrier comprises any one of the amino acid sequences set forth in SEQ ID NOs: 131-135 or 180-184. In some embodiments, the polarized epithelium is a polarized gut epithelium. In some embodiments, the disease is ulcerative colitis, pouchitis, proctitis, Crohn's disease, Multiple sclerosis (MS), Systemic lupus erythematosus (SLE), Graft versus host disease (GVHD), Rheumatoid arthritis, or Psoriasis. In some embodiments, the disease is ulcerative colitis. In some embodiments, the ulcerative colitis is mild-to-moderate or moderate to severe. In some embodiments, the disease is Crohn's disease. In some embodiments, the Crohn's disease is Fistulizing Crohn's disease. In some embodiments, the payload is an interleukin. In some embodiments, the interleukin comprises the amino acid sequence of SEQ ID NOs: 142 or 145.
In one aspect, the present disclosure includes a method of transporting a heterologous payload into a polarized epithelial cell, comprising: (a) contacting the apical membrane of the polarized epithelial cell with a carrier-payload complex; and (b) transporting the carrier-payload complex into the polarized epithelial cell, wherein the carrier-payload complex comprises a carrier derived from a Cholix polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 4-125, or 127-129, or a fragment thereof capable of transporting the carrier-payload complex into the epithelial cell, coupled to the heterologous payload. In some embodiments, contacting the apical membrane of the polarized epithelial cell with the carrier-payload complex comprises interacting of the carrier with the apical entry receptor TMEM132. In some embodiments, interacting of the carrier with the apical entry receptor TMEM132 results in receptor-mediated endocytosis of the carrier-payload complex. In some embodiments, the method further comprises transporting the heterologous payload to an apical compartment or a basal compartment. In some embodiments, the carrier of the carrier-payload complex remains associated with TMEM132 after endocytosis. In some embodiments, the carrier that interacts with TMEM132 comprises amino acid residues 135-151 of SEQ ID NO: 130, or a sequence having at least 90% sequence identity thereto. In some embodiments, the carrier is a Cholix derived polypeptide. In some embodiments, the carrier consists of amino acid residues 1-151, 1-187, 41-187, or 40-205 of a sequence set forth in SEQ ID NO: 130. In some embodiments, the carrier consists of amino acid residues 1-151, 1-187, 41-187, or 40-205 of the sequence set forth in SEQ ID NOs: 1-2. In some embodiments, the carrier consists of any one of the amino acid sequences set forth in SEQ ID NOs: 136-139. In some embodiments, the carrier is non-covalently coupled to the heterologous payload via a nanoparticle. In some embodiments, a ratio of the heterologous payload to the carrier on the nanoparticle is at least 15,000:1. In some embodiments, the heterologous payload is a glucose-lowering agent. In some embodiments, the glucose-lowering agent is non-covalently associated with the nanoparticle. In some embodiments, the heterologous payload is an siRNA. In some embodiments, the siRNA is non-covalently associated with the nanoparticle. In some embodiments, the carrier is covalently-linked to the nanoparticle or is spray-dried on the nanoparticle.
Various features of the present disclosure are set forth with particularity in the appended claims. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative aspects, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:
Provided herein, in certain embodiments, are delivery constructs (e.g., carrier-payload complex) capable of transporting one or more heterologous payload molecules (e.g., one or more therapeutic payloads) into epithelial cells (e.g., polarized gut epithelial cells), e.g., by endocytosis, or across epithelial cells (e.g., polarized gut epithelial cells) by, e.g., by transcytosis. The delivery constructs can comprise a carrier that is coupled to the heterologous payload. The carrier can be capable of transporting the heterologous payload into or across epithelial cells using endogenous trafficking pathways. Utilization of endogenous trafficking pathways, as opposed to use of passive diffusion, can allow the carrier to shuttle the heterologous payload rapidly (e.g., at least 10−6 cm/sec, 10−5 cm/sec) and efficiently (e.g., at least 5%, 10%, 20%, 25%, or 50% of material applied to the apical surface) into or across epithelial cells without impairing the barrier function of these cells or the biological activity of the heterologous payload.
The carrier portion of a delivery construct provided herein can be any molecule (e.g., small molecule, polypeptide, nucleic acid, etc.) capable of increasing the rate and/or amount of a heterologous payload (e.g., a therapeutic payload) delivered into and/or across an epithelium.
A carrier herein can have numerous attributes. In some embodiments, a carrier herein can have a reduced (e.g., at least 50% reduced) or ablated ADP ribosylation activity (e.g., ribosylation of elongation factor 2) relative to a naturally occurring Cholix polypeptide such as SEQ ID NO: 3.
In some embodiments, a carrier herein utilizes an endogenous trafficking pathway to transport a heterologous payload coupled thereto across a polarized epithelial cell. Such carrier can be referred to herein as a transcytosing carrier. In some instances, a carrier herein can utilize an endogenous trafficking pathway to transport a heterologous payload coupled thereto into a polarized epithelial cell. Such carrier can be referred to herein as an endocytosing carrier. Within endocytosing carriers, there can be carriers that deliver a payload coupled thereto into specific regions within the polarized epithelial cells such as an apical compartment, a supranuclear compartment, or a basal compartment.
Any of the carriers herein can transport molecules coupled thereto by interacting and/or co-localizing with one or more endogenous proteins of such epithelium. The one or more endogenous proteins can be receptors or enzymes capable of moving a carrier into or across the epithelial cell. Interacting and/or co-localizing with the one or more endogenous proteins of the epithelial cell can provide a carrier with one or more functions, including endocytosis into the epithelial cell, avoidance of a lysosomal destruction pathway, trafficking from an apical compartment to a basal compartment, and/or exocytosis from the basal membrane of the epithelial cell into a submucosal compartment such as the Lamina propria.
An interaction of such carrier with an endogenous protein can be a selective interaction. Such selective interaction can be a pH-dependent interaction. In instances where a carrier interacts with two or more endogenous proteins, such interactions can be sequential interactions where a first interacting protein hands the carrier off to a second interacting protein. Such sequential interactions can occur at a different pH (e.g., pH 5.5, 7.0, 7.5, etc.). An interaction between a carrier and an endogenous protein can be a covalent or non-covalent interaction. Non-covalent interactions include hydrogen bonding, van der Waals interactions, ionic bonds, π-π-interactions, etc.
In some instances, one of the endogenous proteins that a carrier can interact with can be an apical entry receptor. Such apical entry receptor can be a transmembrane protein 132 (TMEM132). Interaction of a carrier with such apical entry receptor can enable the carrier to enter the epithelial cell through receptor-mediated endocytosis.
A carrier can also interact with a lysosome avoidance receptor. Such interaction with a lysosome avoidance receptor can occur inside the epithelial cell and subsequent to endocytosis. A lysosome avoidance receptor can be a glucose-regulated protein 75 (GRP75, e.g., GRP75B). Interaction of a carrier with such lysosome avoidance receptor can enable the carrier to avoid or circumvent lysosomal degradation. Such ability can allow a carrier to significantly reduce the amount of payload coupled to the carrier reaching a lysosome of a cell, a fate that most therapeutic proteins face once taken up by the gut epithelium.
Furthermore, a carrier can interact with an apical to basal trafficking protein. Such interaction can occur inside the epithelial cell and subsequent to endocytosis. Such apical to basal trafficking protein can be an endoplasmic reticulum Golgi intermediate compartment (ERGIC) protein, such as ERGIC-53. Interaction of a carrier with an ERGIC protein can enable the carrier to move from an apical compartment to a supranuclear compartment or a basal compartment.
A transcytosing carrier can also interact with a basal release protein capable of promoting exocytosis of a carrier from a basal site of an epithelial cell. Such interaction can occur at the basal site of an epithelial cell and subsequent to moving from an apical compartment to a basal compartment. Such basal release protein can be perlecan (also referred to herein as basement membrane-specific heparan sulfate proteoglycan core protein or HSPG). Interaction of a carrier with perlecan can enable the carrier to access a basal recycling system that allows release of the carrier from the basal compartment into a submucosal compartment such as the Lamina propria.
Thus, a transcytosing carrier herein can be a molecule that is capable of interacting with the endogenous proteins TMEM132 (e.g., TMEM132A), GRP75 (e.g., GRP75B), ERGIC (e.g., ERGIC-53), and perlecan (HSPG), enabling such carrier to transport a payload molecule coupled thereto across a polarized epithelium, e.g., a polarized gut epithelium.
An endocytosing carrier herein can be a molecule that is capable of interacting with the endogenous protein TMEM132, allowing apical entry of such carrier. An endocytosing carrier can remain associated with TMEM132 after endocytosis (e.g., compared to a transcytosing carrier that can dissociate from TMEM132 after endocytosis to interact with, e.g., GRP75 or an ERGIC protein) and within apical regions and compartments of the cell (e.g., a polarized epithelial cell). In some cases, such endocytosing carrier can also interact with GRP75. Such interactions with TMEM132 and/or GRP75 can allow the carrier and a payload coupled thereto to avoid, or at least significantly reduce (e.g., less than about 50% compared to the payload molecule when it is not coupled to the carrier), lysosomal degradation. In some instances, an endocytosing carrier can remain in an apical compartment, and not show significant translocation to a basal compartment, for, e.g., at least about 5, 10, 15, 30, 60, or 120 minutes after apical (e.g., luminal) application of the carrier compared to a transcytosing carrier that can show complete transcytosis of nearly all apically applied molecules, e.g., about 5, 10, 15 or 30 minutes after apical (e.g., luminal) application. In some instances, at least about 50%, 75%, or 90% of carrier molecules remain in apical compartments 5 minutes after luminal application of the carrier. In some instances, at least about 50%, 75%, or 90% of carrier molecules remain in apical compartments 10 minutes after luminal application of the carrier. In some instances, at least about 50%, 75%, or 90% of carrier molecules remain in apical compartments 15 minutes after luminal application of the carrier. In some instances, at least about 50%, 75%, or 90% of carrier molecules remain in apical compartments 30 minutes after luminal application of the carrier. The percentage of carrier molecules that remain in the apical compartment of the epithelial cell can be determined by dividing the intensity of the fluorescence signal measured in a basal compartment of the cell by the intensity of the fluorescence signal measured in the apical compartment of the cell at the respective time point.
In other instances, an endocytosing carrier that is capable of transporting a payload to a supranuclear or basal compartment can interact with an ERGIC protein and/or another ER-Golgi trafficking protein complex that can allow the carrier to access such compartments inside an epithelial cell.
An endocytosing or transcytosing carrier can be a polypeptide. Such carrier can be derived from a polypeptide secreted by a bacterium, such as Vibrio cholerae (herein a Cholix derived polypeptide). A carrier can be a chimeric polypeptide derived from two or more different bacterial polypeptides. Such two or more different bacterial polypeptides can be derived from two or more different bacteria (e.g., Vibrio cholerae, Pseudomonas aeruginosa, etc.), and/or derived from two or more different strains of a bacterium (e.g., two or more different strains of Vibrio cholerae, Pseudomonas aeruginosa, etc.).
A carrier can be a naturally or non-naturally occurring polypeptide of a polypeptide secreted by such bacterium.
Non-naturally occurring polypeptides can include those having a C- and/or an N-terminal modification.
In one example, a polypeptide comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid additions relative to a sequence alignment with a naturally occurring polypeptide (e.g., SEQ ID NO: 3) or relative to a sequence alignment with a consensus sequence (e.g., SEQ ID NO: 130).
Examples of substitutions contemplated herein include conservative substitutions of one or more amino acids. The following six groups each contain amino acids that are conservative substitutions for one another: (1) Alanine (A), Serine (S), and Threonine (T); (2) Aspartic acid (D) and Glutamic acid (E); (3) Asparagine (N) and Glutamine (Q); (4) Arginine (R) and Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); and (6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W).
Additionally, or alternatively, mutations in a carrier contemplated herein include one or more of: V1L, L1V, D3E, E4A, E581A, etc., e.g., relative to the sequence set forth in SEQ ID NOs: 1, 2, or 130 (a number designates the amino acid position, a letter before the number designates the modified amino acid, and a letter after the number designate the substituted amino acid). In some cases, a carrier comprises a valine at position 1, a leucine at position 1, an aspartic acid at position 3, a glutamic acid at position 3, a glutamic acid at position 4, or an alanine at position 4 in the carrier (numbering relative to positions in SEQ ID NO: 1).
Examples of deletions include N-terminal truncations and C-terminal truncations.
As used herein, when a C-terminal truncation is referred to as occurring “at” an amino acid position, such amino acid is included in the truncated polypeptide. When an N-terminal truncation is referred to as occurring “at” an amino acid position, such amino acid is excluded from the truncated polypeptide. For example, in one instance, the carrier comprises SEQ ID NO: 1 with a C-terminal truncation at position 386. Such carrier ends at amino acid 386 (A) of SEQ ID NO: 1 at its C terminus. Additionally, the above carrier can be further truncated at position 20 at its N-terminus, thereby having an N-terminal amino acid of proline (P) (which is position 21 in the reference sequence of SEQ ID NO: 1).
