FcRn-binding polypeptides and uses thereof

Abstract

The present invention provides VHH-containing polypeptides that bind to FcRn. Also provided are uses of the VHH-containing polypeptides.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 351,363, filed on June 11, 2022, and U.S. Provisional Application No. 63 / 437,776, filed on January 9, 2023; each of these applications is incorporated herein by reference in its entirety for any purpose. Incorporation by Reference into the Sequence Listing

[0002] This application incorporates by reference the sequence listing in electronic format filed with this application, named 01202-0057-00PCT_Sequence_Listing, created on June 8, 2023, and 54032 bytes in size. Technical Field

[0003] The present invention relates to FcRn-binding polypeptides, including FcRn-binding polypeptides and albumin-binding polypeptides, and methods of using the polypeptides, for example, to treat immune diseases or disorders. Background Art

[0004] Plasma proteins are cleared from the circulation by two major mechanisms: renal filtration of molecules below 60 kDa and micropinocytosis of endothelial cells. Proteins below the renal threshold are rapidly cleared from the circulation with a half-life of 1 day or less, while proteins above the renal threshold are cleared primarily by micropinocytosis with a half-life of about 3 to 5 days. Albumin and immunoglobulin G (IgG) are proteins with longer plasma half-lives, approximately 15 to 30 days, due to their larger size (66 and 150 kDa, respectively) and their ability to be recovered from endothelial micropinocytosis by pH-dependent binding to the neonatal Fc receptor (FcRn).

