Anti-ASGR1 Polypeptides and Uses for Immune Tolerance - Patent application

Abstract

In this specification, Disclosed herein are binding polypeptides that bind to asialoglycoprotein receptor 1 (ASGR1) and methods of using same to induce immune tolerance to an antigen of interest, e.g., to treat, ameliorate, inhibit, or prevent a disease or disorder associated with an unwanted immune response to an antigen of interest, such as an autoimmune disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 268,183, filed February 17, 2022, U.S. Provisional Patent Application No. 63 / 268,190, filed February 17, 2022, U.S. Provisional Patent Application No. 63 / 483,456, filed February 6, 2023, and U.S. Provisional Patent Application No. 63 / 483,466, filed February 6, 2023.

[0002] Sequence Listing Reference This application is filed with an electronic Sequence Listing. The Sequence Listing is provided as an XML file entitled ANOK.048WO_ST26, was created and last modified on February 16, 2023, and is 649,053 bytes in size. The information set forth in the electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] Embodiments of the present disclosure relate to binding polypeptides that bind to ASGR1 and methods of using them to induce immune tolerance to a desired antigen, such as for the prevention or treatment of disease. [Background technology]

[0004] The liver is involved in various tolerogenic processes, such as the development of immune tolerance to non-self antigens absorbed into the bloodstream from the intestine or newly formed antigens from hepatic metabolic activity. Targeting the liver with therapeutic compositions would be beneficial for the induction of antigen-specific immune tolerance. Summary of the Invention

[0005] Antigens, such as non-self antigens absorbed into the bloodstream from the intestine or newly formed antigens through hepatic metabolic activity, fail to induce immune responses in healthy individuals. Antigen-specific immune tolerance and cross-tolerance induction for CD4+ T cells and CD8+ T cells are attributed to various cell types in the liver, including hepatocytes and liver sinusoidal endothelial cells (LSECs), respectively. Hepatocytes are the main cell type that composes the liver parenchyma and can process and present antigens on MHC-I and MHC-II, signaling CD8+ and CD4+ T cells, respectively. LSECs efficiently capture, process, and present soluble antigens in the bloodstream on MHC-I and MHC-II to circulating lymphocytes, typically resulting in the induction of CD4+ regulatory T cells or allergic CD8+ T cells.

[0006] Disclosed herein are asialoglycoprotein receptor 1 (ASGR1)-binding polypeptides. In some embodiments, the ASGR1-binding polypeptide comprises a heavy chain variable region. In some embodiments, the heavy chain variable region comprises one or more of HCDR1, HCDR2, and HCDR3. In some embodiments, the ASGR1-binding polypeptide further comprises a light chain variable region. In some embodiments, the light chain variable region comprises one or more of LCDR1, LCDR2, and LCDR3.

[0007] Also disclosed herein are tolerogenic compounds comprising an ASGR1-binding polypeptide bound to or fused to an antigen to which tolerance is desired. The ASGR1-binding polypeptide and the antigen may be bound or fused via a linker. The ASGR1-binding polypeptide and the antigen may be chemically linked using a chemical linker. The ASGR1-binding polypeptide and the antigen may be recombinantly fused.

[0008] Also disclosed herein are compositions comprising any of the tolerogenic compounds disclosed herein and a pharmaceutically acceptable excipient.

[0009] Also disclosed herein are methods for inducing tolerance to an antigen to which a subject may elicit an unwanted immune response, in some embodiments, the methods comprise administering to the subject any of the tolerogenic compounds or compositions disclosed herein.

[0010] The tolerogenic compounds or compositions disclosed herein are also for use in inducing tolerance to certain antigens to which a subject may elicit an unwanted immune response, or for use in the manufacture of a pharmaceutical product.

[0011] In some embodiments, an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide is provided, comprising a heavy chain variable (VH) region comprising a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), and a third heavy chain complementarity determining region (HCDR3). In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:9. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO:10. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO:11. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:9, HCDR2 comprises the sequence of SEQ ID NO:10, and HCDR3 comprises the sequence of SEQ ID NO:11. In some embodiments, the VH comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15.

[0012] In some embodiments, the ASGR1-binding polypeptide further comprises a light chain variable region (VL) comprising a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3). In some embodiments, LCDR1 comprises the sequence of SEQ ID NO: 25. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO: 26. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 27. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO: 25, LCDR2 comprises the sequence of SEQ ID NO: 26, and LCDR3 comprises the sequence of SEQ ID NO: 27. In some embodiments, the VL comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:31.

[0013] In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO:15 and the light chain variable region comprises the sequence of SEQ ID NO:31.

[0014] In some embodiments, the ASGR1-binding polypeptide is an antibody, a Fab' fragment, a F(ab')2 fragment, a domain antibody (dAb), or an scFv. In some embodiments, the ASGR1-binding polypeptide comprises an Fc domain, and optionally, the Fc domain is silenced.

[0015] In some embodiments, a polynucleotide encoding an ASGR1-binding polypeptide is provided, wherein the polynucleotide comprises one or more of the sequences set forth in SEQ ID NOs: 12-14, or a sequence having at least 95% identity thereto. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 16. In some embodiments, the polynucleotide comprises one or more of the sequences set forth in SEQ ID NOs: 25-27. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 32. In some embodiments, the polynucleotide further comprises the sequence of SEQ ID NO: 16.

[0016] In some embodiments, a tolerogenic compound is provided comprising an ASGR1-binding polypeptide disclosed herein, wherein the ASGR1-binding polypeptide is conjugated or fused to an antigen to which tolerance is desired. In some embodiments, the ASGR1-binding polypeptide and the antigen are conjugated or fused via a linker, optionally a polypeptide linker or a chemical bond linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises glycine and / or serine, and optionally comprises the sequence of SEQ ID NO: 37, or a sequence having at least about 80%, 85%, 90%, or 95% identity thereto. In some embodiments, the antigen is conjugated or fused to the N-terminus or C-terminus of the ASGR1-binding polypeptide.

[0017] In some embodiments, the antigen comprises a food antigen. In some embodiments, the food antigen is associated with celiac disease. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 41. In some embodiments, the antigen comprises SEQ ID NO: 41. In some embodiments, the food antigen is selected from the group consisting of conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6), 31 kda major allergen / disease resistance protein homolog (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 103D (Mal d 1), α-lactalbumin (ALA), lactotransferrin, actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), quiwellin (Act d 5), ovomucoid, ovalbumin, ovotransferrin, and lysozyme, livetin, apovitilin, bosvetin, 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transfer protein (Sin a 3), profilin (Sin a 4), profilin (Api g 4), high molecular weight glycoprotein (Api g 5), tropomyosin (Pen a 1), arginine kinase (Pen m 2), tropomyosin fast isoform, high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, strawberry pathogenesis-associated protein (Fra a 1), profilin (Mus a 1), portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens. In some embodiments, the food antigen is selected from the group consisting of high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens.In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 40-54, or a fragment thereof.

[0018] In some embodiments, the antigen comprises an autoantigen, such as thyroglobulin, thyroperoxidase, thyrotropin receptor, glutamic acid decarboxylase (GAD), 21OH hydroxylase, 17OH hydroxylase, H+ / K+ ATPase, intrinsic factor, transglutaminase, tyrosinase, tyrosinase-related protein-2, myelin basic protein, proteolipid protein, desmoglein, acetylcholine receptor, 2-oxoacid dehydrogenase complex, insulin, proinsulin, preproinsulin, insulinoma-associated protein 2 (IA-2), and selected from insulinoma-associated protein 213 (IA-213), ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocarcinoma-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, duodenal pancreatic homeobox 1, dystrophia myotonica protein kinase (DMPK), islet-specific glucose-6-phosphatase catalytic subunit-associated protein, SST G protein-coupled receptor 1-5, myeloperoxidase (MPO), proteinase-3 / myeloblastin, and portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens.

[0019] In some embodiments, the antigen comprises an antigen associated with an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of multiple sclerosis, type 1 diabetes, rheumatoid arthritis, vitiligo, uveitis, pemphigus vulgaris, neuromyelitis optica, Goodpasture's disease, Parkinson's disease, myasthenia gravis, celiac disease, primary biliary cholangitis, Sjogren's syndrome, autoimmune hepatitis, myocarditis, inflammatory cardiomyopathy, and antineutrophil cytoplasmic antibody-associated vasculitis. In some embodiments, the autoimmune disease is multiple sclerosis. In some embodiments, the antigen comprises a sequence or fragment thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 100, 71-99, 101-106, or 157-159. In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 55-70 or 153-156, or a fragment thereof.

[0020] In some embodiments, a tolerogenic compound is provided comprising an ASGR1-binding polypeptide conjugated or fused to an antigen to which tolerance is desired, wherein the ASGR1-binding polypeptide comprises a heavy chain variable region comprising one, two, or all three of HCDR1, HCDR2, and HCDR3; wherein HCDR1 comprises the sequence of SEQ ID NO: 9; HCDR2 comprises the sequence of SEQ ID NO: 10; and / or HCDR3 comprises the sequence of SEQ ID NO: 11. In some embodiments, the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15. In some embodiments, the ASGR1-binding polypeptide further comprises a light chain variable region comprising one, two, or all three of LCDR1, LCDR2, and LCDR3; wherein LCDR1 comprises the sequence of SEQ ID NO: 25; LCDR2 comprises the sequence of SEQ ID NO: 26; and / or LCDR3 comprises the sequence of SEQ ID NO: 27. In some embodiments, the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 15, and the light chain variable region comprises the sequence of SEQ ID NO: 31. In some embodiments, the ASGR1-binding polypeptide is an antibody, a Fab' fragment, a F(ab')2 fragment, a domain antibody (dAb), or an scFv. In some embodiments, the ASGR1-binding polypeptide comprises an Fc domain, and optionally, the Fc domain is silenced. In some embodiments, the antigen is a polypeptide.

[0021] In some embodiments, the ASGR1-binding polypeptide and the antigen are linked or fused via a linker, optionally a polypeptide linker or a chemical bond linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises glycine and / or serine, and optionally comprises the sequence of SEQ ID NO: 37. In some embodiments, the antigen is linked or fused to the N-terminus or C-terminus of the ASGR1-binding polypeptide. In some embodiments, the ASGR1-binding polypeptide comprises an Fc domain, optionally the Fc domain is silenced, and the antigen is linked or fused to the Fc domain, optionally the antigen is linked or fused to the C-terminus of the Fc domain.

[0022] In some embodiments, the antigen comprises a food antigen. In some embodiments, the food antigen is selected from the group consisting of conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6), 31 kda major allergen / disease resistance protein homolog (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 103D (Mal d 1), α-lactalbumin (ALA), lactotransferrin, actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), quiwellin (Act d 5), ovomucoid, ovalbumin, ovotransferrin, and lysozyme, livetin, apovitilin, bosvetin, 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transfer protein (Sin a 3), profilin (Sin a 4), profilin (Api g 4), high molecular weight glycoprotein (Api g 5), tropomyosin (Pen a 1), arginine kinase (Pen m 2), tropomyosin fast isoform, high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, strawberry pathogenesis-associated protein (Fra a 1), profilin (Mus a 1), portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens. In some embodiments, the food antigen is selected from the group consisting of high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens. In some embodiments, the food antigen is associated with celiac disease. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 40-54, or a fragment thereof.

[0023] In some embodiments, the antigen comprises an autoantigen, such as thyroglobulin, thyroperoxidase, thyrotropin receptor, glutamic acid decarboxylase (GAD), 21OH hydroxylase, 17OH hydroxylase, H+ / K+ ATPase, intrinsic factor, transglutaminase, tyrosinase, tyrosinase-related protein-2, myelin basic protein, proteolipid protein, desmoglein, acetylcholine receptor, 2-oxoacid dehydrogenase complex, insulin, proinsulin, preproinsulin, insulinoma-associated protein 2 (IA-2), and selected from insulinoma-associated protein 213 (IA-213), ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocarcinoma-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, duodenal pancreatic homeobox 1, dystrophia myotonica protein kinase (DMPK), islet-specific glucose-6-phosphatase catalytic subunit-associated protein, SST G protein-coupled receptor 1-5, myeloperoxidase (MPO), proteinase-3 / myeloblastin, and portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens.

[0024] In some embodiments, the antigen comprises an antigen associated with an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of type 1 diabetes, multiple sclerosis, rheumatoid arthritis, vitiligo, uveitis, pemphigus vulgaris, neuromyelitis optica, Goodpasture's disease, Parkinson's disease, myasthenia gravis, celiac disease, primary biliary cholangitis, Sjögren's syndrome, autoimmune hepatitis, myocarditis, inflammatory cardiomyopathy, and antineutrophil cytoplasmic antibody-associated vasculitis. In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOS: 55-70 or 153-156, or a fragment thereof. In some embodiments, the autoimmune disease is multiple sclerosis. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 71-106 or 157-159, or a fragment thereof.In some embodiments, the autoimmune disease is rheumatoid arthritis, wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 107-116, 160-239, or 272-288, or a fragment thereof; Sjogren's syndrome, wherein the antigen comprises a sequence or a fragment thereof having 7%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 119 or 251-270; rheumatic heart disease, autoimmune myocarditis, viral myocarditis, or inflammatory cardiomyopathy, wherein the antigen comprises a sequence or a fragment thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 119 or 251-270; Parkinson's disease, wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NOs: 121 to 129, or a fragment thereof; antineutrophil cytoplasmic antibody-associated vasculitis (ANCA-associated vasculitis), wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NOs: 121 to 129, or a fragment thereof; primary biliary cholangitis, in which the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOS: 130-132 or 271, or a fragment thereof; and autoimmune hepatitis, in which the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOS: 133-140, or a fragment thereof.

[0025] In some embodiments, the antigen comprises an alloantigen, which in some embodiments is selected from the group consisting of subunits of MHC class I and MHC class II haplotype proteins and their complexes with the antigens they present, and the minor blood group antigens RhCE, Kell, Kidd, Duffy, Diego, and MNS.

[0026] Also provided are compositions comprising a tolerogenic compound according to the present disclosure and a pharmaceutically acceptable excipient.

[0027] Provided herein are methods for inducing tolerance to an antigen that may cause an unwanted autoimmune response in a subject, comprising administering to the subject a tolerogenic compound or composition provided herein. In some embodiments, the compound or composition is administered before, after, or both to the subject's exposure to the antigen. In some embodiments, the unwanted immune response is associated with an allergy to a food, animal, plant, or environmental allergen, an autoimmune disease, a therapeutic agent, or graft-versus-host disease.

[0028] In some embodiments, a tolerogenic compound or composition disclosed herein is provided for use in inducing tolerance in a subject to an antigen to which the subject may elicit an unwanted immune response. In some embodiments, the unwanted immune response is associated with an allergy to a food, animal, plant, or environmental allergen, an autoimmune disease, a therapeutic drug, or graft-versus-host disease. In some embodiments, the tolerogenic compound or composition is for use in the manufacture of a medicament. In some embodiments, the composition is for use in a subject in need of induction of immune tolerance in the subject.

[0029] Also provided herein is a method for inducing tolerance to an antigen for which tolerance is desired, the method comprising administering to a subject a compound comprising: (i) an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide comprising a heavy chain variable region including HCDR1, HCDR2, and HCDR3; wherein HCDR1 comprises a sequence having at least 90% identity to SEQ ID NO: 9; HCDR2 comprises a sequence having at least 90% identity to SEQ ID NO: 10; and HCDR3 comprises a sequence having at least 90% identity to SEQ ID NO: 10; and (ii) an antigen for which tolerance is desired, wherein the ASGR1 binding polypeptide is bound or fused to the antigen for which tolerance is desired.

[0030] In some embodiments, the ASGR1 binding polypeptide further comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an array having at least 90% identity to SEQ ID NO: 25, LCDR2 comprises an array having at least 90% identity to SEQ ID NO: 26, and LCDR3 comprises an array having at least 90% identity to SEQ ID NO: 27.

[0031] In some embodiments, the antigen to which tolerance is desired is associated with celiac disease. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 41, 40, 43-54, or a fragment thereof. In some embodiments, the antigen to which tolerance is desired is associated with MS. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 100, 71-99, 101-106, or 157-159, or a fragment thereof. In some embodiments, the antigen to which tolerance is desired is associated with type 1 diabetes. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 55-70 or 153-156, or a fragment thereof.

[0032] In some embodiments, administration is via the intravenous route.

[0033] A method for delivering an antigen to a subject's liver tissue, the method comprising: binding or fusing an antigen to an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide, thereby generating an ASGR1 binding polypeptide-antigen complex; and contacting the ASGR1 binding polypeptide-antigen complex with the subject's liver tissue in situ, wherein the contact causes the ASGR1 binding polypeptide to bind to ASGR1 on the liver tissue, thereby delivering the antigen to the liver tissue.

[0034] In a further embodiment, a method for delivering an antigen to liver tissue of a subject is provided, the method comprising contacting an asialoglycoprotein receptor 1 (ASGR1)-binding polypeptide bound or fused to an antigen with liver tissue of a subject in situ, thereby enabling the ASGR1-binding polypeptide to bind to ASGR1 on the liver tissue, wherein the ASGR1-binding polypeptide comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an array having at least 90% identity to SEQ ID NO: 9; HCDR2 comprises an array having at least 90% identity to SEQ ID NO: 10; HCDR3 comprises an array having at least 90% identity to SEQ ID NO: 11; and the ASGR1-binding polypeptide comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an array having at least 90% identity to SEQ ID NO: 25; LCDR2 comprises an array having at least 90% identity to SEQ ID NO: 26; and LCDR3 comprises an array having at least 90% identity to SEQ ID NO: 27. In some embodiments, tolerance to the antigen is induced in the subject by delivering the antigen in situ to liver tissue of the subject. In some embodiments, the method is for treating an autoimmune disease. In some embodiments, the method is for treating an allergy.

[0035] In some embodiments, an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide is provided, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the sequence of SEQ ID NO: 297; HCDR2 comprises the sequence of SEQ ID NO: 298; HCDR3 comprises the sequence of SEQ ID NO: 299; LCDR1 comprises the sequence of SEQ ID NO: 313; LCDR2 comprises the sequence of SEQ ID NO: 314; and LCDR3 comprises the sequence of SEQ ID NO: 315. In some embodiments, the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 303. In some embodiments, the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 319. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 303 and the light chain variable region comprises the sequence of SEQ ID NO: 319.

[0036] In some embodiments, an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide is provided, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the sequence of SEQ ID NO: 329; HCDR2 comprises the sequence of SEQ ID NO: 330; HCDR3 comprises the sequence of SEQ ID NO: 331; LCDR1 comprises the sequence of SEQ ID NO: 345; LCDR2 comprises the sequence of SEQ ID NO: 346; and LCDR3 comprises the sequence of SEQ ID NO: 347.

[0037] In some embodiments, an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide is provided, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the sequence of SEQ ID NO: 361; HCDR2 comprises the sequence of SEQ ID NO: 362; HCDR3 comprises the sequence of SEQ ID NO: 363; LCDR1 comprises the sequence of SEQ ID NO: 377; LCDR2 comprises the sequence of SEQ ID NO: 378; and LCDR3 comprises the sequence of SEQ ID NO: 379.

[0038] In some embodiments, an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide is provided, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the sequence of SEQ ID NO: 393; HCDR2 comprises the sequence of SEQ ID NO: 394; HCDR3 comprises the sequence of SEQ ID NO: 395; LCDR1 comprises the sequence of SEQ ID NO: 409; LCDR2 comprises the sequence of SEQ ID NO: 410; and LCDR3 comprises the sequence of SEQ ID NO: 411.

[0039] In some embodiments, an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide is provided, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the sequence of SEQ ID NO: 425; HCDR2 comprises the sequence of SEQ ID NO: 426; HCDR3 comprises the sequence of SEQ ID NO: 427; LCDR1 comprises the sequence of SEQ ID NO: 441; LCDR2 comprises the sequence of SEQ ID NO: 442; and LCDR3 comprises the sequence of SEQ ID NO: 443.

[0040] In some embodiments, an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide is provided, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the sequence of SEQ ID NO: 457; HCDR2 comprises the sequence of SEQ ID NO: 458; HCDR3 comprises the sequence of SEQ ID NO: 459; LCDR1 comprises the sequence of SEQ ID NO: 473; LCDR2 comprises the sequence of SEQ ID NO: 474; and LCDR3 comprises the sequence of SEQ ID NO: 475.

[0041] In some embodiments, an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide is provided, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the sequence of SEQ ID NO: 489; HCDR2 comprises the sequence of SEQ ID NO: 490; HCDR3 comprises the sequence of SEQ ID NO: 491; LCDR1 comprises the sequence of SEQ ID NO: 505; LCDR2 comprises the sequence of SEQ ID NO: 506; and LCDR3 comprises the sequence of SEQ ID NO: 507.

[0042] In some embodiments, an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide is provided, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the sequence of SEQ ID NO: 521; HCDR2 comprises the sequence of SEQ ID NO: 522; HCDR3 comprises the sequence of SEQ ID NO: 523; LCDR1 comprises the sequence of SEQ ID NO: 537; LCDR2 comprises the sequence of SEQ ID NO: 538; and LCDR3 comprises the sequence of SEQ ID NO: 539.

[0043] In some embodiments, an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide is provided, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3; wherein HCDR1 comprises the sequence of SEQ ID NO: 553; HCDR2 comprises the sequence of SEQ ID NO: 554; HCDR3 comprises the sequence of SEQ ID NO: 555; LCDR1 comprises the sequence of SEQ ID NO: 569; LCDR2 comprises the sequence of SEQ ID NO: 570; and LCDR3 comprises the sequence of SEQ ID NO: 571.

[0044] In some embodiments, an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide is provided, comprising a light chain variable region comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3; wherein HCDR1 comprises the sequence of SEQ ID NO:9; HCDR2 comprises the sequence of SEQ ID NO:10; and HCDR3 comprises the sequence of SEQ ID NO:11. In some embodiments, the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:15.

[0045] In some embodiments, the ASGR1-binding polypeptide further comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence of SEQ ID NO: 25, LCDR2 comprises the sequence of SEQ ID NO: 26, and LCDR3 comprises the sequence of SEQ ID NO: 27. In some embodiments, the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 15, and the light chain variable region comprises the sequence of SEQ ID NO: 31.

[0046] In some embodiments, the ASGR1-binding polypeptide is optionally an antibody, a Fab' fragment, a F(ab')2 fragment, a domain antibody (dAb), or an scFv. In some embodiments, the ASGR1-binding polypeptide comprises an Fc domain, and optionally, the Fc domain is silenced.

[0047] Also provided herein are polynucleotides encoding ASGR1-binding polypeptides, which polynucleotides comprise one or more of the sequences set forth in SEQ ID NOs: 12-14 or 25-27. In some embodiments, the polynucleotides comprise the sequences set forth in SEQ ID NOs: 16 or 32, or both. In some embodiments, the polynucleotides encode ASGR1-binding polypeptides according to the disclosures herein.

[0048] In some embodiments, tolerogenic compounds are provided that include an ASGR1-binding polypeptide conjugated or fused to an antigen to which tolerance is desired. In some embodiments, the ASGR1-binding polypeptide comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, where HCDR1 comprises the sequence of SEQ ID NO:9, HCDR2 comprises the sequence of SEQ ID NO:10, and HCDR3 comprises the sequence of SEQ ID NO:11. In some embodiments, the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:15.

[0049] In some embodiments, the tolerogenic compounds provided herein also comprise a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence of SEQ ID NO: 25, LCDR2 comprises the sequence of SEQ ID NO: 26, and LCDR3 comprises the sequence of SEQ ID NO: 27. In some embodiments, the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 15, and the light chain variable region comprises the sequence of SEQ ID NO: 31.

[0050] In some embodiments of the tolerogenic compounds provided herein, the ASGR1-binding polypeptide is an antibody, a Fab' fragment, a F(ab')2 fragment, a domain antibody (dAb), or an scFv. In some embodiments, the ASGR1-binding polypeptide of the tolerogenic compounds provided herein comprises an Fc domain, and optionally the Fc domain is silenced.

[0051] In some embodiments of the tolerogenic compounds provided herein, the antigen is a polypeptide. In some embodiments, the ASGR1-binding polypeptide and the antigen are linked or fused via a linker, optionally a polypeptide linker or a chemical bond linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises glycine and / or serine, and optionally comprises the sequence of SEQ ID NO: 37. In some embodiments, the antigen is linked or fused to the N-terminus or C-terminus of the ASGR1-binding polypeptide. In some embodiments of the tolerogenic compounds provided herein, the ASGR1-binding polypeptide comprises an Fc domain, optionally the Fc domain is silenced, and the antigen is linked or fused to the Fc domain, optionally the antigen is linked or fused to the C-terminus of the Fc domain.

[0052] In some embodiments of the tolerogenic compounds provided herein, the antigen comprises a food antigen. In some embodiments, the food antigen is selected from the group consisting of conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6), 31 kda major allergen / disease resistance protein homolog (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 1.03D (Mal d 1), α-lactalbumin (ALA), lactotransferrin, actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), quiwellin (Act d 5), ovomucoid, ovalbumin, ovotransferrin, and lysozyme, livetin, apovitilin, bosbetin, 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transfer protein (Sin a 3), profilin (Sin a 4), profilin (Api g 4), high molecular weight glycoprotein (Api g 5), tropomyosin (Pen a 1), arginine kinase (Pen m 2), tropomyosin fast isoform, high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, strawberry-derived pathogenesis-associated protein (Fra a 1), profilin (Mus a 1), portions of any of the above antigens, and mimetics of any of the above antigens.

[0053] In some embodiments, the food antigen is selected from the group consisting of high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens.

[0054] In some embodiments, the food antigen is associated with celiac disease. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 40-54, or a fragment thereof.

[0055] In some embodiments of the tolerogenic compounds provided herein, the antigen comprises an autoantigen. In some embodiments, the autoantigen is selected from the group consisting of thyroglobulin, thyroperoxidase, thyrotropin receptor, glutamic acid decarboxylase (GAD), 21OH hydroxylase, 17OH hydroxylase, H+ / K+ ATPase, intrinsic factor, transglutaminase, tyrosinase, tyrosinase-related protein-2, myelin basic protein, proteolipid protein, desmoglein, acetylcholine receptor, 2-oxoacid dehydrogenase complex, insulin, proinsulin, preproinsulin, insulinoma-associated protein 2 (IA-2), and selected from insulinoma-associated protein 213 (IA-213), ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocarcinoma-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, duodenal pancreatic homeobox 1, dystrophia myotonica protein kinase (DMPK), islet-specific glucose-6-phosphatase catalytic subunit-associated protein, SST G protein-coupled receptor 1-5, myeloperoxidase (MPO), proteinase-3 / myeloblastin, and portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens.

[0056] In some embodiments of the tolerogenic compounds provided herein, the antigen comprises an antigen associated with an autoimmune disease, hi some embodiments, the autoimmune disease is selected from the group consisting of type 1 diabetes, multiple sclerosis, rheumatoid arthritis, vitiligo, uveitis, pemphigus vulgaris, neuromyelitis optica, Goodpasture's disease, Parkinson's disease, myasthenia gravis, celiac disease, primary biliary cholangitis, Sjogren's syndrome, autoimmune hepatitis, myocarditis, inflammatory cardiomyopathy, and antineutrophil cytoplasmic antibody-associated vasculitis.

[0057] In some embodiments of the tolerogenic compounds provided herein, the autoimmune disease is type 1 diabetes. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 55-70 or 153-156, or a fragment thereof.

[0058] In some embodiments of the tolerogenic compounds provided herein, the autoimmune disease is multiple sclerosis. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 71-106 or 157-159, or a fragment thereof.