N-terminal truncations include those that remove up to 10, 20, 30, 39, or 40 amino acids at the N-terminal of a Cholix sequence herein (e.g., any one of SEQ ID NOs: 1-3, or 130). C-terminal truncations can be those described herein. Such N- and/or C-terminal truncations can result in different functions. Truncations can be described as relative to a wild-type sequence (e.g., SEQ ID NO: 3), relative to a non-naturally occurring sequence (e.g., SEQ ID NO: 1), or relative to a consensus sequence (e.g., SEQ ID NO: 130), wherein the residues are numbered from the N-terminus to the C-terminus, starting with position 1 an the N-terminus. For example, a carrier with a C-terminal truncation at position 266 relative to SEQID NO: 1 comprises amino acid residues 1-266 of SEQ ID NO: 1.
Examples of additions include: a signal peptide sequence, a purification peptide sequence, or other N-terminal modifications. A signal peptide sequence can comprise 1 to about 40 amino acids. In some cases, a carrier comprises an N-terminal methionine. The term “about,” as used herein in the context of a numerical value or range, generally refers to ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the numerical value or range recited or claimed, unless otherwise specified.
A carrier can have a substantial sequence identity (e.g., about, or greater than, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity, or 100% sequence identity) to a naturally occurring polypeptide (e.g., SEQ ID NO: 3), a non-naturally occurring polypeptide (e.g., SEQ ID NOs: 1-2), or to any of the functional fragments described herein (e.g., SEQ ID NOs: 160-168).
The term “sequence identity” or a percent (%) of sequence identity, as used herein is the percentage of residues in a candidate sequence that are identical with the residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
A carrier (e.g., an endocytosing or a transcytosing carrier) herein can be derived from a polypeptide secreted from a Vibrio cholerae bacterium (e.g., those comprising a sequence of any one of SEQ ID NOs: 3-125 or 127-129). Such carrier can be referred to as a Cholix derived polypeptide. A carrier derived from a Cholix polypeptide can include naturally and non-naturally occurring Cholix polypeptide sequences, as well as those sequences that have at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to a naturally (e.g., SEQ ID NO: 3-78) or non-naturally (e.g., SEQ ID NO: 1-2) occurring Cholix polypeptide described herein. A Cholix polypeptide derived carrier can also include endocytosing and/or transcytosing fragments (e.g., N- and/or C-terminal truncations of Cholix polypeptide) of naturally and non-naturally occurring Cholix polypeptide sequences, wherein such endocytosing and/or transcytosing fragments can have at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any of such naturally or non-naturally occurring Cholix polypeptide sequences.
TABLE 1 provides exemplary full-length Cholix and Cholix derived sequences.
TABLE 2 provides a consensus sequence (SEQ ID NO: 130, FORMULA I) of Cholix derived polypeptides that can be used as carriers herein.
Carriers can include all Cholix derived polypeptides having a reduced or ablated ADP ribosylation activity (e.g., ribosylation of elongation factor 2) relative to a naturally occurring Cholix polypeptide such as one with a sequence of SEQ ID NO: 3. Such carriers can be referred to as non-toxic carriers. Examples of such Cholix derived polypeptides include any of the polypeptides with a sequence set forth in any one of SEQ ID NO: 1-10 that can have a deletion at position 581 (e.g., an E581 deletion as, e.g., in SEQ ID NO: 189), a substitution at position 581 (e.g., a E581A substitution), or an alternative deletion or substitution that renders the carrier non-toxic. In some instances, a carrier has an amino acid sequence of SEQ ID NO: 130 with a mutation at position 581. In some instances, a carrier has an amino acid sequence of any one of SEQ ID NOs: 1-3 with a mutation at position 581.
In some instances, a carrier comprises, consists essentially of, or consists of an amino acid sequence of any one of TABLE 1 or TABLE 2 with a C-terminal deletion, substitution and/or addition thereby resulting in a reduced or ablated ADP ribosylation activity. Such deletion, substitution, and/or addition maintains a transport functionality such as transcytosis or endocytosis. As such, some of the carriers herein transcytose, either alone or along with a heterologous payload, while some of the carriers herein endocytose, either alone, or along with a heterologous payload.
In some instances, a first carrier derived from a first Cholix polypeptide can have improved properties compared to a second carrier derived from a second Cholix polypeptide. Such properties can include the ability of a carrier to transport a heterologous payload across a polarized epithelial cell (or cell layer), a stability of a carrier (e.g., an in vivo stability or an ex vivo stability such as shelf-life), the ability of a carrier to be functionally expressed in an expression system such as E. coli or CHO cells, the ability of a carrier to be purified, e.g., using chromatographical methods, and the ability to multimerize (e.g., dimerize) when, e.g., associated (e.g., coupled) with a heterologous payload. It can surprisingly be shown that a first carrier derived from a Cholix polypeptide having the sequence of SEQ ID NO: 1 can have one or more of the following properties compared to a second carrier derived from a Cholix polypeptide having the sequence of SEQ ID NO: 3 and/or 126: (i) an enhanced transcytosis function (e.g., an increased transport rate for transporting a payload across epithelial cells); (ii) improved functional expression (e.g., higher production yields) in E. coli and/or CHO cells; (iii) improved purification properties (e.g., higher purities and/or recovery yields); and (iv) an enhanced in vivo stability (e.g., an increased protease and/or pH stability, melting temperature). Such in vivo stability can be determined by incubating the carrier with, e.g., Caco-2 cells or by administering (e.g., injecting into a lumen) the carrier to a mammal (e.g., a rodent or a human). Enhanced transcytosis function, e.g., a speed or velocity of transportability can be determined, e.g., as described in EXAMPLE 1 by determining the amount of transported carrier and/or payload in a basolateral chamber at certain time points (e.g., 1, 5, 10, 15, 20, or 30 minutes after apical application of the carrier or delivery construct). In some cases, the stability of a carrier against a protease can be determined by incubating the carrier with the protease (e.g., trypsin) at certain molar ratios (e.g., 1:1) and at ambient temperature, and measuring the amount of intact carrier at various time points (e.g., 5, 10, 15, 30, or 45 minutes) using, e.g., size-exclusion chromatography. The stability of the carrier at a certain pH can be determined by incubating the carrier with a buffer having the appropriate pH (e.g., pH 5.5, 6, 6.5, 7, etc.) and measuring the amount of intact carrier at various time points (e.g., 5, 10, 15, 30 min.) using, e.g., size-exclusion chromatography.
A transcytosing carrier can transport a payload, coupled thereto, across an epithelial cell. Such transport can occur in vitro, e.g., using epithelial cell monolayers such as Caco-2 or SMI-100 cell monolayers. In other instances, such transport can occur in vivo, e.g., across a gut epithelium of a subject (e.g., a rodent or a human) into submucosal compartments such as the Lamina propria.
The mechanism of action of a transcytosing carrier can involve one or more of: receptor-mediated endocytosis into a polarized epithelial cell through interaction with TMEM132, LMAN1 and/or GPR75, avoidance of the lysosomal destruction pathway through interaction with GRP75, apical to basal transport through interaction with an ERGIC receptor (e.g., ERGIC-53), and release from the basal membrane into submucosal compartments such as the Lamina propria through interaction with perlecan (HSPG).
A transcytosing carrier can be one that interacts with one or more of the following endogenous proteins: TMEM132A, GPR75, ERGIC protein, and perlecan (HSPG) as shown in EXAMPLES 10 and 12. Interaction of a transcytosing carrier with an apical entry receptor such as TMEM132A can enable apical entry of the carrier into a polarized epithelial cell. Interaction of a transcytosing carrier with a lysosome avoidance receptor such as GRP75 can enable the carrier and a payload coupled thereto to avoid lysosomal degradation, thereby allowing transport of unaltered and functionally intact carrier and payload. Interaction of a transcytosing carrier with an apical to basal transport protein such as an ERGIC protein (e.g., ERGIC-53) can allow the carrier, once endocytosed, to move to the basal site of the epithelial cell. Interaction of a transcytosing carrier with a basal release protein such as perlecan can enable the carrier to enter basal recycling systems and exocytosis of the carrier into basolateral compartments, such as submucosal compartments (e.g., Lamina propria).
Moreover, a transcytosing carrier can co-localize with any one or more of coating protein I (COPI) early endosome antigen 1 (EEA1) for hijacking the endogenous apical to basal transport machinery, and with Ras-related protein 11 a (Rab11a) at the basal side of the epithelial cell for entering basal secretion systems as shown in EXAMPLE 10.
In some instances, a transcytosing carrier does not co-localize with Ras-related protein 7 (Rab7) and/or lysosomal-associated membrane protein 1 (LAMP1) during transport across such epithelial cell, enabling such carrier to avoid lysosomal degradation as shown in EXAMPLE 10.
Examples of transcytosing carriers include those having a C-terminal truncation of any of SEQ ID NOs 1-78 or 130, wherein the C-terminal truncation can occur at the C-terminus of the polypeptide at any amino acid position after the C-terminal residue at position 195 (e.g., truncation at any one of positions 195-634 of SEQ ID NOs: 1-2). Amino acid positions for truncation can be determined using sequence alignment to consensus sequence SEQ ID NO: 130 or any of reference sequences SEQ ID NO: 1, 2 or 3. TABLE 3 below illustrates exemplary carriers by identifying various amino acid residue sequences of such carriers and C-terminal positions that SEQ ID NOs 1-78, or 130 can be truncated at. In some instances, transcytosing carriers include those having a C-terminal truncation of any of SEQ ID NOs 1-2 or 4-78.
Such transcytosing carriers can further be truncated at their N-terminus at an amino acid position up to N-terminal position 20 (e.g., SEQ ID NO: 79 which is truncated at the N-terminal position 17 (starts with position 18)).
Also contemplated herein are transcytosing carriers such having at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity, to any of the carrier sequences shown in TABLE 3.
In one instance the carrier comprises SEQ ID NO: 1 with a C-terminal truncation at position 386. In one instance the carrier comprises SEQ ID NO: 2 with a C-terminal truncation at position 386. In one instance the carrier comprises SEQ ID NO: 4-79 with a C-terminal truncation at position 386. In one instance the carrier comprises SEQ ID NO: 130 with a C-terminal truncation at position 386. In such instances, the sequence does not include SEQ ID NO: 3 with a C-terminal truncation at position 386. In such instances, the sequence does not include SEQ ID NO: 126.
When a Cholix derived carrier has a C-terminal truncation at position 386, it can be referred to herein as Cholix386. The “386” designates the C-terminal truncation after the amino acid that most closely aligns with position 386 when the sequence is part of or aligned with SEQ ID NO: 130. A cholix386 does not necessitate that the polypeptide has 386 amino acids in it. For example, a truncation of SEQ ID NO: 79 at position 386 results in a carrier that is shorter than 386 amino acid residues. Examples of Cholix386 carrier molecules include any one of SEQ ID NOs: 1-79 or 130 truncated at position 386 as it is aligned for the highest sequence identity with SEQ ID NO 130 or it is aligned with any of SEQ ID NOs: 1-3 for highest sequence identity, e.g., ending with the amino acid residues “AQA.”
A Cholix386 can also include polypeptides maintaining substantially the same function as SEQ ID NO: 180 but with one or more additions/deletion/substitutions, and having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the Cholix386 molecules described herein.
Another example of a transcytosing Cholix derived carrier is Cholix266. In one instance, a Cholix266 consists of an amino acid sequence of SEQ ID NO: 181. Other Cholix266 fragments can include those of any of SEQ ID NO: 1-78 truncated at amino acid position 266 as it is aligned for the highest sequence identity with SEQ ID NO 130 or it is aligned with any of SEQ ID NOs 1-3 for highest sequence identity. Alternatively, a carrier can have any sequence of FORMULA I (SEQ ID NO: 130) truncated at the C-terminus at position 266.
Another example of a transcytosing Cholix derived carrier is Cholix251. In one instance, a Cholix251 consists of an amino acid sequence of SEQ ID NO: 182. Other Cholix251 fragments can include those of any of SEQ ID NO: 1-78 truncated at amino acid position 251 as it is aligned for the highest sequence identity with SEQ ID NO 130 or it is aligned with any of SEQ ID NOs 1-3 for highest sequence identity. Alternatively, a carrier can have any sequence of FORMULA I (SEQ ID NO: 130) truncated at the C-terminus at position 251.
Another example of a transcytosing Cholix derived carrier is Cholix245. In one instance, a Cholix245 consists of an amino acid sequence of SEQ ID NO: 183. Other Cholix245 fragments include those of any of SEQ ID NO: 1-79 truncated at amino acid position 245 as it is aligned for the highest sequence identity with SEQ ID NO 130 or it is aligned with any of SEQ ID NOs 1-3 for highest sequence identity. Alternatively, a carrier can have any sequence of FORMULA I (SEQ ID NO: 130) truncated at the C-terminal at position 245.
Another example of a transcytosing Cholix derived carrier is Cholix206. In one instance, a Cholix206 consists of an amino acid sequence of SEQ ID NO: 184. Other Cholix206 fragments include those of any of SEQ ID NO: 1-78 truncated at amino acid position 206 as it is aligned for the highest sequence identity with SEQ ID NO 130 or it is aligned with any of SEQ ID NOs 1-3 for highest sequence identity. Alternatively, a carrier can have any sequence of FORMULA I (SEQ ID NO: 130) truncated at the C-terminal at position 206.
Other examples of carriers include those having a C-terminal truncation at any one of amino acid position 195-634 of the sequence set forth in FORMULA I (SEQ ID NO: 130). Preferably, such truncation is at an amino acid position of any one of 195-347 of the sequence set forth in FORMULA I (SEQ ID NO: 130).
