FcRn-binding polypeptide and its use

Abstract

The present specification provides a VHH-containing polypeptide that binds to FcRn. The use of the VHH-containing polypeptide is also provided.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 351,363, filed on Jun. 11, 2022, and U.S. Provisional Application No. 63 / 437,776, filed on Jan. 9, 2023, the entire contents of which are incorporated herein by reference for all purposes.

[0002] [Incorporation by Reference of a Sequence Listing] This application incorporates by reference a Sequence Listing of 54,032 bytes in size, entitled 01202 - 0057 - 00PCT_Sequence_Listing, created on Jun. 8, 2023, and filed in electronic format together with this application.

[0003] The present invention relates to an FcRn - binding polypeptide comprising an FcRn and an albumin - binding polypeptide, and methods of using the polypeptide, for example, for treating immune diseases or immune disorders.

Background Art

[0004] Plasma proteins are removed from circulation by two main mechanisms: renal filtration of molecules less than 60 kDa and micropinocytosis by endothelial cells. Proteins below the renal threshold are rapidly removed from circulation, resulting in a half - life of less than 1 day, while proteins larger than the renal threshold are removed mainly by micropinocytosis and have a half - life of around 3 to 5 days. Albumin and immunoglobulin G (IgG) are proteins with long plasma half - lives of around 15 to 30 days due to their large size (66 kDa and 150 kDa, respectively) and the ability to be recycled from endothelial micropinocytosis via pH - dependent binding to the neonatal Fc receptor (FcRn).

[0005] A long plasma half - life is useful for therapeutic agents to precisely control plasma drug concentrations, optimize efficacy while limiting toxicity, and reduce the frequency and amount of drug required.

[0006] Therefore, there is a need for FcRn-binding polypeptides with improved plasma half-lives. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0007] Embodiment 1. A polypeptide comprising at least one VHH domain that binds to FcRn, wherein the at least one VHH domain that binds to FcRn comprises a CDR1 sequence selected from SEQ ID NO: 80 and SEQ ID NO: 81, a CDR2 sequence selected from SEQ ID NO: 83 and SEQ ID NO: 84, and a CDR3 sequence of SEQ ID NO: 85.

[0008] Embodiment 2. The polypeptide according to Embodiment 1, wherein each VHH domain that binds to FcRn independently comprises a CDR1 sequence selected from SEQ ID NO: 80 and SEQ ID NO: 81, a CDR2 sequence selected from SEQ ID NO: 83 and SEQ ID NO: 84, and a CDR3 sequence of SEQ ID NO: 85.

[0009] Embodiment 3. The polypeptide according to Embodiment 1 or 2, wherein the at least one VHH domain that binds to FcRn comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO: 80, SEQ ID NO: 83, and SEQ ID NO: 85; SEQ ID NO: 81, SEQ ID NO: 83, and SEQ ID NO: 85; and SEQ ID NO: 81, SEQ ID NO: 84, and SEQ ID NO: 85.

[0010] Embodiment 4. The polypeptide according to Embodiment 3, wherein each VHH domain that binds to FcRn independently comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO: 80, SEQ ID NO: 83, and SEQ ID NO: 85; SEQ ID NO: 81, SEQ ID NO: 83, and SEQ ID NO: 85; and SEQ ID NO: 81, SEQ ID NO: 84, and SEQ ID NO: 85.

[0011] Embodiment 5. The polypeptide according to any one of Embodiments 1 to 4, wherein at least one VHH domain that binds to FcRn is humanized.

[0012] Embodiment 6. The polypeptide according to Embodiment 5, wherein each VHH domain that binds to FcRn is humanized.

[0013] Embodiment 7. The polypeptide according to any one of Embodiments 1 to 6, wherein at least one VHH domain that binds to FcRn comprises a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 86 to 93.

[0014] Embodiment 8. The polypeptide according to Embodiment 7, wherein each VHH domain that binds to FcRn comprises a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 86 to 93.

[0015] Embodiment 9. The polypeptide according to any one of Embodiments 1 to 7, wherein at least one VHH domain that binds to FcRn comprises a sequence selected from SEQ ID NOs: 86 to 93.

[0016] Embodiment 10. The polypeptide according to any one of Embodiments 1 to 9, wherein each VHH domain that binds to FcRn comprises a sequence selected from SEQ ID NOs: 86 to 93.

[0017] Embodiment 11. The polypeptide according to any one of Embodiments 1 to 10, wherein at least one VHH domain that binds to FcRn binds to human FcRn with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

[0018] Embodiment 12. The polypeptide according to any one of Embodiments 1 to 11, wherein each VHH domain that binds to FcRn binds to human FcRn with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

[0019] Embodiment 13. The at least one VHH domain that binds to FcRn is the polypeptide according to any one of Embodiments 1 to 12 that binds to human FcRn at pH 6 and pH 7.4.

[0020] Embodiment 14. Each VHH domain that binds to FcRn is the polypeptide according to any one of Embodiments 1 to 13 that binds to human FcRn at pH 6 and pH 7.4.

[0021] Embodiment 15. The at least one VHH domain that binds to FcRn is the polypeptide according to any one of Embodiments 1 to 14 that blocks the binding of human IgG to human FcRn.

[0022] Embodiment 16. Each VHH domain that binds to FcRn is the polypeptide according to any one of Embodiments 1 to 15 that blocks the binding of human IgG to human FcRn.

[0023] Embodiment 17. The polypeptide is the polypeptide according to any one of Embodiments 1 to 16 that includes at least one VHH domain that binds to albumin.

[0024] Embodiment 18. The at least one VHH domain that binds to albumin is the polypeptide according to Embodiment 17, including a CDR1 sequence selected from SEQ ID NOs: 5 to 8, a CDR2 sequence selected from SEQ ID NOs: 9 to 21, and a CDR3 sequence of SEQ ID NO. 22.

[0025] Embodiment 19. Each VHH domain that binds to albumin independently includes a CDR1 sequence selected from SEQ ID NOs: 5 to 8, a CDR2 sequence selected from SEQ ID NOs: 9 to 21, and a CDR3 sequence of SEQ ID NO. 22, which is the polypeptide according to Embodiment 17.

[0026] Embodiment 20. At least one VHH domain that binds to albumin comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO:5, SEQ ID NO:9, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:10, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:11, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:12, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:13, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:14, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:7, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:8, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:16, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:17, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:18, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:19, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:20, and SEQ ID NO:22; and SEQ ID NO:6, SEQ ID NO:21, and SEQ ID NO:22, and is the polypeptide according to Embodiment 17 or 18.

[0027] Embodiment 21. Each VHH domain that binds to albumin independently comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO:5, SEQ ID NO:9, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:10, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:11, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:12, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:13, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:14, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:7, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:8, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:16, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:17, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:18, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:19, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:20, and SEQ ID NO:22; and SEQ ID NO:6, SEQ ID NO:21, and SEQ ID NO:22, and is the polypeptide according to Embodiment 17 or 18.

[0028] Embodiment 22. The at least one VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 17 to 21, which is humanized.

[0029] Embodiment 23. Each VHH domain that binds to albumin is the polypeptide according to Embodiment 22, which is humanized.

[0030] Embodiment 24. The at least one VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 17 to 23, which comprises a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence selected from SEQ ID NOs: 23 to 43 and SEQ ID NOs: 97 to 100.

[0031] Embodiment 25. Each VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 17 to 23, which comprises a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence selected from SEQ ID NOs: 23 to 43 and SEQ ID NOs: 97 to 100.

[0032] Embodiment 26. The at least one VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 17 to 23, which comprises a sequence selected from SEQ ID NOs: 23 to 43 and SEQ ID NOs: 97 to 100.

[0033] Embodiment 27. Each VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 17 to 23, which comprises a sequence selected from SEQ ID NOs: 23 to 43 and SEQ ID NOs: 97 to 100.

[0034] Embodiment 28. The polypeptide is the polypeptide according to any one of Embodiments 17 to 26, comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence selected from SEQ ID NOs: 69 to 77.

[0035] Embodiment 29. The polypeptide is the polypeptide according to any one of Embodiments 17 to 26, comprising a sequence selected from SEQ ID NOs: 69 to 77.

[0036] Embodiment 30. At least one VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 17 to 29, which binds to human albumin and at least one albumin selected from cynomolgus monkey, mouse, and rat albumin.

[0037] Embodiment 31. Each VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 17 to 29, which binds to human albumin and at least one albumin selected from cynomolgus monkey, mouse, and rat albumin.

[0038] Embodiment 32. At least one VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 17 to 29, which binds to human, cynomolgus monkey, mouse, and rat albumin.

[0039] Embodiment 33. Each VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 17 to 29, which binds to human, cynomolgus monkey, mouse, and rat albumin.

[0040] Embodiment 34. At least one VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 17 to 33, which binds to human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

[0041] Embodiment 35. The polypeptide according to any one of Embodiments 17 to 34, wherein at least one VHH domain that binds to albumin binds to each of human, cynomolgus monkey, mouse, and rat albumins with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

[0042] Embodiment 36. The polypeptide according to any one of Embodiments 17 to 35, wherein each VHH domain that binds to albumin binds to human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

[0043] Embodiment 37. The polypeptide according to any one of Embodiments 17 to 36, wherein each VHH domain that binds to albumin binds to each of human, cynomolgus monkey, mouse, and rat albumins with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

[0044] Embodiment 38. The polypeptide according to any one of Embodiments 17 to 37, wherein each VHH domain that binds to albumin does not bind to albumin domain 3.

[0045] Embodiment 39. The polypeptide according to any one of Embodiments 17 to 38, wherein each VHH domain that binds to albumin does not interfere with the binding of albumin to FcRn.

[0046] Embodiment 40. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide comprises at least two, at least three, or at least four VHH domains that bind to FcRn and at least one VHH domain that binds to albumin.

[0047] Embodiment 41. The polypeptide according to Embodiment 40, wherein the polypeptide comprises two, three, or four VHH domains that bind to FcRn and one VHH domain that binds to albumin.

[0048] Embodiment 42. The polypeptide is the polypeptide according to any one of Embodiments 1 to 41, which contains at least one binding domain that binds to a protein other than albumin or FcRn.

[0049] Embodiment 43. The polypeptide according to Embodiment 42, wherein at least one binding domain that binds to a protein other than albumin or FcRn is a VHH.

[0050] Embodiment 44. The polypeptide according to Embodiment 43, wherein each binding domain that binds to a protein other than albumin or FcRn is a VHH.

[0051] Embodiment 45. The polypeptide according to Embodiment 42 or 43, wherein at least one binding domain that binds to a protein other than albumin or FcRn contains a heavy chain variable region and a light chain variable region.

[0052] Embodiment 46. The polypeptide according to Embodiment 45, wherein each binding domain that binds to a protein other than albumin or FcRn contains a heavy chain variable region and a light chain variable region.

[0053] Embodiment 47. The polypeptide according to any one of Embodiments 42 to 46, wherein at least one binding domain that binds to a protein other than albumin or FcRn is a binding domain of a therapeutic antibody.

[0054] Embodiment 48. The polypeptide according to Embodiment 47, wherein each binding domain that binds to a protein other than albumin is a binding domain of a therapeutic antibody.

[0055] Embodiment 49. The therapeutic antibody is useful for the treatment of a disease or disorder selected from autoimmune diseases or autoimmune disorders, inflammatory diseases or inflammatory disorders, infectious diseases, and cancers, and the polypeptide according to Embodiment 47 or 48.

[0056] Embodiment 50. The polypeptide is the polypeptide according to any one of Embodiments 1 to 41, which contains the amino acid sequence of a therapeutic protein.

[0057] Embodiment 51. The therapeutic protein is the polypeptide according to Embodiment 50, which is useful for the treatment of a disease or disorder selected from autoimmune diseases or disorders, inflammatory diseases or disorders, infectious diseases, and cancers.

[0058] Embodiment 52. The polypeptide is the polypeptide according to any one of the preceding embodiments, wherein the half-life of the polypeptide is longer than that of the same polypeptide lacking the VHH domain that binds to albumin or FcRn.

[0059] Embodiment 53. A pharmaceutical composition comprising the polypeptide according to any one of Embodiments 1 to 52 and a pharmaceutically acceptable carrier.

[0060] Embodiment 54. An isolated nucleic acid encoding the polypeptide according to any one of Embodiments 1 to 52.

[0061] Embodiment 55. A vector comprising the nucleic acid according to Embodiment 54.

[0062] Embodiment 56. A host cell comprising the nucleic acid according to Embodiment 54 or the vector according to Embodiment 55.

[0063] Embodiment 57. A host cell expressing the polypeptide according to any one of Embodiments 1 to 52.

[0064] Embodiment 58. A method for producing the polypeptide according to any one of Embodiments 1 to 52, comprising incubating the host cell according to Embodiment 56 or 57 under conditions suitable for the expression of the polypeptide.