[0059] In some embodiments of the tolerogenic compounds provided herein, the autoimmune disease is rheumatoid arthritis. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 107-116, 160-239, or 272-288, or a fragment thereof.

[0060] In some embodiments of the tolerogenic compounds provided herein, the autoimmune disease is Sjogren's syndrome. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 117-118 or 240-245, or a fragment thereof.

[0061] In some embodiments of the tolerogenic compounds provided herein, the autoimmune disease is rheumatic heart disease, autoimmune myocarditis, viral myocarditis, or inflammatory cardiomyopathy. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 119 or 251-270, or a fragment thereof.

[0062] In some embodiments of the tolerogenic compounds provided herein, the autoimmune disease is Parkinson's disease. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 120 or 247-250, or a fragment thereof.

[0063] In some embodiments of the tolerogenic compounds provided herein, the autoimmune disease is antineutrophil cytoplasmic antibody-associated vasculitis (ANCA-vasculitis). In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 121-129, or a fragment thereof.

[0064] In some embodiments of the tolerogenic compounds provided herein, the autoimmune disease is primary biliary cholangitis. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 130-132 or 271, or a fragment thereof.

[0065] In some embodiments of the tolerogenic compounds provided herein, the autoimmune disease is autoimmune hepatitis. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 133-140, or a fragment thereof.

[0066] In some embodiments of the tolerogenic compounds provided herein, the antigen comprises an alloantigen, hi some embodiments, the alloantigen is selected from the group consisting of complexes of subunits of MHC class I and MHC class II haplotype proteins and the antigens they present, and the minor blood group antigens RhCE, Kell, Kidd, Duffy, Diego, and MNS.

[0067] Also provided herein, in some embodiments, are compositions comprising a tolerogenic compound according to the present disclosure and a pharmaceutically acceptable excipient.

[0068] Also provided herein are methods for inducing tolerance to an antigen that may cause an unwanted autoimmune response in a subject, comprising administering to the subject a tolerogenic compound according to the present disclosure or a composition according to the present disclosure.

[0069] In some embodiments, the tolerogenic compound or composition is administered before, after, or both, the subject is exposed to the antigen.

[0070] In some embodiments, the unwanted immune response is associated with an allergy to a food, animal, plant, or environmental allergen, an autoimmune disease, a therapeutic drug, or graft-versus-host disease.

[0071] Also provided herein are tolerogenic compounds of the present disclosure and / or compositions of the present disclosure for use in inducing tolerance in a subject to an antigen to which the subject may elicit an unwanted immune response, in some embodiments, the unwanted immune response is associated with an allergy to a food, animal, plant, or environmental allergen, an autoimmune disease, a therapeutic drug, or graft-versus-host disease.

[0072] Also provided herein are tolerogenic compounds of the present disclosure and / or compositions of the present disclosure for use in the manufacture of a medicament. Further provided herein are compounds of the present disclosure and / or compositions of the present disclosure for use in a subject in need of inducing immune tolerance.

[0073] In some embodiments, a method for inducing tolerance to an antigen to which tolerance is desired comprises administering to a subject: (i) an asialoglycoprotein receptor (ASGR1)-binding polypeptide comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, HCDR1 comprises a sequence having at least 90% identity to SEQ ID NO:9; HCDR2 comprises a sequence having at least 90% identity to SEQ ID NO: 10; and HCDR3 comprises a sequence having at least 90% identity to SEQ ID NO: 11; and (ii) Antigen to which tolerance is desired wherein the ASGR1-binding polypeptide is conjugated or fused to an antigen to which tolerance is desired.

[0074] In some embodiments, the ASGR1 binding polypeptide further comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an array having at least 90% identity to SEQ ID NO: 25, LCDR2 comprises an array having at least 90% identity to SEQ ID NO: 26, and LCDR3 comprises an array having at least 90% identity to SEQ ID NO: 27.

[0075] In some embodiments of the provided methods, the antigen to which tolerance is desired is associated with celiac disease. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 40-54, or a fragment thereof.

[0076] In some embodiments of the provided methods, the antigen to which tolerance is desired is associated with multiple sclerosis (MS). In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 71-106 or 157-159, or a fragment thereof.

[0077] In some embodiments of the provided methods, the antigen to which tolerance is desired is associated with type 1 diabetes. In some embodiments, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 55-70 or 153-156, or a fragment thereof.

[0078] In some embodiments of the provided methods, administration is by intravenous route.

[0079] Also provided herein is a method for delivering an antigen to a subject's liver tissue, the method comprising binding or fusing the antigen to an asialoglycoprotein receptor 1 (ASGR1) binding polypeptide, thereby generating an ASGR1 binding polypeptide-antigen complex, and contacting the ASGR1 binding polypeptide-antigen complex with the subject's liver tissue in situ, whereby the ASGR1 binding polypeptide binds to ASGR1 on the liver tissue, thereby delivering the antigen to the liver tissue.

[0080] Further provided is a method for delivering an antigen to liver tissue of a subject, the method comprising contacting an asialoglycoprotein receptor 1 (ASGR1)-binding polypeptide bound or fused to an antigen in situ with liver tissue of a subject, thereby enabling the ASGR1-binding polypeptide to bind to ASGR1 on the liver tissue, wherein the ASGR1-binding polypeptide comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an sequence having at least 90% identity to SEQ ID NO: 9, HCDR2 comprises an sequence having at least 90% identity to SEQ ID NO: 10, and HCDR3 comprises an sequence having at least 90% identity to SEQ ID NO: 11; and the ASGR1-binding polypeptide comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an sequence having at least 90% identity to SEQ ID NO: 25, LCDR2 comprises an sequence having at least 90% identity to SEQ ID NO: 26, and LCDR3 comprises an sequence having at least 90% identity to SEQ ID NO: 27. In some embodiments, delivery of an antigen to the liver tissue of a subject induces tolerance to the antigen in situ in the subject. In some embodiments, such methods are for treating autoimmune diseases. In some embodiments, such methods are for treating allergies. [Brief explanation of the drawings]

[0081] In addition to the features described herein, further features and modifications will be readily apparent from the following drawings and description of the embodiments, it being understood that these drawings depict various embodiments and are not intended to limit the scope thereof.

[0082] [Figure 1A] Non-limiting heavy chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0083] [Figure 1B] Non-limiting heavy chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0084] [Figure 1C] Non-limiting heavy chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0085] [Figure 1D] Non-limiting light chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the DNA sequences provided encode the peptide sequences provided, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0086] [Figure 1E] Non-limiting light chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0087] [Figure 1F] Non-limiting examples of light chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0088] [Figure 2A]1 shows the sequences of human asialoglycoprotein receptor 1 (ASGR1) (SEQ ID NO: 33), its extracellular domain (ECD) spanning amino acids Q62-L291 (SEQ ID NO: 34), and a His-Avi tagged variant (SEQ ID NO: 35) that can be used for antigen production and purification.

[0089] [Figure 2B] Shown are p31 mimotope peptides that induce T1D pathology in the BDC2.5 model, conjugable p31 variants with cysteine ​​linkers to attach ASGR1-binding polypeptides, non-limiting examples of glycine-serine linkers, and sequences of glycine-serine linker-p31 variants that can be used for antibody fusion.

[0090] [Figure 3] Survival curves are shown demonstrating that liver-targeted antigens protect mice from T1D in the pre-activation BDC2.5 model. NOD.SCID mice were adoptively transferred intravenously with 3x105 activated BDC2.5 T cells on day 0 and then intravenously administered αASGR1-p31 at 50 pmol / g, 50 pmol / g p31, or saline on days 0 and 4. Blood glucose levels were measured two to three times weekly for 105 days. Mice were considered diabetic after two consecutive blood glucose measurements of 250 mg / dL or higher. Experiments were performed with 7 to 8 mice per group.

[0091] [Figure 4] 1 shows diabetes onset survival curves for NOD.SCID mice adoptively transferred with BCD2.5 splenocytes treated with p31 tolerogen fused with anti-ASGR1 antibody mAb-60819 (819-p31), p31 peptide alone, or saline control.

[0092] [Figure 5]Figure 1 shows the EAE severity curves for EAE model mice injected with encephalitogenic T cells and the liver-targeted tolerogen MOG10 fused with mAb-60819 (819-MOG10). 819-MOG10 was administered at either 2 pmol / g ("dose level 1") or 10 pmol / g ("dose level 2").

[0093] [Figure 6A] These represent non-limiting heavy chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0094] [Figure 6B] The following represent non-limiting heavy chain complementarity-determining region (CDR) sequences that may be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0095] [Figure 6C]These represent non-limiting heavy chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0096] [Figure 6D] These represent non-limiting light chain framework sequences that may be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0097] [Figure 6E] The following represent non-limiting light chain complementarity-determining region (CDR) sequences that may be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that share at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences provided herein.

[0098] [Figure 6F]These represent non-limiting light chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that share at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences provided herein.

[0099] [Figure 7] 1 shows diabetes onset survival curves for NOD.SCID mice adoptively transferred with BCD2.5 splenocytes treated with p31 tolerogen fused with anti-ASGR1 antibody mAb-60856 (856-p31), p31 peptide alone, or saline control.

[0100] [Figure 8A] These represent non-limiting heavy chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0101] [Figure 8B] The following represent non-limiting heavy chain complementarity-determining region (CDR) sequences that may be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0102] [Figure 8C] These represent non-limiting heavy chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0103] [Figure 8D] These represent non-limiting light chain framework sequences that may be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0104] [Figure 8E] The following represent non-limiting light chain complementarity-determining region (CDR) sequences that may be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that share at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences provided herein.

[0105] [Figure 8F]These represent non-limiting light chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that share at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences provided herein.

[0106] [Figure 9A] These represent non-limiting heavy chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0107] [Figure 9B] The following represent non-limiting heavy chain complementarity-determining region (CDR) sequences that may be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0108] [Figure 9C]These represent non-limiting heavy chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0109] [Figure 9D] These represent non-limiting light chain framework sequences that may be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0110] [Figure 9E] The following represent non-limiting light chain complementarity-determining region (CDR) sequences that may be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that share at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences provided herein.

[0111] [Figure 9F]These represent non-limiting light chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that share at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences provided herein.

[0112] [Figure 10] 1 shows diabetes onset survival curves for NOD.SCID mice adoptively transferred with BCD2.5 splenocytes treated with p31 tolerogen fused with anti-ASGR1 antibody mAb-60869 (869-p31), p31 peptide alone, or saline control.

[0113] [Figure 11A] These represent non-limiting heavy chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0114] [Figure 11B] The following represent non-limiting heavy chain complementarity-determining region (CDR) sequences that may be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0115] [Figure 11C] Non-limiting heavy chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0116] [Figure 11D] Non-limiting light chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0117] [Figure 11E] Non-limiting light chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0118] [Figure 11F]The following shows non-limiting light chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0119] [Figure 12A] Non-limiting heavy chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0120] [Figure 12B] Non-limiting heavy chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0121] [Figure 12C]Non-limiting heavy chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0122] [Figure 12D] Non-limiting light chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0123] [Figure 12E] Non-limiting light chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0124] [Figure 12F]The following shows non-limiting light chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0125] [Figure 13A] Non-limiting heavy chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0126] [Figure 13B] Non-limiting heavy chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0127] [Figure 13C]Non-limiting heavy chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0128] [Figure 13D] Non-limiting light chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0129] [Figure 13E] Non-limiting light chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0130] [Figure 13F]The following shows non-limiting light chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0131] [Figure 14A] Non-limiting heavy chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0132] [Figure 14B] Non-limiting heavy chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0133] [Figure 14C]Non-limiting heavy chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0134] [Figure 14D] Non-limiting light chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0135] [Figure 14E] Non-limiting light chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0136] [Figure 14F]The following shows non-limiting light chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0137] [Figure 15A] Non-limiting heavy chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0138] [Figure 15B] Non-limiting heavy chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0139] [Figure 15C]Non-limiting heavy chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0140] [Figure 15D] Non-limiting light chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0141] [Figure 15E] Non-limiting light chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0142] [Figure 15F]The following shows non-limiting light chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0143] [Figure 16A] Non-limiting heavy chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0144] [Figure 16B] Non-limiting heavy chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0145] [Figure 16C]Non-limiting heavy chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0146] [Figure 16D] Non-limiting light chain framework sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0147] [Figure 16E] Non-limiting light chain complementarity-determining region (CDR) sequences that can be used in the anti-ASGR1 antibodies provided herein are shown. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein.

[0148] [Figure 16F]The following shows non-limiting light chain variable region sequences that can be used in the anti-ASGR1 antibodies provided herein. While the provided DNA sequences encode the provided peptide sequences, other DNA sequences that can encode the same peptide sequences due to codon degeneracy are also contemplated. Also contemplated are peptide and DNA sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences provided herein. DETAILED DESCRIPTION OF THE INVENTION

[0149] Immune responses to various antigens can be a significant cause of morbidity and mortality. Immune responses can adversely affect an individual's health and well-being, reduce the effectiveness of treatments the individual is receiving, and even lead to reactions against endogenous molecules that naturally occur or are present in the individual. Broad immunosuppression is used in certain settings to address certain immune responses, but can lead to a general susceptibility to infection and disease. Therefore, more tailored approaches, such as those described herein, are advantageous in that they can target antigen-specific immune responses. Some embodiments disclosed herein take advantage of the role of the liver and its various cell types in developing immune tolerance to specific antigens. For example, in some embodiments, a specific antigen, its immunogenic fragment, and / or mimic thereof (collectively referred to as antigen unless otherwise indicated as a particular type, e.g., fragment) is linked or conjugated to a molecule configured to target the liver (or specific cells in or associated with the liver), thereby processing the specific antigen, its immunogenic fragment, and / or mimic thereof and recalibrating the immune system, allowing the immune response to be reduced, ameliorated, or otherwise eliminated. For example, in some embodiments, the compositions provided herein target delivery to (and uptake by) the liver, such as hepatocytes or other cells expressing scavenger receptors (e.g., asialoglycoprotein receptor (ASGPR)).

[0150] Some embodiments disclosed herein demonstrate that hepatocytes can be engineered using synthetic constructs, such as the compositions disclosed herein, to actively induce immunological tolerance in antigen-specific CD4+ or CD8+ T cells, for example, by presenting or cross-presenting extracellular antigens. Hepatocytes, considered "non-professional antigen-presenting cells," are promising cell candidates for antigen presentation to stimulate tolerogenic T cell responses. Hepatocytes account for 80% of the liver and are in direct contact with circulating T lymphocytes. Hepatocytes do not express immunological costimulatory molecules. Therefore, whether hepatocytes contribute to peripheral tolerance by presenting or cross-presenting blood-borne antigens has been investigated. As demonstrated herein and according to some embodiments, hepatocytes can be engineered in situ (e.g., by targeting hepatocytes with constructs according to embodiments disclosed herein) to present and cross-present antigens to T cells, thereby contributing to peripheral tolerance.

[0151] Unlike other organs, circulating lymphocytes only transmigrate and access the parenchyma when inflammation occurs. The liver microvasculature has a unique palisaded endothelium lacking a basement membrane, allowing direct physical contact between circulating T lymphocytes and hepatic MHC+ parenchymal cells, including hepatocytes. Hepatocytes have a low in vitro cross-presentation capacity compared to other hepatocytes, particularly LSECs. Nevertheless, direct antigen expression achieved by transgenesis and / or viral vector transduction, followed by MHC-I-dependent antigen presentation in hepatocytes in vitro and in vivo, can lead to immune tolerance, primarily through suboptimal activation of antigen-specific CD8+ T lymphocytes due to the lack of CD28 costimulation, leading to clonal deletion of T cells. The induction of CD4+CD25+FoxP3+ Treg cells also occurs through lentivirus-mediated hepatocyte-dependent antigen presentation, suggesting that other antigen-presenting cells (APCs) may be involved in hepatocyte-driven tolerogenic mechanisms, as hepatocytes express low levels of MHC-II to interact with CD4+ T cells.

[0152] Hepatocytes outnumber other cellular components of the liver and are in close contact with blood components. In some embodiments disclosed herein, hepatocytes are used to establish CD4+ and CD8+ T cell peripheral tolerance through the mechanism of extracellular antigen uptake and presentation or cross-presentation. In other embodiments, the constructs and compositions disclosed herein are used alone or in combination with antigen presentation or cross-presentation to induce tolerance through other mechanisms. Hepatocytes possess (among other things) lectin receptors, including the asialoglycoprotein receptor (ASGPR). The apoptotic process activates neuraminidase, which desialylates glycoproteins, exposing terminal N-acetylgalactosamine residues that bind to ASGPR. Hepatocyte-dependent antigen presentation or cross-presentation (among other mechanisms induced by liver-resident immune cells after administration of the constructs disclosed herein (including induction of regulatory T cells) and related methods) can be used in methods of inducing immune tolerance more generally through T cell deletion and / or anergy. In some embodiments, hepatocytes are useful as target cells for tolerogenic prophylactic or therapeutic intervention.

[0153] In general, the compositions provided herein comprise an antigen of interest (e.g., an antigenic fragment of a larger molecule to which immune tolerance is desired, or in some embodiments, multiple antigens / fragments thereof), a targeting moiety (e.g., a molecule, particularly a binding polypeptide, or an antibody, that specifically targets or is recognized by the liver, a cell type within the liver, or another target organ or cell, such as a lymph node and / or spleen), where the antigen of interest and the targeting moiety are linked by a linker or the like. In some embodiments, mimetics of those antigens can be used in place of the antigen or antigen fragment.

[0154] Approaches for connecting an antigen of interest and a targeting moiety are generally known in the art. For example, if the antigen of interest and the targeting moiety are both proteins (e.g., the targeting moiety is an antibody), the antigen of interest and the targeting moiety can be fused together in different configurations, such as at the termini of either protein. In some embodiments, the antigen of interest and the targeting moiety can be recombinantly fused together in different configurations. In some embodiments, the antigen of interest and the targeting moiety can be fused using conventional recombinant cloning techniques. The antigen of interest and the targeting moiety can be connected by a linker, as generally understood in the art. In some embodiments, the linker is advantageously designed and / or configured to release the antigen (or antigen fragment or mimetic thereof) in a native or substantially native form (e.g., prior to being attached or fused to the linker) in vivo. In some embodiments, the antigenic portion of the molecule is attached to the linker via a degradable bond. Thus, in some embodiments, the antigen of interest is liberated at or near the liver (or other target site), processed, and presented to the immune system in such a manner that the immune system recognizes the native antigen (or antigen fragment or mimic thereof) as self and reduces or eliminates the immune response to that antigen.

[0155] In some embodiments, the antigen can be endogenous (e.g., a self-antigen or autoantigen) or exogenous (e.g., a foreign antigen), including, but not limited to, a foreign alloantigen that elicits an unwanted immune response in a transplant recipient (e.g., graft-versus-host disease or transplant rejection), a foreign food, animal, plant, or environmental antigen that elicits an unwanted immune response in a patient (e.g., allergic or hypersensitivity), a therapeutic agent that elicits an unwanted immune response in a patient (e.g., hypersensitivity and / or reduced therapeutic activity), a self-antigen that elicits an unwanted immune response in a patient (e.g., an autoimmune disease), or a tolerogenic portion (e.g., a fragment or epitope) thereof. The compositions provided herein are useful for inducing tolerogenesis to antigens and for treating unwanted immune responses, e.g., graft-versus-host disease, transplant rejection, immune responses to therapeutic agents, autoimmune diseases, and / or allergies, depending on the embodiment.

[0156] Also provided is a pharmaceutical composition comprising a therapeutically effective amount of the compound of the present disclosure.In some embodiments, the compound is mixed with at least one pharmaceutically acceptable excipient.In another aspect, the present disclosure provides a method for treating unwanted immune responses, such as graft-versus-host disease, transplant rejection, reaction to therapeutic agents, autoimmune disease, or allergy.

[0157] term In the following detailed description, reference is made to the accompanying drawings. In the drawings, like symbols generally refer to like elements unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0158] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter belongs. The terms used in the description of the invention specific matters in this specification are for the purpose of describing particular embodiments only and are not intended to limit the invention specific matters.

[0159] The articles "a" and "an" are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0160] "About" means an amount, level, value, number, frequency, proportion, dimension, size, amount, weight, or length that varies by as much as 10% from a reference amount, level, value, number, frequency, proportion, dimension, size, amount, weight, or length.

[0161] In some embodiments, an antigen-binding polypeptide comprises one or more complementarity-determining regions ("CDRs"; as used herein, the term "CDR" is given its ordinary meaning and also refers to a complementarity-determining region (also called a "minimal recognition unit" or "hypervariable region"). CDRs enable an antigen-binding protein to specifically bind to a particular antigen of interest. There are three heavy chain variable region CDRs (CDRH1, CDRH2, and CDRH3) and three light chain variable region CDRs (CDRL1, CDRL2, and CDRL3). Typically, the CDRs of the two chains are aligned by framework regions to form a structure that specifically binds to a particular epitope or domain on the target protein. From the N-terminus to the C-terminus, naturally occurring light and heavy chain variable regions generally follow the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A system has been devised to number the amino acids occupying the positions in each of these domains.

[0162] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of several well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikanira et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallumra et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol. 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Pliickthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70, ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272, ("AbM" numbering scheme).

[0163] The boundaries of a given CDR or FR may vary depending on the scheme used to identify it. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Both Kabat and Chothia numbering are based on the sequence length of the most common antibody regions, with insertions represented by an insertion letter, e.g., "30a," and deletions occurring in some antibodies. The two systems place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme. The AbM scheme, based on that used by Oxford Molecular's AbM antibody modeling software, is a compromise between the Kabat and Chothia definitions.

[0164] Throughout this specification, unless the context clearly dictates otherwise, the words "comprise," "comprises," and "comprising" are understood to mean the inclusion of the recited step or element or steps or elements, but not the exclusion of other steps or elements or steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are necessary or essential, and that no other elements may be present. "Consisting essentially of" means including the elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or function specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or essential, but that other elements may or may not be present, depending on whether they materially affect the activity or function of the recited elements.

[0165] As used herein, the terms "individual," "subject," or "patient" shall have their plain and ordinary meaning as understood in light of the present specification, and refer to a human or non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate, or bird, such as a chicken, as well as other vertebrates or invertebrates. The term "mammal" is used in its ordinary biological sense. Thus, specifically, it includes, but is not limited to, primates, including primates (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, etc.

[0166] As used herein, the term "isolated," as understood in light of the present specification, shall have its plain and ordinary meaning and refer to substances and / or entities that are (1) separated from at least some of the components with which they were associated when originally created (whether in nature and / or in an experimental setting) and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or equal to, about, at least, at least about, less than, less than about (or ranges including and / or spanning the aforementioned values) of other components with which they were originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or a range including and / or spanning the aforementioned values), is about that pure, is at least about that pure, is less than that pure, or is about that pure. As used herein, an "isolated" material can be "pure" (e.g., substantially free of other components). As used herein, the term "isolated cell" can refer to a cell that is not contained in a multicellular organism or tissue.

[0167] As used herein, "in vivo" is given its plain and ordinary meaning as understood in light of the present specification and means performing a method that is inside a living organism, usually an animal, a mammal, including a human, and a plant, as opposed to a tissue extract or a dead organism.

[0168] As used herein, "ex vivo" is to be given its plain and ordinary meaning as understood in light of the present specification and means that the method is carried out outside the body, with little alteration of natural conditions.

[0169] As used herein, "in vitro" is to be given its plain and ordinary meaning as understood in light of the present specification and refers to the performance of a method under other than biological conditions, e.g., in a petri dish or test tube.

[0170] As used herein, the terms "nucleic acid" or "nucleic acid molecule" shall have their plain and ordinary meaning as understood in light of the present specification and refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those naturally occurring in cells, fragments produced by polymerase chain reaction (PCR), and fragments produced by ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (e.g., DNA or RNA), analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have alterations in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications can include, for example, replacing one or more hydroxyl groups with halogens, alkyl groups, amines, or azide groups, or functionalizing the sugar as an ether or ester. Additionally, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars or carbocyclic sugar analogs. Examples of modifications of the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or their analogs. Phosphodiester bond analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates, and phosphoramidates. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain natural or modified nucleobases attached to a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded. "Oligonucleotide" can be used interchangeably with nucleic acid and can refer to double-stranded or single-stranded DNA or RNA.The nucleic acid or nucleic acids are contained in a nucleic acid vector or nucleic acid construct (e.g., a plasmid, virus, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, E. coli artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC)), which can be used to amplify and / or express the nucleic acid or nucleic acids in various biological systems. Typically, the vector or construct also contains elements including, but not limited to, a promoter, an enhancer, a terminator, an inducer, a ribosome binding site, a translation initiation site, a start codon, a stop codon, a polyadenylation signal, an origin of replication, a cloning site, a multiple cloning site, a restriction enzyme site, an epitope, a reporter gene, a selection marker, an antibiotic selection marker, a target sequence, a peptide purification tag, or an accessory gene, or any combination thereof.

[0171] A nucleic acid or nucleic acid molecule can contain one or more sequences encoding different peptides, polypeptides, or proteins, which may be adjacent in the same nucleic acid or nucleic acid molecule or joined by additional nucleic acid between them, such as linkers, repeats, or restriction enzyme sites, or any other sequence that is about, at least, at least about, less than, about less than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or a sequence of a length within a range defined by any two of the foregoing lengths. The term "downstream" in a nucleic acid as used herein shall have its plain and ordinary meaning as understood in the context of this specification, and refers to the sequence on the strand containing the coding sequence (sense strand) that is after the 3' end of the preceding sequence, if the nucleic acid is double-stranded. The term "upstream" in a nucleic acid as used herein shall have its plain and ordinary meaning as understood in the context of this specification, and refers to the sequence on the strand containing the coding sequence (sense strand) that is before the 5' end of the following sequence, if the nucleic acid is double-stranded. The term "grouped" as used herein with respect to nucleic acids shall have its plain and ordinary meaning as understood in light of the present specification and shall refer to two or more sequences that are contiguous, either directly or separated by additional nucleic acid, e.g., a linker or restriction enzyme site, or any other sequence that is about, at least, at least about, less than, about less than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or a sequence of a length in the range defined by any two of the foregoing lengths, but generally without any intervening sequence encoding a functional or catalytic polypeptide, protein, or protein domain.

[0172] The nucleic acids described herein contain nucleobases. The primary, basic, natural or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other bases include purines, pyrimidines, modified nucleobases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.

[0173] As used herein, the terms "peptide," "polypeptide," and "protein," as understood in light of the present specification, shall have their plain and ordinary meaning and refer to a polymer comprising amino acids linked by peptide bonds. Numerous functions of peptides, polypeptides, and proteins are known in the art, including, but not limited to, enzymatic, structural, transport, defensive, hormonal, or signal transduction. Peptides, polypeptides, and proteins are often produced biologically by ribosomal complexes using nucleic acid templates, although chemical synthesis is also possible. Nucleic acid templates can be manipulated to produce multiple peptide, polypeptide, and protein mutations, such as substitutions, deletions, truncations, additions, duplications, and fusions. These fusions of one or more peptides, polypeptides, or proteins can be joined adjacently within the same molecule, or by a linker, repeat, epitope, tag, or extra amino acid between sequences of about, at least, at least about, less than, about, or any other sequence length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or a range of lengths defined by any two of the aforementioned lengths. As used herein, the term "downstream" with respect to a polypeptide shall have its plain and ordinary meaning as understood in light of this specification, and refers to the sequence following the C-terminus of the preceding sequence. As used herein, the term "upstream" on a polypeptide shall have its plain and ordinary meaning as understood in light of the present specification, and shall refer to sequences preceding the N-terminus of the subsequent sequence.