At the N-terminus, a transcytosing carrier can have any of amino acids 1-20 of SEQ ID NOs: 1-78, or 130. In some embodiments, the N-terminus of the carrier has amino acid residues 1-20 of SEQ ID NO: 1 or 2 (100% sequence identity at positions 1-20), or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity to amino acid residues 1-20 of SEQ ID NO: 1 or 2. In some embodiments, the first four amino acids at the N-terminus are VEEA (SEQ ID NO: 185). In some embodiments, such carrier does not comprise SEQ ID NO: 126. In some embodiments, the N-terminus of the carrier has the amino acid residues 1-20 of FORMULA I (SEQ ID NO: 130). Any of such carrier can optionally have an N-terminal modification as described herein. Such N-terminal modification can be an N-terminal methionine. Examples of such carriers are those comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 131-135.
As such, a transcytosing carrier can comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or have 100% sequence identity, to the amino acid residues from position 1 to any of the amino acid residues at any one of the positions 205-275 of the amino acid sequence set forth in FORMULA I (SEQ ID NO: 130).
In such instances, a transcytosing carrier consists, consists essentially of, or comprises amino acid residues 1-275, 1-266, 1-265, 2-265, 3-265, 4-265, 5-265, 1-251, 1-250, 2-250, 3-250, 4-250, 5-250, 1-245, 2-245, 3-245, 4-245, 5-245, 1-206, 1-205, 2-205, 3-205, 4-205, or 5-205 of the amino acid residues set forth in FORMULA I (SEQ ID NO: 130). In various instances, such carrier can consist or consist essentially of amino acid residues 1-275, 1-266, 1-265, 2-265, 3-265, 4-265, 5-265, 1-251, 1-250, 2-250, 3-250, 4-250, 5-250, 1-245, 2-245, 3-245, 4-245, 5-245, 1-206, 1-205, 2-205, 3-205, 4-205, or 5-205 of the same amino acid residues set forth in any one of SEQ ID NOs: 1-78.
Specifically, in some instances, such transcytosing carrier can comprise, consist essentially of, or consist of the amino acid residues 1-275, 1-266, 1-265, 2-265, 3-265, 4-265, 5-265, 1-251, 1-250, 2-250, 3-250, 4-250, 5-250, 1-245, 2-245, 3-245, 4-245, 5-245, 1-206, 1-205, 2-205, 3-205, 4-205, or 5-205 of the amino acid sequence set forth in SEQ ID NO: 1.
Alternatively, in some instances, such transcytosing carrier can comprise, consist essentially of, or consist of the amino acid residues 1-275, 1-266, 1-265, 2-265, 3-265, 4-265, 5-265, 1-251, 1-250, 2-250, 3-250, 4-250, 5-250, 1-245, 2-245, 3-245, 4-245, 5-245, 1-206, 1-205, 2-205, 3-205, 4-205, or 5-205 of the amino acid sequence set forth in SEQ ID NO: 2.
A carrier can be further modified at its N-terminus. Such N-terminus modifications include functional groups that can enhance expression and/or stability of the polypeptide. Such terminal modifications can be illustrated by the following designation “FG-Carrier”, wherein FG is a functional group attached to the N-terminus of the carrier. Examples of functional groups contemplated herein include a methionine for bacterial expression and other signal sequences for expression in CHO cells or HEK-293 cells. Examples of transcytosing carriers with an N-terminal methionine include those having an amino acid sequence set forth in any one of SEQ ID NOs: 131-135 (TABLE 4). It should be noted that functional groups can also be coupled to the C-terminus of a carrier.
Using crystal structure information of a Cholix derived carrier having an amino acid sequence of SEQ ID NO: 178, a handful of regions were identified that can play a role in endocytosis and transcytosis. Such regions are referred to herein as X1, X2, X3, X4, X5. X1 spans amino acid residues 17-25, has an amino acid sequence of SEQ ID NO: 160, and can play a role in apical to basal transport of a carrier, e.g., by allowing interaction of a carrier with an ERGIC protein (e.g., ERGIC-53). X2 spans amino acid residues 170-176, has an amino acid sequence of SEQ ID NO: 161, and can play a role in carrier access to supranuclear compartments and to move from the apical to the basal site of an epithelial cell, e.g., by allowing interaction of a carrier with an ERGIC protein. X3 spans amino acid residues 186-202, has an amino acid sequence of SEQ ID NO: 162, and can play a role in basal release of the carrier into basolateral compartments, e.g., by allowing interaction of a carrier with a basal release protein such as perlecan. X4 spans amino acid residues 31-39, has an amino acid sequence of SEQ ID NO: 163, and can play a role in carrier movement from the apical site an epithelial cell to the basal site, e.g., by allowing interaction of a carrier with an ERGIC protein. X5 spans amino acid residues 135-139, has an amino acid sequence of SEQ ID NO: 164, and can play a role in apical entry of a carrier into an epithelial cell, e.g., by allowing interaction of a carrier with an apical entry receptor such as TMEM132.
Thus, in some embodiments, a transcytosing carrier includes the amino acid residues of any one of SEQ ID NOs: 1-78, or 130 at X1, X2, X3, X4, and/or X5. For example, a carrier herein can have amino acids 1-266 of SEQ ID NO: 1 or 2, or a sequence having at substantial sequence identity thereto; provided however that any one or more of X1, X2, X3, X4, and X5 are identical to those of SEQ ID NO: 1 or 2. In one embodiment, all of X1, X2, X3, X4, X5 are identical to those of SEQ ID NO: 1 or 2. The same can be said for all other carriers and Cholix-derived carriers described herein, such as those provided in TABLE 3 or TABLE 4.
In some embodiments, transcytosing carriers exclude those having a sequence of any one or more of the Cholix sequence polypeptides set forth in SEQ ID NOs: 1-78 or 130. In some instances, those polypeptides are excluded that have a sequence set forth in SEQ ID NO: 3 or a truncated SEQ ID NO: 3 with a C-terminal truncation at residue 425-348, 291, 266, 265, 251, 250, 245, 244, 234, 206, 205, 187, 186, 151, 150, 134, 133 as well as the fragment consisting of residues 40-187 of SEQ ID NO: 3. In some embodiments, transcytosing carriers exclude those comprising, consisting essentially, or consisting of a sequence of SEQ ID NO: 126.
Surprisingly, it has also been identified that carriers shorter than the transcytosing carriers can transport a heterologous payload into a polarized epithelial cell without significant transport of such payload across the epithelial cell. Such carriers can be referred to herein as “endocytosing carriers”. Endocytosing carriers can end up in an intracellular vesicle or cytosol of the epithelial cell.
Examples of endocytosing carriers include those having amino acid residues from any one of the positions 1-40 to any one of the positions 145-194 of FORMULA I (SEQ ID NO: 130). In some embodiments, an endocytosing carrier has an amino acid sequence of any of SEQ ID NOs: 1-80 or 82-120, having a C-terminal truncation at any one of the amino acid positions of 145-194.
Moreover, any of the endocytosing carriers can further have an N-terminal truncation. Such N-terminal truncation can remove up to 40 amino acids from the N-terminus of the carrier. For example, contemplated herein are carriers having amino acids VLYY (SEQ ID NO: 186) or GVLYY (SEQ ID NO: 187) at the N-terminus which is representative of amino acid position 41-44 and 40-44 of SEQ ID NO: 130, respectively. In some instances, a carrier comprises a higher degree of sequence disparity at positions 1-40 as compared to amino acids 41 to a C terminus (e.g., a C-terminus at an amino acid residue selected from positions 145-194) of the amino acid sequence set forth in any one of SEQ ID NOs: 1-80 or 82-120, or SEQ ID NO: 130. A carrier most preferably comprises, consists essentially of, or consists of the amino acid residues from any one of the positions 1-20 to any one of the positions 150-187, or from any one of the amino acid residues at positions 21-41 to the amino acid residue at position 187 or 205 of the amino acid sequence set forth in FORMULA I (SEQ ID NO: 130). TABLE 5 provides exemplary amino acid residues that an endocytosing carrier can comprise, consist essentially of, or consist of
In some instances, such carrier has an N-terminal truncation at position 39 of FORMULA I (SEQ ID NO: 130). In other instances, such carrier has an N-terminal truncation at position 40 of FORMULA I (SEQ ID NO: 130). When such carrier is also truncated at its C-terminus at any one of the amino acid residues at positions 145-206 (i.e., having the C-terminal residue of any one of residues 145-206) of the sequence set forth in FORMULA I (SEQ ID NO: 130), such carrier can be used for endocytosis of a payload into an epithelial cell and transporting such payload to apical compartment(s) of such epithelial cell (see, e.g., EXAMPLE 4). Any of these endocytosing carriers described herein can also comprise an N-terminal modification such as an N-terminal methionine. Examples of such carrier are those comprising, consisting essentially of, or consisting of the amino acid residues 41-187 of SEQ ID NO: 1 (SEQ ID NO: 137), or of the amino acid residues 40-205 of SEQ ID NO: 1 (SEQ ID NO: 138).
In some embodiments, an endocytosing carrier can comprise, consist essentially of, or consist of the amino acid residues 1-150 of any of the sequences set forth in SEQ ID NOs: 1-78, or 130. In some embodiments, an endocytosing carrier can comprise, consist essentially of, or consist of the amino acid residues 1-151 of any of the sequences set forth in SEQ ID NOs: 1-78, or 130. In some embodiments, an endocytosing carrier can comprise, consist essentially of, or consist of the amino acid residues 1-186 of any of the sequences set forth in SEQ ID NOs: 1-78, or 130. In some embodiments, an endocytosing carrier can comprise, consist essentially of, or consist of the amino acid residues 1-187 of any of the sequences set forth in SEQ ID NOs: 1-78, or 130. In one instance, the carrier has amino acids 1-150, 1-151, 1-186, or 1-187 of SEQ ID NO: 1. In another instance, the carrier has amino acids 1-150, 1-151, 1-186, 1-187 of SEQ ID NO: 2.
Any of the endocytosing carriers herein can have a functional group (such as a methionine) attached to their N-terminus. Examples of endocytosing carriers with an N-terminal methionine include those with sequences set forth in any one of SEQ ID NOs: 136 or 139.
In other cases, such endocytosing carrier comprises the amino acid residues from position 40 or 41 to any one of the amino acid residues at positions 187-206 of the amino acid sequence set forth in FORMULA I (SEQ ID NO: 130).
In such instances, an endocytosing carrier can comprise, consist essentially of, or consist of the amino acid residues 40-187 or 41-187 of any one of the sequences set forth in SEQ ID NOs: 1-80, 82-120, or 130. In other instances, a carrier capable of transporting a heterologous payload into a polarized epithelial cell can comprise, consist essentially of, or consist of the amino acid residues 40-205 or 41-205 of any one of the sequences set forth in SEQ ID NOs: 1-80, 82-120, or 130.
Such carrier can comprise, consist essentially of, or consist of the amino acid residues 40-187, 41-187, 40-205, or 41-205 of SEQ ID NO: 1. Exemplary amino acid sequences of such carriers include those that consist of, consist essentially of, or comprise an amino acid sequence set forth in SEQ ID NOs: 137 and 138. Such carriers can be capable of transporting a payload to an apical compartment of a polarized epithelial cell, but not to a basal compartment as those carriers lack amino acid residues 1-39 that can play a role in apical to basal transport.
An endocytosing carrier can comprise the endocytosis fragment that consists of the amino acid residues from position 134 to position 151 of the amino acid sequence set forth in Formula I (SEQ ID NO: 130). In some cases, such functional fragment has the sequence set forth in SEQ ID NO: 165, or a high (e.g., >90%) sequence identity thereto. Exemplary carriers include those that can consist of, consist essentially of, or comprise an amino acid sequence set forth in any one of SEQ ID NOs: 136-139 (TABLE 6). Carriers that lack one or both functional fragments with SEQ ID NOs: 166-167 (TABLE 11) can transport heterologous payload to apical compartments of epithelial cells that can include locations in apical vesicles, in the apical cytosol of the cell, and/or in the apical recycling systems such as apical recycling endosomes. Such carrier can interact with an apical entry receptor such as TMEM132 (e.g., TMEM132A), but not or not significantly with basal trafficking proteins or perlecan (HSPG) and can co-localize with Rab11a in apical compartments of the epithelial cell following endocytosis. In some instances, such carrier can consist of, consist essentially of, or comprise an amino acid sequence set forth in any one of SEQ ID NOs: 137-139 (TABLE 6).
In other embodiments, an endocytosing carrier can comprise the functional fragments that consist of the amino acid residues from position 1 to position 40 and position 152 to position 187, respectively, of the amino acid sequence set forth in FORMULA I (SEQ ID NO: 130). In some cases, such fragments have the amino acid sequences set forth in SEQ ID NO: 166 and 167, respectively, or a high (e.g., >90%) sequence identity to one or both of such functional fragments. Such carrier can interact with an apical to basal trafficking receptor, but not significantly with perlecan. Such trafficking receptor enables such carrier to transport a payload to a supranuclear and/or a basal compartment of a polarized epithelial cell. Such carrier can consist of, consist essentially of, or comprise amino acids 1-187 of SEQ ID NO: 130. An example of such carrier is one that has the sequence set forth in SEQ ID NO: 136.