[0065] Embodiment 59. The method according to Embodiment 58, further comprising isolating an antibody or a polypeptide.

[0066] Embodiment 60. A method comprising administering to a subject the polypeptide according to any one of Embodiments 1 to 52, or the pharmaceutical composition according to Embodiment 53.

[0067] Embodiment 61. A method for treating a disease or disorder, the method comprising administering to a subject suffering from the disease or disorder a pharmaceutically effective amount of the polypeptide according to any one of Embodiments 1 to 52, or the pharmaceutical composition according to Embodiment 53.

[0068] Embodiment 62. The method according to Embodiment 61, wherein the disease or disorder is selected from autoimmune diseases or disorders, inflammatory diseases or disorders, infectious diseases, and cancers.

[0069] Embodiment 63. The method according to Embodiment 61 or 62, wherein the disease or disorder is an autoantibody-mediated disease or disorder.

[0070] Embodiment 64. The method according to any one of Embodiments 61 to 63, wherein the disease or disorder is pemphigus vulgaris, lupus nephritis, myasthenia gravis, Guillain-Barré syndrome, antibody-mediated rejection, antiphospholipid antibody syndrome, chronic inflammatory demyelinating polyneuropathy, immune complex-mediated vasculitis, glomerulonephritis, channelopathy, neuromyelitis optica, autoimmune encephalitis, autoimmune Graves' disease, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, immune neutropenia, dilated cardiomyopathy, or serum sickness.

[0071] Embodiment 65. The method according to any one of Embodiments 60 to 64, wherein the polypeptide is administered subcutaneously.

Brief Description of the Drawings

[0072]

FIG. 1A-1I

FIG. 2A-2D

FIG. 3A-3B

FIG. 4A-4B

FIG. 5

FIG. 6A-6G

FIG. 7A-7D

FIG. 8

FIG. 9A-9D

FIG. 10A-10C

FIG. 11A-11C

FIG. 12A-12B

[0073] The embodiments provided herein relate to FcRn-binding polypeptides comprising FcRn and albumin-binding polypeptides, and their use.

[0074] Definitions and Various Embodiments The section headings used herein are for the purpose of organization only and are not to be construed as limiting the subject matter described.

[0075] All references cited herein, including patent applications, patent publications, and Genbank accession numbers, are hereby incorporated by reference as if each individual reference were specifically and individually indicated as being incorporated by reference in its entirety to form a part of this specification.

[0076] The techniques and procedures described or referenced in this specification are generally well understood and commonly employed, for example, by Sambrook et al., Molecular Cloning: A Laboratory Manual 3 rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)), the series METHODS IN ENZYMOLOGY (Academic Press, Inc.), PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)), Oligonucleotide Synthesis (M. J. Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press, Animal Cell Culture (R. I. Freshney, ed., 1987), Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press, Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell eds., 1993 - 8) J. Wiley and Sons, Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C. A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company, 1993) and the conventional methodologies by those skilled in the art such as the widely used methodologies described in their latest editions are used.

[0077] Unless otherwise specified, scientific and technical terms used in connection with the present disclosure shall have the meanings generally understood by those skilled in the art. Further, unless the context otherwise requires or specifically indicates to the contrary, singular nouns shall include the plural and plural nouns shall include the singular. In case of a conflict in definitions between various sources or references, the definitions set forth in this specification shall prevail.

[0078] Generally, the numbering of residues in immunoglobulin heavy chains is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibody.

[0079] The embodiments of the invention described herein are understood to include "consisting" embodiments and / or "consisting essentially of" embodiments. As used herein, the singular forms "a", "an", and "the" include plural references unless otherwise indicated. The use of the term "or" in this specification is not to be construed as meaning that the alternatives are mutually exclusive.

[0080] In this application, the use of "or" means "and / or" unless specifically specified otherwise or not understood by those skilled in the art. In the context of multiple dependent claims, the use of "or" refers to the citation of two or more preceding independent or dependent claims.

[0081] The terms "reference sample", "reference cell", or "reference tissue" refer to a sample having at least one known characteristic that can be used for comparison with a sample having at least one unknown characteristic. In some embodiments, the reference sample can be used as a positive or negative indicator. By using a reference sample, it is possible to establish, for example, the levels of proteins and / or mRNAs present in a sample having unknown characteristics relative to the levels of proteins and / or mRNAs present in healthy tissue. In some embodiments, the reference sample is a sample derived from a different part of the subject than the part being tested, but from the same subject. In some embodiments, the reference sample is a sample derived from a tissue region surrounding or adjacent to cancer. In some embodiments, the reference sample is not derived from the subject being tested, but is a sample derived from a subject known to have or not have the disorder of interest. In some embodiments, the reference sample is from the same subject, but is a sample at a time point before the subject develops cancer. In some embodiments, the reference sample is a sample derived from a benign cancer sample from the same or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule of interest in the negative reference sample indicates a level at which one of ordinary skill in the art, considering the present disclosure, would recognize that the molecule is absent and / or that a low level of the molecule is present. When a positive reference sample is used for comparison, the expression level or amount of the molecule of interest in the positive reference sample indicates a level at which one of ordinary skill in the art, considering the present disclosure, would recognize that a certain level of the molecule is present.

[0082] As used herein in the context of a subject receiving a benefit from or responding to administration of a therapeutic agent, the terms "benefit", "clinical benefit", "responsiveness", and "therapeutic responsiveness" can be gauged by assessing various endpoints, such as inhibition of disease progression to some extent, including deceleration and complete arrest; reduction in the number of disease episodes and / or symptoms; reduction in lesion size; inhibition (i.e., reduction, deceleration, or complete arrest) of infiltration of diseased cells into adjacent peripheral organs and / or peripheral tissues; inhibition (i.e., reduction, deceleration, or complete arrest) of disease spread; some alleviation of one or more symptoms associated with the disorder; disease-free presentation after treatment, such as an increase in the length of the progression-free survival period, an extension of the overall survival period, a higher response rate, and / or a decrease in the mortality rate at a given time point after treatment. A "non-responsive" or "non-responding" subject or cancer is one that does not meet the above conditions for "responding".

[0083] The terms "nucleic acid molecule", "nucleic acid", and "polynucleotide" are used interchangeably and can refer to a polymer of nucleotides. Such a polymer of nucleotides can include natural and / or non-natural nucleotides and can include, but is not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0084] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues include natural or unnatural amino acid residues and, without limitation, may include peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition includes both full-length proteins and fragments thereof. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Further, for the purposes of the present disclosure, "polypeptide" refers to a protein that includes modifications (generally conservative with respect to nature) such as deletions, additions, and substitutions to a native sequence, so long as the protein maintains the desired activity. These modifications can be intentional, such as by site-directed mutagenesis, or can be accidental, such as by mutations in the host producing the protein or errors in PCR amplification.

[0085] As used herein, "albumin" refers to any natural mature albumin resulting from the processing of albumin precursors within cells. This term includes albumin from any vertebrate origin, including, without limitation, mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. This term also includes naturally occurring variants of albumin, such as splice variants or allelic variants. A non-limiting exemplary mature human albumin amino acid sequence is shown, for example, in UniProt accession number P02768.2. See SEQ ID NO: 1. Non-limiting exemplary mouse, cynomolgus monkey, and rat albumin amino acid sequences are shown in SEQ ID NOs: 2-4.

[0086] As used herein, "FcRn" refers to any native neonatal Fc receptor (FcRn) resulting from the processing of the FcRn precursor intracellularly. This term includes FcRn from any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. This term also includes naturally occurring variants of FcRn, such as splice variants or allelic variants. A non-limiting exemplary mature human FcRn amino acid sequence is shown, for example, in UniProt accession number P55899. See SEQ ID NO: 92. Non-limiting exemplary mouse, cynomolgus monkey, and rat FcRn amino acid sequences are shown in SEQ ID NOs: 93 to 95.

[0087] The term "specifically binds to" an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" when it reacts or associates more frequently, more rapidly, for a longer duration, and / or with higher affinity with a particular cell or substance than with another cell or substance. A single domain antibody (sdAb) or VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target when it binds with higher affinity, avidity, more readily, and / or for a longer duration than to other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to an FcRn epitope is an sdAb or VHH-containing polypeptide that binds to this epitope with higher affinity, avidity, more readily, and / or for a longer duration than to other FcRn epitopes or non-FcRn epitopes. Also, by interpreting this definition, it is understood that, for example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. In general, references to binding, although not always, mean preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0088] As used herein, the term "inhibits" with respect to the activity of a target protein refers to a decrease in the activity of the protein. In some embodiments, "inhibits" refers to a decrease in activity compared to the protein in the absence of the modulator.

[0089] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an sdAb or a VHH-containing polypeptide) binds. Epitopes often include chemically active surface configurations of molecules such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural features and specific charge features. Epitopes can be formed from both contiguous residues and / or juxtaposed non-contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are usually retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes can contain, but are not limited to, at least 3, at least 5, or 8 to 10 residues (e.g., amino acids or nucleotides). In some embodiments, the epitope is less than 20 residues (e.g., amino acids or nucleotides) in length, less than 15 residues in length, or less than 12 residues in length. Two antibodies can bind to the same epitope within an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope can be defined by a certain minimum distance from CDR residues on the antigen-binding molecule. In some embodiments, an epitope can be defined by such distance and can be further limited to those residues involved in a bond (e.g., a hydrogen bond) between a residue of the antigen-binding molecule and an antigen residue. Epitopes can also be defined in a similar manner by various scans. For example, an alanine scan or an arginine scan can identify one or more residues with which the antigen-binding molecule can interact. Unless explicitly stated otherwise, a set of residues as an epitope does not exclude other residues since it is part of the epitope for a particular antigen-binding molecule. Rather, the presence of such a set indicates a minimal epitope sequence (or set of types). Thus, in some embodiments, a set of residues identified as an epitope does not represent an exclusive list of residues for the epitope on the antigen, but rather indicates the minimal epitope associated with the antigen.

[0090] The term "antibody" is used in the broadest sense and is not limited, but includes conventional antibodies (typically including at least one heavy chain and at least one light chain), single domain antibodies (sdAb, including at least one VHH domain and optionally an Fc region), VHH-containing polypeptides (polypeptides containing at least one VHH domain), and antibody-like antigen-binding domains including any of the above fragments as long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody includes a dimerization domain. Such dimerization domains include, but are not limited to, heavy chain constant domains (including CH1, hinge, CH2, and CH3, where CH1 typically pairs with the light chain constant domain CL and the hinge mediates dimerization) and Fc regions (including hinge, CH2, and CH3, where the hinge mediates dimerization).

[0091] The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as camel (including llama), shark, mouse, human, cynomolgus monkey, etc.

[0092] As used herein, the term "antigen-binding domain" refers to the portion of an antibody that is sufficient to bind an antigen. In some embodiments, the antigen-binding domain of a conventional antibody comprises three heavy-chain CDRs and three light-chain CDRs. Thus, in some embodiments, the antigen-binding domain comprises a heavy-chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen, and a light-chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen. In some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises the three CDRs of the VHH domain. Thus, in some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises a VHH domain comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen.

[0093] As used herein, the term "VHH" or "VHH domain" or "VHH antigen-binding domain" refers to the antigen-binding portion of a single-domain antibody such as a camelid antibody or shark antibody. In some embodiments, a VHH comprises three CDRs and four framework regions designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N-terminus and / or C-terminus such that it comprises only partial FR1 and / or FR4 or lacks one or both of those framework regions, so long as the VHH substantially maintains antigen binding and specificity.

[0094] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody comprising at least one monomeric domain, such as a VHH domain, that lacks a light chain and optionally an Fc region. In some embodiments, the sdAb is a dimer of two polypeptides, each polypeptide comprising at least one VHH domain and an Fc region. As used herein, the terms "single domain antibody" and "sdAb" encompass polypeptides comprising multiple VHH domains, e.g., polypeptides having the structure VHH1-VHH2, VHH1-VHH2-Fc, VHH1-VHH2-VHH3 or VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different.

[0095] The term "VHH-containing polypeptide" refers to a polypeptide comprising at least one VHH domain. In some embodiments, the VHH polypeptide comprises two, three, or four or more VHH domains, where each VHH domain may be the same or different. In some embodiments, the VHH-containing polypeptide comprises an Fc region. In some such embodiments, the VHH-containing polypeptide may be referred to as an sdAb. Further, in some such embodiments, the VHH polypeptide may form a dimer. Non-limiting structures of the VHH-containing polypeptide, also referred to as sdAb, include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of such structures, one VHH may be linked to another VHH by a linker, or one VHH may be linked to Fc by a linker. In some such embodiments, the linker comprises from 1 to 20 amino acids, preferably from 1 to 20 amino acids consisting mainly of glycine and optionally serine. In some embodiments, when the VHH-containing polypeptide comprises Fc, it forms a dimer. Thus, the structure VHH1-VHH2-Fc is considered tetravalent when it forms a dimer (i.e., the dimer has four VHH domains). Similarly, the structure VHH1-VHH2-VHH3-Fc is considered hexavalent when it forms a dimer (i.e., the dimer has six VHH domains).