[0174] As used herein, "antigen" shall have its plain and ordinary meaning and shall refer to any substance that serves as a target for a receptor of the innate or adaptive immune response, such as a T-cell receptor, major histocompatibility complex class I and II, a B-cell receptor, or an antibody. In some embodiments, the antigen may originate from within the body (e.g., "self," "auto," or "endogenous"). In further embodiments, the antigen may originate from outside the body ("non-self," "foreign," or "exogenous"), for example, introduced by inhalation, ingestion, injection, implantation, transdermal administration, etc. In some embodiments, exogenous antigens may be biochemically modified within the body. Foreign antigens include, but are not limited to, food antigens, animal antigens, plant antigens, environmental antigens, therapeutic agents, and antigens present in allografts.

[0175] As used herein, the term "epitope," also known as antigenic determinant, shall have its plain and ordinary meaning and shall refer to a segment of a macromolecule (e.g., a protein) that is recognized by the immune system, such as an antibody, B cell, major histocompatibility complex molecule, or T cell. An epitope may be recognized, for example, by an antibody or B cell and may include a portion or segment of a macromolecule that is capable of binding to an antibody or antigen-binding fragment thereof. In this regard, the term "bind" particularly relates to specific binding. For some embodiments of the present invention, the term "epitope" preferably refers to a segment of a protein or polyprotein that is recognized by the immune system. In some embodiments, an "antigen" used in the constructs disclosed herein may include one or more epitopes. In some embodiments, where one or more epitopes are included, the additional epitopes may be from the same antigen or different antigens.

[0176] A peptide that specifically binds to a particular target is called a "ligand" for that target.

[0177] "Specific binding," as a term commonly used in the biological arts, refers to a molecule that binds to a target with relatively high affinity compared to non-target tissues, and generally involves multiple non-covalent interactions such as electrostatic interactions, van der Waals interactions, hydrogen bonding, etc. Specific binding interactions characterize antibody-antigen binding, enzyme-substrate binding, and certain protein-receptor interactions; although such molecules may sometimes bind to tissues other than their specific target, to the extent that such non-target binding is insignificant, high affinity binding pairs can still fall within the definition of specific binding.

[0178] As used herein, the term "conservative change" shall have its plain and ordinary meaning and refer to changes that can generally be made to an amino acid sequence without altering activity. These changes are referred to as "conservative substitutions" or mutations; that is, an amino acid belonging to a group of amino acids having a particular size or characteristic can be replaced with another amino acid. Substitutes for an amino acid sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such substitutions are not expected to substantially affect the apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point. Conservative substitutions also include replacing an optical isomer of a sequence with another optical isomer, specifically substituting a D-amino acid for an L-amino acid at one or more residues in the sequence. Furthermore, all amino acids in a sequence can undergo D-to-L isomeric substitution. Examples of conservative substitutions include, but are not limited to, substituting Lys for Arg to maintain a positive charge, or vice versa; Glu for Asp to maintain a negative charge; Ser for Thr to maintain a free OH; and Gln for Asn to maintain a free NH. Yet another type of conservative substitution involves the introduction of an amino acid with a desired chemical reactivity to provide a reactive site for chemical conjugation reactions when the need for chemical derivatization arises. Such amino acids include, but are not limited to, Cys (to insert a sulfhydryl group), Lys (to insert a primary amine), Asp and Glu (to insert a carboxylic acid group), or unusual non-canonical amino acids containing ketone, azide, alkyne, alkene, and tetrazine side chains. Conservative substitutions or additions of amino acids with free NH2 or SH are particularly advantageous for chemical conjugation to a linker.Additionally, point mutations, deletions, and insertions of the polypeptide or corresponding nucleic acid sequence can be made, in some cases, without impairing the function of the polypeptide or nucleic acid fragment. Substitutions can include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues (including any number of substitutions therebetween as described). Variants usable in the present invention may exhibit up to 200 changes (e.g., replacements, insertions, deletions, N-terminal truncations, and / or C-terminal truncations) in amino acid sequence (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200, including any number therebetween). In some embodiments, the number of changes is greater than 200. Furthermore, in some embodiments, variants comprise polypeptide sequences or corresponding nucleic acid sequences that exhibit a degree of functional equivalence to a reference sequence (e.g., an unmodified or native sequence). In some embodiments, variants exhibit about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% functional equivalence (and any degree of functional equivalence therebetween) to the unmodified or native reference sequence. The amino acid residues described herein use either single-letter amino acid designators or three-letter abbreviations in accordance with standard polypeptide nomenclature. All amino acid residue sequences are represented herein in formulae oriented from left to right in the conventional direction from amino terminus to carboxy terminus.

[0179] The term "sequence identity" is used in relation to the sequence comparison of polypeptides or nucleic acids. This expression particularly refers to the percentage of sequences that are identical to each other, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, after aligning the sequences with each other and, if necessary, introducing gaps, and achieving the maximum percentage of sequence identity without considering conservative substitutions as part of the sequence identity. Alignment to determine the percent identity of amino acid sequences can be performed by various known methods, for example, using publicly available computer software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared.

[0180] As used herein, the term "purity" of any given substance, compound, or material shall have its plain and ordinary meaning as understood in light of this specification, and refers to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, a substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimal points therebetween. Purity may be affected by unwanted impurities, including, but not limited to, nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell debris, small molecules, degradation products, solvents, carriers, vehicles, contaminants, or combinations thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process-related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious pathogens. Purity can be measured using techniques including, but not limited to, electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, ultraviolet-visible spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetry, titration, or a combination thereof.

[0181] As used herein, the term "yield" of any given substance, compound, or material shall have its plain and ordinary meaning as understood in light of this specification, and refers to the actual total amount of a substance, compound, or material relative to the expected total amount. For example, the yield of a substance, compound, or material may be about, at least, at least about, less than, or less than about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, including all decimal points therebetween. Yield may be affected by reaction or process efficiency, unwanted side reactions, decomposition, quality of input substances, compounds, or materials, or loss of desired substances, compounds, or materials at any stage of production.

[0182] As used herein, the term "effective amount" or "effective dose" shall have its plain and ordinary meaning as understood in light of this specification, and refers to an amount of the reference composition or compound that produces an observable effect. For example, an effective amount can refer to the amount of a composition or compound that improves a subject's condition by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Actual dosage levels of the active ingredients in the active compositions of the presently disclosed subject matter can be varied to administer an amount of the active composition or compound effective to achieve the desired response for a particular subject and / or application. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the composition, formulation, route of administration, coadministration with other drugs or treatments, the severity of the condition being treated, and the physical condition and medical history of the subject being treated. In some embodiments, a minimal dose is administered, and in the absence of dose-limiting toxicity, the dosage is titrated up to the minimum effective amount. Determination and adjustment of the effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0183] As used herein, the terms "function" and "functional" shall have their plain and ordinary meaning as understood in light of this specification, meaning biological, enzymatic, or therapeutic function.

[0184] The term "inhibition," as used herein, shall have its plain and ordinary meaning as understood herein and may refer to a reduction or prevention of biological activity. The percentage reduction may be about, at least, at least about, less than, or less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount within a range defined by any two of the foregoing values. The term "delay," as used herein, shall have its plain and ordinary meaning as understood herein and refers to delaying, postponing, or deferring a biological event until a later time than would otherwise be expected. The delay may be about, at least, at least about, less than, or less than about 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the foregoing values. The terms inhibition or delay do not necessarily indicate 100% inhibition or delay; partial inhibition or delay may also be achieved.

[0185] As used herein, "pharmaceutically acceptable" shall have its plain and ordinary meaning as understood in light of the present specification and refers to a carrier, excipient, and / or stabilizer that is nontoxic or has an acceptable level of toxicity to cells or mammals exposed thereto at the dosages and concentrations employed. As used herein, "pharmaceutically acceptable," "diluent," "excipient," and / or "carrier" shall have its plain and ordinary meaning as understood in light of the present specification and is intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, absorption delaying agents, and the like, that are compatible with administration to a human, feline, canine, or other vertebrate host. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are diluents, excipients, and / or carriers approved by a regulatory agency of the federal government, state government, or other regulatory body, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in humans and animals, including non-human mammals such as cats and dogs. The terms diluent, excipient, and / or "carrier" can refer to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water, saline, and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution.Physiologically acceptable carriers can also contain one or more of the following: antioxidants such as ascorbic acid; proteins such as low molecular weight (less than about 10 residues) polypeptides, serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; carbohydrates such as amino acids, glucose, mannose, and dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The compositions, if desired, can also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. The formulation will usually be appropriate for the method of administration.

[0186] Cryoprotectants are additives that improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Cryoprotectants include, but are not limited to, DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methyl-formamide, dimethyl-formamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch. For example, the at least one cryoprotectant can be at a concentration of 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, about that percentage, at least that percentage, less than that percentage, about less than that percentage, or any percentage within a range defined by any two of the foregoing numbers.

[0187] Additional excipients with desirable properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate, sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or combinations thereof. Some excipients may be residual amounts or contaminants from the manufacturing process, including, but not limited to, serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, growth media components, or combinations thereof. The amount of excipient can be, as a percentage of the composition, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w percentage, about that percentage, at least that percentage, less than that percentage, about less than that percentage, or any percentage by weight in the range defined by any two of the foregoing numbers.

[0188] The term "pharmaceutically acceptable salts," as understood in light of this specification, shall have its plain and ordinary meaning and include, but are not limited to, relatively non-toxic, inorganic and organic acid or base addition salts of compositions or excipients, including analgesics, therapeutic agents, other materials, etc. Examples of pharmaceutically acceptable salts include those derived from mineral acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Examples of inorganic bases suitable for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, classes of such organic bases can include, but are not limited to, mono-, di-, and trialkylamines such as methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines such as mono-, di-, and triethanolamine; amino acids such as glycine, arginine, and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; and trihydroxymethylaminoethane.

[0189] Appropriate formulation depends on the selected administration route.The techniques for formulating and administering the compounds described herein are known to those skilled in the art.There are multiple techniques for administering compounds in the art, including enteral, oral, rectal, topical, sublingual, buccal, intrathecal, epidural, transdermal, aerosol, parenteral delivery (including but not limited to intramuscular, subcutaneous, intraarterial, intravenous, intraportal, intraarticular, intradermal, intraperitoneal, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injection).Pharmaceutical compositions are generally tailored to the specific intended administration route.

[0190] As used herein, "carrier" shall have its plain and ordinary meaning as understood in light of the present specification, and means a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and / or uptake of a compound into cells, tissues, and / or body organs.

[0191] As used herein, "diluent" shall have its plain and ordinary meaning as understood in light of this specification, and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the volume of a potent drug whose mass is too small for manufacture and / or administration. A diluent may also be a liquid for dissolving a drug to be administered by injection, ingestion, or inhalation. Common forms of diluents in the art are buffered aqueous solutions, such as, but not limited to, phosphate-buffered saline, which mimic the composition of human blood.

[0192] As used herein, the terms "treat" or "treating" or "treatment" shall have its plain and ordinary meaning and refer to any type of action that imparts a modulating effect, which may be, for example, a beneficial effect, to a subject suffering from a disorder, disease, or condition, including improving the subject's condition (e.g., improvement in one or more symptoms), slowing or reducing the progression of the condition, and / or changing the clinical parameters, disease, or condition, curing the condition, etc. In some embodiments, treating may include one or more of preventing or protecting against a disease or disorder, preventing clinical symptoms from developing, inhibiting a disease or disorder, preventing or suppressing the onset of clinical symptoms, alleviating a disease or disorder, and / or causing regression of clinical symptoms. In certain embodiments, treating a subject achieves, for example, one, two, three, four, or more of the following effects: (i) reducing or ameliorating the severity of a disease state or symptoms associated therewith; (ii) shortening the duration of symptoms associated with the disease or immune response; (iii) protecting against progression of the disease or symptoms associated therewith; (iv) regression of the disease or symptoms associated therewith; (v) developing or protecting against the development of symptoms associated with the disease; (vi) protecting against recurrence of symptoms associated with the disease; (vii) reducing the subject's hospitalization; (viii) reducing the length of hospitalization; (ix) increasing the survival time of a subject with the disease; (x) reducing the number of symptoms associated with the disease; or (xi) enhancing, improving, supplementing, complementing, or enhancing the prophylactic or therapeutic effect of another therapy.

[0193] As used herein, the term "tolerogen" refers to a molecule, composition, or substance that induces immune tolerance in an individual to that molecule, composition, substance, or antigenic portion thereof. Typically, when a substance (e.g., an allergen, foreign peptide, organism, or antigenic portion thereof) is introduced into an individual, an immune response to the substance is initiated. This is relevant in diseases in which the immune system inappropriately reacts to self-proteins, such as autoimmune diseases (e.g., type 1 diabetes, multiple sclerosis, etc.). Administration of a tolerogen ("tolerogen therapy") aims to reduce or alleviate this unwanted immune response, either as a treatment or prophylaxis. The liver plays an important immunoregulatory role, as hepatocytes and other liver-resident cells exhibit the ability to internalize and induce immune tolerance to molecules with normal antigenicity or, in the case of autoimmune diseases, aberrant antigenicity.

[0194] As used herein, the term "liver targeting moiety" refers to a moiety capable of delivering a drug (e.g., an immune tolerance-inducing construct, a polypeptide, etc.) to the liver. The liver contains various cell types, including, but not limited to, hepatocytes, sinusoidal epithelial cells, Kupffer cells, stellate cells, and / or dendritic cells. Typically, a liver targeting moiety directs a polypeptide to one or more of these cells. Each liver cell surface has a receptor that recognizes and specifically binds to the liver targeting moiety. Liver targeting can be achieved, for example, by chemically binding an antigen or ligand to a galactosylated or glycosylated moiety, by desialylating the antigen or ligand to expose the underlying galactosyl or glycosyl moiety, or by specifically binding an antibody to a liver-associated antigen or ligand (e.g., ASGPR).

[0195] As used herein, the term "operably linked" shall be given its ordinary meaning. In some embodiments, by way of example, when two groups are operably linked, the groups are linked in a manner that provides for one or more of the linked groups to retain their original reactivity or activity without substantial loss. In some embodiments, the antigens disclosed herein are operably linked to a linking agent and a targeting agent.

[0196] As used herein, the term "undesired immune response" refers to a reaction by a subject's immune system that is undesirable in a given situation. Typically, the immune system's reaction, when directed against an inappropriate target, can cause, enhance, or worsen disease. For example, an undesired immune response can include, but is not limited to, transplant rejection, immune responses to therapeutic drugs, autoimmune diseases, allergies, or hypersensitivity.

[0197] The term "variant" should be understood as a protein (or nucleic acid) that differs from the protein (or nucleic acid strand) from which it was derived by one or more changes in its length, sequence, or structure. The polypeptide from which a protein variant is derived is also known as the parent polypeptide or polynucleotide. The term "variant" includes "fragments" or "derivatives" of the parent molecule. Typically, "fragments" are smaller in length or size than the parent molecule, while "derivatives" have one or more differences in sequence or structure compared to the parent molecule. It also includes, but is not limited to, modified molecules such as post-translationally modified proteins (e.g., glycosylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids such as methylated DNA. The term "variant" also encompasses mixtures of different molecules, including, but not limited to, RNA-DNA hybrids. Naturally occurring variants and artificially constructed variants are understood to be encompassed by the term "variant" as used herein. Furthermore, variants usable in the present invention can be derived from homologs, orthologs, paralogs, or artificially constructed variants of a parent molecule, provided that the variant exhibits at least one biological activity of the parent molecule (e.g., is functionally active). A variant is characterized by a degree of sequence identity with the parent polypeptide from which it is derived. More precisely, a protein variant in the context of the present disclosure can exhibit at least 80% sequence identity with its parent polypeptide. Preferably, the sequence identity of a protein variant is over a contiguous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, or more amino acids. As noted above, in some embodiments, a variant exhibits about 80%, 85%, 90%, 95%, 97%, 98%, or 99% functional equivalence (and any degree of functional equivalence therebetween) to the unmodified or native reference sequence.

[0198] As used herein, the term "% w / w" or "% wt / wt" shall have its plain and ordinary meaning as understood in light of the present specification and shall mean the percentage of the weight of a component or agent relative to the total weight of the composition, expressed as 100. As used herein, the term "% v / v" or "% vol / vol" shall have its plain and ordinary meaning as understood in light of the present specification and shall mean the percentage of the liquid volume of a compound, substance, component, or agent relative to the total liquid volume of the composition, expressed as 100.

[0199] The disclosure herein describes many embodiments, generally in positive terms, and includes embodiments in which all or part of the subject matter, such as substances or materials, method steps or conditions, protocols, or procedures, is excluded.

[0200] Liver-specific binding polypeptide Disclosed herein are compositions of binding polypeptides that bind to the asialoglycoprotein receptor (ASGPR) or a component thereof. In some embodiments, the component of ASGPR is asialoglycoprotein receptor 1 (ASGR1) or asialoglycoprotein receptor 2 (ASGR2).

[0201] Further anti-ASGPR antibodies are described in US Pat. Nos. 9,771,427 and 10,358,497.

[0202] Further liver-targeting compositions for immune tolerance, which may or may not be polypeptides, are discussed in U.S. Patent Nos. 10,046,056, 10,821,157, 10,940,209, 10,946,079, and 10,953,101, each of which is expressly incorporated by reference in its entirety. Further liver-targeting compositions for immune tolerance, which may or may not be polypeptides, are discussed in WO2021 / 053589, which is expressly incorporated by reference in its entirety.

[0203] Provided herein are ASGR1-binding polypeptides. In some embodiments, the ASGR1-binding polypeptide comprises a heavy chain variable region. In some embodiments, the heavy chain variable region comprises heavy chain complementarity-determining region 1 (HCDR1). In some embodiments, the heavy chain variable region comprises HCDR2. In some embodiments, the heavy chain variable region comprises HCDR3. In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:9. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:297. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:329. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:361. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:393. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:425. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:457. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:489. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:521. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO:553. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 10. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 298. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 330. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 362. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 394. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 426. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 458. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 490. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 522. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 554. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 11. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 299. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 331. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 363. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 395.In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 427. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 459. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 491. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 523. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 555. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 9, 297, 329, 361, 393, 425, 457, 489, 521, or 553, HCDR2 comprises the sequence of SEQ ID NO: 10, 298, 330, 362, 394, 426, 458, 490, 522, or 554, and HCDR3 comprises the sequence of SEQ ID NO: 11, 299, 331, 363, 395, 427, 459, 491, 523, or 555. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 303. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 335. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 367.In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 399. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 431. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 463. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 495. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 527. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 559. In some embodiments, the ASGR1-binding polypeptide comprises a light chain variable region (or further comprises in combination with a heavy chain variable region). In some embodiments, the light chain variable region comprises light chain CDR1 (LCDR1). In some embodiments, the light chain variable region comprises LCDR2. In some embodiments, the light chain variable region comprises LCDR3. In some embodiments, the light chain variable region comprises light chain CDRs LCDR1, LCDR2, and LCDR3.In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:25. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:313. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:345. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:377. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:409. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:441. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:473. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:505. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:537. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:569. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO:26. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO:314. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO:346. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO:378. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO:410. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO:442. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO: 474. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO: 506. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO: 538. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO: 570. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 27. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 315. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 347. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 379. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 411. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 443. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 475. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 507. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 539. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 571.In some embodiments, LCDR1 comprises the sequence of SEQ ID NO: 25, 313, 345, 377, 409, 441, 473, 505, 537, or 569, LCDR2 comprises the sequence of SEQ ID NO: 26, 314, 346, 378, 410, 442, 474, 506, 538, or 570, and LCDR3 comprises the sequence of SEQ ID NO: 27, 315, 347, 379, 411, 443, 475, 507, 539, or 571. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 319. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 351. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 383. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 415. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 447.In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 479. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 511. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 543. In some embodiments, the heavy chain variable region comprises a sequence having 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 575. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 15, and the light chain variable region comprises the sequence of SEQ ID NO: 31. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 303, and the light chain variable region comprises the sequence of SEQ ID NO: 319. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 335, and the light chain variable region comprises the sequence of SEQ ID NO: 351. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 367, and the light chain variable region comprises the sequence of SEQ ID NO: 383. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 399, and the light chain variable region comprises the sequence of SEQ ID NO: 415. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 431, and the light chain variable region comprises the sequence of SEQ ID NO: 447. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 463, and the light chain variable region comprises the sequence of SEQ ID NO: 479. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 495, and the light chain variable region comprises the sequence of SEQ ID NO: 511. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 527, and the light chain variable region comprises the sequence of SEQ ID NO: 543. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 559, and the light chain variable region comprises the sequence of SEQ ID NO: 575. In some embodiments, the ASGR1-binding polypeptide is an antibody, a Fab' fragment, a F(ab')2 fragment, a domain antibody (dAb), or an scFv. In some embodiments, the ASGR1-binding polypeptide comprises an Fc domain. In some embodiments, the Fc domain is silenced. In some embodiments, the Fc domain is partially silenced.

[0204] Also disclosed herein are polynucleotides encoding ASGR1-binding polypeptides. In some embodiments, the polynucleotides comprise one or more of the sequences set forth in SEQ ID NOs: 12-14 or 25-27. In some embodiments, the polynucleotides comprise one or more of the sequences set forth in SEQ ID NOs: 300-302 or 313-315. In some embodiments, the polynucleotides comprise one or more of the sequences set forth in SEQ ID NOs: 332-334 or 345-347. In some embodiments, the polynucleotides comprise one or more of the sequences set forth in SEQ ID NOs: 364-366 or 377-379. In some embodiments, the polynucleotides comprise one or more of the sequences set forth in SEQ ID NOs: 396-398 or 409-411. In some embodiments, the polynucleotides comprise one or more of the sequences set forth in SEQ ID NOs: 428-430 or 441-443. In some embodiments, the polynucleotides comprise one or more of the sequences set forth in SEQ ID NOs: 460-462 or 473-475. In some embodiments, the polynucleotides comprise one or more of the sequences set forth in SEQ ID NOs: 492-494 or 505-507. In some embodiments, the polynucleotide comprises one or more of the sequences set forth in SEQ ID NOs: 524-526 or 537-539. In some embodiments, the polynucleotide comprises one or more of the sequences set forth in SEQ ID NOs: 556-558 or 569-571. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 16, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 15 for a heavy chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 304, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 303 for a heavy chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 336, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 335 for a heavy chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 368, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 367 for a heavy chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 400, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 399 for a heavy chain variable region.In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 432, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 431 for a heavy chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 464, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 463 for a heavy chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 496, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 495 for a heavy chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 528, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 527 for a heavy chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 560, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 559 for a heavy chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 32, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 31 for a light chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 320, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 319 of a light chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 352, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 351 of a light chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 384, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 383 of a light chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 416, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 415 of a light chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 448, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 447 of a light chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 480, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 479 of a light chain variable region.In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 512, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 511 for the light chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 544, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 543 for the light chain variable region. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 576, which is a non-limiting example of a polynucleotide sequence that encodes the sequence of SEQ ID NO: 575 for the light chain variable region. In some embodiments, the polynucleotide encodes any of the ASGR1-binding polypeptides disclosed herein.

[0205] As applied to the encoded ASGR1-binding polypeptides and polynucleotides disclosed herein, the ASGR1-binding polypeptides may further comprise immunoglobulin frameworks. These immunoglobulin frameworks may be conventionally known in the art. For example, in some embodiments, each heavy chain variable region and light chain variable region framework used has four framework sequences (FW-1, FW-2, FW-3, FW-4) that provide three CDRs.