In some instances, an endocytosing carrier is not a fragment of the sequence set forth in SEQ ID NO: 3. In some instances, such carrier is not a fragment of any one of the sequences set forth in SEQ ID NOs: 4-80, or 82-120. In such instances, a carrier does not comprise or consist of amino acid residues 1-151, 1-187, or 40-187 of SEQ ID NO: 3.
D. Functional Sequence Regions of Carriers
In any of the embodiments herein, one or more functional sequence region(s) within a carrier sequence can have a localized high (e.g., >90%) sequence identity to the amino acid residues found in those regions in FORMULA I (SEQ ID NO: 130) in order to maintain functionality across numerous polypeptides and embodiments. In some cases, the amino acid residues in those region(s) can be restricted to those found in naturally occurring Cholix polypeptides such as those having the sequence set forth in any one of SEQ ID NOs: 1-80 or 82-120.
1. Endocytosing Domain
In some instances, a carrier comprises a domain that allows the carrier to enter an epithelial cell (e.g., a polarized epithelial cell) on the apical site. In some instances, such domain allows the carrier to interact with an apical entry receptor. Such apical entry receptor can be TMEM132 or TMEM132A. Such TMEM132 interacting domain may have a consensus sequence of amino acid residues 135-151 of FORMULA I (SEQ ID NO: 130) or of amino acid residues of positions 135-151 of any of SEQ ID NOs: 1-120. TMEM132A is a transmembrane protein that is responsible for apical entry of a carrier into an epithelial cell. In order to maintain carrier interaction with TMEM132A, in some embodiments, a carrier herein includes a SEQ ID NO: 165 which sets forth a consensus sequence for a TMEM132A interacting domain. Alternatively, a carrier herein can include the amino acid residues 135-151 of any one or more of SEQ ID NOs: 1-120, or 130 as its TMEM132 interacting domain.
Moreover, it is surprisingly assumed that subregions within the TMEM132 interacting domain may be particularly relevant for apical entry of the carrier. Thus, a carrier herein may interact with an apical entry receptor such as TMEM132 via an interacting region with an amino acid motif having amino acid residues 135-139 of SEQ ID NO: 130 (e.g., “DQQRN” of SEQ ID NO: 1; SEQ ID NO: 164). Thus, any of the carriers herein (including polypeptides thereof) may include such motif, or the entire TMEM132 interacting domain (e.g., SEQ ID NO: 165) in order to provide entry into the epithelial cells on the apical side. As such regions can have functions within the carrier, the amino acid sequences in such regions may be conserved, or has only up to 1, 2, or 3, amino acid residues that are substitutions, insertions, and/or deletions.
Any of the carriers herein may include a TMEM132 interacting domain or receptor interacting regions in order to enter the epithelial cell. For example, a carrier comprising amino acid residues 1-151 of SEQ ID NO: 1, which includes a TMEM132 interacting domain of SEQ ID NO: 165 can enter epithelial cells on the apical side. Similarly, a Cholix266 carrier, such as a carrier comprising amino acid residues 1-266 of SEQ ID NO: 1 (e.g., SEQ ID NO: 181), includes the TMEM132 domain and can both enter the apical side of the epithelial cells via the TMEM132 domain and transport a heterologous payload across a polarized epithelial cell. Other exemplary carriers comprising a TMEM132 interacting domain are Cholix187, Cholix206, Cholix245, Cholix251, and Cholix386 (e.g., SEQ ID NOs: 136, and 180-184). In some instances, a carrier is one having amino acid residues 1-187, 1-206, 1-245, 1-251, 1-266, or 1-386 of SEQ ID NO: 1.
On the other hand, a carrier lacking a TMEM132 interacting domain, such as M-Cholix134 having SEQ ID NO: 140 can remain in the intestinal lumen and does not, or does not significantly (e.g., less than 10% of carrier material that was applied to the apical surface), enter an epithelial cell (see, e.g., EXAMPLE 6).
2. Supranuclear and Basal Compartment Targeting Domains
In some instances, a carrier comprises N-terminal amino acid residues 1-40 of the sequence set forth in FORMULA I (SEQ ID NO: 130). Such carrier can be used to transport a heterologous payload from an apical to a supranuclear or basal compartment (and into submucosal compartments, if such carrier can also interact with a basal release protein such as perlecan) of an epithelial cell following endocytosis of the carrier into the cell. Examples of such carriers include those comprising amino acid sequences set forth in SEQ ID NOs: 131-135 (TABLE 4).
In some instances, a carrier comprises amino acid residues 17-25 and/or 31-39 of the sequence set forth in FORMULA I (SEQ ID NO: 130). Such regions can be restricted to those amino acid residues found in a naturally occurring Cholix polypeptide or include up to 1, 2, 3, or 4 amino acid substitutions, insertions, and/or deletions. The substitution(s) can be one or more conservative or non-conservative substitutions. The 1, 2, 3, or 4 amino acid substitutions, insertions and/or deletions can preserve a function of amino acids 17-25 and/or 31-39. In some instance, a carrier has a naturally occurring sequence at 17-25 and/or 31-39. In such instances, amino acids 17-25 of a carrier can have the sequence of SEQ ID NO: 160, and/or amino acids 31-39 of the carrier can have the sequence of SEQ ID NO: 163. The function of amino acids 17-25 and 31-39 can be apical to basal transport. Such function can be determined as described elsewhere herein, e.g., by generating carriers comprising N-terminal truncations at such residues and compare to the apical to basal transport capabilities of these carrier to those that do not have an N-terminal truncation (see, e.g., EXAMPLES 5 and 6). Exemplary carriers comprising such regions and being capable of apical to supranuclear and/or basal transport can comprise, consist essentially of, or consist of amino acid residues 1-187, 1-206, 1-245, 1-251, 1-266, and 1-386 of the sequence set forth in SEQ ID NO: 1. In such cases, a carrier can consist of the amino acid sequence set forth in any one of SEQ ID NOs: 131-136.
In some instances, a carrier uses an additional domain to access supranuclear and/or basal compartment. Such domain can have a consensus sequence of amino acid residues 152-187 of FORMULA I (SEQ ID NO: 130) or of amino acid residues of positions 152-187 of any of SEQ ID NOs: 1-120. The function of this domain can be to access supranuclear regions within an epithelial cell, reach basal compartments within an epithelial cell, and/or for co-localization of the carrier with elements of the trans-Golgi network such as coating protein I (COPI).
For example, carriers such as Cholix187 or Cholix206, e.g., carriers with sequence set forth in SEQ ID NOs: 136 and 131, respectively, which include such supranuclear and basal targeting region are capable of accessing supranuclear regions and basal compartments within an epithelial cell, whereas a carrier without such domain, such as a Cholix151 (e.g., SEQ ID NO: 139) remains at the apical side of the epithelial cell and does not significantly access supranuclear regions or basal compartments (see, e.g., EXAMPLE 6).
It is believed that sequence of amino acid residues 170-176 of SEQ ID NO: 130 (e.g., “TRPEHNI,” SEQ ID NO: 161) may be of particular relevance for a carrier to access such supranuclear and basal compartments and co-localize with COPI.
Hence, in some instances, a carrier comprises a supranuclear and basal targeting domain, or consensus sequence of amino acid residues 170-176 of Formula I (SEQ ID NO: 130), or SEQ ID NO: 161 (corresponding to amino acid residues 170-176 of SEQ ID NO: 1). Such carrier can also include a TMEM132A interacting domain as described above.
The supranuclear and basal targeting domain is one that can have minimal sequence variations. As such, a carrier herein can comprise a supranuclear and basal targeting domain that has amino acid residues of naturally occurring polypeptides such as those with SEQ ID NOs: 3-120, or can comprise only up to 1, 2, or 3, amino acid residues that are substitutions, insertions, and/or deletions relative to the residues of naturally occurring polypeptides such as those with SEQ ID NOs: 3-120.
Exemplary carriers comprising a supranuclear targeting domain are Cholix187, Cholix206, Cholix245, Cholix251, Cholix266 and Cholix386. In some instances, a carrier is one having amino acid residues 1-187, 1-206, 1-245, 1-251, 1-266, or 1-386 of SEQ ID NO: 1.
3. Transcytosis Domain
In some instances, a carrier comprises a transcytosis domain. Such domain preferably can include a consensus sequence of amino acid residues 188-206 of FORMULA I (SEQ ID NO: 130) or of amino acid residues of positions 188-206 of any of SEQ ID NOs: 1-120. The function of amino acid residues 188-206 can be multifold and can play a role in transcytosis. The transcytosis region can allow interaction of a carrier with transport receptor like interaction partners (also referred to herein as “TRIPs”), endoplasmic reticulum Golgi intermediate compartment 53 (ERGIC-53, also referred to herein as LMAN1), glucose-regulated protein 75 (GRP75), and perlecan in a pH-dependent and/or sequential manner. Such interactions can allow the carrier to access basal recycling systems that release the carrier (along with any heterologous payload coupled thereto) from the basal membrane of the epithelial cell into the basolateral compartment (e.g., Lamina propria).
Examples of carriers capable of transcytosis include Cholix derived carriers such as Cholix206, Cholix245, Cholix251, Cholix266, and Cholix386. In some instances, such carriers have an amino acid sequence of those with amino acid residues 1-206, 1-245, 1-251, 1-266, and 1-386 of SEQ ID NO: 130. In some instances, such carriers have an amino acid sequence of those with amino acid residues 1-206, 1-245, 1-251, 1-266, and 1-386 of SEQ ID NO: 1. In some instances, such carriers have an amino acid sequence of those with amino acid residues 1-206, 1-245, 1-251, 1-266, and 1-386 of SEQ ID NO: 2. Examples of such carriers with function group, N-terminal methionine are provided in SEQ ID NO: 131-SEQ ID NO: 135. Such carrier (e.g., Cholix206, Cholix245, Cholix251, Cholix266, and Cholix386) can be used for rapid (e.g., at least 10−6 cm/sec, 10−5 cm/sec) and efficient (e.g., at least 5%, 10%, 20%, 25%, or 50% of material applied to the apical surface) transport of a payload across an epithelial cell (see, e.g., EXAMPLE 5).
It is postulated that the sequence of amino acid residues 188-206 (e.g., “AQKEGSRHKRWAHWHTGLA,” SEQ ID NO: 168) with its one or more histidine residues can act as a pH-switch, thereby allowing the carrier to interact with TRIPs such as TMEM132, LMAN1, GRP75, and perlecan in a sequential and/or pH-dependent manner.
TABLE 7 below shows additional Cholix derived polypeptide sequences described herein.
Heterologous payloads contemplated herein can be of any nature, including therapeutic, diagnostic, and imaging. A payload can be part of a delivery construct. A delivery construct can include a carrier coupled to a heterologous payload. The payload can be directly or indirectly, covalently or non-covalently, coupled to the carrier. When covalently attached, a payload can be directly attached to a carrier or via a spacer.
The heterologous payload can be a small molecule, a nucleic acid, a polypeptide, a protein, a nanoparticle, or a combination thereof.
In some instances, the therapeutic payload is a polypeptide such as, e.g., a cytokine, a hormone, a growth factor, a therapeutic antibody, a nucleic acid, an antigen, an enzyme, clotting factor, neurotransmitter, or a polymer.
Cytokines provided herein include chemokines and interleukins (also abbreviated herein as “ILs”). The interleukin can be an IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40.
In some instances, the interleukin is an IL-10 or an IL-22. The interleukin can be from any species (e.g., from a human or a rodent), and is preferably from the organism to which it is intended to administer the payload or delivery construct comprising such payload. Thus, in some instances, the interleukin is a human interleukin. An interleukin provided herein can be a precursor to a mature, secreted interleukin. Such a precursor interleukin can comprise a signal peptide sequence. For example, in some instances, a therapeutic payload can be a precursor of a mature, secreted protein. In other instances, the therapeutic payload is the secreted protein. For example, in some instances, the payload comprises, consists essentially of, or consists of SEQ ID NO 141, which is a full length, precursor of IL-22. In other instances, the payload comprises, consists essentially of, or consists of SEQ ID NO: 142, which is a secreted form of IL-22. In another example, the payload comprises, consists essentially of, or consists of SEQ ID NO 144, which is a full length, precursor of IL-10. In other instances, the payload comprises, consists essentially of, or consists of SEQ ID NO: 145, which is a secreted form of IL-10.
A heterologous payload can comprise an N-terminal methionine. For example, an IL-22 payload can comprise an N-terminal methionine. In such instances, the IL-22 can have a sequence of SEQ ID NO: 143.
In some embodiments, the therapeutic payload is an IL-22. The IL-22 can comprise, consist essentially of, or consist of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99%% sequence identity to the amino acid sequence set forth in SEQ ID NO: 141 or SEQ ID NO: 142, or a functional fragment thereof. In some embodiments, the therapeutic payload comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 142.
In some embodiments, the therapeutic payload is an IL-10. The IL-10 can comprise, consist essentially of, or consist of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99%% sequence identity to the amino acid sequence set forth in SEQ ID NO: 144 or SEQ ID NO: 145, or a functional fragment thereof. In some embodiments, the therapeutic payload comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 145.