[0096] The term "monoclonal antibody" refers to antibodies (including sdAb or VHH-containing polypeptides) of a substantially homogeneous antibody population. That is, the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Further, typically, in contrast to polyclonal antibody preparations that contain different antibodies against different determinants (epitopes), each monoclonal antibody is an antibody against a single determinant on the antigen. Thus, a sample of monoclonal antibodies can bind to the same epitope on the antigen. The qualifying phrase "monoclonal" indicates the nature of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies can be produced by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods as described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries produced using, for example, the techniques described in McCafferty et al., 1990, Nature 348:552-554.

[0097] The term "CDR" refers to complementarity-determining regions defined by at least one particular pattern for one of ordinary skill in the art. In some embodiments, the CDRs can be defined according to any of the Chothia numbering scheme, Kabat numbering scheme, combination of Kabat and Chothia, AbM definition, and / or contact definition. VHH contains three CDRs designated CDR1, CDR2, and CDR3.

[0098] As used herein, the term "heavy chain constant region" refers to at least three heavy chain constant domains, namely, C H 1, hinge, C H 2, and C HRefers to a region containing 3. Of course, deletions and modifications that do not change the function within the domain are included within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to one antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. Furthermore, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. A particular isotype can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 antibody (containing γ1 constant region), IgG2 antibody (containing γ2 constant region), IgG3 antibody (containing γ3 constant region), and IgG4 antibody (containing γ4 constant region); IgA antibodies include, but are not limited to, IgA1 antibody (containing α1 constant region) and IgA2 antibody (containing α2 constant region); IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0099] As used herein, "Fc region" refers to a portion of the heavy chain constant region that includes CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In various embodiments, when the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0100] As used herein, "acceptor human framework" refers to a heavy chain variable domain (V... derived from a human immunoglobulin framework or a human consensus framework, as discussed herein. H) It is a framework comprising the amino acid sequence of the framework. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence or can comprise changes in the amino acid sequence. In some embodiments, the number of amino acid changes is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3 across all human frameworks within a single antigen-binding domain such as a VHH.

[0101] "Affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody such as an sdAb or a VHH-containing polypeptide) and its binding partner (e.g., an antigen). The affinity or apparent affinity of a molecule X for its partner Y is generally represented by the dissociation constant (K D ) or K D(見かけ) . Affinity can be measured by conventional methods known in the art, including the methods described herein (e.g., ELISA K D , KinExA, flow cytometry, and / or surface plasmon resonance devices, etc.). Such methods include, but are not limited to, BIAcore®, Octet®, or methods requiring flow cytometry.

[0102] As used herein, the term "K D " refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. When the term "K D " is used herein, it includes K D and K D(見かけ) .

[0103] In some embodiments, the K D of an antigen-binding molecule is measured by flow cytometry using an antigen-expressing cell line and fitting the mean fluorescence measured at each antibody concentration to a non-linear one-site binding equation (graphpad's Prism Software). In some such embodiments, KD is K D(見かけ) is.

[0104] The term "biological activity" refers to any one or more biological properties of a molecule (whether occurring naturally as seen in vivo, or provided or made possible by recombinant means). Biological properties include, but are not limited to, ligand binding, induction or increase of cell proliferation, and induction or increase of cytokine expression.

[0105] An "agonist" antibody or "activating" antibody is an antibody that increases and / or activates the biological activity of a target antigen. In some embodiments, an agonist antibody binds to an antigen and increases its biological activity by at least about 20%, 40%, 60%, 80%, 85% or more.

[0106] An "antagonist" antibody, "blocking" antibody, or "neutralizing" antibody is an antibody that inhibits, reduces and / or inactivates the biological activity of a target antigen. In some embodiments, a neutralizing antibody binds to an antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99% or more.

[0107] An "affinity matured" sdAb or VHH-containing polypeptide refers to an sdAb or VHH-containing polypeptide having one or more such modifications in one or more CDRs as compared to a parental sdAb or VHH-containing polypeptide that has no modifications that result in an improvement in the affinity of the sdAb or VHH-containing polypeptide for the antigen.

[0108] As used herein, "humanized VHH" refers to a VHH in which one or more framework regions are substantially replaced with human framework regions. In some cases, certain framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Further, a humanized VHH may contain residues that are not found in the original VHH or in the human framework sequence, but are included to further improve and optimize the performance of the sdAb or VHH-containing polypeptide. In some embodiments, the humanized sdAb or VHH-containing polypeptide comprises a human Fc region. As will be appreciated, a humanized sequence can be identified by its primary sequence and does not necessarily indicate the process by which the antibody was made.

[0109] An "effector-positive Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding, Clq binding, and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Such effector functions generally require combination of the Fc region with a binding domain (e.g., an antibody variable domain) and can be evaluated using a variety of assays.

[0110] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include the native sequence human IgG1 Fc region (non-A allotype and A allotype), the native sequence human IgG2 Fc region, the native sequence human IgG3 Fc region, and the native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0111] The "mutant Fc region" includes an amino acid sequence that is different from the amino acid sequence of the Fc region of the native sequence due to at least one amino acid modification. In some embodiments, the "mutant Fc region" includes an amino acid sequence that is different from the amino acid sequence of the Fc region of the native sequence due to at least one amino acid modification, but retains at least one effector function of the Fc region of the native sequence. In some embodiments, the mutant Fc region has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the Fc region of the native sequence or the Fc region of the parent polypeptide, as compared to the Fc region of the native sequence or the Fc region of the parent polypeptide. In some embodiments, the mutant Fc regions herein have at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the Fc region of the native sequence and / or the Fc region of the parent polypeptide.

[0112] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR binds to IgG antibodies (gamma receptors) and is a receptor of the FcγRI subclass, FcγRII subclass, and FcγRIII subclass, including allelic variants and alternatively spliced forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences but mainly differ in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and plays a role in the regulation of immunoglobulin homeostasis.Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997), Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997), Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004), WO 2004 / 92219 (Hinton et al.)).

[0113] As used herein, the terms "substantially similar" or "substantially the same" indicate a sufficiently high degree of similarity between two or more numerical values such that one of ordinary skill in the art would consider the difference between the two or more values to have little or no biological significance and / or statistical significance within the context of the biological characteristics measured by the above values. In some embodiments, two or more substantially similar values differ by no more than one of the approximate values of 5%, 10%, 15%, 20%, 25%, or 50%.

[0114] A polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity to a native sequence polypeptide, after aligning the sequences and introducing gaps as necessary to achieve maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, the variant has at least about 80% amino acid sequence identity. In some embodiments, the variant has at least about 90% amino acid sequence identity. In some embodiments, the variant has at least about 95% amino acid sequence identity to the native sequence polypeptide.

[0115] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide sequence, polypeptide sequence, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, without regard to conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in various ways within the skill of the art, using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. One of skill in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve the maximum alignment over the full length of the sequences being compared.

[0116] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Non-limiting exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into the antibody of interest and the product screened for retention / improvement of a desired activity, such as antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC.

[0117] [Table 1]

[0118] Amino acids can be grouped according to common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues affecting chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0119] Non-conservative substitutions involve exchanging one member of these classes for another.

[0120] The term "vector" is used to describe a polynucleotide that can be manipulated to contain a cloned polynucleotide(s) that can be propagated within a host cell. A vector can contain one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., a promoter and / or enhancer, etc.) that regulate the expression of the polypeptide of interest, and / or one or more selectable marker genes (e.g., an antibiotic resistance gene and a gene that can be used in a colorimetric assay, e.g., β-galactosidase, etc.). The term "expression vector" refers to a vector that is used to express a polypeptide of interest in a host cell.

[0121] "Host cell" refers to a cell that can be or has been a recipient of a vector or an isolated polynucleotide. A host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate animal cells, fungal cells such as yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6™ cells (Crucell), as well as 293 cells and CHO cells, and their derivatives, such as 293-6E cells, CHO-DG44 cells, CHO-K1 cells, CHO-S cells, and CHO-DS cells. Host cells include the progeny of a single host cell, but due to natural mutations, accidental mutations, or intentional mutations, the progeny are not necessarily identical in all respects (in morphology or genomic DNA complement) to the original parent cell. Host cells also include cells that have been transfected in vivo with the polynucleotide(s) provided herein.

[0122] As used herein, the term "isolated" refers to a molecule that is separated from at least some of the components that are typically found together or produced together in nature. For example, a polypeptide is said to be "isolated" when it is separated from at least a portion of the components of the cell that produced it. When a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is "isolated" when it is not part of a larger polynucleotide (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) in which it is typically found in nature, or when, for example, in the case of an RNA polynucleotide, it is separated from at least a portion of the components of the cell that produced it. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

[0123] The terms "individual" and "subject" are used interchangeably herein to refer to an animal, such as a mammal. In some embodiments, but not limited to, methods of treating mammals including humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets are provided. In some examples, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject to be treated can be a patient who has been identified as having or being at sufficient risk of having a disorder associated with the treatment.

[0124] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0125] The term "autoimmune disorder" typically refers to a disease or disorder accompanied by a non-anaphylactic hypersensitivity reaction (type II, III, and / or type IV hypersensitivity reactions) caused by the subject's own humoral and / or cellular immune responses to one or more immunogenic substances of generally endogenous / exogenous origin.

[0126] The term "inflammatory disorder" refers to a disorder accompanied by inflammation, including but not limited to chronic or acute inflammatory diseases, and explicitly includes inflammatory autoimmune diseases and inflammatory allergic conditions.

[0127] The terms "infectious disease" and "infectious disease or infectious disorder" refer to a disease or disorder caused by exogenous infectious agents such as, but not limited to, bacteria, viruses, fungi, protozoa, and parasites.

[0128] The terms "cancer" and "tumor" include solid cancers and hematologic / lymphoid cancers, and also include malignant tumors such as dysplasia, premalignant tumors, and benign tumors. Exemplary cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal carcinoma, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), melanoma, multiple myeloma, neuroblastoma, oral cancer (lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and other carcinomas and sarcomas, as well as post-transplant lymphoproliferative disorder (PTLD), and angiogenic abnormalities associated with nevus flammeus, edema (such as edema associated with brain tumors), and the Meigs syndrome.

[0129] In some embodiments, "increase" or "decrease" each refers to a statistically significant increase or decrease. As will be apparent to those skilled in the art, "modulation" also includes a change (which can be either an increase or a decrease) in the affinity, avidity, specificity, and / or selectivity of a target or antigen for one or more of its ligands, binding partners, homomultimers or heteromultimers, partners or substrates that associate to the target or antigen, compared to the same conditions except for the presence of the test agent, and / or a change (which can be either an increase or a decrease) in the sensitivity of the target or antigen to one or more conditions (such as pH, ionic strength, presence of cofactors, etc.) in the medium or environment in which the target or antigen is present, and / or can include cell proliferation or cytokine production. This can be determined by any suitable method known per se or described herein and / or using any suitable assay, depending on the target involved.

[0130] As used herein, "treatment" is a procedure for obtaining a beneficial or desired clinical outcome. "Treatment" as used herein is directed to any administration or application of a therapeutic agent for a disease in a mammal, including a human. For the purposes of the present disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the degree of the disease, prevention or delay of the progression of the disease (e.g., metastasis, e.g., metastasis to the lung or lymph nodes), prevention or delay of the recurrence of the disease, delay or slowing of the progression of the disease, improvement of the disease state, inhibition of the disease or the progression of the disease, inhibition or slowing of the disease or its progression, prevention of its development, and remission (partial or complete). "Treatment" also includes reduction of the pathological consequences of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. Accordingly, the term treatment does not require the complete removal of all aspects of the disorder by 100 percent.

[0131] "Improvement" means that one or more symptoms are alleviated or improved as compared to the case where no therapeutic agent is administered. "Improvement" also includes shortening or reduction of the duration of the symptoms.

[0132] The term "anticancer agent" is used herein in its broadest sense to refer to an agent used in the treatment of one or more cancers. Exemplary classes of such agents include, but are not limited to, chemotherapeutic agents, anticancer biologics (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiation therapy agents, CAR-T therapy agents, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNA), and oncolytic viruses.

[0133] The term "biological sample" means an amount of material from a living or formerly living organism. Such materials include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, white blood cells, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0134] The term "control" or "reference" refers to a composition known to be free of the analyte ("negative control") or a composition known to contain the analyte ("positive control"). A positive control may contain the analyte at a known concentration.