[0005] Longer plasma half-lives are useful for therapeutic agents to accurately and 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-life. Summary of the invention Embodiment 1. A polypeptide comprising at least one FcRn-binding VHH domain, wherein the at least one FcRn-binding VHH domain comprises a CDR1 sequence selected from SEQ ID NOs: 80-81, a CDR2 sequence selected from SEQ ID NOs: 83-84, and a CDR3 sequence of SEQ ID NO: 85. Embodiment 2. The polypeptide of embodiment 1, wherein each VHH domain that binds to FcRn independently comprises a CDR1 sequence selected from SEQ ID NOs: 80-81, a CDR2 sequence selected from SEQ ID NOs: 83-84, and a CDR3 sequence of SEQ ID NO: 85. Embodiment 3. The polypeptide of embodiment 1 or embodiment 2, wherein at least one FcRn-binding VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 80, 83, and 85; SEQ ID NOs: 81, 83, and 85; and SEQ ID NOs: 81, 84, and 85. Embodiment 4. The polypeptide of embodiment 3, wherein each VHH domain that binds to FcRn independently comprises a CDR1, CDR2, and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 80, 83, and 85; SEQ ID NOs: 81, 83, and 85; and SEQ ID NOs: 81, 84, and 85. Embodiment 5. The polypeptide of any one of embodiments 1 to 4, wherein at least one FcRn-binding VHH domain is humanized. Embodiment 6. The polypeptide of embodiment 5, wherein each VHH domain that binds to FcRn is humanized. Embodiment 7. A polypeptide according to any one of embodiments 1 to 6, wherein at least one FcRn-binding VHH domain 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: 86-93. Embodiment 8. A polypeptide as described in 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%, at least 99% identical to a sequence selected from SEQ ID NOs: 86-93. Embodiment 9. The polypeptide of any one of embodiments 1 to 7, wherein at least one FcRn-binding VHH domain comprises a sequence selected from the group consisting of SEQ ID NOs: 86-93. Embodiment 10. The polypeptide of any one of embodiments 1 to 9, wherein each VHH domain that binds to FcRn comprises a sequence selected from SEQ ID NOs: 86-93. Embodiment 11. The polypeptide of any one of embodiments 1 to 10, wherein at least one FcRn-binding VHH domain binds human FcRn with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM. Embodiment 12. The polypeptide of any one of embodiments 1 to 11, wherein each VHH domain that binds FcRn binds human FcRn with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM. Embodiment 13. The polypeptide of any one of embodiments 1 to 12, wherein at least one FcRn-binding VHH domain binds human FcRn at pH 6 and pH 7.4. Embodiment 14. The polypeptide of any one of embodiments 1 to 13, wherein each VHH domain that binds FcRn binds human FcRn at pH 6 and pH 7.4. Embodiment 15. The polypeptide of any one of embodiments 1 to 14, wherein at least one FcRn-binding VHH domain blocks the binding of human IgG to human FcRn. Embodiment 16. The polypeptide of any one of embodiments 1 to 15, wherein each VHH domain that binds to FcRn blocks the binding of human IgG to human FcRn. Embodiment 17. The polypeptide of any one of embodiments 1 to 16, wherein the polypeptide comprises at least one VHH domain that binds albumin. Embodiment 18. The polypeptide of embodiment 17, wherein at least one albumin-binding VHH domain comprises a CDR1 sequence selected from the group consisting of SEQ ID NOs: 5-8, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 9-21, and a CDR3 sequence of SEQ ID NO: 22. Embodiment 19. The polypeptide of embodiment 17, wherein each VHH domain that binds to albumin independently comprises a CDR1 sequence selected from SEQ ID NOs: 5-8, a CDR2 sequence selected from SEQ ID NOs: 9-21, and a CDR3 sequence of SEQ ID NO: 22. Embodiment 20. A polypeptide according to embodiment 17 or embodiment 18, wherein at least one albumin binding VHH domain comprises a CDR1, CDR2 and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 5, 9 and 22; SEQ ID NOs: 5, 10 and 22; SEQ ID NOs: 5, 11 and 22; SEQ ID NOs: 5, 12 and 22; SEQ ID NOs: 5, 13 and 22; SEQ ID NOs: 5, 14 and 22; SEQ ID NOs: 5, 15 and 22; SEQ ID NOs: 6, 15 and 22; SEQ ID NOs: 7, 15 and 22; SEQ ID NOs: 8, 15 and 22; SEQ ID NOs: 6, 16 and 22; SEQ ID NOs: 6, 17 and 22; SEQ ID NOs: 6, 18 and 22; SEQ ID NOs: 6, 19 and 22; SEQ ID NOs: 6, 20 and 22; and SEQ ID NOs: 6, 21 and 22. Embodiment 21. A polypeptide according to embodiment 17 or embodiment 18, wherein each VHH domain that binds to albumin independently comprises a CDR1, CDR2 and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 5, 9 and 22; SEQ ID NOs: 5, 10 and 22; SEQ ID NOs: 5, 11 and 22; SEQ ID NOs: 5, 12 and 22; SEQ ID NOs: 5, 13 and 22; SEQ ID NOs: 5, 14 and 22; SEQ ID NOs: 5, 15 and 22; SEQ ID NOs: 6, 15 and 22; SEQ ID NOs: 7, 15 and 22; SEQ ID NOs: 8, 15 and 22; SEQ ID NOs: 6, 16 and 22; SEQ ID NOs: 6, 17 and 22; SEQ ID NOs: 6, 18 and 22; SEQ ID NOs: 6, 19 and 22; SEQ ID NOs: 6, 20 and 22; and SEQ ID NOs: 6, 21 and 22. Embodiment 22. The polypeptide of any one of embodiments 17 to 21, wherein at least one albumin binding VHH domain is humanized. Embodiment 23. A polypeptide according to embodiment 22, wherein each VHH domain that binds to albumin is humanized. Embodiment 24. A polypeptide according to any one of embodiments 17 to 23, wherein at least one albumin-binding VHH domain 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: 23-43 and 97-100. Embodiment 25. A polypeptide as described in any one of embodiments 17 to 23, wherein each VHH domain that binds to albumin 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: 23-43 and 97-100. Embodiment 26. The polypeptide of any one of embodiments 17 to 23, wherein at least one albumin binding VHH domain comprises a sequence selected from the group consisting of SEQ ID NOs: 23-43 and 97-100. Embodiment 27. The polypeptide of any one of embodiments 17 to 23, wherein each VHH domain that binds to albumin comprises a sequence selected from the group consisting of SEQ ID NOs: 23-43 and 97-100. Embodiment 28. A polypeptide as described in any one of embodiments 17 to 26, wherein the polypeptide 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: 69-77. Embodiment 29. The polypeptide of any one of embodiments 17 to 26, wherein the polypeptide comprises a sequence selected from SEQ ID NO: 69-77. Embodiment 30. The polypeptide of any one of embodiments 17 to 29, wherein at least one albumin binding VHH domain binds to human albumin and at least one albumin