Hormones provided herein can include peptide and polypeptide hormones. Such hormones can include growth hormones, e.g., human growth hormone (also referred to herein as hGH or somatotropin); pituitary hormones, e.g., chorionic gonadotropin, cosyntropin, menotropins, iorticotropin, protirelin, thyrotropin, vasopressin, lypressin; parathyroid hormones; thyroid hormones; testicular hormones; gastrointestinal hormones, e.g., gastric inhibitory polypeptide, epidermal growth factor-urogastrone, gastric inhibitory polypeptide, gastrin-releasing peptide, gastrins, pentagastrin, tetragastrin, motilin, neuropeptide Y, peptide YY, secretin, vasoactive intestinal peptide, sincalide; incretin hormones, e.g., glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP); metabolic hormones, e.g., insulin; and any derivatives or fragments thereof.
In some embodiments, the hormone is a human growth hormone comprising, consisting essentially of, or consisting of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 146 or 190, or a fragment thereof.
In some embodiments, the therapeutic payload is glucose-lowering agent. In such instances, the payload can be a GLP-1 agent or a GLP-1 agonist. Such agonist can Exenatide or Liraglutide. In other instances, the glucose-lowering agent can be an incretin, a glucagon proprotein, a glucagon-like peptide (e.g., other than GLP-1), a glicentin-related polypeptide, an exendin-3, an exendin-4, lixisenatide (tradenames Adlyxin®, and Lyxumia®, Sanofi), liraglutide (tradename Victoza®, Novo Nordisk A/S), semaglutide (tradename Ozempic®, Novo Nordisk A/S), albiglutide (tradename Tanzeum®, GlaxoSmithKline; GLP-1 dimer fused to albumin), dulaglutide (tradename Trulicity®, Eli Lilly), a glucose-dependent insulinotropic polypeptide, Tirzepatide (Eli Lilly), Dual Amylin Calcitonin Receptor Agonist DACRA-089, Glargine/Lantus®, Glulisin/Apidra®, Glarine/Toujeo®, Insuman®, Detemir/Levemir®, Lispro/Humalog®/Liprolog®, Humulin®, Linjeta, SuliXen®, NN1045, Insulin plus Symlin™, PE0139, fast-acting and short-acting insulins (e.g. Linjeta, PH20, NN1218, HinsBet), (APC-002) hydrogel, oral, inhalable, transdermal and sublingual insulins (e.g. Exubera®, Nasulin®, Afrezza®, Tregopil®, TPM 02, Capsulin, Oral-lyn®, Cobalamin®, oral insulin, ORMD-0801, NN1953, NN1954, NN1956, VIAtab, and Oshadi oral insulin).
In some instances, the therapeutic payload is a therapeutic antibody or a binding fragment thereof. Therapeutic antibodies can include anti-TNFα antibodies. Such anti-TNFα antibodies can be humanized or human antibodies. Anti-TNFα agents can include infliximab (Remicade®), adalimumab (Humira®), or etanercept (ENBREL®).
In some instances, the payload is an antineoplastic agent. Antineoplastic agents can include nitrosoureas, e.g., carmustine, lomustine, semustine, strepzotocin; methylhydrazines, e.g., procarbazine, dacarbazine; steroid hormones, e.g., glucocorticoids, estrogens, progestins, androgens, tetrahydrodesoxycaricosterone; immunoactive compounds such as immunosuppressives, e.g., pyrimethamine, trimethopterin, penicillamine, cyclosporine, azathioprine; and immunostimulants, e.g., levamisole, diethyl dithiocarbamate, enkephalins, endorphins; antimicrobial compounds such as antibiotics, e.g., beta-lactam, penicillin, cephalosporins, carbapenims and monobactams, beta-lactamase inhibitors, aminoglycosides, macrolides, tetracyclins, spectinomycin; antimalarials, amebicides; antiprotazoals; antifungals, e.g., amphotericin-beta, antivirals, e.g., acyclovir, idoxuridine, ribavirin, trifluridine, vidarbine, gancyclovir; parasiticides; antihalmintics; radiopharmaceutics; gastrointestinal drugs; hematologic compounds; immunoglobulins; blood clotting proteins, e.g., anti-hemophilic factor, factor IX complex; anticoagulants, e.g., dicumarol, heparin Na; fibrolysin inhibitors, e.g., tranexamic acid; cardiovascular drugs; peripheral anti-adrenergic drugs; centrally acting antihypertensive drugs, e.g., methyldopa, methyldopa HCl; antihypertensive direct vasodilators, e.g., diazoxide, hydralazine HCl; drugs affecting renin-angiotensin system; peripheral vasodilators, e.g., phentolamine; anti-anginal drugs; cardiac glycosides; inodilators, e.g., amrinone, milrinone, enoximone, fenoximone, imazodan, sulmazole; antidysrhythmics; calcium entry blockers; drugs affecting blood lipids, e.g., ranitidine, bosentan, rezulin; respiratory drugs; sypathomimetic drugs, e.g., albuterol, bitolterol mesylate, dobutamine HCl, dopamine HCl, ephedrine sodium (So), epinephrine, fenfluramine HCl, isoproterenol HCl, methoxamine HCl, norepinephrine bitartrate, phenylephrine HCl, ritodrine HCl; cholinomimetic drugs, e.g., acetylcholine HCl; anticholinesterases, e.g., edrophonium chloride (Cl); cholinesterase reactivators; adrenergic blocking drugs, e.g., acebutolol HCl, atenolol, esmolol HCl, labetalol HCl, metoprolol, nadolol, phentolamine mesylate, propanolol HCl; antimuscarinic drugs, e.g., anisotropine methylbromide, atropine, clinidium bromide (Br), glycopyrrolate, ipratropium Br, scopolamine HBr; neuromuscular blocking drugs; depolarizing drugs, e.g., atracurium besylate, hexafluorenium Br, metocurine iodide, succinylcholine Cl, tubocurarine Cl, vecuronium Br; centrally acting muscle relaxants, e.g., baclofen; neurotransmitters and neurotransmitter agents, e.g., acetylcholine, adenosine, adenosine triphosphate; amino acid neurotransmitters, e.g., excitatory amino acids, GABA, glycine; biogenic amine neurotransmitters, e.g., dopamine, epinephrine, histamine, norepinephrine, octopamine, serotonin, tyramine; neuropeptides, nitric oxide; antiparkinson drugs, e.g., amaltidine HCl, benztropine mesylate, carbidopa; diuretic drugs, e.g., dichlorphenamide, methazolamide, bendroflumethiazide, polythiazide; antimigraine drugs, e.g., carboprost tromethamine mesylate, or methysergide maleate, or a functional derivative thereof.
In some instances, the payload is an enzyme such as hyaluronidase, streptokinase, tissue plasminogen activator, urokinase, PGE-adenosine deaminase; intravenous anesthetics such as droperidol, etomidate, fetanyl citrate/droperidol, hexobarbital, ketamine HCl, methohexital Na, thiamylal Na, thiopental Na; antiepileptics, e.g., carbamazepine, clonazepam, divalproex Na, ethosuximide, mephenyloin, paramethadione, phenyloin, primidone. In various embodiments, the biologically active payload is an enzyme selected from hyaluronidase, streptokinase, tissue plasminogen activator, urokinase, or PGE-adenosine deaminase.
TABLE 8 shows amino acid sequences of exemplary therapeutic payloads provided herein.
A carrier can be coupled to a heterologous payload via a spacer. A spacer provided herein can provide steric flexibility, accurate folding, and/or proper biological activity and function of both the carrier and the payload.
A spacer can comprise one or more amino acid residues. In some instances, the spacer is an amino acid-based spacer. Such spacer can comprise, consist essentially of, or consist of at least about 5, 10, 15, 20, 25, 35, 50, 75, or 100 amino acid residues. In some instances, a spacer comprises, consists essentially of, or consists of at most about 30, 25, 20, 15, or 10 amino acid residues. In some instances, the majority (e.g., more than 90%) of these amino acid residues are glycine and/or serine residues.
A spacer can be a cleavable or non-cleavable spacer. In some instances, a cleavable spacer can be cleaved by an enzyme, e.g., a protease. A non-cleavable spacer may not be cleavable by such enzyme. For example, a non-cleavable spacer can be used in cases where higher systemic concentrations of a payload are an objective.
A spacer can comprise one or more repeats of glycine-serine oligopeptide sequences. Thus, in some instances, a carrier comprises, consists essentially of, or consists of the amino acid sequences (GS)x (SEQ ID NO: 169), (GGS)x (SEQ ID NO: 170), (GGGS)x (SEQ ID NO: 171), (GGGGS)x (SEQ ID NO: 172), or (GGGGGS)x (SEQ ID NO: 173), wherein x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some cases, a spacer comprises, consists essentially of, or consists of the amino acid sequence (GGGGS)x (SEQ ID NO: 174), wherein x=1, 2, 3, 4 or 5. In such instances, a spacer can consist of 5, 10, 15, 20, or 25 amino acids.
Examples of spacers provided herein are those comprising, consisting essentially of, or consisting of an amino acid sequence having at least 50%, 75%, 90%, or 99% sequence identity to GGGGSGGGGSGGGGS (SEQ ID NO: 175), GGGGSGGGGSGGGG (SEQ ID NO: 176), or GGGGSNLQGGLRQPR (SEQ ID NO: 177), a fragment of any of the above, or a combination of any of the above. In some instances, the spacer consists of the amino acid sequence set forth in any one or SEQ ID NO: 196 (GGGGS) or SEQ ID NO: 197 (GGGGSGGGGSGGGGSGGGGSGGGGS). In some instances, any of the above spacer can comprise an additional glycine or serine residue at either the N- and/or C-terminal.
In some embodiments, the spacer coupling a carrier to a therapeutic payload comprises an amino acid sequence having at least 50%, 75%, 90%, or 99% sequence identity to SEQ ID NOs: 175-176.
Provided herein are delivery constructs (e.g., a carrier-payload complex) that can comprise a carrier coupled to a heterologous payload. A carrier can be coupled to such payload covalently or non-covalently (e.g., via ionic interactions, van der Waals interactions, π-π interactions, etc.). A carrier can be coupled directly or indirectly to a heterologous payload.
A heterologous payload can be coupled to an N- and/or C-terminus of a carrier. In some instances, a heterologous payload is directly and covalently coupled to a C-terminus of a carrier by forming a covalent amide bond between the C-terminal carboxyl group of the carrier and the N-terminal amine of the heterologous payload. In some instances, a heterologous payload is indirectly and covalently coupled to the carrier via a spacer.
Thus, in some instances, when a carrier is covalently coupled to a payload, the delivery construct can be represented according to Formula II: C−S−P or Formula III: P−S−C, wherein C is a carrier, S is a spacer, or optionally a bond, and P is a heterologous payload. A delivery construct can further comprise one or more modifications on its N-terminus and/or C-terminus. Such a modification(s) can include an N-terminal methionine residue. Thus, Formula II and Formula III can also include an N-terminal methionine (e.g., M+C−S−P) or (e.g., M+P−S−C).
A carrier can be coupled to a heterologous payload via chemical/synthetic conjugation (e.g., using amide coupling reactions) or by recombinant expression in a bacterial (e.g., in E. coli) or mammalian (e.g., Chinese Hamster Ovary (CHO)) cell as a fusion protein.
A delivery construct, or part thereof (e.g., the carrier and/or spacer), can be a polypeptide. The term “polypeptide,” as used herein, can include both natural and unnatural amino acids.
Delivery constructs provided herein can transcytose across polarized epithelial cells with a high flux rate through one or more moderate-affinity, high-capacity dynamic and/or pH-dependent interactions of the carrier with one or more transport receptor-like interaction partners (TRIPs). Such TRIPs can be elements of an endogenous trafficking pathways, and as such, can allow a carrier to transport heterologous payload across the epithelial cell barrier without impairing the barrier itself and without significantly altering (e.g., chemically/enzymatically modifying) the carrier or the payload.
Furthermore, interactions with TRIPs can allow a carrier to transport a payload across an intact epithelium (e.g., a polarized gut epithelium) with transport rates of at least about 10−6 cm/sec, 10−5 cm/sec, or 10−4 cm/sec.
In some instances, a carrier is indirectly and non-covalently coupled to a payload. In such instances, nanoparticles (e.g., liposomes, metallic nanoparticles, polymer-based nanoparticles, etc.) can be loaded (e.g., on the inside and/or on the surface of the particle) with payload molecules (e.g., IL-10, IL-22, GLP-1, etc.), and Cholix derived carrier molecule(s) can be coupled to such nanoparticles (e.g., onto its surface). In some instances, a ratio of payload to carrier can be at least about 15000:1, 10000:1, 5000:1, 2500:1, 1000:1, 500:1, 250:1, 100:1, 50:1, 25:1, 10:1, 5:1, 2.5:1, 1:1. This can allow transport of such payload-containing nanoparticles into or across polarized epithelial cells (e.g., polarized gut epithelial cells) using the Cholix derived carriers attached to the surface. In some cases, a nanoparticle can release the payload following transcytosis or intracellular delivery. In cases where the nanoparticle is transported across epithelial cells, the released payload can bind to receptors within submucosal tissue (e.g., Lamina propria) and/or can enter the systemic circulation and thus provide a certain function (e.g., a therapeutic or diagnostic function) systemically. In other cases, where a nanoparticle releases the payload inside an epithelial cell, the payload (e.g., a nucleic acid) may provide certain intracellular functions, e.g., production of transgenes within these cells, modulation of gene expression, etc.