[0206] A non-limiting example of a heavy chain framework 1 (H-FR1) is provided in SEQ ID NO: 1 and may be encoded by the polynucleotide provided in SEQ ID NO: 5. Another non-limiting example of a heavy chain framework 1 (H-FR1) is provided in SEQ ID NO: 289 and may be encoded by the polynucleotide provided in SEQ ID NO: 293. Another non-limiting example of a heavy chain framework 1 (H-FR1) is provided in SEQ ID NO: 321 and may be encoded by the polynucleotide provided in SEQ ID NO: 325. Another non-limiting example of a heavy chain framework 1 (H-FR1) is provided in SEQ ID NO: 353 and may be encoded by the polynucleotide provided in SEQ ID NO: 357. Another non-limiting example of a heavy chain framework 1 (H-FR1) is provided in SEQ ID NO: 385 and may be encoded by the polynucleotide provided in SEQ ID NO: 389. Another non-limiting example of a heavy chain framework 1 (H-FR1) is provided in SEQ ID NO: 417 and may be encoded by the polynucleotide provided in SEQ ID NO: 421. A non-limiting example of another heavy chain framework 1 (H-FR1) is provided in SEQ ID NO:449 and may be encoded by the polynucleotide provided in SEQ ID NO:453. A non-limiting example of another heavy chain framework 1 (H-FR1) is provided in SEQ ID NO:481 and may be encoded by the polynucleotide provided in SEQ ID NO:485. A non-limiting example of another heavy chain framework 1 (H-FR1) is provided in SEQ ID NO:513 and may be encoded by the polynucleotide provided in SEQ ID NO:517. A non-limiting example of another heavy chain framework 1 (H-FR1) is provided in SEQ ID NO:545 and may be encoded by the polynucleotide provided in SEQ ID NO:549. A non-limiting example of a heavy chain framework 2 (H-FR2) is provided in SEQ ID NO:2 and may be encoded by the polynucleotide provided in SEQ ID NO:6. A non-limiting example of a heavy chain framework 2 (H-FR2) is provided in SEQ ID NO:290 and may be encoded by the polynucleotide provided in SEQ ID NO:294. A non-limiting example of heavy chain framework 2 (H-FR2) is provided in SEQ ID NO:322 and can be encoded by the polynucleotide provided in SEQ ID NO:326.A non-limiting example of a heavy chain framework 2 (H-FR2) is provided in SEQ ID NO: 354 and may be encoded by the polynucleotide provided in SEQ ID NO: 358. A non-limiting example of a heavy chain framework 2 (H-FR2) is provided in SEQ ID NO: 386 and may be encoded by the polynucleotide provided in SEQ ID NO: 390. A non-limiting example of a heavy chain framework 2 (H-FR2) is provided in SEQ ID NO: 418 and may be encoded by the polynucleotide provided in SEQ ID NO: 422. A non-limiting example of a heavy chain framework 2 (H-FR2) is provided in SEQ ID NO: 450 and may be encoded by the polynucleotide provided in SEQ ID NO: 454. A non-limiting example of a heavy chain framework 2 (H-FR2) is provided in SEQ ID NO: 482 and may be encoded by the polynucleotide provided in SEQ ID NO: 486. A non-limiting example of a heavy chain framework 2 (H-FR2) is provided in SEQ ID NO: 514 and may be encoded by the polynucleotide provided in SEQ ID NO: 518. A non-limiting example of a heavy chain framework 2 (H-FR2) is provided in SEQ ID NO: 546 and may be encoded by the polynucleotide provided in SEQ ID NO: 550. A non-limiting example of a heavy chain framework 3 (H-FR3) is provided in SEQ ID NO: 3 and may be encoded by the polynucleotide provided in SEQ ID NO: 7. A non-limiting example of a heavy chain framework 3 (H-FR3) is provided in SEQ ID NO: 291 and may be encoded by the polynucleotide provided in SEQ ID NO: 295. A non-limiting example of a heavy chain framework 3 (H-FR3) is provided in SEQ ID NO: 323 and may be encoded by the polynucleotide provided in SEQ ID NO: 327. A non-limiting example of a heavy chain framework 3 (H-FR3) is provided in SEQ ID NO: 355 and may be encoded by the polynucleotide provided in SEQ ID NO: 359. A non-limiting example of a heavy chain framework 3 (H-FR3) is provided in SEQ ID NO: 387 and may be encoded by the polynucleotide provided in SEQ ID NO: 391. A non-limiting example of heavy chain framework 3 (H-FR3) is provided in SEQ ID NO:419 and can be encoded by the polynucleotide provided in SEQ ID NO:423.A non-limiting example of a heavy chain framework 3 (H-FR3) is provided in SEQ ID NO: 451 and may be encoded by the polynucleotide provided in SEQ ID NO: 455. A non-limiting example of a heavy chain framework 3 (H-FR3) is provided in SEQ ID NO: 483 and may be encoded by the polynucleotide provided in SEQ ID NO: 487. A non-limiting example of a heavy chain framework 3 (H-FR3) is provided in SEQ ID NO: 515 and may be encoded by the polynucleotide provided in SEQ ID NO: 519. A non-limiting example of a heavy chain framework 3 (H-FR3) is provided in SEQ ID NO: 547 and may be encoded by the polynucleotide provided in SEQ ID NO: 551. A non-limiting example of a heavy chain framework 4 (H-FR4) is provided in SEQ ID NO: 4 and may be encoded by the polynucleotide provided in SEQ ID NO: 8. A non-limiting example of a heavy chain framework 3 (H-FR3) is provided in SEQ ID NO: 292 and may be encoded by the polynucleotide provided in SEQ ID NO: 296. A non-limiting example of a heavy chain framework 4 (H-FR4) is provided in SEQ ID NO: 324 and may be encoded by the polynucleotide provided in SEQ ID NO: 328. A non-limiting example of a heavy chain framework 4 (H-FR4) is provided in SEQ ID NO: 356 and may be encoded by the polynucleotide provided in SEQ ID NO: 360. A non-limiting example of a heavy chain framework 4 (H-FR4) is provided in SEQ ID NO: 388 and may be encoded by the polynucleotide provided in SEQ ID NO: 392. A non-limiting example of a heavy chain framework 4 (H-FR4) is provided in SEQ ID NO: 420 and may be encoded by the polynucleotide provided in SEQ ID NO: 424. A non-limiting example of a heavy chain framework 4 (H-FR4) is provided in SEQ ID NO: 452 and may be encoded by the polynucleotide provided in SEQ ID NO: 456. A non-limiting example of a heavy chain framework 4 (H-FR4) is provided in SEQ ID NO: 484 and may be encoded by the polynucleotide provided in SEQ ID NO: 488. A non-limiting example of a heavy chain framework 4 (H-FR4) is provided in SEQ ID NO: 516 and may be encoded by the polynucleotide provided in SEQ ID NO: 520.A non-limiting example of a heavy chain framework 4 (H-FR4) is provided in SEQ ID NO: 548 and may be encoded by the polynucleotide provided in SEQ ID NO: 552. A non-limiting example of a light chain framework 1 (L-FR1) is provided in SEQ ID NO: 17 and may be encoded by the polynucleotide provided in SEQ ID NO: 21. A non-limiting example of a light chain framework 1 (L-FR1) is provided in SEQ ID NO: 305 and may be encoded by the polynucleotide provided in SEQ ID NO: 309. A non-limiting example of a light chain framework 1 (L-FR1) is provided in SEQ ID NO: 337 and may be encoded by the polynucleotide provided in SEQ ID NO: 341. A non-limiting example of a light chain framework 1 (L-FR1) is provided in SEQ ID NO: 369 and may be encoded by the polynucleotide provided in SEQ ID NO: 373. A non-limiting example of a light chain framework 1 (L-FR1) is provided in SEQ ID NO: 401 and may be encoded by the polynucleotide provided in SEQ ID NO: 405. A non-limiting example of a light chain framework 1 (L-FR1) is provided in SEQ ID NO: 433 and may be encoded by the polynucleotide provided in SEQ ID NO: 437. A non-limiting example of a light chain framework 1 (L-FR1) is provided in SEQ ID NO: 465 and may be encoded by the polynucleotide provided in SEQ ID NO: 469. A non-limiting example of a light chain framework 1 (L-FR1) is provided in SEQ ID NO: 497 and may be encoded by the polynucleotide provided in SEQ ID NO: 501. A non-limiting example of a light chain framework 1 (L-FR1) is provided in SEQ ID NO: 529 and may be encoded by the polynucleotide provided in SEQ ID NO: 533. A non-limiting example of a light chain framework 1 (L-FR1) is provided in SEQ ID NO: 561 and may be encoded by the polynucleotide provided in SEQ ID NO: 565. A non-limiting example of a light chain framework 2 (L-FR2) is provided in SEQ ID NO: 18 and may be encoded by the polynucleotide provided in SEQ ID NO: 22. A non-limiting example of a light chain framework 2 (L-FR2) is provided in SEQ ID NO:306 and can be encoded by the polynucleotide provided in SEQ ID NO:310.A non-limiting example of a light chain framework 2 (L-FR2) is provided in SEQ ID NO: 338 and may be encoded by the polynucleotide provided in SEQ ID NO: 342. A non-limiting example of a light chain framework 2 (L-FR2) is provided in SEQ ID NO: 370 and may be encoded by the polynucleotide provided in SEQ ID NO: 374. A non-limiting example of a light chain framework 2 (L-FR2) is provided in SEQ ID NO: 402 and may be encoded by the polynucleotide provided in SEQ ID NO: 406. A non-limiting example of a light chain framework 2 (L-FR2) is provided in SEQ ID NO: 434 and may be encoded by the polynucleotide provided in SEQ ID NO: 438. A non-limiting example of a light chain framework 2 (L-FR2) is provided in SEQ ID NO: 466 and may be encoded by the polynucleotide provided in SEQ ID NO: 470. A non-limiting example of a light chain framework 2 (L-FR2) is provided in SEQ ID NO: 498 and may be encoded by the polynucleotide provided in SEQ ID NO: 502. A non-limiting example of a light chain framework 2 (L-FR2) is provided in SEQ ID NO: 530 and may be encoded by the polynucleotide provided in SEQ ID NO: 534. A non-limiting example of a light chain framework 2 (L-FR2) is provided in SEQ ID NO: 562 and may be encoded by the polynucleotide provided in SEQ ID NO: 566. A non-limiting example of a light chain framework 3 (L-FR3) is provided in SEQ ID NO: 19 and may be encoded by the polynucleotide provided in SEQ ID NO: 23. A non-limiting example of a light chain framework 3 (L-FR3) is provided in SEQ ID NO: 307 and may be encoded by the polynucleotide provided in SEQ ID NO: 311. A non-limiting example of a light chain framework 3 (L-FR3) is provided in SEQ ID NO: 339 and may be encoded by the polynucleotide provided in SEQ ID NO: 343. A non-limiting example of a light chain framework 3 (L-FR3) is provided in SEQ ID NO: 371 and may be encoded by the polynucleotide provided in SEQ ID NO: 375. A non-limiting example of a light chain framework 3 (L-FR3) is provided in SEQ ID NO:403 and can be encoded by the polynucleotide provided in SEQ ID NO:407.A non-limiting example of a light chain framework 3 (L-FR3) is provided in SEQ ID NO: 435 and may be encoded by the polynucleotide provided in SEQ ID NO: 439. A non-limiting example of a light chain framework 3 (L-FR3) is provided in SEQ ID NO: 467 and may be encoded by the polynucleotide provided in SEQ ID NO: 471. A non-limiting example of a light chain framework 3 (L-FR3) is provided in SEQ ID NO: 499 and may be encoded by the polynucleotide provided in SEQ ID NO: 503. A non-limiting example of a light chain framework 3 (L-FR3) is provided in SEQ ID NO: 531 and may be encoded by the polynucleotide provided in SEQ ID NO: 535. A non-limiting example of a light chain framework 3 (L-FR3) is provided in SEQ ID NO: 563 and may be encoded by the polynucleotide provided in SEQ ID NO: 567. Light chain framework 4 (L-FR. A non-limiting example of a light chain framework 4 (L-FR4) is provided in SEQ ID NO: 20 and may be encoded by the polynucleotide provided in SEQ ID NO: 24. A non-limiting example of a light chain framework 4 (L-FR4) is provided in SEQ ID NO: 308 and may be encoded by the polynucleotide provided in SEQ ID NO: 312. A non-limiting example of a light chain framework 4 (L-FR4) is provided in SEQ ID NO: 340 and may be encoded by the polynucleotide provided in SEQ ID NO: 344. A non-limiting example of a light chain framework 4 (L-FR4) is provided in SEQ ID NO: 372 and may be encoded by the polynucleotide provided in SEQ ID NO: 376. A non-limiting example of a light chain framework 4 (L-FR4) is provided in SEQ ID NO: 404 and may be encoded by the polynucleotide provided in SEQ ID NO: 408. A non-limiting example of a light chain framework 4 (L-FR4) is provided in SEQ ID NO: 436 and may be encoded by the polynucleotide provided in SEQ ID NO: 440. A non-limiting example of a light chain framework 4 (L-FR4) is provided in SEQ ID NO: 468 and may be encoded by the polynucleotide provided in SEQ ID NO: 472. A non-limiting example of a light chain framework 4 (L-FR4) is provided in SEQ ID NO: 500 and may be encoded by the polynucleotide provided in SEQ ID NO: 504. A non-limiting example of a light chain framework 4 (L-FR4) is provided in SEQ ID NO: 532 and may be encoded by the polynucleotide provided in SEQ ID NO: 536. A non-limiting example of a light chain framework 4 (L-FR4) is provided in SEQ ID NO: 564 and may be encoded by the polynucleotide provided in SEQ ID NO: 568. As will be appreciated by those of skill in the art, it is contemplated that alternative frameworks may be substituted for any of the frameworks disclosed herein.

[0207] In some embodiments, the ASGR1-binding polypeptides disclosed herein bind to ASGR1. By binding to ASGR1, these ASGR1-binding polypeptides can also bind to the asialoglycoprotein receptor complex (ASGPR), which is composed of ASGR1 and ASGR2 subunits. In some embodiments, the ASGR1-binding polypeptides provided herein do not bind to ASGR2 (e.g., they are specific for ASGR1).

[0208] Tolerogenic Compounds and Compositions Embodiments disclosed herein relate to compounds, compositions (e.g., pharmaceutical compositions), or constructs related to immune tolerance. Immune tolerance can be induced to various antigens based on the teachings disclosed herein. For example, antigens can be endogenous (e.g., autoantigens) or exogenous (e.g., foreign antigens or alloantigens), including, but not limited to, alloantigens that cause an unwanted immune response in a transplant recipient, such as graft-versus-host disease or transplant rejection; foreign food, animal, plant, or environmental antigens that cause an unwanted immune response in a patient, such as allergy or hypersensitivity; therapeutic agents that cause an unwanted immune response in a patient, such as hypersensitivity and / or reduced therapeutic activity; autoantigens that cause an unwanted immune response in a patient, such as autoimmune disease; or tolerogenic portions, such as fragments or epitopes, of any type of antigen.

[0209] Disclosed herein are tolerogenic compounds. In some embodiments, the tolerogenic compound comprises an ASGR1-binding polypeptide conjugated or fused to an antigen for which tolerogenicity is desired. In some embodiments, the ASGR1-binding polypeptide and the antigen are conjugated or fused via a linker, optionally a peptide linker or a chemically conjugated linker. In some embodiments, the ASGR1-binding polypeptide is chemically conjugated to the antigen. In some embodiments, the ASGR1-binding polypeptide is recombinantly fused to the antigen. In some embodiments, the ASGR1-binding polypeptide is any of the ASGR1-binding polypeptides disclosed herein. In some embodiments, the ASGR1-binding polypeptide comprises a heavy chain variable region. In some embodiments, the heavy chain variable region comprises one or more of the heavy chain CDRs HCDR1, HCDR2, and HCDR3. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 9. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 297. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 329. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 361. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 393. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 425. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 457. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 489. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 521. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 553. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 10. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 298. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 330. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 362. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 394. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 426. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 458. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 490. In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 522.In some embodiments, HCDR2 comprises the sequence of SEQ ID NO: 554. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 11. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 299. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 331. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 363. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 395. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 427. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 459. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 491. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 523. In some embodiments, HCDR3 comprises the sequence of SEQ ID NO: 555. In some embodiments, HCDR1 comprises the sequence of SEQ ID NO: 9, 297, 329, 361, 393, 425, 457, 489, 521, or 553, HCDR2 comprises the sequence of SEQ ID NO: 10, 298, 330, 362, 394, 426, 458, 490, 522, or 554, and HCDR3 comprises the sequence of SEQ ID NO: 11, 299, 331, 363, 395, 427, 459, 491, 523, or 555. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 303. In some embodiments, the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:335.In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 367. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 399. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 431. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 463. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 495. In some embodiments, the heavy chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 527. In some embodiments, the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 559. In some embodiments, the ASGR1-binding polypeptide comprises a light chain variable region (or further comprises in combination with a heavy chain variable region).In some embodiments, the light chain variable region comprises one or more of the light chain CDRs LCDR1, LCDR2, and LCDR3. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:25. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:313. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:345. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:377. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:409. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:441. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:473. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:505. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:537. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO:569. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO:26. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO:314. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO:346. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO:378. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO: 410. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO: 442. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO: 474. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO: 506. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO: 538. In some embodiments, LCDR2 comprises the sequence of SEQ ID NO: 570. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 27. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 315. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 347. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 379. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 411. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 443. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 475. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 507. In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 539.In some embodiments, LCDR3 comprises the sequence of SEQ ID NO: 571. In some embodiments, LCDR1 comprises the sequence of SEQ ID NO: 25, 313, 345, 377, 409, 441, 473, 505, 537, or 569, LCDR2 comprises the sequence of SEQ ID NO: 26, 314, 346, 378, 410, 442, 474, 506, 538, or 570, and LCDR3 comprises the sequence of SEQ ID NO: 27, 315, 347, 379, 411, 443, 475, 507, 539, or 571. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 319. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 351. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 383. In some embodiments, the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:415.In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 447. In some embodiments, the light chain variable region comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 479. In some embodiments, the light chain variable region is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% identical to SEQ ID NO:511. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 543. In some embodiments, the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 543. In some embodiments, the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 575. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 15, and the light chain variable region comprises the sequence of SEQ ID NO: 31. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 303, and the light chain variable region comprises the sequence of SEQ ID NO: 319. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 335, and the light chain variable region comprises the sequence of SEQ ID NO: 351. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 367, and the light chain variable region comprises the sequence of SEQ ID NO: 383. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 399, and the light chain variable region comprises the sequence of SEQ ID NO: 415. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 431, and the light chain variable region comprises the sequence of SEQ ID NO: 447. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 463, and the light chain variable region comprises the sequence of SEQ ID NO: 479. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 495, and the light chain variable region comprises the sequence of SEQ ID NO: 511. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 527, and the light chain variable region comprises the sequence of SEQ ID NO: 543. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 559, and the light chain variable region comprises the sequence of SEQ ID NO: 575. In some embodiments, the ASGR1-binding polypeptide is an antibody, a Fab' fragment, a F(ab')2 fragment, a domain antibody (dAb), or an scFv. In some embodiments, the ASGR1-binding polypeptide comprises an Fc domain.In some embodiments, the Fc domain is silenced. In some embodiments, the Fc domain is partially silenced.

[0210] In some embodiments of any of the tolerogenic compounds disclosed herein, the ASGR1-binding polypeptide and the antigen are linked or fused together. In some embodiments, the ASGR1-binding polypeptide and the antigen are linked or fused together via a linker. In some embodiments, the ASGR1-binding polypeptide and the antigen are chemically linked, such as with a chemically conjugated linker. In some embodiments, the ASGR1-binding polypeptide and the antigen are recombinantly fused together. In some embodiments, the ASGR1-binding polypeptide and the antigen are recombinantly fused together with a linker. In some embodiments, the linker is a polypeptide linker (e.g., including, but not limited to, when the ASGR1-binding polypeptide and the antigen are both polypeptides). In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises glycine and / or serine. The use of glycine and serine linkers is commonly known in the art. In some embodiments, the linker comprises the sequence of SEQ ID NO: 37. In some embodiments, the antigen is conjugated or fused to the N-terminus or C-terminus of the ASGR1-binding polypeptide, for example, to the N-terminus or C-terminus of the heavy chain, heavy chain variable region, light chain, or light chain variable region of the ASGR1-binding polypeptide when the ASGR1-binding polypeptide is in the form of an antibody. In some embodiments, the ASGR1-binding polypeptide comprises an Fc domain. In some embodiments, the Fc domain is silenced or partially silenced, for example, so that the Fc domain exhibits substantially reduced or no binding to FcRn or other FcRγ receptors. In some embodiments, silencing the Fc domain inhibits the antibody from recycling out of the cell after uptake. In some embodiments, the antigen is conjugated or fused to the Fc domain. In some embodiments, the antigen is conjugated or fused to the C-terminus of the Fc domain.

[0211] In some embodiments, the antigen of any of the tolerogenic compounds disclosed herein is a drug capable of inducing an unwanted immune response in a subject. In some embodiments, the antigen can be a protein, peptide, or polypeptide. In some embodiments, the antigen can be a complete or partial therapeutic agent, a full-length autoantigen or portion thereof, a full-length alloantigen or portion thereof, a full-length allergen or portion thereof, or a mimic of any of the foregoing antigens. Nucleic acids encoding any of the foregoing antigens or nucleic acids that themselves act as antigens (e.g., as nucleic acid autoantigens) are also contemplated. In some embodiments, multiple antigens, or portions or fragments thereof, can be used in combination. For example, if a long peptide identified as P has antigenic regions A, B, C, and D, a composition disclosed herein for inducing tolerance to P can contain any one or more of A, B, C, and D, and / or any one or more repeats of A, B, C, and D. The listing of a particular antigen in one category or its association with a particular disease or reaction does not preclude that antigen from being considered part of another category or from being associated with another disease or reaction.

[0212] In some embodiments, the antigens to which tolerance is desired include one or more foreign antigens, such as food, animal, plant, and environmental antigens, to which the patient experiences an unwanted immune response. Therapeutic proteins can also be considered foreign antigens due to their foreign origin, but for clarity of exposition in this disclosure, such therapeutic agents are described as separate groups. Similarly, plant and animal antigens can be eaten and can be considered food antigens, while environmental antigens can be derived from plants, but are still considered foreign antigens. For the sake of brevity, no attempt will be made to distinguish and define all of these potentially overlapping groups, as one of ordinary skill in the art will recognize antigens that can be employed in the compositions of the present disclosure, especially in light of the detailed description and examples.

[0213] In some embodiments, the antigen comprises one or more therapeutic agents that are proteins, peptides, antibodies, antibody-like molecules (including antibody fragments and fusion proteins with antibodies and antibody fragments), or gene therapy vectors. These include human, non-human (e.g., murine), non-natural (e.g., artificial) proteins, antibodies, chimeric antibodies, humanized antibodies, viruses, and virus-like particles, as well as non-antibody binding scaffolds such as fibronectin, DARPins, and knottins. In some embodiments, human allotransplantation antigens are used that elicit an unwanted immune response in transplant recipients. In some embodiments, the antigen comprises one or more autoantigens that elicit an unwanted autoimmune response. While autoantigens are endogenous in patients with autoimmune disease, according to some embodiments, the polypeptides employed in the disclosed compositions are exogenously synthesized (as opposed to purified and enriched from a source).

[0214] In some embodiments, the antigens to which tolerance is desired include one or more foreign antigens, such as food, animal, plant, and environmental antigens, to which the patient experiences an unwanted immune response. Therapeutic proteins can also be considered foreign antigens due to their foreign origin, but for clarity of exposition in this disclosure, such therapeutic agents are described as separate groups. Similarly, plant and animal antigens can be eaten and can be considered food antigens, while environmental antigens can be derived from plants, but are still considered foreign antigens. For the sake of brevity, no attempt will be made to distinguish and define all of these potentially overlapping groups, as one of ordinary skill in the art will recognize antigens that can be employed in the compositions of the present disclosure, especially in light of the detailed description and examples.

[0215] In some embodiments, the antigen is selected from the group consisting of insulin, proinsulin, preproinsulin, gluten, gliadin, myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein, desmoglein-3, desmoglein-1, alpha-synuclein, acetylcholine receptor, factor VIII, factor IX, asparaginase, uricase, adeno-associated virus (AAV), and fragments of any of the foregoing. In some embodiments, the antigen is not a full-length protein. For example, in some embodiments, the antigen is not full-length gliadin, insulin, or proinsulin. In some embodiments, the antigen is not full-length myelin basic protein, full-length myelin oligodendrocyte protein, or full-length proteolipid protein. In some embodiments, the antigen is not a fragment of a protein. As described in more detail below, there are a variety of antigens to which tolerance may be desired. These include, but are not limited to, exogenous antigens that provoke a deleterious immune response when a subject is exposed to the antigen. In some embodiments, the adverse immune response can be the result of ingestion of an antigen, for example, via oral, nasal, or some other mucosal route. Such routes are the case, for example, for food antigens. In some embodiments, the antigen can be intentionally administered to the subject, for example, in conjunction with administration of a therapeutic composition to treat a disease or condition from which the subject suffers. In still additional embodiments, the antigen can be produced by the subject, for example, an autoimmune antigen. For example, in some embodiments, the antigen comprises a foreign transplant antigen, or a tolerogenic portion thereof, to which the transplant recipient elicits an unwanted immune response. In some embodiments, the antigen comprises a foreign food, animal, plant, or environmental antigen, or a tolerogenic portion thereof, to which the patient elicits an unwanted immune response. In some embodiments, the antigen comprises a foreign therapeutic agent, or a tolerogenic portion thereof, to which the patient elicits an unwanted immune response. In some embodiments, the antigen comprises a synthetic autoantigen, or a tolerogenic portion thereof, to which the endogenous version elicits an unwanted immune response.

[0216] In further detail, in some embodiments, the antigen is a food antigen. In some such embodiments, the antigen is one or more of conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6), α-lactalbumin (ALA), lactotransferrin, Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m 2), tropomyosin fast isoform, high molecular weight glutenin, low molecular weight glutenin, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein, secalin, and avenin. In some embodiments, fragments of any of these antigens and / or mimotopes of any of these antigens are also used. In some embodiments, the antigen is selected from the group consisting of gluten, high molecular weight glutenin, low molecular weight glutenin, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein, secalin, and avenin, and fragments thereof. In some embodiments, the antigen is selected from the group consisting of gluten, high molecular weight glutenin, low molecular weight glutenin, alpha-gliadin, gamma-gliadin, and omega-gliadin, and fragments thereof. In some embodiments, the antigen is gluten or a fragment thereof. In some embodiments, the antigen is gliadin or a fragment thereof.

[0217] In some embodiments, compounds are provided wherein the antigen is a therapeutic agent. In some embodiments, the antigen is selected from the group consisting of factor VII, factor VIII, factor IX, asparaginase, uricase, adeno-associated virus (AAV), and fragments of any thereof. In some embodiments, the antigen is a therapeutic agent selected from the group consisting of factor VII, factor IX, and fragments thereof. In some embodiments, the antigen is a therapeutic agent selected from the group consisting of factor VIII or a fragment thereof. In some embodiments, when the antigen is a therapeutic agent, the compounds can be used in treating, preventing, reducing, or otherwise ameliorating an immune response to a therapeutic agent for hemophilia. As discussed herein, mimotopes of any antigenic portion of the above antigens can be used in some embodiments.

[0218] In some embodiments, the antigen comprises asparaginase or a fragment thereof. In some embodiments, the antigen comprises uricase or a fragment thereof. In some such embodiments, the compounds can be used in treating, preventing, reducing, or otherwise ameliorating immune responses to anti-neoplastic agents. As discussed herein, mimotopes of any antigenic portion of the above antigens can be used in some embodiments.

[0219] In some embodiments, the antigen is associated with an autoimmune disease, for example, in some embodiments, the associated autoimmune disease is one or more of type 1 diabetes, multiple sclerosis, rheumatoid arthritis, vitiligo, uveitis, pemphigus vulgaris, celiac disease, myasthenia gravis, and neuromyelitis optica.

[0220] In some embodiments, the autoimmune disease is type 1 diabetes and the antigen comprises insulin or a fragment thereof. In some embodiments, the autoimmune disease is type 1 diabetes and the antigen comprises proinsulin or a fragment thereof. In some embodiments, the autoimmune disease is type 1 diabetes and the antigen comprises preproinsulin or a fragment thereof. As discussed herein, mimotopes of antigenic portions of any of the above antigens can be used in some embodiments. In some embodiments, combinations of these antigens can be incorporated into tolerogenic compounds, which may help reduce immune responses to autoantigens at multiple points along the insulin pathway.

[0221] In some embodiments, the autoimmune disease is multiple sclerosis and the antigen comprises myelin basic protein or a fragment thereof. In some embodiments, the autoimmune disease is multiple sclerosis and the antigen comprises myelin oligodendrocyte glycoprotein or a fragment thereof. In some embodiments, the autoimmune disease is multiple sclerosis and the antigen comprises proteolipid protein or a fragment thereof. As discussed herein, mimotopes of antigenic portions of any of the above antigens can be used in some embodiments. In some embodiments, combinations of these antigens can be incorporated into tolerogenic compounds (e.g., mixtures of antigens or fragments of MOG, MBP, and / or PLP), which may help reduce immune responses to autoantigens at multiple points along the pathways controlling myelination or myelin repair.

[0222] As discussed herein, mimotopes of any antigenic portion of the above autoantigens (or portions otherwise disclosed herein) may be used in some embodiments.

[0223] In some embodiments, the pharmaceutically acceptable composition consists of or consists essentially of a compound wherein the antigen is a food antigen, a therapeutic agent, an autoantigen, or a fragment thereof, an optional linker, and a liver targeting moiety disclosed herein.

[0224] Antigens may be complete proteins, portions of complete proteins, peptides, etc., and may be derivatized, e.g., linked with linkers. In some embodiments, antigens may be variants compared to those found in nature, and / or may include conservative substitutions, particularly maintaining sequence identity, and / or may be desialylated (or otherwise modified).

[0225] In some embodiments, the antigen comprises a food antigen. In some embodiments, the food antigen is selected from the group consisting of conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6), 31 kda major allergen / disease resistance protein homolog (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 1.03D (Mal d 1), α-lactalbumin (ALA), lactotransferrin, actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), quiwellin (Act d 5), ovomucoid, ovalbumin, ovotransferrin, and lysozyme, livetin, apovitilin, bosbetin, 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transfer protein (Sin a 3), profilin (Sin a 4), profilin (Api g 4), high molecular weight glycoprotein (Api g 5), tropomyosin (Pen a 1), arginine kinase (Pen m 2), tropomyosin fast isoform, high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, strawberry pathogen-associated protein (Fra a 1), profilin (Mus a 1), portions of any of the above antigens, and mimetics of any of the above antigens.

[0226] In some embodiments, the food antigen is selected from the group consisting of gluten, high molecular weight glutenin, low molecular weight glutenin, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein, secalin, and avenin, portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens. In some embodiments, the food antigen is selected from gluten, high molecular weight glutenin, low molecular weight glutenin, alpha-gliadin, gamma-gliadin, and omega-gliadin, portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens. In some embodiments, the food antigen is gluten or a portion or mimetic thereof. Generally, these antigens may be associated with gluten intolerance, gluten-sensitive enteropathy, and / or celiac disease. In some embodiments, the antigen is associated with the HLA-DQ2 serogroup.