[0135] The term "inhibit" or "inhibiting" refers to a decrease or cessation of any phenotypic characteristic, or a decrease or cessation of the incidence, degree, or likelihood of that characteristic. "Reducing" or "inhibiting" means decreasing, reducing, or ceasing the activity, function, and / or amount as compared to a reference. In some embodiments, "reducing" or "inhibiting" means the ability to cause an overall decrease of 10% or more. In some embodiments, "reducing" or "inhibiting" means the ability to cause an overall decrease of 50% or more. In some embodiments, "reducing" or "inhibiting" means the ability to cause an overall decrease of 75%, 85%, 90%, 95% or more. In some embodiments, the above amounts are inhibited or decreased over a period of time as compared to a control over the same period of time.

[0136] As used herein, "delaying the onset of a disease" means delaying, preventing, decelerating, retarding, stabilizing, suppressing, and / or extending the onset of a disease (such as cancer). This delay can be of various lengths depending on the medical history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or substantial delay may in fact encompass prevention in the sense that the individual does not develop the disease. For example, it is possible to delay advanced cancer such as the occurrence of metastasis.

[0137] "Prevention" as used herein includes providing prevention against the occurrence or recurrence of a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed with the disease. Unless otherwise indicated, the terms "reduce," "inhibit," or "prevent" do not indicate or require complete prevention over the entire period, but rather indicate or require prevention only over the period measured.

[0138] The "therapeutically effective amount" of a substance / molecule, agonist or antagonist can vary depending on factors such as the individual's disease state, age, gender, and weight, as well as the ability of the substance / molecule, agonist or antagonist to induce the desired response in the individual. The therapeutically effective amount is also an amount where the therapeutically beneficial effect exceeds any toxic or harmful effects of the substance / molecule, agonist or antagonist. The therapeutically effective amount can be delivered in one or more administrations. The therapeutically effective amount refers to an amount effective to achieve the desired therapeutic and / or prophylactic result over the required dosage amount and over the required time.

[0139] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation in a form that enables the biological activity of the active ingredient(s) to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is administered. Such formulations can be sterilized.

[0140] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art used with a therapeutic agent that together with a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation employed.

[0141] Administration "in combination" with one or more additional therapeutic agents includes simultaneous (co-administration) and sequential administration in any order.

[0142] As used herein, the term "in combination" refers to administration of two or more therapeutic agents where at least a portion of the administrations overlap in time, or where the administration of one therapeutic agent is included in a short period relative to the administration of the other therapeutic agent, or where the therapeutic effects of both therapeutic agents overlap for at least some period of time.

[0143] As used herein, the term "sequentially" refers to administration of two or more therapeutic agents where the administrations do not overlap in time, or where the therapeutic effects of the therapeutic agents do not overlap.

[0144] As used herein, "in combination with" refers to adding one therapy to the administration of another. Thus, "in combination with" refers to administering one therapy before, during, or after another therapy is administered to an individual.

[0145] The term "package insert" is used to refer to the instructions customarily included in the commercial package of a therapeutic product that includes information regarding instructions for use, method of use, dosage, administration, combination therapies, contraindications and / or warnings regarding the use of such therapeutic product.

[0146] "Manufactured product" means any manufactured product (e.g., a package or container) or kit that includes at least one reagent, such as a medicament for treating a disease or disorder (e.g., cancer), or a probe that specifically detects a biomarker described herein. In some embodiments, the manufactured product or kit is advertised, distributed, or sold as a unit for performing the methods described herein.

[0147] The terms "label" and "detectable label" mean a moiety that, for example, binds to an antibody or antigen and enables the detection of a reaction (e.g., binding) between members of a specific binding pair. The labeled member of the specific binding pair is referred to as "detectably labeled." Thus, the term "labeled binding protein" refers to a protein into which a label that provides for the identification of the binding protein has been incorporated. In some embodiments, the label is a detectable marker that can generate a signal detectable visually or by instrumental means, such as the incorporation of a radioactively labeled amino acid or the binding of a biotinylated moiety to a polypeptide that can be detected by a labeled avidin (e.g., streptavidin that includes an enzyme activity detectable by a fluorescent marker or by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153(Sm), chromogen, fluorescent label (e.g., FITC, rhodamine, lanthanide phosphor), enzyme label (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent marker, biotinyl group, predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for a secondary antibody, metal binding domain, epitope tag), and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include a light-emitting moiety, e.g., an acridinium compound, and a fluorescence-emitting moiety, e.g., fluorescein. In this regard, although the moiety itself may not be detectably labeled, it may become detectable upon reacting with another moiety.

[0148] Exemplary FcRn-binding polypeptide Single domain antibodies (sdAbs) comprising a VHH domain that binds to FcRn are provided herein. In some embodiments, the VHH domain that binds to FcRn has an affinity (K D ) for binding of 0.01 nM to 5 nM, or 0.01 nM to 2 nM, or 0.01 nM to 1 nM, 0.01 nM to 0.5 nM, 0.05 nM to 5 nM, or 0.05 nM to 2 nM, or 0.05 nM to 1 nM, or 0.05 nM to 0.5 nM.

[0149] In various embodiments, polypeptides comprising at least one VHH domain that binds to FcRn are provided. In some embodiments, polypeptides comprising 1, 2, 3, 4, 5, 6, 7, or 8 VHH domains that bind to FcRn are provided. In some embodiments, the polypeptides provided herein comprise 1, 2, 3, or 4 VHH domains that bind to FcRn. Such polypeptides may also include one or more additional VHH domains that bind to one or more target proteins other than FcRn.

[0150] In various embodiments, a polypeptide comprising one or more VHH domains that bind to FcRn also comprises a therapeutic antigen-binding domain and / or a therapeutic polypeptide. Such therapeutic antigen-binding domains include, but are not limited to, antigen-binding domains of conventional antibodies comprising heavy chain variable regions and light chain variable regions, as well as antigen-binding domains of single-domain antibodies such as VHH domains. Non-limiting exemplary therapeutic polypeptides include, for example, receptor extracellular domains, enzymes, and ligands. In various embodiments, a polypeptide comprising at least one VHH domain that binds to FcRn has a longer half-life in vivo when fused to at least one VHH domain that binds to albumin than the same polypeptide that is not fused to at least one VHH domain that binds to albumin. In some embodiments, the half-life is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold longer than the half-life of a polypeptide that does not have a VHH domain that binds to albumin.

[0151] In some embodiments, a polypeptide comprising at least one VHH domain that binds to FcRn comprises an Fc region. In some embodiments, the polypeptides provided herein comprise one, two, three, or four VHH domains that bind to FcRn and an Fc region. In some embodiments, the Fc region mediates dimerization of the polypeptide under physiological conditions.

[0152] In various embodiments, a VHH domain that binds to FcRn comprises a CDR1 sequence selected from SEQ ID NO: 80 and SEQ ID NO: 81, a CDR2 sequence selected from SEQ ID NO: 83 and SEQ ID NO: 84, and a CDR3 sequence of SEQ ID NO: 85. In various embodiments, a VHH domain that binds to FcRn comprises CDR1, CDR2, and CDR3 sequences selected from SEQ ID NO: 80, SEQ ID NO: 83, and SEQ ID NO: 85; SEQ ID NO: 81, SEQ ID NO: 83, and SEQ ID NO: 85; and SEQ ID NO: 81, SEQ ID NO: 84, and SEQ ID NO: 85.

[0153] In some embodiments, the VHH domain that binds to FcRn comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence selected from SEQ ID NO: 86 to SEQ ID NO: 93. In some embodiments, the VHH domain that binds to FcRn comprises an amino acid sequence selected from SEQ ID NO: 86 to SEQ ID NO: 93.

[0154] In some embodiments, a VHH domain that binds to FcRn is provided, which competes for binding to FcRn with a VHH domain comprising an amino acid sequence selected from SEQ ID NO: 86 to SEQ ID NO: 93.

[0155] Exemplary albumin-binding polypeptides Polypeptides comprising a VHH domain that binds to albumin are provided herein. In some embodiments, the VHH domain that binds to albumin does not interfere with the binding of albumin to FcRn. In some embodiments, the VHH domain that binds to albumin does not bind to domain 3 of albumin. In some embodiments, the VHH domain that binds to albumin binds with an affinity (K D ) of 0.01 nM to 5 nM, or 0.01 nM to 2 nM, or 0.01 nM to 1 nM, 0.01 nM to 0.5 nM, 0.05 nM to 5 nM, or 0.05 nM to 2 nM, or 0.05 nM to 1 nM, or 0.05 nM to 0.5 nM.

[0156] In various embodiments, a polypeptide is provided that comprises at least one VHH domain that binds to albumin. In some embodiments, a polypeptide is provided that comprises one, two, three, four, five, six, seven, or eight VHH domains that bind to albumin. In some embodiments, the polypeptides provided herein comprise one, two, three, or four VHH domains that bind to albumin. Such polypeptides may also comprise one or more additional VHH domains that bind to one or more target proteins other than albumin.

[0157] In various embodiments, a polypeptide that comprises one or more VHH domains that bind to albumin also comprises a therapeutic antigen-binding domain and / or a therapeutic polypeptide. Such therapeutic antigen-binding domains include, but are not limited to, the antigen-binding domains of conventional antibodies that comprise a heavy-chain variable region and a light-chain variable region, and the antigen-binding domains of single-domain antibodies such as VHH domains. Non-limiting exemplary therapeutic polypeptides include, for example, receptor extracellular domains, enzymes, and ligands. In various embodiments, a polypeptide that comprises at least one VHH domain that binds to albumin has a longer half-life in vivo than the same polypeptide that does not comprise at least one VHH domain that binds to albumin. In some embodiments, the half-life is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold longer than the half-life of a polypeptide that does not comprise a VHH domain that binds to albumin.

[0158] In some embodiments, a polypeptide that comprises at least one VHH domain that binds to albumin comprises an Fc region. In some embodiments, the polypeptides provided herein comprise one, two, three, or four VHH domains that bind to albumin and an Fc region. In some embodiments, the Fc region mediates dimerization of the polypeptide under physiological conditions.

[0159] In various embodiments, the VHH domain that binds to albumin comprises a CDR1 sequence selected from SEQ ID NO: 5 to SEQ ID NO: 8, a CDR2 sequence selected from SEQ ID NO: 9 to SEQ ID NO: 21, and a CDR3 sequence of SEQ ID NO: 22. In various embodiments, the VHH domain that binds to albumin comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 12, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 14, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 7, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 8, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 16, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 17, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 18, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 19, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 20, and SEQ ID NO: 22; and SEQ ID NO: 6, SEQ ID NO: 21, and SEQ ID NO: 22. In some embodiments, the VHH domain that binds to albumin comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of SEQ ID NO: 6, SEQ ID NO: 21, and SEQ ID NO: 22.

[0160] In some embodiments, the VHH domain that binds to albumin comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence selected from SEQ ID NO: 23 to SEQ ID NO: 43 and SEQ ID NO: 97 to SEQ ID NO: 100. In some embodiments, the VHH domain that binds to albumin comprises an amino acid sequence selected from SEQ ID NO: 23 to SEQ ID NO: 43 and SEQ ID NO: 97 to SEQ ID NO: 100. In some embodiments, the VHH domain that binds to albumin comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 99. In some embodiments, the VHH domain that binds to albumin comprises the amino acid sequence of SEQ ID NO: 99.

[0161] In some embodiments, there is provided a VHH domain that binds to albumin, which competes for binding to albumin with a VHH domain comprising an amino acid sequence selected from SEQ ID NO: 23 to SEQ ID NO: 43 and SEQ ID NO: 97 to SEQ ID NO: 100.

[0162] Exemplary FcRn and albumin-binding polypeptides In some embodiments, there is provided a polypeptide comprising at least one VHH domain that binds to FcRn and at least one VHH domain that binds to albumin. In various embodiments, the VHH domain that binds to FcRn and / or the VHH domain that binds to albumin is a VHH domain provided herein.

[0163] In some embodiments, there is provided a polypeptide comprising one, two, three, four, five, six, seven, or eight VHH domains that bind to FcRn and at least one VHH domain that binds to albumin. In some embodiments, the polypeptides provided herein comprise one, two, three, or four VHH domains that bind to FcRn and at least one VHH domain that binds to albumin. A non-limiting exemplary polypeptide comprising at least one VHH domain that binds to FcRn and at least one FcRn that binds to albumin is shown in FIG. 5.