In various embodiments, a delivery construct or carrier-payload complex comprises (a) a carrier comprising a Cholix polypeptide that does not comprise SEQ ID NO: 179, and does not consist of SEQ ID NO: 126, complexed with (b) a heterologous payload, wherein the carrier is capable of (i) transcytosing the heterologous payload across a polarized epithelial cell; or (ii) transporting the heterologous payload into the polarized epithelial cell.
In some embodiments, a delivery construct comprises a Cholix derived carrier comprising, consisting essentially of, or consisting of at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid residues 1-386 of the amino acid sequence set forth in SEQ ID NO: 1 or 2.
In some embodiments, a delivery construct comprises a Cholix derived carrier comprising, consisting essentially of, or consisting of at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid residues from any one of the positions 1-40 to any one of the amino acid residues at positions 150-347 of the amino acid sequence set forth in SEQ ID NO: 1. In some instances, such carrier comprises, consists essentially of, or consists of at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid residues from positions 1-151, 1-187, 41-187, 1-206, 1-245, 1-251, or 1-266 of the amino acid sequence set forth in SEQ ID NO: 1. In other instances, a carrier comprises, consists essentially of, or consists of at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid residues from positions 1-151, 1-187, 41-187, 1-206, 1-245, 1-251, or 1-266 of the amino acid sequence set forth in SEQ ID NO: 2.
Any of such carriers can be coupled to a therapeutic payload comprising, consisting essentially of, or consisting of at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 141, 142, 144, 145, and 146.
Such therapeutic payload can be coupled to a Cholix derived carrier via a spacer comprising, consisting essentially of, or consisting of at least 66%, 73%, 80%, 86%, 93%, or 100% sequence identity to the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 175).
Thus, in some instances, a delivery construct comprises a carrier comprising, consisting essentially of, or consisting of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 134 coupled via a spacer to a therapeutic payload comprising, consisting essentially of, or consisting of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 142. In some instances, the spacer comprises, consists essentially of, or consists of an amino acid sequence having at least 66%, 73%, 80%, 86%, 93%, or 100% sequence identity to SEQ ID NO: 175.
In other instances, a delivery construct comprises a carrier comprising, consisting essentially of, or consisting of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 135 coupled via a spacer to a therapeutic payload comprising, consisting essentially of, or consisting of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 142. In some instances, the spacer comprises, consists essentially of, or consists of an amino acid sequence having at least 66%, 73%, 80%, 86%, 93%, or 100% sequence identity to SEQ ID NO: 175.
In some instances, a delivery construct comprises a carrier comprising, consisting essentially of, or consisting of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 134 via a spacer comprising, consisting essentially of, or consisting of an amino acid sequence having at least 66%, 73%, 80%, 86%, 93%, or 100% sequence identity to SEQ ID NO: 175 to a therapeutic payload. Such therapeutic payload can be a cytokine, a hormone, or a therapeutic antibody or a functional binding fragment thereof. In some instances, the therapeutic payload comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 142, SEQ ID NO: 145, or SEQ ID NO: 146.
In some instances, a delivery construct comprises a carrier comprising, consisting essentially of, or consisting of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 135 via a spacer comprising, consisting essentially of, or consisting of an amino acid sequence having at least 66%, 73%, 80%, 86%, 93%, or 100% sequence identity to SEQ ID NO: 176 to a therapeutic payload. Such therapeutic payload can be a cytokine, a hormone, or a therapeutic antibody or a functional binding fragment thereof. In some instances, the therapeutic payload comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 145.
In some instances, a delivery construct comprises, consists essentially of, or consists of at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 147-150, 152-159, or 188.
In some embodiments, a delivery construct consists of the amino acid sequence set forth in SEQ ID NO: 147.
In some embodiments, a delivery construct consists of the amino acid sequence set forth in SEQ ID NO: 149.
In some embodiments, a delivery construct consists of the amino acid sequence set forth in SEQ ID NO: 188.
Amino acid sequences of exemplary delivery constructs herein are shown in TABLE 9.
Provided herein, in some embodiments, are delivery constructs comprising a carrier coupled to a heterologous payload. The carriers provided herein can be used to transport such payload (e.g., a therapeutic payload) to various locations inside an epithelial cell such as the apical side (e.g., an apical recycling system), the basal side, and/or supranuclear compartment(s). Delivery across a polarized gut epithelium can include delivery to submucosal compartments (e.g., Lamina propria and/or other submucosal intestinal compartments) and/or systemic circulation (e.g., via the hepatic portal system).
A. Methods of Treatment
The high flux transport capacities of carriers provided herein across intact epithelial barriers (e.g., a polarized gut epithelium) can be used to deliver therapeutic and/or diagnostic payload molecules to a subject in need thereof (e.g., a human or a rodent). For example, delivery of therapeutic payload to submucosal compartments, e.g., the Lamina propria, can allow for treatment and/or diagnosis of diseases or conditions located at and/or originated from such locations in the GI tract, whereas systemic delivery of payload can be used to provide therapeutically effective concentrations in various cell(s), tissue(s), or organ(s) within an organism.
Diseases that can be treated using a delivery construct of this disclosure can include inflammatory diseases, autoimmune diseases, cancer, metabolic diseases, neurodegenerative diseases and neurological diseases, viral disease or infections, and cardiovascular disease.
In some instances, the inflammatory disease can include inflammatory bowel disease, psoriasis, bacterial sepsis, Crohn's disease (e.g., fistulizing Crohn's disease), ulcerative colitis (e.g., moderate-to-severe ulcerative colitis or mild-to-moderate ulcerative colitis), collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminate colitis, pancreatitis, liver inflammation (e.g., a hepatitis), pouchitis, proctitis, and epithelial cell injury.
In some instances, the autoimmune disease can include systemic lupus erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia, thrombocytopenia purpura, Grave's disease, Sjogren's disease, dermatomyositis, Hashimoto's disease, polymyositis, multiple sclerosis, diabetes mellitus, rheumatoid arthritis, and scleroderma.
In some instances, the cancer can include non-Hodgkin's lymphomas (NHL), Hodgkin's lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, carcinomas of the bladder, kidney, ovary, cervix, breast, lung, or nasopharynx cancer, malignant melanoma, rituximab resistant NHL, and leukemia.
In some instances, the metabolic disorder can include diabetes, diabetes as a consequence of obesity, hyperglycemia, dyslipidemia, hypertriglyceridemia, syndrome X, insulin resistance, impaired glucose tolerance (IGT), diabetic dyslipidemia, hyperlipidemia, fatty liver disease, nonalcoholic steatohepatitis, obesity, impaired glucose tolerance, raised fasting glucose, insulin resistance, urinary albumin secretion, central obesity, hypertension, elevated triglycerides, elevated LDL cholesterol and/or reduced HDL cholesterol, hyperglycemia, hyperinsulinemia, dyslipidemia, ketosis, hypertriglyceridemia, syndrome X, insulin resistance, impaired fasting glucose, impaired glucose tolerance (IGT), diabetic dyslipidemia, gluconeogenesis, excess glycogenolysis, diabetic ketoacidosis, hypertriglyceridemia, hypertension, diabetic nephropathy, renal insufficiency, renal failure, hyperphagia, muscle wasting, diabetic neuropathy, diabetic retinopathy, diabetic coma, arteriosclerosis, coronary heart disease, peripheral artery disease, and hyperlipidemia.
In some instances, the cardiovascular disease can include vascular disease, heart disease, and stroke.
Other diseases and conditions that can be treated using a delivery construct of this disclosure can include growth hormone deficiency (GHD), Turner syndrome (TS), Noonan syndrome, Prader-Willi syndrome, short stature homeobox-containing gene (SHOX) deficiency, chronic renal insufficiency, idiopathic short stature, short bowel syndrome, allergy, graft-vs-host disease, anemia, disorders of hematopoietic cells, and diseases of the endocrine system or reproductive systems.
Furthermore, a delivery construct can be administered as a pharmaceutical composition to a subject in need thereof. A delivery construct herein can be formulated into a pharmaceutical composition for increased therapeutic efficacy. For example, a delivery construct can be formulated such that it is being released at specific location(s) in or around the GI tract of a subject. In some instances, a delivery construct can be formulated to increase its biological activity for engaging immune cells in the various part in or around the GI tract, such as the ileum.
A delivery construct can be administered via various administration routes. In some cases, administration includes oral administration of the delivery construct. In some instances, a delivery construct is orally administered as a tablet or a capsule.
B. Experimental Methods
Methods are provided herein for transcytosis testing and evaluation of Cholix Carrier interacting proteins (e.g., TRIPs).
1. Transcytosis Testing
The transcytosis function of an isolated delivery constructs can be tested as a function of the delivery construct's ability to pass through an epithelial membrane (e.g., a polarized gut epithelium) via transcytosis. The delivery construct's transcytosis activity can be tested by any method known by one of skill in the art, without limitation. In various embodiments, transcytosis activity can be tested by assessing the ability of a delivery construct to enter a non-polarized cell to which it binds. In cases of a Cholix derived carrier, and without intending to be bound to any particular theory or mechanism of action, it is described herein that the transcytosis function that allows a delivery construct to pass through a polarized epithelial cell and the function to enter non-polarized cells resides in the same domain or region, i.e., amino acid residues 1-266 of SEQ ID NO: 1. Thus, the delivery construct's ability to enter the cell can be assessed, for example, by detecting the physical presence of the construct in the interior of the cell. For example, the delivery construct can be labeled with, for example, a fluorescent marker, and the delivery construct exposed to the cell. Then, the cells can be washed, removing any delivery construct that has not entered the cell, and the amount of label remaining in the cell(s) can be determined. Detecting the label within these cells, e.g., using microscopy, indicates that the delivery construct has entered the cell.
The delivery construct's transcytosis ability can be tested by assessing a delivery construct's ability to pass through a polarized epithelial cell. For example, the delivery construct can be labeled with, for example, a fluorescent marker (e.g., RFP) and contacted to the apical membranes of a layer of epithelial cells. In another example, the delivery construct can be detected using antibodies (e.g., monoclonal and/or polyclonal antibodies) directed against the delivery construct, or a portion thereof such as a Cholix derived carrier or a payload. Fluorescence detected on the basolateral side of the membrane formed by the epithelial cells (e.g., a basolateral chamber as illustrated in
In vivo transcytosis can be tested using male Wistar rats. Male Wistar rats can be housed 3-5 per cage with a 12/12 h light/dark cycle and can be 225-275 g (approximately 6-8 weeks old) when placed on study. Experiments can be conducted during the light phase using a non-recovery protocol that uses continuous isoflurane anesthesia. A 4-5 cm midline abdominal incision that exposes mid-jejunum regions can be conducted. Stock solutions at 3.86×10−5M of test articles can be prepared in phosphate buffered saline (PBS), with 50 μL (per 250 g rat) being administered by intraluminal injection (ILI) using a 29-gauge needle. The injection site mesentery can then be marked with a permanent marker. At study termination, a 3-5 mm region that captured the marked intestine segment can be isolated and processed for microscopic assessment. In vivo experiments can be performed in accordance with the U.K. Animals (Scientific Procedures) Act of 1986, the European Communities Council Directive of 1986 (86/609/EEC), and the University of Bath's ethical review procedures.
2. Evaluation of Cholix Carrier Interacting Proteins (i.e., TRIPs)
In order to identify Cholix interacting partners (e.g., receptors, enzymes, etc.) and establish the vesicular compartments where they interact with Cholix polypeptides (e.g., residues 1-266 of a Cholix sequence or a truncated version thereof), a series of pull-down assays can be performed to identify potential interaction partners that can be followed by in silico associations using surface plasmon resonance, in vitro transcytosis studies using polarized Caco-2 human intestinal epithelial cells where genetic knockdown of specific targets can be achieved, and in vivo transcytosis studies where Cholix elements and specific receptors can be co-localized in established vesicular structures. Without being bound to any theory, it is assumed that a transcytosis process can involve elements that are normally restricted within specific vesicular elements of polarized intestinal epithelial cells but can be recruited or “hijacked” by, e.g., Cholix derived carriers, to leave the late endosome and avoid lysosomal degradation following release from the cell into a basolateral compartment (e.g., via apical recycling mechanisms, apical receptor-mediated exocytosis, etc.).
3. Measuring Co-Localization of Carriers with Cellular Proteins
Co-localization of a carrier or carrier-payload complex described herein with one or more cellular proteins can be determined by fluorescence microscopy. For example, a Cholix derived carrier can be applied to the apical membrane of a polarized epithelial cell(s) (e.g., Caco-2) or to intestinal epithelial tissue. Following receptor-mediated endocytosis, the update of the carrier into the cell can be determined by fluorescence microscopy, e.g., by using labeled anti-Cholix carrier antibodies or dye-labeled carriers, or by using anti-payload antibodies. Samples or tissue sections can further be stained with markers specific for cellular proteins such as Rab7, Rab11, e.g., as described in EXAMPLE 7. Various image analysis techniques can then be used to determine the relative position of the carrier to the cellular protein (see e.g., EXAMPLE 6).
The following examples merely illustrate the disclosure and are not intended to limit the disclosure in any way.
In this Example, the preparation of a delivery construct as a single amino acid sequence comprising a Cholix carrier sequence, a spacer sequence, and a therapeutic payload is described.