[0227] In celiac disease, major antigens include, but are not limited to, tissue transglutaminase and native and deamidated forms of gluten or gluten-like proteins such as alpha-, gamma-, omega-gliadins, glutenin, hordein, secalin, and avenin. For example, those skilled in the art will understand that antigens associated with gluten intolerance can be converted into more immunogenic antigens in the body by deamidation by tissue glutaminase, which converts the glutamine in the antigen to glutamic acid. Thus, antigens associated with gluten intolerance can be considered foreign food antigens in their original form, but can also be considered self-antigens after modification in the body.

[0228] In some embodiments, sequences for wheat gluten proteins, as well as other proteins associated with gluten intolerance and / or celiac disease, are generally known in the art and are described, for example, in Bromilow et al., "A curated gluten protein sequence database to support development of proteomics methods for determination of gluten-free foods," J. Proteomics (2017) 163:67-75, which is expressly incorporated herein by reference in its entirety.

[0229] In some embodiments, peptides or epitopes useful in the tolerogenic compounds disclosed herein for use in preventing unwanted immune responses to proteins involved in gluten intolerance and / or celiac disease comprise some or all of the following sequences, alone or in combination:

[0230] HLA-DQ-2.5 related, alpha-gliadin 33-mer native: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 40)

[0231] HLA-DQ-2.5-associated, alpha-gliadin 33-mer deamidated: LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 41)

[0232] HLA-DQ-8 related, alpha-gliadin: QQYPSGQGSFQPSQQNPQ (SEQ ID NO: 42)

[0233] HLA-DQ-8 related, omega gliadin (wheat, U5UA46): QPFPQPEQPFPW (SEQ ID NO: 43)

[0234] Alpha-gliadin "15-mer" fragment: ELQPFPQPELPYPQP (SEQ ID NO: 44)

[0235] Gliadin with a linker: GGGPQPQPFPSQQPY (SEQ ID NO: 45)

[0236] Gliadin with linker and cysteine ​​complex moiety: GCRGGGPQPQPFPSQQPY (SEQ ID NO: 46)

[0237] Extended Gliadin: PQPQPFPSQQPYLQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 47)

[0238] Gliadin with a cysteine ​​complex moiety: GCGPQPQPFPSQQPYLQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 48)

[0239] Deamidated extended gliadin: PQPQPFPSQQPYLQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 49)

[0240] Deamidated extended gliadin: GCGPQPQPFPSQQPYLQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 50)

[0241] DQ-8, alpha-gliadin: FQQPQQQYPSGEGSFQPSQENPQAQ (SEQ ID NO: 51)

[0242] DQ-8, alpha-gliadin with a cysteine ​​complex moiety: GCFQQPQQQYPSGEGSFQPSQENPQAQ (SEQ ID NO: 52)

[0243] DQ-8, Extended Alpha-Gliadin: FQQPQQQYPSGEGSFQPSQENPQAQGSVQPQQLPQFEEIRN (SEQ ID NO: 53)

[0244] DQ-8, alpha-gliadin extended with a cysteine ​​complex moiety: GCFQQPQQQYPSGEGSFQPSQENPQAQGSVQPQQLPQFEEIRN (SEQ ID NO: 54)

[0245] For any of the peptide or epitope sequences provided herein for use as tolerogens (e.g., SEQ ID NOS: 40-54 or fragments thereof), embodiments may also include sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity, which are contemplated to have the same or similar potency. Some of the sequences provided herein may include a cysteine ​​linking moiety for chemical conjugation. Other moieties for chemical conjugation are also contemplated. In another embodiment, the sequences provided herein can be fused to an antibody sequence without an additional linking moiety, for example, by recombinant cloning techniques.

[0246] In some embodiments, the antigen is a foreign antigen associated with an animal, plant, or environmental allergen, toxin, or irritant. Some non-limiting examples include, but are not limited to, antigens, allergens, toxins, or irritants derived from cats, mice, dogs, horses, bees, dust, mold, trees, and plants:

[0247] weeds (including ragweed allergens Amb a 1, 2, 3, 5, and 6, and Amb t 5; ragweed Che a 2 and 5; and other weed allergens Par j 1, 2, and 3, and Par o 1);

[0248] grass (including major allergens Cyn d 1, 7, and 12, Dac g 1, 2, 5, Hol I 1.01203, Lol p 1, 2, 3, 5, and 11, Mer a 1, Pha a 1, Poa p 1, 5);

[0249] Pollen from ragweed and other weeds (including longhorn dock, lamb's quarter, common ragweed, plantain, red sorrel, and sagebrush), grasses (including Bermuda, Johnson, Kentucky, orchard, silvergrass, and timothy), and trees (including catalpa, elm, hickory, olive, pecan, sycamore, and walnut);

[0250] Dust (Der p 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 18, 20, 21, and 23 from Dermatophagoides pteronyssinus species; Der f 1, 2, 3, 6, 7, 10, 11, 13, 14, 15, 16, 18, 22, and 24 from Dermatophagoides farina species; Blo t 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 19, and 21 from Blomia tropicalis species; and Euroglyphus maynei allergen Eur m 2, and the allergen Tyr p 13 from Tyrophagus putrescentiae, and the allergens Bla g 1, 2, and 4; Per a 1, 3, and 7); from cockroaches.

[0251] Pets (including cats, dogs, rodents, and livestock; major cat allergens include Fel d 1–8, feline IgA, BLa g 2, and feline albumin; major dog allergens include Can f 1–6 and canine albumin);

[0252] Bee stings, including the major allergens API m 1-12; and

[0253] Molds containing allergens derived from the genera Aspergillus and Penicillium, as well as the species Alternaria alternatus, Davidiella tasiana, and Trichophyton rubrum.

[0254] In some embodiments, the antigen comprises an autoantigen. In some embodiments, the autoantigen is selected from the group consisting of thyroglobulin, thyroperoxidase, thyrotropin receptor, glutamic acid decarboxylase (GAD), 21OH hydroxylase, 17OH hydroxylase, H+ / K+ ATPase, intrinsic factor, transglutaminase, tyrosinase, tyrosinase-related protein-2, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, desmoglein, desmoglein-1, desmoglein-3, desmoglein-4, alpha-synuclein, acetylcholine receptor, 2-oxoacid dehydrogenase complex, insulin, proinsulin, The antigen is selected from preproinsulin, insulinoma-associated protein 2 (IA-2), insulinoma-associated protein 213 (IA-213), ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, duodenal pancreatic homeobox 1, dystrophia myotonica protein kinase (DMPK), islet-specific glucose-6-phosphatase catalytic subunit-associated protein, SST G protein-coupled receptor 1-5, myeloperoxidase (MPO), proteinase-3 / myeloblastin, and fragments or portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens. In some embodiments, the antigen comprises an antigen associated with an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of type 1 diabetes, multiple sclerosis, rheumatoid arthritis, vitiligo, uveitis, pemphigus vulgaris, neuromyelitis optica, Goodpasture's disease, Parkinson's disease, myasthenia gravis, celiac disease, primary biliary cholangitis, Sjogren's syndrome, autoimmune hepatitis, myocarditis, inflammatory cardiomyopathy, and antineutrophil cytoplasmic antibody-associated vasculitis.

[0255] In some embodiments, the antigen to which tolerance is desired is a viral antigen, e.g., a viral antigen from a therapeutic viral vector such as an adeno-associated viral vector (AAV). In some embodiments, the antigen to which tolerance is desired comprises or is an immunogenic fragment from AAV serotype 2 capsid protein 1 (SEQ ID NO: 141). In some embodiments, the antigen to which tolerance is desired comprises or is an immunogenic fragment from AAV serotype 2 capsid protein 2 (SEQ ID NO: 142). In some embodiments, the antigen to which tolerance is desired comprises or is an immunogenic fragment from AAV serotype 2 capsid protein 3 (SEQ ID NO: 143). In some embodiments, the antigen to which tolerance is desired comprises or is an immunogenic fragment from AAV serotype 9 capsid protein 1 (SEQ ID NO: 144). In some embodiments, the antigen to which tolerance is desired comprises or is an immunogenic fragment from AAV serotype 9 capsid protein 2 (SEQ ID NO: 145). In some embodiments, the antigen to which tolerance is desired comprises or is an immunogenic fragment derived from AAV serotype 9 capsid protein 3 (SEQ ID NO: 146).

[0256] MART1 (Melanoma Antigen Recognized by T Cells 1, Melan-A), including exogenously derived forms that are useful in the compositions of the present disclosure, has the following sequence (Uniprot #Q16655): MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGVLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEKNCEPVVPNAPPAYEKLSAEQSPPPYSP (SEQ ID NO: 147).

[0257] Tyrosinase, including exogenously obtained forms, that are useful in the compositions of the present disclosure has the following sequence (Uniprot #P14679): (SEQ ID NO: 148).

[0258] The melanocyte protein PMEL (gp100), including exogenously derived forms, that are useful in the compositions of the present disclosure has the following sequence (Uniprot #P40967): (SEQ ID NO: 149).

[0259] Aquaporin-4, including exogenously obtained forms, that are useful in the compositions of the present disclosure has the following sequence (Uniprot #P55087): MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV (SEQ ID NO: 150).

[0260] In uveitis, the main antigens include retinal S antigen or "S-arrestin," and interphotoreceptor retinoid-binding protein (IRBP) or retinol-binding protein 3.

[0261] S-arrestin, including exogenously obtained forms, that are useful in the compositions of the present disclosure has the following sequence (Uniprot #P10523): MAASGKTSKSEPNHVIFKKISRDKSVTIYLGNRDYIDHVSQVQPVDGVVLVDPDLVKGKKVYVTLTCAFRYGQEDIDVIGLTFRRDLYFSRVQVYPPVGAASTPTKLQESLLKKLGSNTYPFLLTFPDYLPCSVMLQPAPQDSGKSCGVDFEVKAFATDSTDAEEDKIPKKSSVRLLIRKVQHAPLEMGPQPRAEAAWQFFMSDKPLHLAVSLNKEIYFHGEPIPVTVTVTNNTEKTVKKIKAFVEQVANVVLYSSDYYVKPVAMEEAQEKVPPNSTLTKTLTLLPLLANNRERRGIALDGKIKHEDTNLASSTIIKEGIDRTVLGILVSYQIKVKLTVSGFLGELTSSEVATEVPFRLMHPQPEDPAKESYQDANLVFEEFARHNLKDAGEAEEGKRDKNDVDE (SEQ ID NO: 151).

[0262] IRBP, including exogenously obtained forms, that are useful in the compositions of the present disclosure has the following sequence (Uniprot #P10745):

[0263] In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the antigen is insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65), GAD-67, glucose-6-phosphatase 2, islet-specific glucose-6-phosphatase catalytic subunit-associated protein (IGRP), insulinoma-associated protein 2 (IA-02), insulinoma-associated protein 2 beta (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen 38, GLIMA38, chromogranin-A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular-entero-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regenerating gene II, pancreaticoduodenal homeobox 1, dystrophy myotonica kinase, and SST G protein-coupled receptor 1-5, or a fragment, portion, or mimetic thereof. In some embodiments, combinations of these antigens can be incorporated into tolerogenic compounds, resulting in a synergistic effect that reduces immune responses to autoantigens at multiple points along the insulin pathway. It should be noted that recombinant insulin and its derivatives, used therapeutically in the treatment of diabetes, are included in embodiments of the present disclosure. It should be noted that insulin is an example of an antigen that can be characterized as both an autoantigen and a therapeutic protein antigen. For example, rHu insulin and bovine insulin are therapeutic protein antigens (subject to unwanted immune attack), while endogenous human insulin is an autoantigen (subject to unwanted immune attack). Because endogenous human insulin is not available for pharmaceutical compositions, recombinant forms are utilized in certain embodiments of the compositions of the present disclosure.

[0264] Human insulin, including exogenously derived forms, that are useful in the compositions of the present disclosure has the following sequence (Uniprot #P01308): MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 153).

[0265] GAD-65, including exogenously obtained forms, useful in the compositions of the present disclosure has the following sequence (Uniprot #Q05329): (SEQ ID NO: 154).

[0266] IGRP, including exogenously derived forms, that are useful in the compositions of the present disclosure has the following sequence (Uniprot #QN9QR9): MDFLHRNGVLIIQHLQKDYRAYYTFLNFMSNVGDPRNIFFIYFPLCFQFNQTVGTKMIWVAVIGDWLNLIFKWILFGHRPYWWVQETQIYPNHSSPCLEQFPTTCETGPGSPSGHAMGASCVWYVMVTAALSHTVCGMDKFSITLHRLTWSFLWSVFWLIQISVCISRVFIATHFPHQVILGVIGGMLVAEAFEHTPGIQTASLGTYLKTNLFLFLFAVGFYLLLRVLNIDLLWSVPIAKKWCANPDWIHIDTTPFAGLVRNLGVLFGLGFAINSEMFLLSCRGGNNYTLSFRLLCALTSLTILQLYHFLQIPTHEEHLFYVLSFCKSASIPLTVVAFIPYSVHMLMKQSGKKSQ (SEQ ID NO: 155).

[0267] In some embodiments, human proinsulin, including exogenously obtained forms, that are useful in the compositions of the present disclosure has the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 156).

[0268] In some embodiments, peptides or epitopes useful in the tolerogenic compounds disclosed herein for use in preventing unwanted immune responses against proteins involved in the insulin pathway and / or for treating type 1 diabetes include some or all of the following sequences, individually or in combination:

[0269] Human Proinsulin 1-70: FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQ (SEQ ID NO: 55);

[0270] Human Proinsulin 9-70: SHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQ (SEQ ID NO: 56);

[0271] Human proinsulin 9-38 SHLVEALYLVCGERGFFYTPKTRREAEDLQ (SEQ ID NO: 57);

[0272] Human proinsulin 1-38: FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQ (SEQ ID NO: 58);

[0273] Human proinsulin 9-23 SHLVEALYLVCGERG (SEQ ID NO: 59);

[0274] Human proinsulin 45-71(C13-A6): GGGPGAGSLQPLALEGSLQKRGIVEQC (SEQ ID NO: 60);

[0275] Human proinsulin C24-A1 LALEGSLQKRG (SEQ ID NO: 61);

[0276] Human Proinsulin C19-A3 GSLQPLALEGSLQKRGIV (SEQ ID NO: 62);

[0277] Human proinsulin C13-32 GGGPGAGSLQPLALEGSLQK (SEQ ID NO: 63);

[0278] Human proinsulin B9-C4 SHLVEALYLVCGERGFFYTPKTRREAED (SEQ ID NO: 64);

[0279] Human proinsulin C22-A5 QPLALEGSLQKRGIVEQ (SEQ ID NO: 65);

[0280] Human IA-2 654-674: AEGPPEPSRVSSVSSQFSDAAQASPSSHSSTPSWCEEPA (SEQ ID NO: 66)

[0281] Human IA-2 718-782: AYQAEPNTCATAQGEGNIKKNRHPDFLPYDHARIKLKVESSPSRSDYINASPIIEHDPRMPAYIA (SEQ ID NO: 67);

[0282] Human IA-2 785-819: GPLSHTIADFWQMVWESGCTVIVMLTPLVEDGVKQ (SEQ ID NO: 68);

[0283] Human IA-2 828-883: GASLYHVYEVNLVSEHIWCEDFLVRSFYLKNVQTQETRTLTQFHFLSWPAEGTPAS (SEQ ID NO: 69);

[0284] Human IA-2 943-979 EHVRDQRPGLVRSKDQFEFALTAVAEEVNAILKALPQCG (SEQ ID NO: 70).

[0285] For any of the peptide or epitope sequences provided herein for use as tolerogens (e.g., SEQ ID NOS: 55-70 or fragments thereof), embodiments can also include sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity, which are contemplated to have the same or similar potency. It is also contemplated that the sequences can include a cysteine ​​conjugation moiety or any other alternative moiety for chemical conjugation. In alternative embodiments, the sequences provided herein can be fused to an antibody sequence without an additional conjugation moiety, for example, by recombinant cloning techniques.

[0286] In some embodiments, the autoimmune disease is multiple sclerosis. In some embodiments, the antigen comprises myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, or a fragment, portion, or mimic thereof. In some embodiments, combinations of these antigens can be incorporated into tolerogenic compounds, resulting in a synergistic effect that reduces the immune response to autoantigens at multiple points along the pathological pathway and promotes disease resolution.

[0287] In some embodiments, peptides or epitopes useful in the tolerogenic compounds disclosed herein are disclosed for preventing unwanted immune responses against proteins involved in myelination, myelin repair, and / or for use in treating multiple sclerosis, where the primary antigens include, but are not limited to, myelin basic protein ("MBP"), myelin oligodendrocyte glycoprotein ("MOG"), and myelin proteolipid protein ("PLP").

[0288] MBP, including exogenously obtained forms, that are useful in the compositions of the present disclosure has the following sequence (Uniprot #P02686): MGNHAGKRELNAEKASTNSETNRGESEKKRNLGELSRTTSEDNEVFGEADANQNNGTSSQDTAVTDSKRTADPKNAWQDAHPADPGSRPHLIRLFSRDAPGREDNTFKDRPSESDELQTIQEDSAATSESLDVMASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKDSHHPARTAHYGSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFKLGGRDSRSGSPMARR (SEQ ID NO: 157).

[0289] MOG, including exogenously derived forms, that are useful in the compositions of the present disclosure has the following sequence (Uniprot #Q16653): MASLSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEGGFTCFFRDHSYQEEAAMELKVEDPFYWVSPGVLVLLAVLPVLLLQITVGLIFLCLQYRLRGKLRAEIENLHRTFDPHFLRVPCWKITLFVIVPVLGPLVALIICYNWLHRRLAGQFLEELRNPF (SEQ ID NO: 158).

[0290] PLP, including exogenously obtained forms, that are useful in the compositions of the present disclosure has the following sequence (Uniprot #P60201): MGLLECCARCLVGAPFASLVATGLCFFGVALFCGCGHEALTGTEKLIETYFSKNYQDYEYLINVIHAFQYVIYGTASFFFLYGALLLAEGFYTTGAVRQIFGDYKTTICGKGLSATVTGGQKGRGSRGQHQAHSLERVCHCLGKWLGHPDKFVGITYALTVVWLLVFACSAVPVYIYFNTWTTCQSIAFPSKTSASIGSLCADARMYGVLPWNAFPGKVCGSNLLSICKTAEFQMTFHLFIAAFVGAAATLVSLLTFMIAATYNFAVLKLMGRGTKF (SEQ ID NO: 159).

[0291] In some embodiments, peptides or epitopes useful in the tolerogenic compounds disclosed herein include some or all of the following sequences, individually or in combination, derived from myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or myelin proteolipid protein (PLP):

[0292] MBP 1-39: GCASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDS (SEQ ID NO: 71);

[0293] MBP 13-32: KYLATASTMDHARHGFLPRH (SEQ ID NO: 72);

[0294] MBP 76-106: SHGRTQDENPVVHFFKNIVTPRTPPPSQGKGCG (SEQ ID NO: 73);

[0295] MBP 83-99 (modified peptide ligand): ENPWHFFKNIVTPRTP (SEQ ID NO: 74);

[0296] MBP 102-136: SQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSCG (SEQ ID NO: 75)

[0297] MBP 111-129: LSRFSWGAEGQRPGFGYGG (SEQ ID NO: 76);

[0298] MBP 146-170: AQGTLSKIFKLGGRDSRSGSPMARR (SEQ ID NO: 77);

[0299] MBP 146-170 with cysteine ​​binding moiety: AQGTLSKIFKLGGRDSRSGSPMARRCG (SEQ ID NO: 78);

[0300] MBP 83-99: ENPVVHFFKNIVTPRTP (SEQ ID NO: 79);

[0301] MBP 82-98: DENPVVHFFKNIVTPRT (SEQ ID NO: 80);

[0302] MBP 82-99: DENPVVHFFKNIVTPRTP (SEQ ID NO: 81);

[0303] MBP 82-106: DENPVVHFFKNIVTPRTPPPSQGKG (SEQ ID NO: 82);

[0304] MBP 87-106: VHFFKNIVTPRTPPPSQGKG (SEQ ID NO: 83);

[0305] MBP 131-155: ASDYKSAHKGLKGVDAQGTLSKIFK (SEQ ID NO: 84);

[0306] MBP 131-170 with cysteine ​​binding moiety: ASDYKSAHKGFKGVDAQGTLSKIFKLGGRDSRSGSPMARRCG (SEQ ID NO: 85);

[0307] MBP 76-136: SHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSCG (SEQ ID NO: 86);

[0308] MOG 1-20: GQFRVIGPRHPIRALVGDEV (SEQ ID NO: 87);

[0309] MOG 1-27: GQFRVIGPRHPIRALVGDEVELPCRIS (SEQ ID NO: 88);

[0310] Alternative MOG 35-55: MEVGWYRPPFSRWHLYRNGK (SEQ ID NO: 89);

[0311] MOG 30-60: KNATGMEVGWYRSPFSRVVHLYRNGKDQDAE (SEQ ID NO: 90);

[0312] MOG 34-56: GMEVGWYRSPFSRVVHLYRNGKD (SEQ ID NO: 91);

[0313] MOG 35-55: MEVGWYRPPFSRVVHLYRNGK (SEQ ID NO: 92);

[0314] MOG 35-55 (mouse): MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 93);

[0315] MOG 33-62: TGMEVGWYRPPFSRVVHLYRNGKDQDGDQA (SEQ ID NO: 94);

[0316] MOG 11-30: PIRALVGDEVELPCRISPGK (SEQ ID NO: 95);

[0317] MOG 18-62: DEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQA (SEQ ID NO: 96);

[0318] MOG 21-40: ELPCRISPGKNATGMEVGWY (SEQ ID NO: 97);

[0319] MOG 64-86: EYRGRTELLKDAIGEGKVTLRIR (SEQ ID NO: 98);

[0320] MOG 1-60: GQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGD (SEQ ID NO: 99);

[0321] MOG 1-62: GQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQA (SEQ ID NO: 100);

[0322] PLP 41-58: GTEKLIETYFSKNYQDYE (SEQ ID NO: 101);

[0323] PLP 89-106: GFYTTGAVRQIFGDYKTT (SEQ ID NO: 102);

[0324] PLP 95-116: AVRQIFGDYKTTICGKGLSATV (SEQ ID NO: 103);

[0325] PLP 178-197: NTWTTCQSIAFPSKTSASIG (SEQ ID NO: 104);

[0326] PLP 190-209: SKTSASIGSLCADARMYGVL (SEQ ID NO: 105); and

[0327] PLP 139-154: HCLGKWLGHPDKFVGI (SEQ ID NO: 106).

[0328] For any of the peptide or epitope sequences provided herein for use as tolerogens (e.g., SEQ ID NOS: 75-106), embodiments may also include sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity, which are expected to have the same or similar potency. Some of the sequences provided herein may include a cysteine ​​linking moiety for chemical conjugation. Other moieties for chemical conjugation are also contemplated. In other embodiments, the sequences provided herein can be fused to antibody sequences without an additional linking moiety, for example, by recombinant cloning techniques.

[0329] In some embodiments, the antigen is a therapeutic agent. In some embodiments, the antigen is selected from the group consisting of factor VIII, factor IX, asparaginase, uricase, adeno-associated virus (AAV) (i.e., for use in gene therapy), and mimetics, fragments, or portions of any of the foregoing antigens. In some embodiments, the antigen is associated with hemophilia.

[0330] In embodiments in which the antigen is a therapeutic protein, peptide, antibody, or antibody-like molecule, the specific antigen may be selected from the following: abatacept, abciximab, adalimumab, adenosine deaminase, adotrastuzumab emtansine, agalsidase alfa, agalsidase beta, aldesulquin, alglucerase, alglucosidase alfa, alpha-1-proteinase inhibitors, anakinra, anistreplase (anisoylated plasminogen streptokinase activator complex), antithrombin III, antithymocyte globulin, atelectasis inhibitors, and the like. Prolactin, bevacizumab, bivalirudin, botulinum toxin type A, botulinum toxin type B, C1 esterase inhibitors, canakinumab, carboxypeptidase G2 (glucarpidase and voraxase), certolizumab pegol, cetuximab, collagenase, crotalidae immune Fab, darbepoetin-α, denosumab, digoxin immune Fab, dornase alfa, eculizumab, etanercept, factor VIIa, factor VIII, factor IX, factor XI, factor XIII, fibrinogen, filgrastim, galsulfase, golimumab, acetic acid Acid histrelin, hyaluronidase, idursulfase, imiglucerase, infliximab, insulin [including recombinant human insulin ("rHu insulin") and bovine insulin], interferon-α2a, interferon-α2b, interferon-β1a, interferon-β1b, interferon-γ1b, ipilimumab, L-arginase, L-asparaginase, L-methionase, lactase, laronidase, lepirudin / hirudin, mecasermin, mecasermin rinfabate, methoxynatalizumab, octocopherol Reotide, ofatumumab, oprelvekin, pancreatic amylase, pancreatic lipase, papain, peg-asparaginase, peg-doxorubicin hydrochloride, peg-epoetin-β, pegfilgrastim, peg-interferon-α2a, peg-interferon-α2b, peglotidase, pegvisomant, phenylalanine ammonia-lyase (PAL), protein C, rasburicase (uricase), sacrosidase, salmon calcitonin, sargramostim, streptokinase, tenecteplase, teriparatide, tocilizumab (atlizumab),Trastuzumab, type 1 alpha-interferon, ustekinumab, vW factor. Therapeutic proteins can be obtained from natural sources (e.g., enriched and purified) or synthetic, e.g., recombinant, and include antibody therapeutics that are typically IgG monoclonals or fragments or fusions.

[0331] Specific therapeutic proteins, peptides, antibodies, or antibody-like molecules include, but are not limited to, abciximab, adalimumab, agalsidase alfa, agalsidase beta, aldesulquin, alglucosidase alfa, factor VIII, factor IX, infliximab, insulin (including rHu insulin), L-asparaginase, laronidase, natalizumab, octreotide, phenylalanine ammonia lyase (PAL), or rasburicase (uricase), and generally various forms of IgG monoclonal antibodies.

[0332] In some embodiments, hemostatic agents (e.g., Factor VIII and Factor IX), insulin (including rHu insulin), and the therapeutic molecules uricase, phenylalanine ammonia lyase (PAL), and asparaginase (which may be of non-human origin) are used.

[0333] In some embodiments, therapeutic agents are delivered, for example, via the use of gene therapy vectors. In some such embodiments, an immune response can be generated against a portion of such vectors and / or their cargo (e.g., therapeutic agents). Thus, in some embodiments, antigens to which tolerance is desired include gene therapy vectors, including, but not limited to, adenoviruses and adeno-associated viruses (and corresponding variants -1, -2, -5, -6, -8, -9, and / or other parvoviruses), lentiviruses, and retroviruses.

[0334] In some embodiments, exemplary antigens for use in tolerogenic compounds for autoimmune thyroid diseases, including Hashimoto's thyroiditis and Graves' disease, include, but are not limited to, thyroglobulin (TG), thyroid peroxidase (TPO), thyrotropin receptor (TSHR), sodium iodide symporter (NIS), or megalin. In some embodiments, for thyroid-associated ophthalmopathy and dermatosis, insulin-like growth factor 1 receptor is another exemplary antigen. In some embodiments, for hypoparathyroidism, calcium-sensing receptor is another exemplary antigen.

[0335] In some embodiments, examples of antigens for use in tolerogenic compounds for Addison's disease include, but are not limited to, 21OH hydroxylase, 17OH hydroxylase, cytochrome P450 side chain cleavage enzyme (P450scc) and / or P450c21 or P450c17, or adrenocorticotropic hormone receptor (ACTH receptor).

[0336] In some embodiments, examples of antigens for use in tolerogenic compounds for premature ovarian failure include, but are not limited to, FSH receptor and alpha-enolase.