[0164] In various embodiments, the polypeptides provided herein may comprise one or more additional antigen-binding domains that bind to one or more target antigens other than albumin or FcRn. In various embodiments, polypeptides comprising one or more VHH domains that bind to albumin and one or more VHH domains that bind to FcRn also comprise a therapeutic antigen-binding domain and / or a therapeutic polypeptide. Such therapeutic antigen-binding domains include, but are not limited to, antigen-binding domains of conventional antibodies comprising heavy-chain variable regions and light-chain variable regions, and antigen-binding domains of single-domain antibodies such as VHH domains. Non-limiting exemplary therapeutic polypeptides include, for example, receptor extracellular domains, enzymes, and ligands. The VHH domain that binds to FcRn and / or the VHH domain that binds to albumin may be linked to the antigen-binding domain or therapeutic polypeptide at the N-terminus, or C-terminus, of the antigen-binding domain or therapeutic polypeptide (e.g., VHH-VHH-antigen-binding domain or antigen-binding domain-VHH-VHH), or may be linked at different termini (e.g., VHH-antigen-binding domain-VHH).

[0165] In some embodiments of the polypeptide, adjacent portions of the polypeptide, such as a first VHH and a second VHH or a VHH and an antigen-binding domain, may be linked to each other by a linker. In some such embodiments, the linker comprises 1 to 20, 1 to 12, or 1 to 8 amino acids and is preferably composed mainly of glycine and optionally serine. A non-limiting example of such a linker is GSGGGS (SEQ ID NO: 101). In some embodiments of the polypeptide, the C-terminal VHH may be extended by one or more additional amino acid residues. In certain embodiments, such residues are glycine residues. In some embodiments, the C-terminal VHH is extended by two or more glycine residues.

[0166] In some embodiments, a polypeptide comprising at least one VHH domain that binds to albumin and one VHH domain that binds to FcRn does not contain an Fc region. In some embodiments, a polypeptide comprising at least one VHH domain that binds to albumin and one VHH domain that binds to FcRn contains an Fc region. In some embodiments, the polypeptides provided herein comprise one, two, three, or four VHH domains that bind to albumin, one, two, three, or four VHH domains that bind to FcRn, and an Fc region. In some embodiments, the Fc region mediates dimerization of the polypeptide under physiological conditions.

[0167] In various embodiments, a polypeptide comprising at least one VHH domain that binds to albumin and FcRn has a longer half-life in vivo than the same polypeptide that does not comprise at least one VHH domain that binds to albumin. In some embodiments, the half-life is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold longer than the half-life of a polypeptide that does not have a VHH domain that binds to albumin.

[0168] In some embodiments, a polypeptide comprising at least one VHH domain that binds to albumin and at least one VHH domain that binds to FcRn comprises a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NO: 69 to SEQ ID NO: 77. In some embodiments, a polypeptide comprising at least one VHH domain that binds to albumin and one VHH domain that binds to FcRn comprises a sequence selected from SEQ ID NO: 69 to SEQ ID NO: 77.

[0169] In some embodiments, a polypeptide comprising at least one VHH domain that binds to albumin and one VHH domain that binds to FcRn comprises a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NO: 69 to SEQ ID NO: 74 and SEQ ID NO: 77. In some embodiments, a polypeptide comprising at least one VHH domain that binds to albumin and one VHH domain that binds to FcRn comprises a sequence selected from SEQ ID NO: 69 to SEQ ID NO: 74 and SEQ ID NO: 77.

[0170] Humanized VHH domain In some embodiments, the VHH domain is humanized. A humanized VHH domain (e.g., in an sdAb or VHH-containing polypeptide) is useful as a therapeutic molecule because it reduces or eliminates the human immune response to non-human antibodies that could otherwise generate an immune response to the antibody therapy and reduce the effectiveness of the treatment. Generally, a humanized VHH comprises a CDR (or a portion thereof) from a non-human antibody and an FR (or a portion thereof) from a human antibody sequence. In some embodiments, some FR residues in the humanized VHH are replaced with corresponding residues from a non-human antibody (e.g., the VHH from which the CDR residues are derived) to, for example, restore or improve the specificity or affinity of the VHH.

[0171] Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and further described, for example, in Riechmann et al., (1988) Nature 332:323-329, Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033, U.S. Patent No. 5,821,337, U.S. Patent No. 7,527,791, U.S. Patent No. 6,982,321, and U.S. Patent No. 7,087,409, Kashmiri et al., (2005) Methods 36:25-34, Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"), Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling"), and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0172] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296), framework regions derived from consensus sequences of human antibodies of certain subgroups of the heavy chain variable region (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285, and Presta et al. (1993) J. Immunol, 151:2623), human mature (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633), and framework regions obtained from screening of FR libraries (see, e.g., Baca et al., (1997) J. Biol. Chem. 272: 10678-10684, and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, humanized VHHs are produced by replacing the FR region of the VHH with a human FR region. In some embodiments, replacing certain FR residues of the human FR improves one or more properties of the humanized VHH. A VHH domain having such replaced residues is also further referred to herein as "humanized."

[0173] Fc domain In various embodiments, the Fc region contained in the polypeptide is a human Fc region or is derived from a human Fc region.

[0174] In some embodiments, the Fc region contained in the polypeptide is derived from a human Fc region and contains mutations designed for heterodimerization, referred to herein as "knob" and "hole". In some embodiments, the "knob" Fc region contains the mutation T366W. In some embodiments, the "hole" Fc region contains the mutations T366S, L368A, and Y407V. In some embodiments, the Fc region used for heterodimerization contains additional mutations such as the mutation S354C on the first member of the heterodimeric Fc pair, which forms an asymmetric disulfide bond with the corresponding mutation Y349C on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair contains a modified H435R or H435K to prevent binding to Protein A while maintaining FcRn binding. In some embodiments, one member of the heterodimeric Fc pair contains a modified H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc region contains a modified H435R or H435K (when the modification is H435R, sometimes referred to as "hole-R"), while the knob Fc region does not contain it. In some cases, the hole-R mutation improves the purification of the heterodimer over the homodimeric hole Fc region that may be present.

[0175] Non-limiting exemplary Fc regions that can be used in the polypeptide include Fc regions comprising the amino acid sequences of SEQ ID NOs: 47 to 68.

[0176] Expression and production of the polypeptide Nucleic acid molecules are provided that contain polynucleotides encoding the polypeptides provided herein. In some embodiments, the nucleic acid molecule can also encode a leader sequence that directs the secretion of the polypeptide, which leader sequence is typically cleaved so that it is not present in the secreted polypeptide. The leader sequence can be a native heavy chain (or VHH) leader sequence or another heterologous leader sequence.

[0177] Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0178] Vectors are provided that contain nucleic acids encoding the polypeptides provided herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, a vector optimized for expression of the polypeptide in a desired cell type, such as a CHO cell or a CHO-derived cell, or an NSO cell, is selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0179] In some embodiments, the polypeptides provided herein can be expressed in prokaryotic cells such as bacterial cells, or in eukaryotic cells such as fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used for expression of the polypeptide include, but are not limited to, COS cells including COS7 cells, 293 cells including 293-6E cells, CHO cells including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells, PER.C6™ cells (Crucell), and NSO cells. In some embodiments, the polypeptide can be expressed in yeast. See, for example, U.S. Patent Application Publication No. 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform desired post-translational modifications on the polypeptide. For example, in some embodiments, CHO cells produce a polypeptide with a higher level of sialylation than the same polypeptide produced in 293 cells.

[0180] Introduction of one or more nucleic acids (such as vectors) into a desired host cell can be achieved by any method including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press (2001). The nucleic acid can be transiently or stably transfected into the desired host cell according to any suitable method.

[0181] Also provided are host cells containing any of the nucleic acids or vectors described herein. In some embodiments, host cells that express the polypeptides described herein are provided. The polypeptides expressed in the host cells can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include agents that bind to the ROR1 ECD and Fc region. For example, Protein A, Protein G, Protein A / G, or an antibody affinity column can be used to purify polypeptides containing an Fc region by binding to the Fc region. Hydrophobic interaction chromatography, such as butyl columns or phenyl columns, may also be suitable for purifying some polypeptides such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be suitable for purifying some polypeptides such as antibodies. Mixed mode chromatography (e.g., reverse phase / anion exchange, reverse phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for purifying some polypeptides such as antibodies. Many methods for purifying polypeptides are known in the art.

[0182] In some embodiments, the polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009), Spirin, Trends Biotechnol. 22: 538-45 (2004), Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0183] In some embodiments, polypeptides produced by the above methods are provided. In some embodiments, the polypeptide is produced in a host cell. In some embodiments, the polypeptide is produced in a cell-free system. In some embodiments, the polypeptide is purified. In some embodiments, a cell culture medium containing the polypeptide is provided.

[0184] In some embodiments, compositions comprising antibodies produced by the above methods are provided. In some embodiments, the composition comprises a polypeptide produced in a host cell. In some embodiments, the composition comprises a polypeptide produced in a cell-free system. In some embodiments, the composition comprises a purified polypeptide.

[0185] Exemplary methods of treating a disease using an FcRn-binding polypeptide In some embodiments, provided herein is a method of treating a disease in an individual, comprising administering a therapeutic polypeptide comprising an FcRn binding domain provided herein. Such diseases include any disease for which treatment with a therapeutic polypeptide comprising an FcRn binding domain is beneficial. Non-limiting exemplary diseases that can be treated with the therapeutic polypeptides comprising an FcRn binding domain provided herein include infectious diseases, immune diseases or immune disorders (e.g., autoimmune diseases or autoimmune disorders), inflammatory diseases or inflammatory disorders, and cancer. In some embodiments, the disease or disorder is an autoantibody-mediated disease or autoantibody-mediated disorder. In some embodiments, the disease or disorder is pemphigus vulgaris, lupus nephritis, myasthenia gravis, Guillain-Barré syndrome, antibody-mediated rejection, antiphospholipid antibody syndrome, chronic inflammatory demyelinating polyneuropathy, immune complex-mediated vasculitis, glomerulonephritis, channelopathy, neuromyelitis optica, autoimmune encephalitis, autoimmune Graves' disease, idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia (AIHA), immune neutropenia, dilated cardiomyopathy, or serum sickness. The method comprises administering to the individual an effective amount of a therapeutic polypeptide comprising an FcRn binding domain provided herein. In some embodiments, the method of treatment promotes clearance of autoantibodies in the subject. In some embodiments, the method of treatment blocks an immune response in the subject (e.g., blocks activation of an immune response based on immune complexes). Such methods of treatment can be performed in humans or animals. In some embodiments, provided is a method of treating a human.

[0186] The therapeutic polypeptides containing the FcRn binding domain provided herein can be administered to a subject as needed. The determination of the frequency of administration can be made by those skilled in the art, such as a attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health status of the subject being treated, etc. In some embodiments, an effective dose of the therapeutic polypeptide is administered to the subject one or more times. In some embodiments, an effective dose of the therapeutic polypeptide containing the FcRn binding domain is administered to the subject daily, twice a week, weekly, every two weeks, once a month, etc. An effective dose of the therapeutic polypeptide containing the FcRn binding domain is administered to the subject at least once. In some embodiments, an effective dose of the therapeutic polypeptide can be administered in multiple doses over at least one month, at least six months, or at least one year.

[0187] In some embodiments, the pharmaceutical composition is administered in an amount effective to treat the disease. The therapeutically effective amount typically depends on the body weight of the subject being treated, the physical or health status of that subject, the extent of the condition being treated, or the age of the subject being treated.

[0188] In some embodiments, the therapeutic polypeptide can be administered in vivo by various routes including, but not limited to, intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. Appropriate formulations and routes of administration can be selected according to the intended use.

[0189] Pharmaceutical composition In some embodiments, the composition containing the polypeptide is provided in a formulation containing a variety of pharmaceutically acceptable carriers (e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th 3rd ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd rd ed., Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers are available, including vehicles, adjuvants, and diluents. Additionally, a variety of pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, etc. are also available. Non-limiting exemplary carriers include physiological saline, buffered physiological saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0190] Non-limiting exemplary methods of diagnosis and treatment In some embodiments, the methods described herein are useful for evaluating a subject and / or a sample from a subject (e.g., a patient suffering from an autoimmune disease or a cancer patient). In some embodiments, the evaluation is one or more of diagnosis, prognosis, and / or therapeutic efficacy.

[0191] In some embodiments, the methods described herein include evaluating the presence, absence, or level of a protein. In some embodiments, the methods described herein include evaluating the presence, absence, or level of expression of a nucleic acid. The compositions described herein can be used for these measurements. In some embodiments, the evaluation can indicate treatment (including treatment with the polypeptides described herein).

[0192] Kit Also provided are articles of manufacture and kits comprising any of the polypeptides described herein and appropriate packaging. In some embodiments, the invention includes kits comprising (i) a polypeptide provided herein and (ii) instructions for using the kit to administer the polypeptide to an individual.

[0193] Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These manufactured articles can be further sterilized and / or sealed. Unit dosage forms containing the compositions described herein are also provided. These unit dosage forms can be stored in suitable packaging in single or multiple unit dosages and can be further sterilized and sealed. The instructions provided in the kits of the present invention are typically instructions written on a label or accompanying document (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions held on a magnetic storage disk or an optical storage disk) are also acceptable. Instructions for use regarding the use of an antibody generally include information regarding dosage, dosing schedule, and route of administration for the intended therapeutic or industrial use. The kit can further include an explanation for selecting an appropriate individual treatment.