First, the gene of the delivery construct was amplified by PCR, incorporating restriction enzymes pairs of NdeI and EcoRI, PstI and PstI, AgeI and EcoRI, or PstI and EcoRI sites at two ends of the PCR products. After restriction enzyme digestion, the PCR products were cloned into an appropriate plasmid for cellular expression, which was digested with the corresponding restriction enzyme pairs. The resulting construct comprised the amino acid sequence set forth in SEQ ID NO: 147 and was also tagged with a 6-His motif (SEQ ID NO: 205) at the N-terminus of the protein to facilitate purification. The final plasmids were verified by restriction enzyme digestions and DNA sequencing.
The delivery constructs were expressed as follows: E. coli BL21(DE3) pLysS competent cells (Novagen, Madison, Wis.) were transformed using a standard heat-shock method in the presence of the appropriate plasmid to generate delivery construct expression cells, selected on ampicillin-containing media, and isolated and grown in Luria-Bertani broth (Difco; Becton Dickinson, Franklin Lakes, N.J.) with antibiotic, then induced for protein expression by the addition of 1 mM isopropyl-D-thiogalactopyranoside (IPTG) at OD 0.6. Two hours following IPTG induction, cells were harvested by centrifugation at 5,000 rpm for 10 min. Inclusion bodies were isolated following cell lysis and proteins were solubilized in the buffer containing 100 mM Tris-HCl (pH 8.0), 2 mM EDTA, 6 M guanidine HCl, and 65 mM dithiothreitol. Solubilized delivery construct was refolded in the presence of 0.1 M Tris, pH=7.4, 500 mM L-arginine, 0.9 mM GSSG, 2 mM EDTA. The refolded protein (SEQ ID NO: 147) was purified by Q sepharose Ion Exchange and Superdex 200 Gel Filtration chromatography (Amersham Biosciences, Inc., Sweden). The purity of proteins was assessed by SDS-PAGE and analytic HPLC (Agilent, Inc. Palo Alto, Calif.).
The delivery construct was evaluated to verify the proper folding with regard to its anticipated molecular size. Following induction, expressed protein was collected from inclusion bodies. The extent of expression of the delivery construct was verified by western blot, and the apparent molecular weight was compared to the calculated mass.
The results demonstrated stable and efficient production of functional delivery construct in high yield and purity.
This example demonstrates an in vitro model designed to evaluate the transport properties of delivery constructs described herein.
For apical to basolateral permeability, test articles (e.g., delivery construct, payload, etc.) were added to the apical (A) side and the amount of permeation was determined on the basolateral (B) side. For basolateral to apical permeability, test articles were added to the basolateral (B) side and amount of permeation was determined on the apical (A) side.
Data can be expressed as permeability (Papp) according to the following equation: Papp=(dQ/dt)/(C0*A). Q/dt is a rate of permeation, C0 is initial concentration of test article, and A is the area of the monolayer. An efflux transport ratio (Re) can be calculated according to the following equation: (Re)=Papp(B−A)/Papp(A−B). Re>2 can indicate a potential substrate for P-gp or other active efflux transporters.
SMI-100 or Caco-2 cells can be used to assess the transcytosis function of a carrier or delivery construct in vitro.
For Caco-2 cells, an ELISA assay was performed to evaluate the ability of a carrier or delivery construct to move across Caco-2 cell monolayers via transcytosis. Caco-2 (ATCC HTB-37™) cells were maintained in 5% CO2 at 37° C. in complete media: Dulbecco's modified Eagle's medium F12 (DMEM F12) supplemented with 10% fetal bovine serum, 2.5 mM glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml (Gibco BRL, Grand Island, N.Y.). Cells were fed every 2 to 3 days with this media (designated complete medium) and passaged every 5 to 7 days. For assays, cells were seeded into 24- or 96-well plates and grown to confluence.
Caco-2 cells were grown as confluent monolayers on collagen-coated 0.4-μm pore size polycarbonate membrane transwell supports (Corning-Costar, Cambridge, Mass.) and used 18-25 days after attaining a trans-epithelial electrical resistance (TER) of >250 Ω·cm2 as measured using a chopstick Millicell-ERS® voltmeter (Millipore). Apical to basolateral (A→B) transport of a carrier or delivery construct across these monolayer was determined by measuring the amount of transported protein at certain time points (e.g., 15, 30, and 45 minutes) after a e.g., 4.7 nM, 23.6 nM and 236 nM apical application of delivery construct at 37° C. TER measurements and the extent of 10 kDa fluorescent dextran (measured using an HPLC size exclusion protocol) were used to verify monolayer barrier properties during the course of the study. The extent of transport of the delivery construct was determined by titration of collected media in the cell-based cytotoxicity assay. Transported delivery construct was measured by enzyme linked immunosorbant assay (ELISA) using antibodies (e.g., anti-carrier or anti-payload, such as an anti-IL-22 antibody) for capture and detection.
Confluent monolayers of human small intestinal tissues (SMI-100, MatTek Corporation; Ashland, Mass., USA) established on cell culture inserts were allowed to stabilize for 24 h at 37° C. prior to use. Only inserts having a trans-epithelial electric resistance (TEER) of >400 Ω·cm2 were considered to have sufficient monolayer integrity for use in studies. A secondary verification of monolayer integrity was performed by assessing suppression of 70 kD dextran transport. The chambers were washed once with transport buffer (PBS). Test molecules (e.g., delivery constructs, payloads, etc.), prepared at a concentration of 20 μg/mL, were applied to the apical surface of inserts in 100 μL volumes. Basolateral volumes of 500 μL PBS were replaced at each time point for transport studies. Each experimental condition was performed in triplicate.
This example demonstrates that a carrier of SEQ ID NO: 134 can transport IL-22 payload (SEQ ID NO: 142) across polarized gut epithelial cells in vitro. This example further demonstrates that the carrier with SEQ ID NO: 134 can transport biologically active IL-22 payload across polarized gut epithelial cells and to the Lamina propria in vivo.
Transport of delivery construct (SEQ ID NO: 147) across Caco-2 cell monolayers and small intestine epithelial tissue (also referred to herein as SMI-100) was tested by applying the delivery construct to the apical membrane of the epithelial cells, according to EXAMPLE 2 and as illustrated in
The data in
For in vivo experiments, transcytosis was tested using male Wistar rats. Male Wistar rats were housed 3-5 per cage with a 12/12 h light/dark cycle and were about 225-275 g (approximately 6-8 weeks old) when placed on study. Experiments were conducted during the light phase using a non-recovery protocol that uses continuous isoflurane anesthesia. A 4-5 cm midline abdominal incision that exposed mid-jejunum regions was conducted. Stock solutions at 3.86×10−5M of delivery construct were prepared in phosphate buffered saline (PBS), with 50 μL (per 250 g rat) being administered by intraluminal injection (ILI) using a 29-gauge needle. The injection site mesentery was then marked with a permanent marker. At study termination, a 3-5 mm region that captured the marked intestine segment was isolated and processed for microscopic assessment.
The results of the transcytosis activity of the delivery construct with SEQ ID NO: 147 are shown in
Moreover,
Follow-on experiments also showed that transcytosis of delivery constructs comprising a Cholix derived carrier can depend on the presence of both GRP75 (
Together, these data demonstrate that the Cholix derived carriers described herein efficiently (e.g., at least 5%, 10%, 20%, 25%, or 50% of material applied to the apical surface) transport therapeutic payload such as IL-22 across polarized epithelial layers, with significantly increased transport rates and overall transport efficiency (e.g., at least about 2-3 fold increase) compared to the payload alone.
This example demonstrates the capability of truncated Cholix derived carriers to transport payload into polarized epithelial cells.
These data demonstrated that Cholix derived carriers with C-terminal truncations at position 187 or 205 of SEQ ID NO: 1, and an N-terminal truncation at positions 40 or 41 of SEQ ID NO: 1, respectively, can transport payload into polarized epithelial cells. These data further suggested that the N-terminal 39 amino acids of Cholix carrier can play a role in transcytosis but are not sufficient for transport to basolateral intracellular vesicles, and that 1-187 is not sufficient for transcytosis but sufficient to transport payload into epithelial cells (e.g., into supranuclear and basal compartments).
This example demonstrates the in vitro apical to basal transcytosis function of various Cholix derived carriers that were coupled to human growth hormone via a spacer using recombinant expression as described above in EXAMPLE 1.
The following carriers were evaluated for their ability to cross polarized human small intestinal epithelial cell monolayers (TABLE 10):
These results suggested that residues 1-266 of Cholix were sufficient for apical-to-basal transport, and that it can function as a transcytosis element to deliver various heterologous payloads across epithelial cells. Additionally, it was demonstrated that elements within the first 206 amino acid residues of the Cholix polypeptide consisting of the sequence set forth in SEQ ID NO: 1 were sufficient for the transcytosis function and thus can be used to efficiently (e.g., at least 5%, 10%, 20%, 25%, or 50% of material applied to the apical surface) shuttle heterologous payload molecules such as therapeutic and/or diagnostic payloads across an epithelial cell layer (e.g., the gut epithelium), thereby enabling oral administration of payload that would otherwise only be administrable via parenteral administration routes (e.g., intravenously of subcutaneously).
This example demonstrates the in vivo apical to basal transcytosis function of various Cholix derived carriers that were coupled to human growth hormone via a spacer using recombinant expression as described above in EXAMPLE 1.
Selected Cholix carriers shown in TABLE 10 of EXAMPLE 5 were examined for their capacity for transcytosis in vivo following ILI into rat jejunum.
The data obtained in these experiments showed the extent of apical to basal transport across polarized gut epithelial cells in rat jejunum of six delivery constructs, each including a different Cholix carrier. Localization of the Cholix carrier (red fluorescence) and hGH (green fluorescence) was demonstrated (
Thus, these results are in line with data obtained from in vitro transcytosis experiments described in EXAMPLE 5 and demonstrated that carriers with a C-terminal truncation at any one of residues 206-266 of the Cholix sequence set forth in SEQ ID NO: 1 can rapidly (e.g., at least 10−6 cm/sec, 10−5 cm/sec) and efficiently (e.g., at least 5%, 10%, 20%, 25%, or 50% of material applied to the apical surface) transport payload molecules (e.g., therapeutic proteins) across epithelial cells (e.g., across polarized gut epithelial cells of a subject). Moreover, these results showed that carriers with a C-terminal truncation at any one of residues 151-187 of the Cholix sequence set forth in SEQ ID NO: 1 and/or an N-terminal truncation at any one of residues 1-40 of SEQ ID NO: 1 can be used to deliver various heterologous payloads into epithelial cells.
Based on these data, the following functional Cholix sequence fragments were identified for carriers derived from the Cholix polypeptide of SEQ ID NO: 1.
135DQQRNIIEV
1VEEALNIFDECRSP
151LDNQTLEQWKTQG
188AQKEGSRHKR
This example demonstrates that Cholix derived carriers co-localize with Ras-related protein Rab11a (Rab11a or Rab11). Co-localization of a carrier with Rab11a can occur on the apical side or the basal side of an epithelial cell. It was shown that co-localization of the carrier with Rab11a at the apical side of an epithelial cell can direct the carrier to apical recycling endosomes and/or into the intestinal lumen. Co-localization of the carrier with Rab11a at the basal side of an epithelial cell indicates that the carrier can utilize basal recycling mechanisms for its release from the basal cell membrane into basolateral compartments (e.g., Lamina propria).
The co-localization of four delivery constructs with Rab11a was tested by intraluminal injection (ILI) of 50 μL of 3.86×10−5 M solutions in PBS of the four different delivery constructs. Such delivery constructs consisted of the amino acid sequences set forth in SEQ ID NOs: 152-154 and SEQ ID NO: 159.
These data demonstrated that a functional fragment of Cholix that enables basal co-localization with Rab11a to access the basal recycling system can reside at least within amino acid residues 187-266 of SEQ ID NO: 1.
Moreover, these data enable the rational design of carriers that can transport payloads to various locations inside an epithelial cell or across such epithelial cell. For examples, Cholix derived carriers comprising or consisting of amino acid residues 1-151, 1-187, 41-187, 41-187, 40-205 or 41-205 of the amino acid sequence set forth in SEQ ID NO: 1 can be used for intra-epithelial payload delivery, whereas Cholix derived carriers comprising amino acid residues 1-266 of SEQ ID NO: 1 can be used to transport payload across such epithelial cell barrier and into the Lamina propria.
This example describes epithelial cell compartment specific protein markers that were used to determine proteins that interact with Cholix derived carriers during endocytosis and/or transcytosis processed.
The following TABLE 12 below shows exemplary cell compartment specific protein markers used herein. For example, Cholix derived delivery constructs comprising an IL-10 as the heterologous payload were followed during experiments using either a monoclonal antibody (mAb) against IL-10 and/or a polyclonal antibody (pAb) raised against the Cholix carrier (e.g., one that comprises residues 1-266 or 1-386 of SEQ ID NO: 1).
This example demonstrates the determination of TRIPs that Cholix derived carriers interact with during transcytosis across polarized epithelial cells. This example further demonstrates critical interaction partners that can play a role in Cholix transcytosis.
Caco-2 cells, parental or K8, HSPG (perlecan), or GRP74 knockdown (KO) stable cells, were seeded at 1.5×105 cells/ml in each transwell. On day 18, 100 μl of PBS containing delivery construct (SEQ ID NO: 150) at 20 μg/ml or equal molar control hGH (SEQ ID NO: 190) was added onto apical side and 500 μl of PBS in the basal chamber. The amount of proteins in the basal solution after 1 hr at 37° C. was analyzed by western blotting. Blot was probed with an anti-hGH mAb.