[0337] In some embodiments, examples of antigens for use in tolerogenic compounds for autoimmune hypophysitis or pituitary autoimmune diseases include, but are not limited to, pituitary-specific protein factors (PGSF) 1a and 2, or type 2 iodothyronine deoxygenase.

[0338] In some embodiments, examples of antigens for use in tolerogenic compounds for rheumatoid arthritis include, but are not limited to, collagen I, collagen II, immunoglobulin-binding protein, double-stranded DNA, fibrin, fibrinogen (including fibrinogen alpha, beta, or gamma chains), vimentin, aggrecan, or alpha-enolase.

[0339] An exemplary sequence of collagen II is provided as Uniprot #P02458 (SEQ ID NO: 107). In some embodiments, the tolerogenic compound is selected from amino acids 1236-1249 (SEQ ID NO: 108), 662-678 (SEQ ID NO: 160), 459-478 (SEQ ID NO: 161), 461-473 (SEQ ID NO: 162), 471-485 (SEQ ID NO: 163), 450-470 (SEQ ID NO: 164), 595-603 (SEQ ID NO: 165), 455-474 (SEQ ID NO: 166), 459-478 (SEQ ID NO: 167), 459-479 (SEQ ID NO: 168), 459-479 (SEQ ID NO: 169), 461-473 (SEQ ID NO: 160), 471-485 (SEQ ID NO: 161), 450-470 (SEQ ID NO: 162), 595-603 (SEQ ID NO: 163), 459-479 (SEQ ID NO: 164), 459-479 (SEQ ID NO: 165), 459-479 (SEQ ID NO: 166), 459-479 (SEQ ID NO: 167), 459-479 (SEQ ID NO: 168), 459-479 (SEQ ID NO: 169), 461-473 (SEQ ID NO: 162), 471-485 (SEQ ID NO: 163), 450-470 (SEQ ID NO: 164), 595-603 ( (SEQ ID NO: 166), 498-511 (SEQ ID NO: 167), 1133-1150 (SEQ ID NO: 168), 1427-1435 (SEQ ID NO: 169), 378-440 (SEQ ID NO: 170), 1350-1362 (SEQ ID NO: 171), 1237-1249 (SEQ ID NO: 172), 511-525 (SEQ ID NO: 173), or 756-764 (SEQ ID NO: 174). In some embodiments, the tolerogenic compound is post-translationally modified, for example, amino acids 461-473 of collagen II are carbamylated at K4 (SEQ ID NO: 272), amino acids 455-474 of collagen II are hydroxylated at P4 and P19 (SEQ ID NO: 273), amino acids 1237-1249 of collagen II are citrullinated at R4 (SEQ ID NO: 274), amino acids 511-525 of collagen II are citrullinated at R5 (SEQ ID NO: 275), or amino acids 756-764 of collagen II are hydroxylated at P6 (SEQ ID NO: 276).

[0340] An exemplary sequence of aggrecan is provided as Uniprot #P16112 (SEQ ID NO: 109). In some embodiments, the tolerogenic compound comprises amino acids 84-103 (SEQ ID NO: 110), 32-64 (SEQ ID NO: 199), 68-82 (SEQ ID NO: 200), 76-110 (SEQ ID NO: 201), 116-130 (SEQ ID NO: 202), 140-169 (SEQ ID NO: 203), 161-177 (SEQ ID NO: 204), 174-188 (SEQ ID NO: 205), 200-215 (SEQ ID NO: 206), 225-230 (SEQ ID NO: 207), 230-240 (SEQ ID NO: 208), 240-250 (SEQ ID NO: 209), 250-260 (SEQ ID NO: 210), 261-270 (SEQ ID NO: 211), 272-280 (SEQ ID NO: 212), 274-282 (SEQ ID NO: 213), 280-290 (SEQ ID NO: 214), 290-300 (SEQ ID NO: 215), 300-310 (SEQ ID NO: 216), 310-320 (SEQ ID NO: 217), 320-330 (SEQ ID NO: 218), 330-340 (SEQ ID NO: 219), 340-350 (SEQ ID NO: 220), 350-360 (SEQ ID NO: 221), 361-370 (SEQ ID NO: 22 244 (SEQ ID NO: 207), 252 to 269 (SEQ ID NO: 208), 287 to 314 (SEQ ID NO: 209), 341 to 355 (SEQ ID NO: 210), 520 to 539 (SEQ ID NO: 211), 553 to 570 (SEQ ID NO: 212), 568 to 583 (SEQ ID NO: 213), 589 to 601 (SEQ ID NO: 214), 620 to 636 (SEQ ID NO: 215), 666 to 674 (SEQ ID NO: 216), 714 to 725 (SEQ ID NO: 217), 866-879 (SEQ ID NO: 218), 988-998 (SEQ ID NO: 219), 1056-1067 (SEQ ID NO: 220), 1352-1366 (SEQ ID NO: 221), 1645-1658 (SEQ ID NO: 222), 1892-1904 (SEQ ID NO: 223), 1785-1798 (SEQ ID NO: 224), 1805-1819 (SEQ ID NO: 225), 1885-1898 (SEQ ID NO: 226), 1937-1950 (SEQ ID NO: 2 27), 1961-1974 (SEQ ID NO: 228), 2018-2031 (SEQ ID NO: 229), 2104-2118 (SEQ ID NO: 230), 2189-2201 (SEQ ID NO: 231), 2282-2296 (SEQ ID NO: 232), 2351-2368 (SEQ ID NO: 233), 2507-2521 (SEQ ID NO: 234), 2513-2528 (SEQ ID NO: 235), or 2516-2530 (SEQ ID NO: 236).In some embodiments, the tolerogenic compound is post-translationally modified, for example, amino acids 161-177 of aggrecan are citrullinated at R3, R10, and R13 (SEQ ID NO: 282), amino acids 200-215 of aggrecan are citrullinated at R11 (SEQ ID NO: 283), amino acids 225-244 of aggrecan are citrullinated at R7 and R12 (SEQ ID NO: 284), amino acids 520-539 of aggrecan are citrullinated at R11 and R16 (SEQ ID NO: 285), amino acids 553-570 of aggrecan are citrullinated at R4 and R9 (SEQ ID NO: 286), amino acids 568-583 of aggrecan are citrullinated at R8 (SEQ ID NO: 287), or amino acids 620-636 of aggrecan are citrullinated at R13 (SEQ ID NO: 288).

[0341] An example sequence for the fibrinogen alpha chain is provided as Uniprot #P02671 (SEQ ID NO: 111). In some embodiments, the tolerogenic compound may include amino acids 78 to 91 (SEQ ID NO: 112), 717 to 725 (SEQ ID NO: 113), 51 to 95 (SEQ ID NO: 175), 138 to 152 (SEQ ID NO: 176), 171 to 185 (SEQ ID NO: 177), 201 to 215 (SEQ ID NO: 178), 300 to 314 (SEQ ID NO: 179), 347 to 361 (SEQ ID NO: 180), 363 to 377 (SEQ ID NO: 181), 420 to 434 (SEQ ID NO: 182), 438 to 452 (SEQ ID NO: 183), 456 to 470 (SEQ ID NO: 184), 542 to 556 (SEQ ID NO: 185), 717 to 725 (SEQ ID NO: 186), or 737 to 751 (SEQ ID NO: 187) of the fibrinogen alpha chain. In some embodiments, the tolerogenic compound is post-translationally modified, for example, amino acids 138-152 of the fibrinogen alpha chain are citrullinated at R6 (SEQ ID NO: 277), amino acids 456-470 of the fibrinogen alpha chain are citrullinated at R3 and R4 (SEQ ID NO: 278), amino acids 717-725 of the fibrinogen alpha chain are citrullinated at R4 (SEQ ID NO: 279), or amino acids 737-751 of the fibrinogen alpha chain are citrullinated at R7 (SEQ ID NO: 280).

[0342] An example sequence of fibrinogen beta chain is provided as Uniprot #P02675 (SEQ ID NO: 188). In some embodiments, the tolerogenic compound may include amino acids 433-411 (SEQ ID NO: 189) or 69-81 (SEQ ID NO: 190) of the fibrinogen beta chain.

[0343] An exemplary sequence of vimentin is provided as Uniprot #P08670 (SEQ ID NO: 114). In some embodiments, a tolerogenic compound can include amino acids 66-78 (SEQ ID NO: 115), 447-455 (SEQ ID NO: 116), 26-49 (SEQ ID NO: 191), 51-88 (SEQ ID NO: 192), 116-122 (SEQ ID NO: 193), 130-138 (SEQ ID NO: 194), 226-242 (SEQ ID NO: 195), 371-387 (SEQ ID NO: 196), 415-443 (SEQ ID NO: 197), or 447-455 (SEQ ID NO: 198) of vimentin. In some embodiments, the tolerogenic compound is post-translationally modified, e.g., amino acids 447-455 of vimentin are citrullinated at R4 (SEQ ID NO: 281).

[0344] An example sequence of alpha enolase is provided as Uniprot #P06733 (SEQ ID NO: 237). In some embodiments, the tolerogenic compound can include amino acids 4-40 (SEQ ID NO: 238) or 326-340 (SEQ ID NO: 239) of alpha enolase.

[0345] In some embodiments, examples of antigens for use in tolerogenic compounds for autoimmune gastritis include, but are not limited to, H+ / K+ ATPase.

[0346] In some embodiments, examples of antigens for use in tolerogenic compounds for pernicious anemia include, but are not limited to, endogenous factors.

[0347] In some embodiments, examples of antigens for use in tolerogenic compounds for vitiligo include, but are not limited to, tyrosinase or tyrosinase-related proteins 1 and 2.

[0348] In some embodiments, examples of antigens for use in tolerogenic compounds for myasthenia gravis include, but are not limited to, nicotinic acetylcholine receptor (AchR), muscle-specific kinase (MuSK), or lipoprotein receptor-related protein 4 (LRP4).

[0349] In some embodiments, examples of antigens for use in tolerogenic compounds for pemphigus vulgaris and its variants include, but are not limited to, desmogelin-1, desmogelin-3, desmogelin-4, desmocollin 1, plectin, plakoglobin, periplakin, desmoplakin I and II, envoplakin, and acetylcholine receptor.

[0350] In some embodiments, examples of antigens for use in tolerogenic compounds for bullous pemphigoid include, but are not limited to, BP180, BP230, plectin, or laminin-5.

[0351] In some embodiments, examples of antigens for use in tolerogenic compounds for dermatitis herpetiformis (Duhring's disease) include, but are not limited to, endomysium or tissue transglutaminase.

[0352] In some embodiments, examples of antigens for use in tolerogenic compounds for epidermolysis bullosa acquisita include, but are not limited to, collagen VII.

[0353] In some embodiments, examples of antigens for use in tolerogenic compounds for systemic sclerosis include, but are not limited to, matrix metalloproteinase 1, matrix metalloproteinase 3, heat shock protein 47, fibrillin 1, PDGF receptor, Scl-70, U1 snRNP, Th / To, Ku, Jo1, NAG-2, centromere proteins, topoisomerase I, nucleolar proteins, RNA polymerase I, II, or III, PM-Scl, fibrillarin, or B23.

[0354] In one embodiment, examples of antigens for use in tolerogenic compounds for mixed connective tissue disease include, but are not limited to, U1 snRNP.

[0355] In some embodiments, exemplary antigens for use in tolerogenic compounds for Sjogren's syndrome include, but are not limited to, nuclear antigens SS-A (Ro) and SS-B (La), fodrin, poly(ADP-ribose) polymerase, topoisomerase, muscarinic receptor, and Fc-gamma receptor IIIb. An exemplary sequence of SS-A is provided as Uniprot #P19474 (SEQ ID NO: 117).

[0356] An exemplary sequence of SS-B is provided as Uniprot #P05455 (SEQ ID NO: 118). In some embodiments, the tolerogenic compound can include amino acids 19-33 (SEQ ID NO: 240), 46-72 (SEQ ID NO: 241), 151-180 (SEQ ID NO: 242), 211-228 (SEQ ID NO: 243), 244-258 (SEQ ID NO: 244), or 304-329 (SEQ ID NO: 245) of SS-B.

[0357] In some embodiments, examples of antigens for use in tolerogenic compounds for systemic lupus erythematosus include, but are not limited to, "Smith antigen," SS-A, high mobility group box 1 (HMGB1), nucleosomes, histone proteins, and double-stranded DNA.

[0358] In some embodiments, examples of antigens for use in tolerogenic compounds for Goodpasture's syndrome include, but are not limited to, glomerular basement membrane protein or collagen IV.

[0359] In some embodiments, exemplary antigens for use in tolerogenic compounds for rheumatic heart disease, autoimmune myocarditis, viral myocarditis, and / or inflammatory cardiomyopathy include, but are not limited to, cardiac myosin and myosin 6. An exemplary sequence for cardiac myosin is provided as Uniprot #P13533 (SEQ ID NO: 119).

[0360] An exemplary sequence of myosin 6 is provided as Uniprot #P12883 (SEQ ID NO:251). In some embodiments, the tolerogenic compound is selected from amino acids 1906-1923 (SEQ ID NO:252), 1828-1845 (SEQ ID NO:253), 1411-1493 (SEQ ID NO:254), 1554-1584 (SEQ ID NO:255), 1170-1181 (SEQ ID NO:256), 1619-1636 (SEQ ID NO:257), 1671-1714 (SEQ ID NO:258), 450-464 (SEQ ID NO:259), 1801-1819 (SEQ ID NO:260), and 1902-2019 (SEQ ID NO:261). 0), 1333-1363 (SEQ ID NO:261), 1867-1897 (SEQ ID NO:262), 1749-1814 (SEQ ID NO:263), 1142-1156 (SEQ ID NO:264), 1372-1389 (SEQ ID NO:265), 1710-1727 (SEQ ID NO:266), 1645-1662 (SEQ ID NO:267), 689-703 (SEQ ID NO:268), 1593-1610 (SEQ ID NO:269), or 111-119 (SEQ ID NO:270).

[0361] In some embodiments, examples of antigens for use in tolerogenic compounds for polyglandular autoimmune syndrome type 1 include, but are not limited to, aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine ​​sulfinic acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, hepatic P450 cytochromes P4501A2 and P4502A6, SOX-9, SOX-10, calcium-sensing receptor protein, or type 1 interferon.

[0362] In some embodiments, examples of antigens for use in tolerogenic compounds for neuromyelitis optica include, but are not limited to, aquaporin 4.

[0363] In some embodiments, examples of antigens for use in tolerogenic compounds for uveitis include, but are not limited to, retinal S antigen, S-arrestin, interphotoreceptor retinoid binding protein (IRBP), or retinol binding protein 3.

[0364] In some embodiments, exemplary antigens for use in tolerogenic compounds for Parkinson's disease include, but are not limited to, alpha-synuclein. An exemplary sequence of alpha-synuclein is provided as Uniprot #P37840 (SEQ ID NO: 120). In some embodiments, the tolerogenic compound can include amino acids 2-23 (SEQ ID NO: 246), 32-46 (SEQ ID NO: 247), 56-85 (SEQ ID NO: 248), 81-110 (SEQ ID NO: 249), or 116-140 (SEQ ID NO: 250) of alpha-synuclein.

[0365] In some embodiments, exemplary antigens for use in tolerogenic compounds for antineutrophil cytoplasmic antibody-associated vasculitis (ANCA-V, ANCA-vasculitis) include, but are not limited to, myeloperoxidase and proteinase-3 / myeloblastin. An exemplary sequence for myeloperoxidase is provided as Uniprot #P05164 (SEQ ID NO:121). In some embodiments, the tolerogenic compound may include amino acids 447-461 (SEQ ID NO:122), 435-454 (SEQ ID NO:123), 409-423 (SEQ ID NO:124), 409-474 (SEQ ID NO:125), 279-341 (SEQ ID NO:126), 341-409 (SEQ ID NO:127), or 598-745 (SEQ ID NO:128) of myeloperoxidase. An exemplary sequence for proteinase-3 is provided as Uniprot #P24158 (SEQ ID NO:129).

[0366] In some embodiments, exemplary antigens for use in tolerogenic compounds for primary biliary cholangitis include, but are not limited to, the pyruvate dehydrogenase complex E2 subunit (PDC-E2). An exemplary sequence of PDC-E2 is provided as Uniprot #P10515 (SEQ ID NO: 130). In some embodiments, the tolerogenic compound can include amino acids 163-176 (SEQ ID NO: 131), 159-167 (SEQ ID NO: 132), or 159-176 (SEQ ID NO: 271) of PDC-E2.

[0367] In some embodiments, exemplary antigens for use in tolerogenic compounds for autoimmune hepatitis include, but are not limited to, cytochrome P450 2D6 (CYP2D6). An exemplary sequence of CYP2D6 is provided as Uniprot #P10635 (SEQ ID NO: 133). In some embodiments, the tolerogenic compound may include amino acids 245-254 (SEQ ID NO: 134), 217-260 (SEQ ID NO: 135), 305-348 (SEQ ID NO: 136), 193-212 (SEQ ID NO: 137), 305-325 (SEQ ID NO: 138), 313-322 (SEQ ID NO: 139), or 393-412 (SEQ ID NO: 140) of CYP2D6.

[0368] In some embodiments, the antigen comprises an alloantigen. In some embodiments, the alloantigen is selected from the group consisting of subunits of MHC class I and MHC class II haplotype proteins and their complexes with antigens (e.g., donor / recipient differences identified by tissue cross-matching), and minor blood group antigens RhCE, Kell, Kidd, Duffy, Ss, Diego, and MNS (including single nucleotide polymorphisms), and mimetics, fragments, or portions thereof. In some embodiments, the antigen is associated with graft-versus-host disease, transplant rejection, or autoimmune aplastic anemia. Such compositions can be individually prepared for a given donor / recipient pair.

[0369] Additional allergens with more detailed descriptions, as well as the nomenclature standardized by the WHO / IUIS Subcommittee on Allergen Nomenclature used herein (e.g., "Aca f 2"), can be found on the World Wide Web (www.allergen.org).

[0370] In some embodiments, the antigen of the tolerogenic composition may include any one or more of the antigen sequences disclosed herein, for example, any one or more of SEQ ID NOs: 40-288 or fragments thereof, or other antigens generally known in the art.

[0371] Also disclosed herein are compositions comprising any one or more of the tolerogenic compounds disclosed herein and a pharmaceutically acceptable excipient.

[0372] The pharmaceutical compositions can be prepared in a manner known per se, for example, by conventional mixing, dissolving, granulating, dragee-making, filling, emulsifying, encapsulating, entrapping, or tabletting processes. In addition, the active ingredients are contained in an amount effective to achieve the intended purpose. Many of the compounds used in the pharmaceutical combinations disclosed herein can be provided as salts with pharmaceutically compatible counterions.

[0373] The compositions can, if desired, be presented in a dispenser device containing one or more unit dosage forms containing the active ingredient. The dispenser device can be accompanied by instructions for administration. The dispenser can also have a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice can reflect approval by the government agency of the drug form for administration to humans or animals. Such notice can be, for example, labeling approved by the U.S. Food and Drug Administration (FDA) for prescription drugs or an approved product insert. Compositions comprising the compounds and / or salts described herein formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Methods for inducing tolerance

[0374] Also disclosed herein are methods for inducing tolerance to an antigen that may cause an unwanted immune response in a subject. In some embodiments, the method comprises administering any one or more of the tolerogenic compounds or compositions disclosed herein. In some embodiments, the tolerogenic compound or composition is administered before the subject is exposed to the antigen, after the subject is exposed to the antigen, or both. In some embodiments, the unwanted immune response is associated with allergies to food, animal, plant, or environmental allergens, autoimmune diseases, therapeutic drugs, or graft-versus-host disease, or other diseases and disorders associated with any of the antigens disclosed herein.

[0375] Additionally, the tolerogenic compounds and compositions disclosed herein are for use in inducing tolerance in a subject to an antigen to which the subject may elicit an unwanted immune response, in some embodiments, the unwanted immune response is associated with an allergy to a food, animal, plant, or environmental allergen, an autoimmune disease, a therapeutic drug, or graft-versus-host disease, or other disease or disorder associated with any of the antigens disclosed herein.

[0376] Also disclosed herein are tolerogenic compounds and compositions for use in the manufacture of medicaments.

[0377] In some embodiments, the compounds or compositions provided herein are used to treat, prevent, reduce, or otherwise modify an immune response to an antigen. In some embodiments, an immune response is occurring or is occurring. In some embodiments, the treatment and use of the compounds or compositions is prophylactic. In some embodiments, administration to a subject is performed before, after, or both before and after exposure of the subject to an antigen. In certain embodiments, administration before exposure exerts a prophylactic effect, substantially avoiding or significantly reducing an unwanted immune response.

[0378] Administration of the composition can be via a variety of methods, including, but not limited to, parenteral, intravenous, infusion, intramuscular, oral, rectal, pulmonary, topical, aerosol, transdermal, intradermal, or other routes of administration. In some embodiments, the composition is delivered in a therapeutically effective amount, for example, by a systemic or local route (e.g., intravenous, intraarterial, topical, intramuscular, subcutaneous, intramedullary, intrathecal, intrathecal, intraperitoneal, intranasal, intraocular, etc.). Administration can be performed yearly, monthly, daily, weekly, multiple times daily, or at less frequent or substantially equal times as needed (e.g., prior to anticipated exposure).

[0379] In some embodiments, the compound or composition is administered in an amount sufficient to induce clonal deletion and / or anergy of T cells specific to an antigen of interest. In some embodiments, the compound or composition is configured to target hepatocytes and / or LSECs. In some embodiments, the compound or composition is administered in an amount sufficient to induce clonal deletion and / or anergy of T cells specific to an antigen of interest. In some embodiments, the compound or composition is configured to target CD4 + CD25 + FOXP3 + In some embodiments, the compound or composition is configured to induce the expansion of a specific population or subpopulation of regulatory T cells, such as regulatory T cells. In some embodiments, the compound or composition is administered in an amount effective to reduce the concentration of an antibody in the patient's blood that is causally involved in any of the diseases or disorders disclosed herein, such as graft-versus-host disease, transplant rejection, immune response to a therapeutic agent, autoimmune disease, hypersensitivity, and / or allergy, by at least 10%, 20%, 30%, 40%, or 50%, or any percentage within a range defined by any two of the foregoing reductions.

[0380] The compounds or compositions described herein can be administered to a patient alone or in combination with other active ingredients, such as in combination therapy.

[0381] In some embodiments, the compound or composition is provided as a unit dose. In some embodiments, the methods and uses disclosed herein comprise administering the unit dose to a patient or subject. In some embodiments, the unit dose comprises 1 μg / kg to 10 mg / kg of the tolerogenic compound relative to the subject's body weight. In some embodiments, the tolerogenic compound relative to body weight per single administration is equal to or less than about 10 μg / kg, about 50 μg / kg, about 75 μg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, about 0.75 mg / kg, about 1.0 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, about 4.0 mg / kg, about 5.0 mg / kg, about 10.0 mg / kg, or a range spanning and / or including the aforementioned values. In some embodiments, the amount of tolerogenic compound administered is a unit dose that is less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg / kg body weight. In some embodiments, a dosing regimen is provided in which a subject receives at least one dose of a composition according to an embodiment disclosed herein. In some embodiments, a subject receives at least two, at least three, at least four, at least five, or more doses of a composition according to an embodiment disclosed herein. In some embodiments, a given subsequent dose is provided at a concentration equal to or less than the preceding dose. For example, if a second dose is administered, and the first dose concentration is 0.5 mg / kg, the second dose can be administered at about 0.25 mg / kg. In further embodiments, the dosage is kept constant over time. Depending on the severity of the underlying immune response (or potential immune response), the dosage can optionally escalate over time. Non-limiting embodiments [1] an asialoglycoprotein receptor 1 (ASGR1)-binding polypeptide, comprising a heavy chain variable (VH) region comprising a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region HCDR2, and a third heavy chain complementarity-determining region HCDR3; HCDR1 comprises the sequence of SEQ ID NO: 9, 297, 329, 361, 393, 425, 457, 489, 521, or 553; HCDR2 comprises the sequence of SEQ ID NO: 10, 298, 330, 362, 394, 426, 458, 490, 522, or 554; and An asialoglycoprotein receptor 1 (ASGR1) binding polypeptide, wherein HCDR3 comprises the sequence of SEQ ID NO: 11, 299, 331, 363, 395, 427, 459, 491, 523, or 555. [2] An ASGR1-binding polypeptide according to embodiment 1, wherein the heavy chain variable region comprises an array having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15, 303, 335, 367, 399, 431, 463, 495, 527, or 559. [3] 2. The ASGR1-binding polypeptide of embodiment 1, further comprising a light chain variable region (VL) comprising a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3); LCDR1 comprises the sequence of SEQ ID NO: 25, 313, 345, 377, 409, 441, 473, 505, 537, or 569; LCDR2 comprises the sequence of SEQ ID NO: 26, 314, 346, 378, 410, 442, 474, 506, 538, or 570; and An ASGR1-binding polypeptide, wherein LCDR3 comprises the sequence of SEQ ID NO: 27, 315, 347, 379, 411, 443, 475, 507, 539, or 571. [4] An ASGR1-binding polypeptide according to embodiment 3, wherein the light chain variable region comprises an array having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 31, 319, 351, 383, 415, 447, 479, 511, 543, or 575. [5] 2. An ASGR1-binding polypeptide according to embodiment 1, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 15, 303, 335, 367, 399, 431, 463, 495, 527, or 559, and the light chain variable region comprises the sequence of SEQ ID NO: 31, 319, 351, 383, 415, 447, 479, 511, 543, or 575. [6] 2. The ASGR1-binding polypeptide of embodiment 1, which is an antibody, a Fab' fragment, a F(ab')2 fragment, a domain antibody (dAb), or an scFv. [7] An ASGR1-binding polypeptide according to embodiment 1, comprising an Fc domain, optionally wherein the Fc domain is silenced. [8] A polynucleotide encoding an ASGR1-binding polypeptide described in embodiment 1, comprising one or more of the sequences of SEQ ID NOs: 12-14, 300-302, 332-334, 364-366, 396-398, 428-430, 460-462, 492-494, 524-526, or 556-558. [9] 9. The polynucleotide of embodiment 8, comprising the sequence of SEQ ID NO: 16, 304, 336, 368, 400, 432, 464, 496, 528, or 560.

[10] A polynucleotide encoding an ASGR1-binding polypeptide described in embodiment 3, comprising one or more of the sequences of SEQ ID NOs: 25-27, 313-315, 345-347, 377-379, 409-411, 441-443, 473-475, 505-507, 537-539, or 569-571.

[11] 11. The polynucleotide of embodiment 10, comprising the sequence of SEQ ID NO: 32, 320, 352, 384, 416, 448, 480, 512, 544, or 576.

[12] 12. The polynucleotide of embodiment 11, further comprising the sequence of SEQ ID NO: 16, 304, 336, 368, 400, 432, 464, 496, 528, or 560.

[13] A tolerogenic compound comprising an ASGR1-binding polypeptide described in any one of embodiments 1 to 12, wherein the ASGR1-binding polypeptide is bound or fused to an antigen to which tolerance is desired.

[14] The tolerogenic compound of embodiment 13, wherein the ASGR1-binding polypeptide and the antigen are linked or fused via a linker, optionally wherein the linker is a peptide linker or a chemical bond linker.

[15] 15. The tolerogenic compound of embodiment 14, wherein the linker is a cleavable linker.

[16] 16. The tolerogenic compound of embodiment 15, wherein the linker comprises glycine and / or serine, and optionally wherein the linker comprises the sequence of SEQ ID NO: 37.