[0194] The container can be a unit dose, bulk packaging (e.g., multi-dose packaging), or sub-unit dose. For example, kits can be provided that contain an amount of a molecule disclosed herein sufficient to provide effective treatment to an individual over a long period of time, such as any approximate period of 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more. The kit can also include multiple unit doses of the molecule and instructions for use and can be packaged in an amount sufficient for storage and use in a pharmacy, e.g., a hospital pharmacy and a dispensing pharmacy. In some embodiments, the kit includes a dry (e.g., lyophilized) composition that can generally form a stable aqueous suspension of the antibody upon reconstitution, resuspension, or rehydration.

Examples

[0195] The examples discussed below are intended to purely illustrate the present invention and should in no way be construed as limiting the present invention. These examples are not intended to represent that the following experiments are all or the only experiments conducted. Efforts have been made to ensure accuracy with respect to the numerical values used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0196] Example 1: Development of an anti-albumin single-domain antibody (sdAb) Single-domain antibodies targeting human albumin were generated by immunizing llamas and / or alpacas with recombinant human serum albumin (SEQ ID NO: 1).

[0197] After the expression of specific anti-albumin antibody titers, llama / alpaca peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of the blood of the immunized animals, total mRNA was isolated using the Qiagen RNeasy Maxi Kit, and subsequently converted to first-strand cDNA using Thermo Superscript IV Reverse Transcriptase and oligo dT priming. The VHH sequences were specifically amplified by PCR using the cDNA as a template and cloned into a yeast surface display vector as a VHH-Fc-AGA2 fusion protein. Fc was human IgG1 Fc, or in some cases, a mutant IgG1 Fc with reduced effector function.

[0198] A yeast library presenting the VHH-Fc-AGA2 fusion protein was enriched using recombinant human albumin by fluorescence-activated cell sorting (FACS) following magnetic bead isolation. The sorted yeast were plated out, and the isolated colonies were picked into 96-well blocks to induce yeast cell surface expression of the VHH-Fc-AGA2 fusion protein. Biotinylated recombinant human albumin or an irrelevant biotinylated protein (albumin-negative) was directly applied to the induced yeast, washed, treated with fluorophore-labeled streptavidin, and analyzed by 96-well flow cytometry.

[0199] The nucleic acid sequence encoding the VHH that binds to biotinylated recombinant human albumin and does not bind to irrelevant biotinylated proteins was cloned into a mammalian expression vector in-frame with the human Fc xELL coding region and expressed by transient transfection in HEK293 Freestyle cells (293F cells) or CHO cells using polyethyleneimine. After 3 to 7 days, the supernatant was collected, and the secreted recombinant protein was purified by protein A chromatography, and the concentration was calculated from the absorbance at 280 nm and the extinction coefficient.

[0200] The anti-albumin sdAb 4A01 was selected for humanization.

[0201] Example 2: Humanization and interspecies cross-reactivity of anti-albumin sdAb 4A01 Various humanized forms of sdAb 4A01 were generated based on the human heavy chain framework VH3-23 * 04. Certain amino acids were back-mutated to donor amino acids, and certain mutations were tested, for example, in CDR2. Figure 1A shows the alignment of the human heavy chain acceptor sequence and the humanized forms of 4A01.

[0202] The binding of the monomeric anti-albumin sdAb 4A01 (“lm4A01”) and its humanized version to human serum albumin, cynomolgus monkey serum albumin, mouse serum albumin, and rat serum albumin was determined by ELISA as follows. Medisorp plates were coated overnight at 4 °C with albumin protein at 2 μg / ml, 50 μl / well (human, mouse, and rat albumin - Sigma, cynomolgus monkey albumin - Abcam). 1-fold Fish Gelatin (blocking agent, Bethyl Laboratories) was added to the wells coated with albumin, followed by incubation at room temperature for 1 hour. Titration of the sdAb fusion protein (starting from 100 nM, 1:3 horizontally or 1:4 vertically) was added and incubated at room temperature for 1 hour. The plates were washed 3 times with 0.1% D-PBST, then anti-human Fc HRP antibody (1:2000 in 0.1% D-PBST, Jackson) was added and incubated at room temperature for 30 minutes. The plates were washed 3 times with 0.1% D-PBST, then TMB substrate was added. Absorbance at 650 nm was read with a plate reader (Molecular Devices), and the data were plotted using a one-site - total binding equation (model: Y = Bmax×X / (Kd+X)+NS×X+background, GraphPad Prism).

[0203] The binding of 4A01 and the humanized form of 4A01 to human albumin is shown in Figures 1B and 1C. All sdAbs bound to human albumin with a K D of 0.10 nM to 0.43 nM. The binding of 4A01 and the humanized form of 4A01 to cynomolgus monkey albumin is shown in Figures 1D and 1E. All sdAbs bound to cynomolgus monkey albumin with a K D of 0.11 nM to 0.34 nM. The binding of 4A01 and the humanized form of 4A01 to mouse albumin is shown in Figures 1F and 1G. All sdAbs bound to mouse albumin with a K D of 0.11 nM to approximately 0.25 nM. The binding of 4A01 and the humanized form of 4A01 to rat albumin is shown in Figures 1H and 1I. All sdAbs bound to rat albumin with a K of 0.14 nM to approximately 0.33 nMD It bound to rat albumin.

[0204] Figures 2A - 2D show the binding of 4A01 and humanized hz4A01v51 to human (2A), cynomolgus monkey (2B), mouse (2C), and rat (2D) albumin. 4A01 and all humanized variants tested bound to all four albumins with an affinity of less than 1 nM. Humanized hz4A01v51 bound to all four albumins with an affinity of less than 0.3 nM and achieved a maximum binding rate of over 90%.

[0205] The framework region of hz4A01v51 was further modified by reverse - mutating certain residues to donor amino acids and / or introducing alternative charged residues, etc. Modified monomeric anti - albumin antibodies (including the VHH domain of hz4A01v51.9, hz4A01v51.11, hz4A01v51.12, or hz4A01v51.13) showed similar binding profiles at pH 6 and pH 7.4, and all showed improved binding compared to that observed for hz4A01v51, especially at pH 6 (data not shown).

[0206] Example 3: Anti - albumin sdAb 4A01 does not bind to albumin domain 3 Albumin binds mainly via domain 3 to the β2 - microglobulin - FcRn complex, and this binding is thought to be involved in improving the half - life of proteins fused with anti - albumin antibodies or albumin itself. To determine whether anti - albumin sdAb 4A01 binds to albumin domain 3, the binding of 4A01 - NNT - hFc was assayed by biolayer interferometry as follows.

[0207] Albumin domain 3 (with mouse Fc tag) was immobilized on an anti-mouse IgG Fc capture biosensor. All buffer / protein formulations were in MBST5 (50 nM MES (pH 5), 150 mM NaCl, 0.025% Tween®). The baseline was established with buffer only. Human albumin domain III with mouse Fc tag (10 μg / ml) was loaded onto an anti-mouse IgG Fc capture biosensor (ForteBio). Then, anti-albumin sdAb 4A01 (4A01-NNT-hFc) and 1C04 (similar format) were loaded, allowed to associate with the captured biotin domain 3, and then dissociated with MBST5. sdAb 1C04 is known to bind to albumin domain 3 and was used as a positive control. See Figure 3A.

[0208] As shown in Figure 3B, 1C04 bound to immobilized albumin domain 3, while 4A01 did not.

[0209] Next, anti-albumin sdAb 4A01 (4A01-NNT-hFc), hz4A01v51, and 1C04 were tested for interference with albumin-FcRn binding as follows. Binding was assessed by biolayer interferometry using biotinylated recombinant FcRn-B2M immobilized on a streptavidin biosensor. Then, the immobilized FcRn-B2M was complexed with recombinant human albumin. All buffer / protein formulations were in MBST5 (50 mM MES (pH 5), 150 mM NaCl, 0.025% Tween®). The baseline was established with buffer only. Biotinylated FcRn-B2M (10 μg / ml, Acro Biosystems) was loaded onto a streptavidin biosensor (ForteBio) to determine a further baseline. Then, 50 μM recombinant human albumin (Sigma) was added and allowed to associate with the immobilized FcRn-B2M. Then, anti-albumin sdAb 4A01 and 4A01v51, and sdAb 1C04 were loaded, allowed to associate with the captured biotin domain 3, and then dissociated with MBST5. See Figure 4A.

[0210] As shown in Figure 3B, both 4A01 and hx4A01v51 bound to albumin associated with FcRn-B2M, while 1C04 did not bind.

[0211] Example 4: Development of FcRn single-domain antibodies (sdAbs) Single-domain antibodies targeting human FcRn were generated via immunization of llamas with a polypeptide comprising human B2m (SEQ ID NO: 79) and the extracellular domain of human FcRn (SEQ ID NO: 78) linked by a flexible linker and fused to a polyhistidine tag for purification. After the expression of specific anti-FcRn antibody titers, llama peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of blood of the immunized animals, total mRNA was isolated using the Qiagen RNeasy Maxi Kit, and then converted to first-strand cDNA using Thermo Superscript IV Reverse Transcriptase and oligo dT priming. The VHH sequences were specifically amplified via PCR using the cDNA as a template and cloned into a yeast surface display vector as a VHH-Fc-AGA2 fusion protein.

[0212] The yeast library presenting the VHH-Fc-AGA2 fusion protein was enriched using the recombinant form of FcRn ECD via magnetic bead isolation followed by fluorescence-activated cell sorting (FACS). The sorted yeast were plated out, and the isolated colonies were picked into 96-well blocks and cultured in a medium that switches the expression from surface-displayed VHH-Fc to secretion into the medium. The supernatant from the 96-well yeast secretion cultures was applied to 293F cells transiently transfected with FcRn (FcRn-positive) or untransfected 293F cells (FcRn-negative), washed, treated with a fluorophore-labeled anti-human IgG1 Fc secondary antibody, and analyzed by 96-well flow cytometry.

[0213] One VHH domain that binds to FcRn (clone B10a) was substantially humanized as described in Example 2. Using the humanized B10a VHH domain (hzB10a.v1-v2.5), bispecific polypeptides targeting human FcRn and albumin having the structures shown in Figure 5(i) (cx11383, cx11558, cx12003, cx12006, cx12007, cx12008, and cx12032) or Figure 5(ii) (cx11385 and cx11642) were generated.

[0214] Example 5: Binding of anti-FcRn VHH Polypeptide to Human FcRn Binding of the anti-FcRn VHH polypeptide to human FcRn at neutral (7.4) or endosomal (6) pH was tested by flow cytometry using a CHO-based overexpression system or isolated human peripheral blood mononuclear cells (PBMC). CHO cells were transiently transfected to overexpress the extracellular domain (ECD) of human FcRn (SEQ ID NO: 78) fused to the transmembrane domain, and intracellular citrate and beta2 microglobulin (SEQ ID NO: 79).

[0215] CHO cells were seeded in 96-well plates at 50,000 cells per well and resuspended in PBS (pH 7.4) or buffer containing 20 mM His, 150 mM NaCl (pH 6). Serial dilutions of the test substance were prepared to twice the final concentration in the above buffer (pH 7.4 or pH 6) and added to the cell suspension. The plates were incubated at 4°C for 60 minutes. After incubation, the cells were washed with their respective buffers and then incubated with a fluorescently labeled (AF647) antibody that detects the VHH polypeptide for 30 minutes at 4°C. Binding of the detection antibody to FcRn on the transfected cells (citrate positive) was measured using an iQue Screener (Sartorius) after the final wash.

[0216] Isolated human PBMCs were seeded at 120,000 cells per well in a 96-well plate and washed with PBS (pH 7.4). The cells were stained with Zombie Aqua to exclude dead cells and then stained with fluorescently labeled specific antibodies against surface lineage markers (CD14, CD16, CD3, CD56, CD19, and HLA-DR) so that different PBMC subpopulations could be gated. After staining of the surface markers, the cells were fixed and permeabilized using a Foxp3 / Transcription Factor Fixation / Permeabilization Kit (eBioscience®) according to the manufacturer's recommended protocol. The cells were then resuspended in the above buffer to generate conditions of pH 7.4 or pH 6, and the test substance was stained as in the case of CHO cells. After the final wash, the binding to FcRn on PBMC subpopulations (monocytes, B cells, or T cells) was measured using a Novocyte-Quanteon flow cytometer (Agilent).