Next, the pH-dependency of the interaction between a Cholix protein and GRP75 was evaluated.
Moreover,
These data demonstrated that GRP75 and perlecan can play a role in transcytosis function of Cholix derived carriers, and the Cholix proteins bind GRP75, perlecan, TMEM132 proteins.
This example demonstrates that carriers derived from a Cholix polypeptide are directed away from lysosomes during transcytosis and thus do not interact with the lysosomal recycling pathway that allows the carrier to transcytose unaltered and fully functional across polarized epithelial cells.
A follow-up experiment showed that Cholix-derived carriers can utilize ERGIC proteins (e.g., ERGIC-53) to traffic from apical to basal compartments following endocytosis.
Altogether, these data showed that Cholix-derived carriers utilized endogenous bacterial trafficking mechanisms to achieve apical to basal transcytosis, allowing such carriers and delivery constructs comprising such carriers to traffic from the intestinal lumen to the Lamina propria without impairing the barrier function of a gut epithelium and without being enzymatically or chemically modified during such transport. This transepithelial transport mechanisms can enable oral administration of a therapeutic delivery construct comprising a Cholix-derived carrier coupled to a therapeutic payload and transport of the therapeutic payload across intact and polarized epithelial membranes.
This example shows structural sequence elements of a Cholix polypeptide with SEQ ID NO: 178.
This structure data shows that functional sequence fragments within the Cholix sequence of SEQ ID NO: 1 can have close proximity to each other such as regions X3 and X4 and regions X1 and X2.
This example demonstrates that Cholix derived carriers can utilize distinct compartments for trafficking into and across (e.g., via transcytosis) epithelial cells using various marker proteins (see e.g., EXAMPLE 8). These data indicates that Cholix derived carrier constructs can utilize or “hijack” specific and endogenous Cholix trafficking pathways (
Additional trafficking experiments utilizing fluorescence microscopy have been conducted to shed light on the transcytosis mechanism of the Cholix carrier with SEQ ID NO: 135 that is included in the delivery construct with SEQ ID NO: 149.
Together, these data demonstrated that Cholix derived carriers can utilize endogenous Cholix trafficking pathways to transcytose across polarized epithelial cells and thus can be used to rapidly and efficiently transport payload (e.g., therapeutic proteins such as interleukins) across epithelial cell barriers without impairing the biological activity of such payload.
This example shows the Cholix polypeptide derived carrier can transport anti-TNFα agents across intact epithelial cell layers.
The delivery constructs are tested for intestinal epithelial transport as follows: wild-type rats (Sprague Dawley®, ˜200-250 grams, ˜6 weeks old, purchased from Charles River) are fasted overnight to clear intestines; prepare microfuge tubes containing 4% formaldehyde, tubes for tissue preservation, microfuge tubes for blood collection, microfuge tubes for serum collection, PBS, and test article; animals are prepped for experiment (anesthetize animals with Isoflurane, and shave abdomen); prepare injections for each animal (each animal receives 4 injections (2× jejunum and 2× colon); opened the abdominal cavity, located and marked sites of injection with distinguishing colors; slowly inject test article into lumen (injection occurs at 10 minutes in colon and 40 minutes in jejunum) and animals receive 35 μg of protein per injection at concentration of 1 μg/μL; animals are euthanized at 50 minutes; terminal blood is collected via cardiac puncture; remove jejunum and colon and placed on plastic-lined work surface; flush contents of jejunum and colon using PBS and discarded; excise 1 cm length of intestine from injection site; cut the excised tissue in half, and place 1 section into 4% formaldehyde. The remaining tissue is then sliced lengthwise and immediately placed in microfuge tube and frozen. This process can be repeated for all injection sites. Remove liver (˜1 cm3) and divide into 2 pieces. Place 1 section of liver in formaldehyde and the second for immediate frozen storage. Intestinal, liver & blood serum samples are collected at 40 min termination. Centrifuge blood samples and transfer serum to container for storage. Samples are transported on dry-ice and stored at −80° C. The dosing strategy was as follows: Chx386-anti-TNFα 100 μL, 490 pmol/98 μg (4.9 uM); Chx415-anti-TNFα 100 μL, 490 pmol/99.5 μg (4.9 uM); anti-TNFα 100 μL, 490 pmol/76 μg (4.9 uM).
Bioanalytical analysis of the intestinal epithelial transport of the Cholix-anti-TNF-α constructs are performed using a mouse IgG1 ELISA kit (Abeam®, Cat #ab133045) as follows: tissue samples are obtained from Brains On-line; 300 μL assay buffer 1× is added to each tube containing tissue sample; tissue is removed from the assay buffer and placed on sterile, clean cell culture lid plate; intestinal samples are gently scrapped with a cell scrapper being careful to avoid collection of the mesentery; liver samples are treated in a similar manner with additional maceration and homogenization; the resulting cellular homogenate is transferred back into the original tube; remaining tissue samples and the work area are rinsed with 100 μL buffer (2×); cellular homogenate solution is centrifuged at maximum force for 5 minutes; supernatant is applied to ELISA plate according to the manufacturer's instructions. Remaining supernatant is stored at −20° C. for later use.
These data demonstrate that Cholix derived carriers are capable of transporting a variety of payload molecules such as anti-TNFα agent across intact and polarized epithelial cell layers.
Exenatide (SEQ ID NO: 195) is a peptide having GLP-1-like biological activity that is stabilized by a C-terminal amine and an N-terminal H. In this Example, two non-naturally occurring isolated constructs comprising: 1) a carrier having SEQ ID NO: 194 processed to a carrier having SEQ ID NO: 192 and crosslinked to SEQ ID NO: 195 and 2) a carrier having SEQ ID NO: 191 processed to carrier having SEQ ID NO: 193 and crosslinked to SEQ ID NO: 195 were prepared and tested for intestinal epithelial transport in vivo. Carriers having SEQ ID NO: 191 and SEQ ID NO: 192 were prepared as described herein and Exenatide (SEQ ID NO: 195) (Cat #HOR-246) was purchased from ProSpec-Tany Technogene Ltd. PO Box 6591, East Brunswick, N.J. 08816. A Pierce™ Controlled Protein-Protein Crosslinking Kit (Cat #23456) comprising the Sulfo-SMCC Crosslinking Agent was purchased from ThermoFisher.
Exenatide (10 mg) was dissolved in 5 mL H2O to form a 2 mg/mL solution. Sulfo-SMCC (2 mg) was dissolved in 2 mL of PBS. Immediately thereafter, 0.088 mL (˜5-fold molar excess) of the Sulfo-SMCC solution was added to 1.0 mL Exenatide solution and incubated for 30 minutes at room temperature. Nonreacted Sulfo-SMCC was removed by applying 1.0 mL of the maleimide-Exenatide reaction mixture to a desalting column equilibrated with PBS, eluting with PBS, and collecting 0.5 mL fractions. The absorbance at 280 nm of each fraction was measured to locate the protein peak. Peak fractions containing most of the protein were pooled. The concentration of the pooled activated Exenatide was determined by comparing its absorbance at 280 nm with the absorbance of the original protein solution.
Carriers having SEQ ID NO: 193 and SEQ ID NO: 194 have a C-terminal extension comprising a TEV cleavage site flanked by two cysteine residues that form a disulfide bond and a C-terminal His6 tag (SEQ ID NO: 205). Carriers having SEQ ID NO: 193 and SEQ ID NO: 194 were purified on a HisTrap column using standard methods. 2 mg protein (200 μL of 10 mg/ml) in PBS at pH 7.4 was activated by treatment with 2 μl of 0.1M dithiothreitol and 5 μl of TEV protease for two hours at 30° C. Cleaved and reduced protein was applied to a 1-ml HisTrap column equilibrated with PBS. The C-terminal fragment bound to the column and the activated N-terminal SEQ ID NO: 191 or SEQ ID NO: 192 product with a free cysteine near its C-terminus was collected in the flow through.
The maleimide-activated Exenatide and carriers (sulfhydryl-SEQ ID NO: 191 protein or sulfhydryl-SEQ ID NO: 192 protein) were mixed in equal molar amounts and then incubated for 60 minutes at room temperature. The purity of the SMCC-crosslinked SEQ ID NO: 192-Exenatide complex was assessed on a Coomassie-stained SDS gel. The complex was approximately the correct molecular weight and had >90% purity (
The delivery constructs were tested for intestinal epithelial transport as follows: wild-type Sprague Dawley® rats (˜200-250 grams, ˜6 weeks old, purchased from Charles River) were fasted overnight to clear their intestines. The following materials were prepared: microfuge tubes containing 4% formaldehyde, tubes for tissue preservation, microfuge tubes for blood collection, microfuge tubes for serum collection, PBS, and test article. Animals were prepped for the experiment by anesthetizing them with Isoflorane and shave their abdomens. Four injections were prepared for each animal (2 per jejunum and 2 per colon). The abdominal cavity was opened. Injection sites were located and marked with distinguishing colors. Test articles were slowly injected into the lumen over 10 minutes for the colon and 40 minutes for the jejunum. Animals received 35 μg of protein per injection at concentration of 1 μg/μL. Animals were euthanized at 50 minutes. Terminal blood was collected via cardiac puncture. The jejunum and colon were removed and placed on a plastic-lined work surface. The contents of the jejunum and colon were flushed using PBS and discarded. A 1 cm length of intestine was excised from the injection site. The excised tissue was cut in half. One section was placed into 4% formaldehyde. The remaining tissue was then sliced lengthwise and immediately placed in a microfuge tube and frozen. This process was repeated for all injection sites. The liver (˜1 cm3) was removed and divided into 2 pieces. For storage, one section of liver was placed in formaldehyde and a second was immediately frozen. Intestinal, liver & blood serum samples were collected at 40 min after injection. Blood samples were centrifuged and the resulting serum was transferred to a container for storage. Samples were transported on dry ice and stored at −80° C. The dosing strategy was as follows: SEQ ID NO: 192-Exenatide 100 μL, 490 pmol/29.4 μg (4.9 μM); SEQ ID NO: 191-Exenatide 100 μL, 490 pmol/30.9 μg (4.9 μM); SEQ ID NO: 195, 100 μL, 490 pmol/2 μg (4.9 μM).
Bioanalytical analysis of the intestinal epithelial transport of SEQ ID NO: 192-Exenatide, SEQ ID NO: 191-Exenatide, and SEQ ID NO: 195 (Exenatide) was performed using an Exendin-4 ELISA kit (Phoenix Pharma, Cat #EK-070-94) as follows: tissue samples were obtained from Brains On-line; 300 μL assay buffer (1×) was added to each tube containing a tissue sample; tissue was removed from the assay buffer and placed on a sterile, clean cell culture lid plate; intestinal samples were gently scrapped with a cell scrapper, being careful to avoid collection of the mesentery; liver samples were treated in a similar manner with additional maceration and homogenization; the resulting cellular homogenate was transferred back into the original tube; remaining tissue samples and the work area were rinsed with 100 μL buffer (2×); cellular homogenate solution was centrifuged at maximum force for 5 minutes; supernatant was applied to an ELISA plate, which was processed according to the manufacturer's instructions; remaining supernatant was stored at −20° C. for later use.
As depicted in
This example shows that amino acid linkers of various lengths and the coupling of a heterologous payload to the N-terminus of a carrier does not significantly impact the payloads ability to bind its target when included into a delivery construct.
The IL-22 receptor dimerization assay was performed by seeding DiscoverX HEK293 cells and incubate the cells for 16 h (5,000 cells per well) using the shown concentrations of agonist (delivery construct containing the IL-22 payload). The endpoint luminescence was read on a on plate reader using PathHunter® eXpress IL22RA1/IL10RB Dimerization Assay.
The pSTAT3 activation assay was conducted using Colo205 cells incubated with 10 μL of agonist (the respective delivery construct or IL-22 control) having the various concentrations for 15 min. The extend of pSTAT3 activation was then read using MSD STAT3 plates (Cat. No. N450SMA-1).
All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of this disclosure have been described in terms of embodiments, it will be apparent to those of skill in the art that variations can be applied to the articles and methods without departing from the spirit and scope of the disclosure. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the disclosure as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the disclosure pertains. All patents, patent applications, and publications are herein incorporated by reference in their entirety for all purposes and to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes. The disclosure illustratively described herein suitably can be practiced in the absence of any element(s) not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the present disclosure has been specifically disclosed by embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims.
This application is a continuation application of International Patent Application No. PCT/US2019/050708, filed Sep. 11, 2019, which claims the benefit of U.S. Provisional Application Nos. 62/888,144, filed Aug. 16, 2019; 62/888,400, filed Aug. 16, 2019; 62/888,133, filed Aug. 16, 2019; 62/816,022, filed Mar. 8, 2019; and 62/756,889 filed Nov. 7, 2018, which applications are incorporated herein by reference in their entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 62888133 | Aug 2019 | US | |
| 62888144 | Aug 2019 | US | |
| 62888400 | Aug 2019 | US | |
| 62816022 | Mar 2019 | US | |
| 62756889 | Nov 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2019/050708 | Sep 2019 | US |
| Child | 16884456 | US |