[17] The tolerogenic compound of embodiment 13, wherein the antigen is conjugated or fused to the N-terminus or C-terminus of the ASGR1-binding polypeptide.

[18] The tolerogenic compound of embodiment 13, wherein the antigen comprises a food antigen.

[19] The tolerogenic compound of embodiment 18, wherein the food antigen is associated with celiac disease.

[20] 20. The tolerogenic compound of embodiment 19, wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:41. [twenty one] 20. The tolerogenic compound of embodiment 19, wherein the antigen comprises SEQ ID NO:41. [twenty two] Food allergens included conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6), 31 kda major allergen / disease resistance protein homolog (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 1.03D (Mal d 1), α-lactalbumin (ALA), lactotransferrin, actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), quiwellin (Act d 5), ovomucoid, ovalbumin, ovotransferrin, and lysozyme, livetin, apovitilin, bosbetin, 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transfer protein (Sin a 3), profilin (Sin a 4), profilin (Api g 4), high molecular weight glycoprotein (Api g 5), tropomyosin (Pen a 1), arginine kinase (Pen m 2), tropomyosin fast isoform, high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, strawberry pathogenesis-associated protein (Fra a 1), profilin (Mus a 1), portions of any of the above antigens, and mimetics of any of the above antigens. [twenty three] 23. The tolerogenic compound of any of embodiments 13 to 19 or 22, wherein the food antigen is selected from the group consisting of high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens. [twenty four] 20. The tolerogenic compound of any of embodiments 13 to 19, wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of SEQ ID NOs: 40 to 54, or a fragment thereof. [twenty five] 18. The tolerogenic compound of any of embodiments 13 to 17, wherein the antigen comprises an autoantigen.

[26] Autoantigens include thyroglobulin, thyroperoxidase, thyrotropin receptor, glutamic acid decarboxylase (GAD), 21OH hydroxylase, 17OH hydroxylase, H+ / K+ ATPase, intrinsic factor, transglutaminase, tyrosinase, tyrosinase-related protein-2, myelin basic protein, proteolipid protein, desmoglein, acetylcholine receptor, 2-oxoacid dehydrogenase complex, insulin, proinsulin, preproinsulin, insulinoma-associated protein 2 (IA-2), insulinoma 26. The tolerogenic compound of embodiment 25, wherein the tolerogenic compound is selected from: IA-213, ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular-intestinal-pancreatic / pancreatic-associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, duodenal pancreatic homeobox 1, dystrophia myotonica protein kinase (DMPK), islet-specific glucose-6-phosphatase catalytic subunit-associated protein, SST G protein-coupled receptor 1-5, myeloperoxidase (MPO), proteinase-3 / myeloblastin, and portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens.

[27] 27. The tolerogenic compound of any of embodiments 13-17 or 25-26, wherein the antigen comprises an antigen associated with an autoimmune disease.

[28] 28. The tolerogenic compound of embodiment 27, wherein the autoimmune disease is selected from the group consisting of multiple sclerosis, type 1 diabetes, rheumatoid arthritis, vitiligo, uveitis, pemphigus vulgaris, neuromyelitis optica, Goodpasture's disease, Parkinson's disease, myasthenia gravis, celiac disease, primary biliary cholangitis, Sjogren's syndrome, autoimmune hepatitis, myocarditis, inflammatory cardiomyopathy, and antineutrophil cytoplasmic antibody-associated vasculitis.

[29] The tolerogenic compound of embodiment 27 or 28, wherein the autoimmune disease is multiple sclerosis.

[30] 30. The tolerogenic compound of embodiment 29, wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 100, 71-99, 101-106 or 157-159, or a fragment thereof.

[31] The tolerogenic compound of embodiment 27 or 28, wherein the autoimmune disease is type 1 diabetes.

[32] 32. The tolerogenic compound of embodiment 31, wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 55-70 or 153-156, or a fragment thereof.

[33] an ASGR1-binding polypeptide conjugated or fused to an antigen to which tolerance is desired, wherein the ASGR1-binding polypeptide comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3; HCDR1 comprises the sequence of SEQ ID NO: 9, 297, 329, 361, 393, 425, 457, 489, 521, or 553; HCDR2 comprises the sequence of SEQ ID NO: 10, 298, 330, 362, 394, 426, 458, 490, 522, or 554; and A tolerogenic compound wherein HCDR3 comprises the sequence of SEQ ID NO: 11, 299, 331, 363, 395, 427, 459, 491, 523, or 555.

[34] 34. The tolerogenic compound of embodiment 33, wherein the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15, 303, 335, 367, 399, 431, 463, 495, 527, or 559.

[35] the ASGR1-binding polypeptide further comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3; LCDR1 comprises the sequence of SEQ ID NO: 25, 313, 345, 377, 409, 441, 473, 505, 537, or 569; LCDR2 comprises the sequence of SEQ ID NO: 26, 314, 346, 378, 410, 442, 474, 506, 538, or 570; and 35. The tolerogenic compound of embodiment 33 or 34, wherein LCDR3 comprises the sequence of SEQ ID NO: 27, 315, 347, 379, 411, 443, 475, 507, 539, or 571.

[36] 36. The tolerogenic compound of embodiment 35, wherein the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 31, 319, 351, 383, 415, 447, 479, 511, 543, or 575.

[37] 37. The tolerogenic compound of any of embodiments 33 to 36, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 15, 303, 335, 367, 399, 431, 463, 495, 527, or 559, and the light chain variable region comprises the sequence of SEQ ID NO: 31, 319, 351, 383, 415, 447, 479, 511, 543, or 575.

[38] 38. The tolerogenic compound of any of embodiments 33 to 37, which is an antibody, a Fab' fragment, a F(ab')2 fragment, a domain antibody (dAb), or an scFv.

[39] 39. The tolerogenic compound of any of embodiments 33 to 38, wherein the ASGR1-binding polypeptide comprises an Fc domain, and optionally the Fc domain is silenced.

[40] The tolerogenic compound of embodiments 34 to 39, wherein the antigen is a polypeptide.

[41] The tolerogenic compound according to any of embodiments 33 to 40, wherein the ASGR1-binding polypeptide and the antigen are linked or fused via a linker, optionally wherein the linker is a peptide linker or a chemical bond linker.

[42] The tolerogenic compound of embodiment 41, wherein the linker is a cleavable linker.

[43] 42. The tolerogenic compound of embodiment 41, wherein the linker comprises glycine and / or serine, and optionally wherein the linker comprises the sequence of SEQ ID NO: 37.

[44] The tolerogenic compound of any of embodiments 33 to 43, wherein the antigen is conjugated or fused to the N-terminus or C-terminus of the ASGR1-binding polypeptide.

[45] A tolerogenic compound described in any of embodiments 33 to 44, wherein the ASGR1-binding polypeptide comprises an Fc domain, optionally wherein the Fc domain is silenced, and wherein the antigen is bound or fused to the Fc domain, optionally wherein the antigen is bound or fused to the C-terminus of the Fc domain.

[46] 36. The tolerogenic compound of any of embodiments 33 to 35, wherein the antigen comprises a food antigen.

[47] Food antigens included conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6), 31 kda major allergen / disease resistance protein homolog (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 103D (Mal d 1), α-lactalbumin (ALA), lactotransferrin, actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), quiwellin (Act d 5), ovomucoid, ovalbumin, ovotransferrin, and lysozyme, livetin, apovitilin, bosbetin, 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transfer protein (Sin a 3). 3), profilin (Sin a 4), profilin (Api g 4), high molecular weight glycoprotein (Api g 5), tropomyosin (Pen a 1), arginine kinase (Pen m 2), tropomyosin fast isoform, high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, strawberry pathogenesis-associated protein (Fra a 1), profilin (Mus a 1), portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens.

[48] 48. The tolerogenic compound of embodiment 46 or 47, wherein the food antigen is selected from the group consisting of high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens.

[49] The tolerogenic compound of any of embodiments 46 to 48, wherein the food antigen is associated with celiac disease.

[50] 50. The tolerogenic compound of embodiment 49, wherein the antigen comprises a sequence or fragment thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 40-54.

[51] 46. ​​The tolerogenic compound of any of embodiments 33 to 45, wherein the antigen comprises an autoantigen.

[52] Autoantigens include thyroglobulin, thyroperoxidase, thyrotropin receptor, glutamic acid decarboxylase (GAD), 21OH hydroxylase, 17OH hydroxylase, H+ / K+ ATPase, intrinsic factor, transglutaminase, tyrosinase, tyrosinase-related protein-2, myelin basic protein, proteolipid protein, desmoglein, acetylcholine receptor, 2-oxoacid dehydrogenase complex, insulin, proinsulin, preproinsulin, insulinoma-associated protein 2 (IA-2), insulinoma 52. The tolerogenic compound of embodiment 51, wherein the tolerogenic compound is selected from: IA-213, ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular-intestinal-pancreatic / pancreatic-associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, duodenal pancreatic homeobox 1, dystrophia myotonica protein kinase (DMPK), islet-specific glucose-6-phosphatase catalytic subunit-associated protein, SST G protein-coupled receptor 1-5, myeloperoxidase (MPO), proteinase-3 / myeloblastin, and portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens.

[53] The tolerogenic compound of any of embodiments 33-45 or 51-52, wherein the antigen comprises an antigen associated with an autoimmune disease.

[54] 54. The tolerogenic compound of embodiment 53, wherein the autoimmune disease is selected from the group consisting of type 1 diabetes, multiple sclerosis, rheumatoid arthritis, vitiligo, uveitis, pemphigus vulgaris, neuromyelitis optica, Goodpasture's disease, Parkinson's disease, myasthenia gravis, celiac disease, primary biliary cholangitis, Sjogren's syndrome, autoimmune hepatitis, myocarditis, inflammatory cardiomyopathy, and antineutrophil cytoplasmic antibody-associated vasculitis.

[55] The tolerogenic compound of embodiment 53 or 54, wherein the autoimmune disease is type 1 diabetes.

[56] 56. The tolerogenic compound of embodiment 55, wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 55-70 or 153-156, or a fragment thereof.

[57] The tolerogenic compound of embodiment 53 or 54, wherein the autoimmune disease is multiple sclerosis.

[58] 58. The tolerogenic compound of embodiment 57, wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 71-106 or 157-159, or a fragment thereof.

[59] 55. The tolerogenic compound of embodiment 53 or 54, wherein the autoimmune disease is: rheumatoid arthritis, wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 107-116, 160-239, or 272-288, or a fragment thereof; Sjogren's syndrome, wherein the antigen comprises a sequence or fragment thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 117-118, or 240-245; rheumatic heart disease, autoimmune myocarditis, viral myocarditis, or inflammatory cardiomyopathy, wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 119 or 251-270, or a fragment thereof; Parkinson's disease, wherein the antigen comprises a sequence or fragment thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 120 or 247-250; antineutrophil cytoplasmic antibody-associated vasculitis (ANCA-associated nephritis), wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 121-129, or a fragment thereof; primary biliary cholangitis, wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 130-132 or 271, or a fragment thereof; or Autoimmune hepatitis, wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 133-140, or a fragment thereof; A tolerogenic compound.

[60] 46. ​​The tolerogenic compound of any of embodiments 33 to 45, wherein the antigen comprises an alloantigen.

[61] The tolerogenic compound of embodiment 60, wherein the alloantigen is selected from the group consisting of subunits of MHC class I and MHC class II haplotype proteins and their complexes with the antigens they present, and the minor blood group antigens RhCE, Kell, Kidd, Duffy, Diego, and MNS.

[62] A composition comprising a tolerogenic compound according to any of embodiments 33 to 61 and a pharmaceutically acceptable excipient.

[63] A method for inducing tolerance to an antigen that may cause an unwanted autoimmune response in a subject, comprising administering to the subject a tolerogenic compound of any of embodiments 33 to 61 or a composition of embodiment 62.

[64] 64. The method of embodiment 63, wherein the tolerogenic compound or composition is administered before, after, or both to the subject's exposure to the antigen.

[65] 65. The method of embodiment 63 or 64, wherein the unwanted immune response is associated with an allergy to a food, animal, plant, or environmental allergen, an autoimmune disease, a therapeutic drug, or graft-versus-host disease.

[66] A tolerogenic compound according to any of embodiments 33 to 45 or a composition according to embodiment 63 for use in inducing tolerance in a subject to an antigen to which the subject may evoke an unwanted immune response.

[67] 67. The tolerogenic compound or composition for use according to embodiment 66, wherein the unwanted immune response is associated with an allergy to a food, animal, plant, or environmental allergen, an autoimmune disease, a therapeutic drug, or graft-versus-host disease.

[68] A tolerogenic compound according to any of embodiments 33 to 45 or a composition according to embodiment 62 for use in the manufacture of a medicament.

[69] The compound of any of embodiments 33 to 45 or the composition of embodiment 62 for use in a subject in need of induction of immune tolerance.

[70] 1. A method for inducing tolerance to an antigen to which tolerance is desired, comprising administering to a subject: (i) an asialoglycoprotein receptor 1 (ASGR1)-binding polypeptide comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, HCDR1 comprises a sequence having at least 90% identity to SEQ ID NO: 9, 297, 329, 361, 393, 425, 457, 489, 521, or 553; HCDR2 comprises a sequence having at least 90% identity to SEQ ID NO: 10, 298, 330, 362, 394, 426, 458, 490, 522, or 554; and HCDR3 comprises a sequence having at least 90% identity to SEQ ID NO: 11, 299, 331, 363, 395, 427, 459, 491, 523, or 555; and (ii) Antigen to which tolerance is desired wherein the ASGR1-binding polypeptide is conjugated or fused to an antigen to which tolerance is desired.

[71] the ASGR1-binding polypeptide further comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3; LCDR1 comprises a sequence having at least 90% identity to SEQ ID NO: 25, 313, 345, 377, 409, 441, 473, 505, 537, or 569; LCDR2 comprises a sequence having at least 90% identity to SEQ ID NO: 26, 314, 346, 378, 410, 442, 474, 506, 538, or 570; and LCDR3 comprises a sequence having at least 90% identity to SEQ ID NO: 27, 315, 347, 379, 411, 443, 475, 507, 539, or 571; The method of embodiment 70.

[72] 72. The method of embodiment 70 or 71, wherein the antigen to which tolerance is desired is associated with celiac disease.

[73] 73. The method of embodiment 72, wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 41, 40, 43-54, or a fragment thereof.

[74] 72. The method of embodiment 70 or 71, wherein the antigen to which tolerance is desired is associated with MS.

[75] 75. The method of embodiment 74, wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 100, 71-99, 101-106 or 157-159, or a fragment thereof.

[76] 72. The method of embodiment 70 or 71, wherein the antigen to which tolerance is desired is associated with type 1 diabetes.

[77] 77. The method of embodiment 76, wherein the antigen comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 55-70 or 153-156, or a fragment thereof.

[78] 78. The method of any of embodiments 70 to 77, wherein administration is by intravenous route.

[79] 1. A method of delivering an antigen to liver tissue in a subject, comprising: conjugating or fusing the antigen to an asialoglycoprotein receptor 1 (ASGR1)-binding polypeptide, thereby generating an ASGR1-binding polypeptide-antigen complex; and contacting the ASGR1-binding polypeptide-antigen complex in situ with liver tissue of a subject; Here, this contact causes the ASGR1-binding polypeptide to bind to ASGR1 on the liver tissue, thereby delivering the antigen to the liver tissue.

[80] 1. A method of delivering an antigen to liver tissue in a subject, comprising: contacting an asialoglycoprotein receptor 1 (ASGR1)-binding polypeptide conjugated or fused to an antigen with liver tissue of a subject in situ, thereby allowing the ASGR1-binding polypeptide to bind to ASGR1 on the liver tissue; wherein the ASGR1-binding polypeptide comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3; HCDR1 comprises a sequence having at least 90% identity to SEQ ID NO: 9, 297, 329, 361, 393, 425, 457, 489, 521, or 553; HCDR2 comprises a sequence having at least 90% identity to SEQ ID NO: 10, 298, 330, 362, 394, 426, 458, 490, 522, or 554; and HCDR3 comprises a sequence having at least 90% identity to SEQ ID NO: 11, 299, 331, 363, 395, 427, 459, 491, 523, or 555; the ASGR1-binding polypeptide comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3; LCDR1 comprises a sequence having at least 90% identity to SEQ ID NO: 25, 313, 345, 377, 409, 441, 473, 505, 537, or 569; LCDR2 comprises a sequence having at least 90% identity to SEQ ID NO: 26, 314, 346, 378, 410, 442, 474, 506, 538, or 570; and The method, wherein LCDR3 comprises a sequence having at least 90% identity to SEQ ID NO: 27, 315, 347, 379, 411, 443, 475, 507, 539, or 571.

[81] 81. The method of embodiment 79 or 80, wherein delivering the antigen to the subject's liver tissue in situ induces tolerance of the subject to the antigen.

[82] The method of any of embodiments 80 to 81 for the treatment of an autoimmune disease.

[83] The method according to any of embodiments 80 to 82 for the treatment of allergies. [Example]

[0382] Additional embodiments are disclosed in more detail in the following examples, which are not intended to limit the scope of the claims in any way. The methods and materials represent non-limiting embodiments of how the compounds disclosed herein can be made, screened, tested, or otherwise evaluated.

[0383] Example 1: Materials and Methods An antibody was generated against the extracellular domain (ECD) of human ASGR1 (SEQ ID NO: 34), which corresponds to amino acids Q62-L291 of the full-length wild-type ASGR1 sequence (SEQ ID NO: 33).

[0384] Preparation of ASGR1 ASGR1 was biotinylated using the EZ-Link Sulfo-NHS-Biotinylation kit (Thermo Scientific, Cat #21425).

[0385] ASGR1 protein was concentrated to ~1 mg / mL and buffer-exchanged into PBS before adding a 1:7.5 molar ratio of biotinylation reagent. The mixture was kept overnight at 4°C before being buffer-exchanged again to remove free biotin in solution. Biotinylation was confirmed via streptavidin sensor binding of the labeled protein on the ForteBio.

[0386] Naive Library Selection Eight naive human synthetic yeast libraries, each with a diversity of approximately 10, were grown as previously described (see, e.g., Y. Xu et al., PEDS 26(10), 663-70 (2013); WO2009036379; WO2010105256; and WO20099568).

[0387] For the first two rounds of selection, magnetic bead sorting using the Miltenyi MACS system was performed as previously described (see, e.g., Siegel et al., J Immunol Methods 286(1-2), 141-153 (2004)). Briefly, yeast cells (approximately 10 cells / library) were incubated with 50 nM biotinylated human ASGR1-His in wash buffer (HEPES-buffered saline (HBS) / 0.1% bovine serum albumin (BSA) / 1 mM CaCl) for 30 min at 30 °C. After washing once with 40 mL of ice-cold wash buffer, the cell pellet was suspended in 20 mL of wash buffer, and streptavidin microbeads (500 μl) were added to the yeast and incubated for 15 min at 4 °C. The yeast were then pelleted, resuspended in 5 mL of wash buffer, and loaded onto a Miltenyi LS column. After loading 5 mL, the column was washed three times with 3 mL of wash buffer. The column was then removed from the magnetic field, and the yeast was eluted with 5 mL of growth medium and grown overnight.

[0388] The following selections were performed using flow cytometry (FACS). Yeast were pelleted, washed three times with wash buffer, and incubated at 30°C with 50 nM biotinylated human ASGR1-His, 50 nM biotinylated cynomolgus monkey ASGR1-His, 50 nM biotinylated mouse ASGR1-His, or 50 nM biotinylated rat ASGR1-His to obtain species cross-reactivity, or with polyspecific reagent (PSR) to remove nonspecific antibodies from the selection. For PSR depletion, the library was incubated with a 1:10 dilution of biotinylated PSR reagent as previously described (see, e.g., Y. Xu et al., PEDS 26(10), 663-70 (2013)). Yeast were also incubated with 50 nM biotinylated human ASGR1-His without CaCl2 to enrich for calcium-dependent antibodies. After various incubations, the yeast cells were washed twice with wash buffer and stained with goat F(ab')2 anti-human kappa-FITC (LC-FITC) (Southern Biotech, Cat # 2062-02) diluted 1:100 and either streptavidin-AF633 (SA-633) (Life Technologies, Cat # S21375) diluted 1:500 or extravidin-phycoerythrin (EA-PE) (Sigma-Aldrich, Cat # E4011) diluted 1:50 for 15 min at 4°C. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.3 mL of wash buffer and transferred to a sort tube with a strainer cap. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and sort gates were determined to select for antibodies with the desired properties. Selection rounds were repeated until a population possessing all the desired properties was obtained. After the final sorting round, the yeast were plated and individual colonies were picked for characterization.

[0389] Light chain batch shuffle diversification The heavy chains obtained from the naive output were used to prepare light chain diversified libraries for additional rounds of selection. These libraries were subjected to one round of MACS and four rounds of FACS as described above. During various FACS selection rounds, the libraries were evaluated for, for example, PSR binding, species cross-reactivity, calcium dependence, and affinity pressure via ASGR1 titration. Sorting was performed to obtain populations with the desired properties. From each FACS selection round, individual colonies were selected for sequencing and characterization.

[0390] Antibody production and purification Yeast clones were grown to saturation and then induced for 48 hours at 30°C with shaking. After induction, yeast cells were pelleted and the supernatant was collected for purification. IgG was purified using a Protein A column and eluted with acetic acid at pH 3.5.

[0391] ForteBio OctetKD Measurement ForteBio affinity measurements were generally performed using Octet HTX as previously described (see, e.g., Estep et al., Mabs 5(2), 270-278 (2013)). Briefly, ForteBio affinity measurements were performed by loading IgG onto the AHC sensor online. The sensor was equilibrated offline in assay buffer for 30 minutes and monitored online for 60 seconds to establish a baseline. The IgG-loaded sensor was exposed to 100 nM ASGR1 for 3 minutes, then transferred to assay buffer for 3 minutes, and off-rate measurements were performed. All kinetics were analyzed using a 1:1 binding model.

[0392] ForteBio epitope binning Epitope binning was performed using a standard sandwich-format cross-blocking assay. Control anti-ASGR1 IgG was loaded onto the AHQ sensor, and unoccupied Fc binding sites on the sensor were blocked with an irrelevant human IgG1 antibody. The sensor was then exposed to 100 nM human ASGR1-His followed by a second anti-ASGR1 antibody. Further binding of the second antibody after ASGR1 binding indicates an unoccupied epitope (non-competing), whereas no binding indicates a blocked epitope (competing).

[0393] Cell binding analysis Approximately 200,000 human ASGR1-overexpressing or parental cells were washed with wash buffer and incubated with 100 μL of 100 nM IgG for 15 minutes at room temperature. Cells were then washed twice with wash buffer and incubated with 100 μL of 1:100 anti-human IgG R-PE (Southern Biotech Cat #2040-09) for 15 minutes on ice. Cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences).

[0394] Example 2: Non-limiting examples of anti-ASGR1 antibodies This process identified several non-limiting examples of anti-ASGR1 antibodies, including those sequences listed in Table 2 and those depicted in Figures 1A-1F, 6A-6F, 8A-8F, 9A-9F, 11A-11F, 12A-12F, 13A-13F, 14A-14F, 15A-15F, and 16A-16F. Based on previous experience, it is expected that some sequence divergence can be tolerated without significantly affecting ASGR1 binding. The anti-ASGR1 antibodies are referred to as mAb-60819, mAb-60856, mAb-60881, mAb-60869, mAb-83198, mAb-83226, mAb-83237, mAb-83245, mAb-83257, mAb-83256, and elsewhere herein by the last three digits, e.g., "819." [Table 1] [Table 2] mAb-60819 was biochemically characterized and showed a 9.29x10 -9 Apparent KD of M (KD app mAb-60856 was biochemically characterized and found to have a 9.29x10 -9 Apparent KD of M (KD app mAb-60881 was biochemically characterized and found to have approximately 3 × 10 -9 mAb-60869 was biochemically characterized and found to have an apparent KD (KDapp) of 3.22 x 10 -9 Apparent KD of M (KD app ) for sugar binding. 2+ The protein has an active site where the sugar chains bind calcium at the active site, and there are also calcium ions at the proximal and distal sites, and all of these calcium ions stabilize the protein structure. Therefore, the binding of anti-ASGR1 antibodies can be measured by surface plasmon resonance using the Biacore system, biolayer interferometry using the Octet system, and cell-based assays, as well as by measuring pH and Ca. 2+ The dependence of mAb-60819 on pH and Ca was evaluated. 2+ mAb-60856, mAb-60881, and mAb-60869 were pH and Ca dependent, with binding significantly reduced in the absence of calcium. 2+ We found that binding of these antibodies to the ASGR1 target was independent of the presence or removal of calcium ions.

[0395] Example 3: Induction of immune tolerance in a type 1 diabetes model A novel liver-targeting immune tolerance platform that specifically targets antigen-binding glycopolymers to the liver to induce antigen-specific immune tolerance has previously been described in different forms and capacities in U.S. Patent No. 10,821,157, U.S. Patent Application Publication No. 2020 / 0101146, and PCT International Publication No. WO 2021 / 053589, each of which is expressly incorporated herein by reference in its entirety. This liver-targeting technology induces immune tolerance by depleting antigen-specific T cells, reducing antigen-specific inflammatory cytokine production, and enhancing regulatory T cells (Tregs). We conceived and developed a novel antigen delivery platform using a monoclonal antibody (mAb) specific for ASGR1 (αASGR1), which is expressed in liver parenchymal cells, including hepatocytes. We tested whether antigens targeted to the liver using the αASGR1 antibody induce immune tolerance in the type 1 diabetes (T1D) mouse model BDC2.5. In this model, targeting antigens to the liver using, as a non-limiting example, the αASGR1 antibody (mAb1) delayed the onset of diabetes, indicating that antigen-specific immune tolerance was induced in this model.

[0396] We used the BDC2.5 TCR transgenic mouse model of T1D (NOD.Cg-Tg (TcraBDC2.5, TcrbBDC2.5)1Doi / DoiJ [Jackson Laboratories]). BDC2.5 TCR transgenic mice express diabetogenic CD4 T cells specific for a region of chromogranin A (ChgA) posttranslationally linked to the c-peptide fragment of proinsulin. + Possessing T cells. BDC2.5 CD4 + T cells express major histocompatibility class II (MHC-II) IA g7 They also recognize the restricted polypeptide p31, which is one of several non-native mimotope peptides recognized by BCD2.5 T cells. g7The αASGR1 mAb induces autoimmune diabetes in non-obese diabetic mice. When adoptively transferred into NOD.SCID mice, BDC2.5 transgenic T cells induce T1D pathology characterized by insulitis and hyperglycemia. As described herein, we evaluated whether p31 delivered to the liver using the αASGR1 mAb induces antigen-specific tolerance and prevents and / or delays disease in this highly aggressive T1D model.

[0397] The efficacy of recombinantly expressed αASGR1-p31 fusion as a tolerogen was evaluated using the BDC2.5 mouse model of T1D. In this model, activated BDC2.5 TCR transgenic splenocytes were adoptively transferred into non-obese diabetic (NOD) mice carrying a severe combined immunodeficiency (SCID) mutation. The onset of diabetes was observed 10–14 days after adoptive transfer in control animals receiving vehicle (saline).

[0398] NOD.SCID(NOD.Cg-Prkdc <scid> / J [Jackson Laboratories]): These immunodeficient mice lack the ability to develop mature T and B lymphocytes due to mutations in the protein kinase PRKDC, which is required for VDJ recombination of antigen receptor genes in germ cells. NOD.SCID mice do not spontaneously develop diabetes unless they receive adoptive transfer of diabetogenic T cells.