[0217] The binding of several anti-FcRn VHH polypeptides to human FcRn at neutral (7.4) pH was also tested by ELISA. A 96-well ELISA plate was coated overnight at 4 °C with human FcRn / B2m heterodimer protein in PBS, washed with PBST, and then blocked with 3% BSA in PBST for 1 hour at room temperature. Serial dilutions of the test substance were prepared in PBST (pH 7.4) and added to the plate. The plate was incubated for 1 hour at room temperature. After incubation, the plate was washed with PBST, and then incubated with a horseradish peroxidase (HRP)-conjugated antibody to detect the polypeptide for 1 hour at room temperature. The plate was washed with PBS-T, followed by the addition of TMB substrate to develop color. The TMB substrate was stopped with an equal volume of stop solution (1 M H2SO4). The absorbance at 450 nm was measured using a 96-well plate reader (Emax, Molecular Devices). All data were plotted and analyzed using GraphPad Prism analysis software.

[0218] As shown in FIGS. 6A and 6B and FIGS. 6D and 6E, all the FcRn target polypeptides tested bind to human FcRn overexpressed in CHO cells with apparent affinities in the low nanomolar to sub-nanomolar range. The binding is pH-independent and is equivalent at pH 7.4 and pH 6. As shown in FIG. 6C, a polypeptide having a bivalent FcRn binding domain (shown in FIG. 5(ii)) binds to FcRn with a higher affinity than its monovalent counterpart (shown in FIG. 5(i)). A polypeptide having monovalent specificity for albumin and a non-mammalian target VHH unit, formatted as shown in FIG. 5(i), does not bind to human FcRn with significant affinity, indicating that the binding is mediated by a VHH unit specific for FcRn. FIGS. 6F and 6G show that pH-independent binding to FcRn is retained in human primary cells. A polypeptide targeting FcRn and albumin (cx11558), formatted as shown in FIG. 5(i), binds to human monocytes expressing FcRn with low nanomolar affinity at pH 7.4 and pH 6, but does not bind to FcRn-negative B cells or T cells.

[0219] Example 6: Binding of Polypeptides to Human Albumin The binding of the humanized VHH-containing polypeptide to human albumin at neutral (7.4) or late endosome (5.5) pH was tested by ELISA. A 96-well ELISA plate was coated overnight at 4 °C with albumin protein in PBS, washed with PBS-T, and then blocked with 5% powdered milk in PBS-T for 1 h at room temperature. Serial dilutions of the test substance were prepared in PBS (pH 7.4) or buffer containing 20 mM His, 150 mM NaCl (pH 5.5) and added to the plate. The plate was incubated for 1 h at room temperature. After incubation, the wells were washed with the respective buffer and then incubated for 30 min at room temperature with an HRP-conjugated antibody that detects the polypeptide. The plate was washed with the respective buffer, TMB substrate was added and color development was allowed. The TMB substrate was stopped with an equal volume of stop solution (1 M H2SO4). Absorbance at 450 nm was measured with a 96-well plate reader. The data were plotted and analyzed using GraphPad Prism analysis software. The results are shown in FIGS. 7A-7D.

[0220] As shown in FIGS. 7A-7D, all of the tested bispecific anti-FcRn / anti-albumin polypeptides bind to human albumin with apparent affinities in the low nanomolar to sub-nanomolar range. The binding is not pH-dependent and is equivalent at pH 7.4 and pH 5.5. Since the polypeptide with monovalent specificity for albumin and the non-mammalian target VHH unit, formatted as shown in FIG. 5(i), do not bind to human albumin with significant affinity, the binding is mediated by the VHH unit specific for albumin.

[0221] Example 7: Blocking of the binding of IgG to human FcRn by a VHH-containing polypeptide Blocking of the binding of human IgG to human FcRn expressed on HEK293 cells by a humanized VHH-containing polypeptide targeting human FcRn was measured by flow cytometry. HEK293 cells were transiently transfected to overexpress the extracellular domain (ECD) of human FcRn (SEQ ID NO: 78) fused to the transmembrane domain and intracellular citrin and beta2 microglobulin (SEQ ID NO: 79). 50,000 transiently transfected HEK293 cells were seeded in a 96-well plate and resuspended in 20 mM His, 150 mM NaCl (pH 6). Serial dilutions of the test substance were prepared at twice the final concentration in the above buffer and added to the cell suspension. The plate was incubated at 4 °C for 60 minutes. After incubation, biotinylated human IgG was added at a final concentration of 1 μM and incubated for an additional 60 minutes. The cells were then washed with the above buffer and then incubated with a fluorescent label (AF647) streptavidin at 4 °C for 30 minutes to detect the binding of IgG to FcRn. The binding of streptavidin to cells expressing FcRn (citrin positive) was measured using an iQue Screener (Sartorius) after the final wash. The data were plotted and analyzed using GraphPad Prism analysis software. The results are shown in Figure 8.

[0222] As shown in Figure 8, increasing the concentration of the test substance of the polypeptide resulted in a decrease in the binding of human IgG to FcRn expressed on HEK293 cells, as measured by a decrease in the mean fluorescence intensity (MFI) of the AF647-conjugated streptavidin reagent used to detect bound biotinylated human IgG. The inhibitory activity (IC50) was similar between the construct (cx11383) formatted as described in Figure 5(i) and the construct (cx11385) formatted as described in Figure 5(ii), and fell within the low nanomolar range for both test substances.

[0223] Example 8: In Vivo Pharmacokinetic Profile and IgG Clearance of Polypeptides Targeting Human FcRn and Albumin The pharmacokinetic (PK) profiles of humanized polypeptides targeting human FcRn and albumin were tested in normal BALB / c mice and transgenic animals expressing human FcRn and human albumin on a C57BL / 6 background. The FcRn-targeting VHH sequences used do not cross-react with mouse FcRn, but the VHH sequences targeting albumin bind to mouse albumin (data not shown). Therefore, the ability to deplete IgG in vivo was assayed only in the human FcRn / albumin transgenic mouse line after injection of human IgG.

[0224] For evaluation of serum exposure in BALB / c mice, animals were intravenously injected with the polypeptide test substance at a single dose of either 30 mg / kg or 0.3 mg / kg. Serum samples were collected at 30 minutes, 6 hours, 24 hours, 96 hours, and 168 hours after injection of the test substance. Human FcRn / albumin transgenic mice were first injected with 500 mg / kg of human IgG and then, 24 hours later, with a single dose of 20 mg / kg of the polypeptide test substance. Serum samples were collected from these mice at 30 minutes, 24 hours, 96 hours, and 168 hours after administration of the test substance. Test substance concentration and human IgG levels in mouse serum were determined by ELISA. For the test substance PK ELISA, human FcRn / B2M heterodimer protein (His-tag, Acro Biosystem) was immobilized on 96-well ELISA plates by incubating a 4 μg / mL protein solution in PBS at 4°C for 12 hours. The next day, the plates were blocked with 3% BSA TBS-T buffer for 2 hours, after which the serum samples were incubated on these plates for 2 hours. Binding of the test substance in the serum samples to the FcRn immobilized on the ELISA plates was detected using a horseradish peroxidase (HRP)-conjugated secondary detection antibody capable of binding to the polypeptide. The secondary antibody was incubated on the plates for 1 hour, binding was visualized using TMB substrate solution, followed by addition of stop solution (1 <H2SO4), and absorbance at 450 nm was measured with an Emax spectrophotometer (Molecular Devices).

[0225] Human IgG levels in mouse sera were also determined by ELISA. Polyclonal goat capture antibodies specific for human IgG were coated onto 96-well ELISA plates at 1 μg / mL in PBS at 4 °C for 12 h. The next day, the plates were blocked with 1% BSA in PBS buffer for 2 h, after which the serum samples were incubated on these plates for 2 h. Binding of human IgG in the sera was detected using a second goat anti-human IgG horseradish peroxidase (HRP)-conjugated detection antibody capable of binding to IgG in addition to the capture antibody. The secondary antibody was incubated on the plates for 1 h and binding was visualized as described above. Absorbance values measured in each ELISA were converted to test substance concentrations in SoftMax Pro using a standard curve from known concentrations of protein analyzed in parallel with each assay. Four-parameter logistic regression was used to fit the standard curve. Data were exported and graphed using GraphPad Prism analysis software.

[0226] As shown in FIGS. 9A and 9B, the 30-minute serum cMax levels and overall serum exposure of a multivalent multispecific polypeptide targeting FcRn and albumin formatted as described in FIGS. 5(i) (cx11558) and 5(ii) (cx11642) are significantly higher than those of a polypeptide without albumin binding (cx11915). Both bispecific formats show similar PK profiles at the tested dose levels. FIG. 9C shows the PK profiles of two similar constructs formatted as described in FIGS. 5(i) (cx11383) and 5(ii) (cx11385), highlighting their serum exposures compared to a nipocalimab (anti-FcRn antibody) sequence analog generated based on publicly available information. All three test substances start at similar cMax serum concentrations. However, the exposure time of the multivalent multispecific single polypeptide is significantly longer compared to nipocalimab, a conventional antibody. Among the two polypeptides, the construct (cx11383) formatted as described in FIG. 5(i) has a longer serum half-life. The extended serum exposure also leads to better removal of human IgG pre-injected in a human FcRn / albumin transgenic mouse system. The complexes of cx11383 and cx11385 both induce enhanced clearance of human IgG compared to the nipocalimab sequence analog, resulting in lower serum IgG residual levels at the end of the study (168 hours). See FIG. 9D.

[0227] Example 9: In Vivo IgG Removal by a Polypeptide Targeting Human FcRn and Albumin in Cynomolgus Monkeys The pharmacodynamic (PD) and pharmacokinetic (PK) profiles of a humanized polypeptide targeting human FcRn and albumin were evaluated in several studies in cynomolgus monkeys. In one study, naive male cynomolgus monkeys aged 4 to 5 years were divided into five treatment groups of 2 animals per group. The first and second groups were treated with 100 mg / kg and 20 mg / kg of cx11558 (monovalent), respectively, the third and fourth groups were treated with 100 mg / kg and 20 mg / kg of cx11642 (bivalent for FcRn binding), respectively, and the fifth group was treated with 20 mg / kg of efgartigimod (NDC 73475-3041-5, lot number AHUC01A, recombinant engineered IgG Fc fragment). All treatments were administered I.V. by infusion at 10 mL / kg for 60 minutes. Serum samples were collected from approximately 2 mL of whole blood from all animals and all groups before dosing, at the end of infusion, and at 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 384, 432, 480, 552, 600, 648, 696, 744, 792, and 840 hours after dosing.

[0228] Cynomolgus IgG concentrations were analyzed according to the manufacturer's protocol using the Cynomolgus IgG ELISA kit (IMNCYIGGKT) from Molecular Innovations. Briefly, cynomolgus IgG standard dilutions were added in duplicate to pre-coated ELISA plates, and 2×10 6 ~3×10 6Serum samples prepared at the final dilution were added in triplicate, and the plates were incubated for 30 minutes. The plates were then washed, incubated with biotinylated anti-cynomolgus IgG primary antibody for 30 minutes, washed, and incubated with horseradish peroxidase-conjugated streptavidin antibody for 30 minutes. The plates were then washed, TMB substrate solution was added to all wells for 2 to 10 minutes, and 1N H2SO4 was added to all wells to stop the reaction. The absorbance at 450 nm was measured using a plate spectrophotometer, and the background was subtracted from all standards and samples. A 450 was plotted against the IgG amount of all standards, and the standard curve was analyzed using a 4-parameter logistic (4PL) curve fit of Prism. The amount of IgG in all samples was determined using the standard curve, and this value was multiplied by the dilution factor used for each serum sample. The IgG concentrations over time (as percent change from baseline) in the cx11558 and efgartigimod treatment groups are plotted in Figure 10A.

[0229] The FcRn occupancy by cx11558 and cx11642 on monocytes was examined by flow cytometry from whole blood samples. Whole blood was treated with RBS lysis buffer and centrifuged. The cell pellet was washed, resuspended in PBS, and divided into three separate wells for staining. All cells were stained with ZombieAqua (Biolegend) and incubated for 10 minutes with cyno IgG (diluted to 0.2 mg / mL) in FACS buffer, followed by the addition of the following antibody panel: CD14 - BV421; CD16 - BV605; CD8 - BV785; CD20 - PE / Cy7; NKG2A - PE; CD3 - PE - Dazzle 594; HLA - DR - APC - Cy7 (supplied by Biolegend or BD) and incubated for an additional 30 minutes at 4°C in FACS buffer. After washing, the cells were treated with eBioscience fix / perm at 4°C for 30 minutes, washed twice with cold perm buffer, and then incubated for 30 minutes at 4°C with saturated concentration of B10a.v2.4 VHH - Avl - His (self - made reagent, to detect free FcRn), saturated concentration of unlabeled test substance (cx11558 or cx11642; to detect total FcRn), or buffer (to detect occupied FcRn). The cells were then washed and incubated for 30 minutes at 4°C with streptavidin - AF647 (to detect B10av2.4VHH - Avi - His) or anti - id - AF647 (self - made reagent, to detect cx11558 and cx11642). After washing, the cells were resuspended in FACS buffer and analyzed using a Novocyte Quanteon flow cytometer. The FcRn occupancy by cx11558 and cx11642 is shown in Figure 10B.