[0399] The primary objective was to test whether intrahepatic administration of p31 peptide using αASGR1 mAb could prevent the onset of diabetes. 5 BDC2.5 spleen cells were preactivated in vitro with 0.5 μM p31 for 4 days and then adoptively transferred intravenously into NOD.SCID mice. On days 0 and 4 after cell transfer, NOD.SCID recipient mice received intravenous injections of αASGR1-p31, free p31 peptide, or saline. Mice were monitored for blood glucose levels two to three times weekly. Onset of diabetes was defined as two consecutive blood glucose measurements of 250 mg / dL or higher.

[0400] Antigen design and preparation of tolerogens The tolerogen used in this experiment was BDC2.5 CD4 + The αASGR1 antibody-p31 tolerogen (mAb1-p31) was designed to contain the p31 mimotope sequence (YVRPLWVRME (SEQ ID NO: 36)) recognized by T cells. The αASGR1 antibody-p31 tolerogen (mAb1-p31) contained a glycine-serine linker (e.g., SEQ ID NO: 37) between the C-terminus of the heavy chain and the p31 antigen (e.g., SEQ ID NO: 39).

[0401] mAb1-p31 p31 was recombinantly expressed and fused to the end of the αASGR1 mAb (mAb1) heavy chain via a short glycine-serine (GS) linker. Other examples of antibodies specific for ASGR1 and / or ASGR2 can be found in U.S. Patent Nos. 9,771,427 and 10,358,497, each of which is expressly incorporated herein by reference in its entirety. An example of a GS linker-p31 peptide sequence that can be fused to the αASGR1 antibody heavy chain (or other acceptable region of an antibody) is shown in SEQ ID NO: 39. DNA sequences corresponding to the GS linker and p31 mimotope were synthesized and cloned into a modified PCDNA3.4 vector containing DNA corresponding to the αASGR1 antibody. αASGR1-p31 was produced by transient transfection of Expi293 cells at a 480 mL scale for 5 days using the manufacturer's protocol. The cell supernatant was collected by centrifugation and filtered through a 0.2 μm aPES membrane filter unit to remove cells and debris before antibody purification.

[0402] Animals and in vivo procedures These experiments followed animal guidelines approved by the Institutional Animal Care and Use Committee. Upon arrival, NOD.SCID mice were randomly removed from their shipping containers and assigned to cages by animal care staff.

[0403] Isolation and activation of splenocytes for adoptive transfer Spleens from BDC2.5 mice were harvested and processed into a cell suspension before activation with p31 peptide. Briefly, spleens were collected in 5 mL of Iscove's Modified Dulbecco's Medium (IMDM) and gently dispersed with a syringe plunger through a 70 μm cell strainer. The cell strainer was then washed with 5 mL of IMDM, and the cell suspension was centrifuged at 500 × g for 5 min at 4 °C. The supernatant was aspirated, and the pellet was disrupted and resuspended in 1 mL of ammonium chloride-potassium (ACK) red blood cell lysis buffer and incubated at room temperature for 2 min. The cells were washed twice with IMDM, transferred through a 30 μm filter to a new 15 mL conical tube, and counted. The cells were centrifuged again and resuspended in IMDM medium. Spleen cells were then cultured with 0.5 μM p31 peptide at a cell density of 1 × 10 cells. 6 cells / mL, 37 o C, Activated BDC2.5 splenocytes were cultured for 4 days at 5% CO2. Washed with IMDM medium and incubated at 3 x 10 6 The cells were resuspended at a density of 0.1 cells / mL, and 0.1 mL of the cell suspension was administered intravenously to mice (adoptive transfer).

[0404] blood sugar measurement Blood was collected from mice by pricking the tip of the tail with an insulin syringe. Blood glucose levels were measured using a portable glucometer (Lifescan) and glucose test strips (GenUltimate). The day of diabetes onset was defined as the first day on which blood glucose levels exceeded 250 mg / dL for two consecutive days. Diabetic mice were then euthanized.

[0405] BDC2.5 model procedure overview Twenty-one NOD.SCID female mice were used as recipients, and one BDC2.5 female mouse was used as a spleen cell donor. The recipient mice were divided into three experimental groups (Table 3). The mice were monitored 2-3 times a week with a portable glucometer. For the in vivo portion of the experiment, the following procedures were performed:

[0406] Day 4: Spleen cells were isolated from BDC2.5 mice and cultured at 1 x 10 6 The cells were cultured at a cell density of 1000 cells / mL with 0.5 μM p31 peptide at 37°C and 5% CO for 4 days.

[0407] Day 0: Recipient NOD.SCID mice were divided into four groups (Table 3). Cultured splenocytes were harvested, washed, and prepared into a single-cell suspension. 3 x 10 5 Cells / mouse were adoptively transferred iv to all recipients.

[0408] On days 0 and 4, mice were intravenously injected with mAb1-p31, p31, or saline (Table 3). The dose was calculated based on the average body weight (21.3 g) of all recipient mice participating in the study. Baseline body weight and blood glucose levels were measured 2 days before the start of the study.

[0409] Days 4-105: Mice were monitored for T1D by twice-weekly blood glucose measurements. Mice were considered diabetic when blood glucose levels were ≥ 250 mg / dL on two consecutive occasions. [Table 3]

[0410] result :

[0411] Liver-targeted p31 antigen induces immune tolerance and prolongs glucose-free survival in the BDC2.5 model of T1D Animals treated with mAb1-p31 demonstrated prolonged diabetes-free survival (median survival time [MST] = 91 days) compared with p31- and saline-treated controls (MST = 9 days and 8 days, respectively) (p = 0.0001, Figure 3, Table 4; compared with p31-treated controls using the Mantel-Cox log-rank test). These data demonstrate that αASGR1 mAb1-p31 protects mice from T1D by inducing p31-specific immune tolerance, even when diabetogenic T cells are highly preactivated prior to adoptive transfer. [Table 4]

[0412] Example 4: Immune tolerance in the BDC2.5 mouse T1D model using non-limiting embodiments of anti-ASGR1 antibodies The anti-ASGR1 antibodies mAb-60819, mAb-60856, and mAb-60869 described herein (e.g., VH having the sequences of SEQ ID NOs: 15, 303, and 367, and VL having the sequences of SEQ ID NOs: 31, 319, and 383, respectively) were used to prepare p31 tolerogens for testing in the BDC2.5 T1D mouse model. The p31 peptide was fused to the C-terminus of the antibody heavy chain via a glycine-serine linker.

[0413] Optionally, the effector functions of the Fc region of the antibody can be silenced so that it does not bind to FcRn or other FcRγ receptors, and the Fc portion of the antibody is not required for the desired immune tolerogenic effect.

[0414] Anti-ASGR1 antibody tolerogens were administered to a NOD.SCID T1D mouse model adoptively transferred with BCD2.5 splenocytes according to the method described in Example 2. As shown in Figures 4, 7, and 10, mAb-60819, mAb-60856, and mAb-60869 significantly delayed the onset of T1D in the NOD.SCID model compared with unconjugated p31 peptide or saline control.

[0415] Example 5: Induction of immune tolerance in the EAE mouse model of multiple sclerosis The tolerogens disclosed herein were also examined for their effect on immune tolerance in a mouse model of human multiple sclerosis (MS). The animal model is an adoptive transfer mouse model of experimental autoimmune encephalomyelitis (EAE).

[0416] Indicators of immune tolerance include changes in the following parameters: 1. disease severity as measured by disease score (paralysis), 2. weight loss, and 3. inflammatory cytokine production by myelin-specific CD4+ T cells.

[0417] EAE is a well-established mouse model of MS that has been widely used in the preclinical development of other MS therapeutics, and it is characterized by activated autoreactive CD4 + The transfer of T cells induces an MS-like disease. Because the transferred cells used in this therapy are highly activated, this model faithfully mimics the therapeutic environment in which patients have circulating activated T cells that recognize self-antigens. These findings may be generalizable to other autoimmune diseases, such as celiac disease, in which immune pathology is driven by autoreactive T cells.

[0418] Briefly, in the EAE adoptive transfer model, donor mice are immunized to generate antigen-specific encephalitogenic T cells. Spleen cells harvested from these mice are activated and expanded in cell culture and then transferred intraperitoneally (ip) to recipient mice, resulting in symptomatic EAE. The antigen used for vaccination in this model is an immunodominant peptide fragment of myelin oligodendrocyte glycoprotein (MOG), spanning amino acid sequence 35-55. The analogous region of human MOG is highly homologous to mouse MOG, and a T cell epitope spanning amino acids 34-56 of human MOG has also been identified in human MS, similar to mouse EAE. As disclosed herein, other fragments of human MOG, such as MOG 1-60, MOG 33-62, or other MOG fragments disclosed herein or otherwise known in the art, may be used in some embodiments.

[0419] Efficacy in the EAE model is measured by reduction in several clinical parameters, including:

[0420] 1) EAE incidence, which is the percentage of mice that develop symptoms at any time during the study, regardless of severity or subsequent remission.

[0421] 2) Mean Maximum Score (MMS). Calculated for each subject as the average of the highest clinical scores reached at any time during the study. It is considered a primary indicator of disease severity. The theoretical range is 0 to 5 based on the degree of mobility.

[0422] 3) Weight loss, which is expected to be positively correlated with increasing disease scores.

[0423] 4) Median date of onset: The median date on which animals in a group showed a clinical score above 0.

[0424] 5) Mean end score. This is the average clinical score for each group at the end of the study. Because disease scores naturally decrease during the study, this measure is not likely to be relevant to overall efficacy.

[0425] Antigen Design The EAE induction model involves vaccinating mice with the immunodominant peptide domain of mouse MOG (MOG 35-55; SEQ ID NO: 79), which induces encephalitogenic CD4 + T cells are induced, expanded ex vivo, and adoptively transferred into recipient mice to mediate disease. The tolerogen can include the immunodominant peptide domain of MOG fused with a linker, such as a cysteine-containing linker, for binding to an ASGR1-binding polypeptide, or other linkers for fusion to an antibody, such as linkers containing glycine and serine residues, as contemplated herein. A homologous immunodominant peptide domain of human MOG (MOG 35-55; SEQ ID NO: 78) can be used.

[0426] EAE was induced in C57BL / 6 recipient mice by i.p. injection of encephalitogenic T cells from B6.SJL donor mice immunized with MOG35-55 on day 0. Test condition mice were also treated with liver-targeted MOG35-55 antigen.

[0427] The negative control group was vehicle (saline) treated mice, which are expected to develop EAE.

[0428] We administered MOG 33-62 ("MOG10") conjugated with mAb-60819 (819-MOG10) to EAE model mice injected with encephalitogenic T cells. 819-MOG10 was administered at doses of 2 pmol / g or 10 pmol / g. As seen in Figure 5, administration of 10 pmol / g of 819-MOG10 was >90% effective up to 20 days after EAE induction. This efficacy was dose-dependent. These data demonstrate that myelin-derived autoantigen (MOG) delivered to the liver by αASGR1 mAb-60819 induces antigen-specific immune tolerance and prevents unwanted immune responses associated with autoimmune diseases (such as MS, a non-limiting example).

[0429] As disclosed herein, in some embodiments, targeting an antigen to which tolerance is desired, such as, by way of non-limiting example, an antigen associated with MS, celiac disease, or type 1 diabetes, to the liver with an anti-ASGR1 antibody results in antigen-specific immune tolerance to prevent unwanted immune responses associated with the disease to which the antigen to which tolerance is desired, such as, by way of non-limiting example, MS, celiac disease, or type 1 diabetes. As disclosed herein, in some embodiments, targeting an antigen to which tolerance is desired, such as, by way of non-limiting example, an antigen associated with MS, celiac disease, or type 1 diabetes, to the liver results in antigen-specific immune tolerance to prevent unwanted immune responses associated with the disease to which the antigen to which tolerance is desired.

[0430] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0431] With respect to the use of virtually any plural and / or singular term herein, those skilled in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be set forth herein for clarity.

[0432] In general, those skilled in the art will understand that terms used herein, particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "comprise" is typically interpreted as "including, but not limited to," the term "having" is typically interpreted as "having at least," the term "include" is typically interpreted as "including, but not limited to," etc.). As those skilled in the art will further understand, if a specific number of introduced claim recitations is intended, such intention will be expressly set forth in the claim; in the absence of such recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases will generally be interpreted as meaning that the introduction of a claim reference by the indefinite article "a" or "an" limits the particular claim that includes such introduced claim reference to embodiments that include only one such reference; e.g., "a" and / or "an" will generally be interpreted as meaning "at least one" or "one or more." Furthermore, even when a specific number of introduced claims is explicitly recited, those skilled in the art will recognize that such recitation will generally be interpreted as meaning at least the recited number (e.g., the base recitation "two recitations," without any other modifier, means at least two recitations, or more than two recitations). Furthermore, when phrases similar to "at least one of A, B, C, etc." are u...

Claims

1. an asialoglycoprotein receptor 1 (ASGR1)-binding polypeptide, comprising a heavy chain variable (VH) region comprising a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), and a third heavy chain complementarity determining region (HCDR3); a light chain variable region (VL) comprising a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3); (i) HCDR1 comprises the sequence of SEQ ID NO:9, HCDR2 comprises the sequence of SEQ ID NO:10, HCDR3 comprises the sequence of SEQ ID NO:11, LCDR1 comprises the sequence of SEQ ID NO:25, LCDR2 comprises the sequence of SEQ ID NO:26, and LCDR3 comprises the sequence of SEQ ID NO:27; or (ii) HCDR1 comprises the sequence of SEQ ID NO:297, HCDR2 comprises the sequence of SEQ ID NO:298, HCDR3 comprises the sequence of SEQ ID NO:299, LCDR1 comprises the sequence of SEQ ID NO:313, LCDR2 comprises the sequence of SEQ ID NO:314, and LCDR3 comprises the sequence of SEQ ID NO:315; or (iii) HCDR1 comprises the sequence of SEQ ID NO: 329, HCDR2 comprises the sequence of SEQ ID NO: 330, HCDR3 comprises the sequence of SEQ ID NO: 331, LCDR1 comprises the sequence of SEQ ID NO: 345, LCDR2 comprises the sequence of SEQ ID NO: 346, and LCDR3 comprises the sequence of SEQ ID NO: 347; or (iv) HCDR1 comprises the sequence of SEQ ID NO: 361, HCDR2 comprises the sequence of SEQ ID NO: 362, HCDR3 comprises the sequence of SEQ ID NO: 363, LCDR1 comprises the sequence of SEQ ID NO: 377, LCDR2 comprises the sequence of SEQ ID NO: 378, and LCDR3 comprises the sequence of SEQ ID NO: 379; or (v) HCDR1 comprises the sequence of SEQ ID NO: 393, HCDR2 comprises the sequence of SEQ ID NO: 394, HCDR3 comprises the sequence of SEQ ID NO: 395, LCDR1 comprises the sequence of SEQ ID NO: 409, LCDR2 comprises the sequence of SEQ ID NO: 410, and LCDR3 comprises the sequence of SEQ ID NO: 411; or (vi) HCDR1 comprises the sequence of SEQ ID NO: 425, HCDR2 comprises the sequence of SEQ ID NO: 426, HCDR3 comprises the sequence of SEQ ID NO: 427, LCDR1 comprises the sequence of SEQ ID NO: 441, LCDR2 comprises the sequence of SEQ ID NO: 442, and LCDR3 comprises the sequence of SEQ ID NO: 443; or (vii) HCDR1 comprises the sequence of SEQ ID NO: 457, HCDR2 comprises the sequence of SEQ ID NO: 458, HCDR3 comprises the sequence of SEQ ID NO: 459, LCDR1 comprises the sequence of SEQ ID NO: 473, LCDR2 comprises the sequence of SEQ ID NO: 474, and LCDR3 comprises the sequence of SEQ ID NO: 475; or (viii) HCDR1 comprises the sequence of SEQ ID NO: 489, HCDR2 comprises the sequence of SEQ ID NO: 490, HCDR3 comprises the sequence of SEQ ID NO: 491, LCDR1 comprises the sequence of SEQ ID NO: 505, LCDR2 comprises the sequence of SEQ ID NO: 506, and LCDR3 comprises the sequence of SEQ ID NO: 507; or (ix) HCDR1 comprises the sequence of SEQ ID NO: 521, HCDR2 comprises the sequence of SEQ ID NO: 522, HCDR3 comprises the sequence of SEQ ID NO: 523, LCDR1 comprises the sequence of SEQ ID NO: 537, LCDR2 comprises the sequence of SEQ ID NO: 538, and LCDR3 comprises the sequence of SEQ ID NO: 539; or (x) HCDR1 comprises the sequence of SEQ ID NO: 553, HCDR2 comprises the sequence of SEQ ID NO: 554, HCDR3 comprises the sequence of SEQ ID NO: 555, LCDR1 comprises the sequence of SEQ ID NO: 569, LCDR2 comprises the sequence of SEQ ID NO: 570, and LCDR3 comprises the sequence of SEQ ID NO: 571; Asialoglycoprotein receptor 1 (ASGR1) binding polypeptide.

2. the heavy chain variable region comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15; and / or the light chain variable region comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 31; 2. The ASGR1-binding polypeptide of claim 1, wherein the heavy chain variable region preferably comprises the sequence of SEQ ID NO: 15 and the light chain variable region comprises the sequence of SEQ ID NO:

31.

3. Antibody, Fab' fragment, F(ab') ２ a fragment, a domain antibody (dAb), or an scFv; or 2. The ASGR1-binding polypeptide of claim 1, comprising an Fc domain, optionally wherein the Fc domain is silenced.

4. Includes the sequences of SEQ ID NOs: 12 to 14 and the sequences of SEQ ID NOs: 25 to 27 A polynucleotide encoding the ASGR1-binding polypeptide of claim 1, preferably comprising the sequences of SEQ ID NO: 16 and SEQ ID NO:

32.

5. A tolerogenic compound comprising the ASGR1-binding polypeptide of claim 1, wherein the ASGR1-binding polypeptide is bound or fused to an antigen to which tolerance is desired. The ASGR1-binding polypeptide and the antigen are linked or fused via a linker, optionally the linker being a peptide linker or a chemical bond linker; Preferably, the linker is a cleavable linker. More preferably, the linker comprises glycine and / or serine, and optionally the linker comprises the sequence of SEQ ID NO: 37, or A tolerogenic compound in which the antigen is conjugated or fused to the N-terminus or C-terminus of an ASGR1-binding polypeptide.

6. the antigen comprises a food antigen; Preferably, the food antigen is associated with celiac disease. More preferably, the antigen comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 41; or the antigen comprises SEQ ID NO: 41, or Food antigens included conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6), 31 kda major allergen / disease resistance protein homolog (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 1.03D (Mal d 1), α-lactalbumin (ALA), lactotransferrin, actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), quiwellin (Act d 5), ovomucoid, ovalbumin, ovotransferrin, and lysozyme, livetin, apovitilin, bosbetin, 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transfer protein (Sin a 3). a 3), profilin (Sin a 4), profilin (Api g 4), high molecular weight glycoprotein (Api g 5), tropomyosin (Pen a 1), arginine kinase (Pen m 2), tropomyosin fast isoform, high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, strawberry pathogenesis-associated protein (Fra a 1), profilin (Mus a 1), portions of any of the above antigens, and / or mimetics of any of the above antigens; the food antigen is selected from the group consisting of high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens; or the antigen comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 40-54, or a fragment thereof; or the antigen comprises an autoantigen; Preferably, the autoantigen is thyroglobulin, thyroperoxidase, thyrotropin receptor, glutamic acid decarboxylase (GAD), 21OH hydroxylase, 17OH hydroxylase, H+ / K+ ATPase, intrinsic factor, transglutaminase, tyrosinase, tyrosinase-related protein-2, myelin basic protein, proteolipid protein, desmoglein, acetylcholine receptor, 2-oxoacid dehydrogenase complex, insulin, proinsulin, preproinsulin, insulinoma-associated protein 2 (IA-2), insulin and / or selected from: linoma-associated protein 213 (IA-213), ICA69, ICA12 (SOX-13), carboxypeptidase H, imogen 38, GLIMA 38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepato-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, duodenal pancreatic homeobox 1, dystrophia myotonica protein kinase (DMPK), islet-specific glucose-6-phosphatase catalytic subunit-associated protein, SST G protein-coupled receptor 1-5, myeloperoxidase (MPO), proteinase-3 / myeloblastin, and portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens; and / or the antigen comprises an antigen associated with an autoimmune disease; Preferably, the autoimmune disease is selected from the group consisting of multiple sclerosis, type 1 diabetes, rheumatoid arthritis, vitiligo, uveitis, pemphigus vulgaris, neuromyelitis optica, Goodpasture's disease, Parkinson's disease, myasthenia gravis, celiac disease, primary biliary cholangitis, Sjogren's syndrome, autoimmune hepatitis, myocarditis, inflammatory cardiomyopathy, and antineutrophil cytoplasmic antibody-associated vasculitis, and / or The autoimmune disease is multiple sclerosis, Preferably, the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 100, 71-99, 101-106, or 157-159, or a fragment thereof; or The autoimmune disease is type 1 diabetes, 6. The tolerogenic compound of claim 5, wherein the antigen preferably comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 55-70 or 153-156, or a fragment thereof.

7. 10. A tolerogenic compound comprising the ASGR1-binding polypeptide of claim 1 conjugated or fused to an antigen to which tolerance is desired, Preferably, the tolerogenic compound wherein the antigen is a polypeptide.

8. The ASGR1-binding polypeptide and the antigen are linked or fused via a linker, optionally the linker being a peptide linker or a chemical bond linker; Preferably, the linker is a cleavable linker, or the linker comprises glycine and / or serine, and optionally the linker comprises the sequence of SEQ ID NO: 37; and / or the antigen is conjugated or fused to the N-terminus or C-terminus of the ASGR1-binding polypeptide; and / or The tolerogenic compound of claim 7, wherein the ASGR1-binding polypeptide comprises an Fc domain, optionally wherein the Fc domain is silenced, and wherein the antigen is bound or fused to the Fc domain, optionally wherein the antigen is bound or fused to the C-terminus of the Fc domain.

9. the antigen comprises a food antigen; Preferably, the food antigen is selected from the group consisting of conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6), 31 kda major allergen / disease resistance protein homologue (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 1.03D (Mal d 1), α-lactalbumin (ALA), lactotransferrin, actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), quiwellin (Act d 5), ovomucoid, ovalbumin, ovotransferrin, and lysozyme, livetin, apovitilin, bosbetin, 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transfer protein (Sin a 3), profilin (Sin a 4), profilin (Api g 4), high molecular weight glycoprotein (Api g 5), tropomyosin (Pen a 1), arginine kinase (Pen m 2), tropomyosin fast isoform, high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, strawberry pathogenesis-associated protein (Fra a 1), profilin (Mus a 1), portions of any of the above antigens, and / or mimetics of any of the above antigens; and / or the food antigen is selected from the group consisting of high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma-, and omega-gliadin, hordein, secalin, avenin, portions of any of the foregoing antigens, and mimetics of any of the foregoing antigens; and / or Food antigens are associated with celiac disease 8. The tolerogenic compound of claim 7, wherein the antigen preferably comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 40-54, or a fragment thereof.

10. the antigen comprises an autoantigen; Preferably, the autoantigen is thyroglobulin, thyroperoxidase, thyrotropin receptor, glutamic acid decarboxylase (GAD), 21OH hydroxylase, 17OH hydroxylase, H+ / K+ ATPase, intrinsic factor, transglutaminase, tyrosinase, tyrosinase-related protein-2, myelin basic protein, proteolipid protein, desmoglein, acetylcholine receptor, 2-oxoacid dehydrogenase complex, insulin, proinsulin, preproinsulin, insulinoma-associated protein 2 (IA-2), insulin 8. The tolerogenic compound of claim 7, wherein the tolerogenic compound is selected from: i) inoma-associated protein 213 (IA-213), ICA69, ICA12 (SOX-13), carboxypeptidase H, imogen 38, GLIMA 38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepato-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, duodenal pancreatic homeobox 1, dystrophia myotonica protein kinase (DMPK), islet-specific glucose-6-phosphatase catalytic subunit-associated protein, SST G protein-coupled receptor 1-5, myeloperoxidase (MPO), proteinase-3 / myeloblastin, and portions of any of the above antigens, and mimetics of any of the above antigens.

11. the antigen comprises an antigen associated with an autoimmune disease; Preferably, the autoimmune disease is selected from the group consisting of type 1 diabetes, multiple sclerosis, rheumatoid arthritis, vitiligo, uveitis, pemphigus vulgaris, neuromyelitis optica, Goodpasture's disease, Parkinson's disease, myasthenia gravis, celiac disease, primary biliary cholangitis, Sjogren's syndrome, autoimmune hepatitis, myocarditis, inflammatory cardiomyopathy, and antineutrophil cytoplasmic antibody-associated vasculitis, and / or wherein the autoimmune disease is type 1 diabetes, and preferably the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 55-70 or 153-156, or a fragment thereof; or The autoimmune disease is multiple sclerosis, and preferably the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 71-106 or 157-159, or a fragment thereof; or Autoimmune diseases: rheumatoid arthritis, wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 107-116, 160-239, or 272-288, or a fragment thereof; Sjogren's syndrome, wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 117-118 or 240-245, or a fragment thereof; rheumatic heart disease, autoimmune myocarditis, viral myocarditis, or inflammatory cardiomyopathy, wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 119 or 251-270, or a fragment thereof; Parkinson's disease, wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 120 or 247-250, or a fragment thereof; antineutrophil cytoplasmic antibody-associated vasculitis (ANCA-associated vasculitis), wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 121-129, or a fragment thereof; primary biliary cholangitis, wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 130-132 or 271, or a fragment thereof; or Autoimmune hepatitis, wherein the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 133-140, or a fragment thereof; 8. The tolerogenic compound of claim 7, wherein

12. the antigen comprises an alloantigen; 8. The tolerogenic compound of claim 7, wherein the alloantigen is selected from the group consisting of complexes of subunits of MHC class I and MHC class II haplotype proteins and the antigens they present, and the minor blood group antigens RhCE, Kell, Kidd, Duffy, Diego, and MNS.

13. A composition comprising a tolerogenic compound according to any one of claims 7 to 12 and a pharmaceutically acceptable excipient.

14. (i) A tolerogenic compound according to any one of claims 7 to 12 or a composition comprising a tolerogenic compound according to any one of claims 7 to 12 and a pharmaceutically acceptable excipient for use in inducing tolerance in a subject to an antigen to which the subject is susceptible to an unwanted immune response, Preferably, the unwanted immune response is associated with an allergy to a food, animal, plant, or environmental allergen, an autoimmune disease, a therapeutic drug, or graft-versus-host disease; or (ii) for use in a subject in need of induction of immune tolerance; preferably the antigen to which tolerance is desired is associated with celiac disease, preferably the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 41, 40, 43-54, or a fragment thereof; or the antigen to which tolerance is desired is associated with MS, preferably the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 100, 71-99, 101-106, or 157-159, or a fragment thereof; or the antigen to which tolerance is desired is associated with type 1 diabetes, preferably the antigen comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 55-70 or 153-156, or a fragment thereof; and / or The tolerogenic compound or composition is administered intravenously; Tolerogenic compounds or compositions.

15. 13. A tolerogenic compound according to any one of claims 7 to 12, (i) for use in delivering an antigen to liver tissue of a subject; contacting the tolerogenic compound with liver tissue of a subject in situ; wherein this contact causes the ASGR1-binding polypeptide to bind to ASGR1 on the liver tissue, thereby delivering the antigen to the liver tissue. Preferably, delivery of the antigen to the subject's liver tissue in situ induces tolerance in the subject to the antigen; or (ii) for use in the treatment of autoimmune diseases; Preferably, for use in the treatment of allergies: Tolerogenic compounds.

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