[0230] The serum levels of cx11558, cx11642, and efgartigimod over time were determined by ELISA. Biotinylated human FcRn was coated onto pre-blocked wells of a Pierce® Streptavidin Coating High Capacity Plate, and standards, controls, and serum samples were diluted and added to the plate in duplicate. After incubation for 90 - 120 minutes, the plate was washed and incubated for 60 minutes with either an anti-id-HRP conjugate (a homemade reagent for detecting cx11558 or cx11642) or a mouse anti-human IgG Fc HRP conjugate (for detecting efgartigimod). The plate was then washed, TMB substrate was added to all wells for 2 - 10 minutes, 1N H2SO4 was added to all wells to stop the reaction, and absorbance was measured at 450 nm. Background (650 nm) was subtracted from all standards and samples using a SpectraMax M5e plate reader, and OD 450-650 was reported. The absorbance and concentration of the standards were plotted and curve-fitted using 4PL regression analysis in Softmax Pro v.5.4.1 and later. The sample concentration was then interpolated from the standard curve regression analysis and reported in ng / mL mass units. The serum concentration over time is plotted in Figure 10C.

[0231] As shown in Figure 10A, cx11558 administered at any dose promoted the clearance of cynomolgus IgG and resulted in keeping the serum IgG residual levels low until at least day 27, compared with efgartigimod. Cx11642 administered at any dose induced the promotion of cynomolgus IgG clearance below the levels observed with efgartigimod, but the serum IgG levels appeared to return to normal levels earlier than those observed with cx11558 at the compatible dose of 100 mg / kg (data not shown). As shown in Figure 10B, cx11558 administered at 20 mg / kg and 100 mg / kg maintained complete occupancy of FcRn on monocytes for about 9 days and about 18 days, respectively, while cx11642 maintained complete occupancy of FcRn for about half the period. As shown in Figure 10C, the serum concentrations of cx11558 administered at doses of 20 mg / kg and 100 mg / kg were maintained above 10 μg / mL (which correlates with an approximate 100% receptor occupancy) for about 9 days and about 18 days, respectively, while the serum concentrations of 20 mg / kg and 100 mg of cx11642 decreased below 10 μg / mL by day 6 and day 8, respectively, and efgartigimod decreased below 10 μg / mL by day 6.

[0232] In further studies, the PD and PK profiles of a humanized polypeptide targeting human FcRn and albumin administered by intravenous (I.V.) or subcutaneous (S.C.) injection were evaluated. In this study, naive male cynomolgus monkeys were divided into three treatment groups of two animals per group. The first and second groups were treated with a single dose of 20 mg / kg and 100 mg / kg of cx12007 by I.V. administration (10 mL / kg infused over 70 minutes), and the third group was treated with a single dose of 100 mg / kg cx12007 by S.C. administration (1 mL / kg). Serum samples were collected from whole blood before dosing, at the end of infusion, and at 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 384, 432, 504, 552, 600, 648, 696, 744, 792, and 840 hours after dosing from all animals and all groups. Cynomolgus IgG concentrations were analyzed using the Meso Scale Discovery NHP isotyping kit (K15203D) according to the manufacturer's protocol. Briefly, calibrator standards and serum samples prepared at a 2×10 5 fold final dilution were added in duplicate to the MSD plates, and the plates were incubated for 2 hours. The plates were then washed and incubated for 2 hours in the detection antibody solution. After additional washing, read buffer was added, and the plates were read immediately on an MSD imager. The standard curve was generated from the 1 / Y of the calibrator values 2Analysis was performed using a four-parameter logistic curve fitting model with a weighting function. The amount of IgG in all samples was determined using a standard curve and this value was multiplied by the dilution factor used for each serum sample. The IgG concentration over time (as percent change from baseline) in each of the treatment groups is plotted in Figure 11A. FcRn occupancy on monocytes was essentially examined by flow cytometry as described above, except that unlabeled cx12007 at saturation concentration (to detect total FcRn) was used. FcRn occupancy by cx12007 is shown in Figure 11B. The serum concentration of cx12007 was essentially determined by ELISA as described above and is plotted over time in Figure 11C.

[0233] As shown in Figure 11A, all treatment groups showed serum IgG removal troughs of a similar magnitude to a maximum removal of approximately 77% and a similar time to reach maximum removal. The shortest time to reach 40% removal was approximately 4 days, and at doses of 20 mg / kg and 100 mg / kg, serum IgG residual levels were maintained low until at least day 21 and day 36, respectively. As shown in Figure 11B, cx12007 administered at doses of 20 mg / kg and 100 mg / kg maintained FcRn occupancy on monocytes completely for 10 days and 19 days, respectively. As shown in Figure 11C, the serum concentration of cx12007 administered at doses of 20 mg / kg and 100 mg / kg was maintained above 10 μg / mL for 10 days and 19 days, respectively. The profile of the 100 mg / kg dose was similar for both the I.V. and S.C. administration routes.

[0234] In further studies, the PK and PD profiles following subcutaneous (S.C.) administration of the humanized polypeptide cx12007 were evaluated in cynomolgus monkeys at different doses. In this study, naive male cynomolgus monkeys were divided into three treatment groups, each containing three animals per group. The first, second, and third groups were treated with a single dose of cx12007 at 20 mg / kg, 40 mg / kg, and 70 mg / kg, respectively, by S.C. administration at 1 mL / kg. Serum samples were collected from whole blood before dosing, at the end of infusion, and at 24, 36, 48, 72, 144, 216, 288, 360, 432, 504, 576, 648, 720, and 792 hours after dosing from all animals and all groups. Cynomolgus IgG concentrations were analyzed via the Meso Scale Discovery NHP isotyping kit (K15203D) as described above. The IgG concentrations over time (as percent change from baseline) for each treatment group are plotted in Figure 12A. Serum concentrations of cx12007 were determined basically as described above by ELISA and are plotted over time in Figure 12A.

[0235] As shown in Figure 12A, all treatment groups showed a serum IgG removal trough of similar degree to that of removal of greater than approximately 70% maximum, and a similar time to reach maximum removal. Cx12007 administered S.C. at all dose levels maintained low serum IgG residual levels throughout the study, with IgG levels increasing more rapidly in the 20 mg / kg group. As shown in Figure 12B, the serum concentrations of cx12007 administered S.C. at 20 mg / kg, 40 mg / kg, and 70 mg / kg doses were maintained above 10 μg / mL for approximately 9 days, at least 12 days, and at least 15 days, respectively.

[0236] In these studies, administration of the humanized polypeptides targeting human FcRn and albumin showed good tolerability with no observed severe or serious adverse events. Overall, these data demonstrate that polypeptides targeting human FcRn and albumin have good tolerability, a longer serum half-life than efgartigimod, more rapidly mediate IgG clearance, and maintain lower serum IgG levels over a longer period. These data also demonstrate that such molecules exhibit similar activity when administered subcutaneously.

[0237] The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the above-described embodiments are to be considered in all respects as illustrative and not restrictive of the present disclosure. Therefore, the scope of the present disclosure is indicated by the appended claims rather than the above detailed description, and all changes within the meaning and scope of the claims of equivalents are intended to be included herein.

[0238] [Table 2] TIFF2025519559000004.tif251170TIFF2025519559000005.tif253170TIFF2025519559000006.tif249170TIFF2025519559000007.tif250170TIFF2025519559000008.tif252170TIFF2025519559000009.tif253170TIFF2025519559000010.tif245170TIFF2025519559000011.tif250170TIFF2025519559000012.tif165170

Claims

1. A polypeptide comprising at least one VHH domain that binds to FcRn, wherein at least one or each of the VHH domains that bind to FcRn comprises a CDR1 sequence selected from SEQ ID NO: 80 and SEQ ID NO: 81, a CDR2 sequence selected from SEQ ID NO: 83 and SEQ ID NO: 84, and a CDR3 sequence of SEQ ID NO:

85.

2. The polypeptide according to claim 1, wherein at least one or each VHH domain bound to FcRn comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NOs. 80, 83, and 85; SEQ ID NOs. 81, 83, and 85; and SEQ ID NOs. 81, 84, and 85.

3. The polypeptide according to claim 1, wherein at least one or each of the VHH domains bound to FcRn is humanized.

4. At least one or each VHH domain that binds to FcRn is (a) A sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from sequence numbers 86 to 91; or (b) Sequence selected from sequence numbers 86 to 93 The polypeptide according to claim 1, comprising:

5. The polypeptide according to claim 1, wherein at least one or each VHH domain that binds to FcRn binds to human FcRn with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

6. The polypeptide according to claim 1, wherein at least one or each VHH domain that binds to FcRn binds to human FcRn at pH 6 and pH 7.

4.

7. The polypeptide according to claim 1, wherein at least one or each VHH domain that binds to FcRn blocks the binding of human IgG to human FcRn.

8. The polypeptide according to claim 1, wherein the polypeptide includes an Fc region.

9. The polypeptide according to claim 8, wherein the Fc region mediates dimerization.

10. The polypeptide according to claim 1, wherein the polypeptide comprises at least one VHH domain that binds to albumin.

11. The polypeptide according to claim 10, wherein at least one or each VHH domain that binds to albumin comprises a CDR1 sequence selected from SEQ ID NOs. 5 to 8, a CDR2 sequence selected from SEQ ID NOs. 9 to 21, and a CDR3 sequence of SEQ ID NO.

22.

12. The polypeptide according to claim 10, wherein at least one or each VHH domain that binds to albumin comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 12, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 14, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 7, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 8, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 16, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 17, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 18, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 19, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 20, and SEQ ID NO: 22; and SEQ ID NO: 6, SEQ ID NO: 21, and SEQ ID NO:

22.

13. The polypeptide according to claim 10, wherein at least one or each VHH domain that binds to FcRn and / or at least one VHH domain that binds to albumin is humanized.

14. At least one or each VHH domain that binds to albumin, (a) A sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from sequence numbers 23 to 43 and sequence numbers 97 to 100; or (b) Sequences selected from sequence numbers 23 to 43 and sequence numbers 97 to 100, The polypeptide according to claim 10, comprising:

15. The aforementioned polypeptide is (a) A sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from sequence numbers 69 to 77; or (b) Sequence selected from sequence numbers 69 to 77, The polypeptide according to claim 10, comprising:

16. The polypeptide according to claim 10, wherein at least one or each VHH domain that binds to albumin binds to human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

17. The polypeptide according to claim 10, wherein each VHH domain that binds to albumin does not bind to the albumin domain 3 and / or does not interfere with the binding of albumin to FcRn.

18. The polypeptide according to claim 1, wherein the polypeptide comprises at least two, at least three, or at least four VHH domains that bind to FcRn and at least one VHH domain that binds to albumin.

19. The polypeptide according to claim 1, wherein the polypeptide comprises at least one binding domain that binds to a protein other than FcRn.

20. (a) at least one or each binding domain that binds to a protein other than FcRn is VHH; and / or (b) At least one or each binding domain that binds to a protein other than FcRn includes a heavy chain variable region and a light chain variable region. The polypeptide according to claim 19.

21. The polypeptide according to claim 19, wherein at least one or each binding domain that binds to a protein other than FcRn is a binding domain for a therapeutic antibody, and the therapeutic antibody is useful for treating a disease or disorder selected from autoimmune diseases or autoimmune disorders, inflammatory diseases or inflammatory disorders, infections, and cancer.

22. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 21 and a pharmaceutically acceptable carrier.

23. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 21, or a vector comprising the isolated nucleic acid.

24. A host cell comprising the nucleic acid or vector according to claim 23.

25. A host cell expressing the polypeptide according to any one of claims 1 to 21.

26. A method for producing a polypeptide according to any one of claims 1 to 21, comprising incubating a host cell according to claim 24 or 25 under conditions suitable for the expression of the polypeptide.

27. The method according to claim 26, further comprising isolating the antibody or the polypeptide.

28. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 21 for treating a disease or disorder in a subject.

29. The pharmaceutical composition according to claim 28, wherein the disease or disorder is selected from autoimmune diseases or autoimmune disorders, inflammatory diseases or inflammatory disorders, infectious diseases, and cancer.

30. The pharmaceutical composition according to claim 28, wherein the disease or disorder is pemphigus vulgaris, lupus nephritis, myasthenia gravis, Guillain-Barré syndrome, antibody-mediated rejection, antiphospholipid syndrome, chronic inflammatory demyelinating polyneuropathy, immune complex-mediated vasculitis, glomerulitis, channel disease, neuromyelitis optica, autoimmune encephalitis, autoimmune Graves' disease, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, immune neutropenia, dilated cardiomyopathy, or serum sickness.

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