selected from cynomolgus monkey, mouse and rat albumin. Embodiment 31. The polypeptide of any one of embodiments 17 to 29, wherein each albumin-binding VHH domain binds to human albumin and at least one albumin selected from cynomolgus monkey, mouse and rat albumin. Embodiment 32. The polypeptide of any one of embodiments 17 to 29, wherein at least one albumin binding VHH domain binds human, cynomolgus monkey, mouse and rat albumin. Embodiment 33. The polypeptide of any one of embodiments 17 to 29, wherein each VHH domain that binds to albumin binds to human, cynomolgus monkey, mouse and rat albumin. Embodiment 34. The polypeptide of any one of embodiments 17 to 33, wherein at least one albumin binding VHH domain binds human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM. Embodiment 35. The polypeptide of any one of embodiments 17 to 34, wherein at least one albumin-binding VHH domain binds to each of human, cynomolgus monkey, mouse and rat albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM. Embodiment 36. The polypeptide of any one of embodiments 17 to 35, wherein each VHH domain that binds albumin binds human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM. Embodiment 37. The polypeptide of any one of embodiments 17 to 36, wherein each VHH domain that binds albumin binds to each of human, cynomolgus monkey, mouse and rat albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM. Embodiment 38. The polypeptide of any one of embodiments 17 to 37, wherein each VHH domain that binds to albumin does not bind to albumin domain 3. Embodiment 39. The polypeptide of 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. Embodiment 40. The polypeptide according to any of the preceding embodiments, wherein the polypeptide comprises at least two, at least three or at least four FcRn binding VHH domains and at least one albumin binding VHH domain. Embodiment 41. The polypeptide of embodiment 40, wherein the polypeptide comprises two, three or four FcRn binding VHH domains and one albumin binding VHH domain. Embodiment 42. The polypeptide of any one of embodiments 1 to 41, wherein the polypeptide comprises at least one binding domain that binds a protein other than albumin or FcRn. Embodiment 43. A polypeptide according to embodiment 42, wherein at least one binding domain that binds a protein other than albumin or FcRn is a VHH. Embodiment 44. A polypeptide according to embodiment 43, wherein each binding domain that binds a protein other than albumin or FcRn is a VHH. Embodiment 45. The polypeptide of embodiment 42 or embodiment 43, wherein at least one binding domain that binds a protein other than albumin or FcRn comprises a heavy chain variable region and a light chain variable region. Embodiment 46. A polypeptide according to embodiment 45, wherein each binding domain that binds a protein other than albumin or FcRn comprises a heavy chain variable region and a light chain variable region. Embodiment 47. The polypeptide of any one of embodiments 42 to 46, wherein at least one binding domain that binds a protein other than albumin or FcRn is a binding domain of a therapeutic antibody. Embodiment 48. A polypeptide according to embodiment 47, wherein each binding domain that binds a protein other than albumin is a binding domain of a therapeutic antibody. Embodiment 49. The polypeptide of embodiment 47 or embodiment 48, wherein the therapeutic antibody is useful for treating a disease or disorder selected from the group consisting of an autoimmune disease or disorder, an inflammatory disease or disorder, an infection, and cancer. Embodiment 50. The polypeptide of any one of embodiments 1 to 41, wherein the polypeptide comprises the amino acid sequence of a therapeutic protein. Embodiment 51. The polypeptide of embodiment 50, wherein the therapeutic protein is useful for treating a disease or condition selected from the group consisting of an autoimmune disease or condition, an inflammatory disease or condition, an infection, and cancer. Embodiment 52. The polypeptide according to any of the preceding embodiments, wherein the half-life of the polypeptide is longer than the half-life of the same polypeptide lacking the albumin binding VHH domain. Embodiment 53. A pharmaceutical composition comprising the polypeptide of any one of embodiments 1 to 52 and a pharmaceutically acceptable carrier. Embodiment 54. An isolated nucleic acid encoding the polypeptide of any one of embodiments 1 to 52. Embodiment 55. A vector comprising the nucleic acid of embodiment 54. Embodiment 56. A host cell comprising the nucleic acid of embodiment 54 or the vector of embodiment 55. Embodiment 57. A host cell expressing the polypeptide of any one of embodiments 1 to 52. Embodiment 58. A method of producing a polypeptide according to any one of embodiments 1 to 52, comprising cultivating a host cell according to embodiment 56 or embodiment 57 under conditions suitable for expressing the polypeptide. Embodiment 59. The method of embodiment 58, further comprising isolating the antibody or polypeptide. Embodiment 60. A method comprising administering a polypeptide according to any one of embodiments 1 to 52 or a pharmaceutical composition according to embodiment 53 to a subject. Embodiment 61. A method of treating a disease or condition, comprising administering a pharmaceutically effective amount of a polypeptide according to any one of embodiments 1 to 52 or a pharmaceutical composition according to embodiment 53 to a subject suffering from the disease or condition. Embodiment 62. The method of embodiment 61, wherein the disease or disorder is selected from the group consisting of an autoimmune disease or disorder, an inflammatory disease or disorder, an infection, and cancer. Embodiment 63. The method of embodiment 61 or embodiment 62, wherein the disease or disorder is an autoantibody-mediated disease or disorder. Embodiment 64. The method of 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, glomerulitis, ion channelopathy, neuromyelitis optica, autoimmune encephalitis, autoimmune Grave's disease, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, immune neutropenia, dilated cardiomyopathy, or serum sickness. Embodiment 65. The method of any one of embodiments 60 to 64, wherein the polypeptide is administered subcutaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1A to FIG. 1I . Figure 1A An alignment of certain 4A01 humanized variants (SEQ ID NOs: 24-43) is shown. Figure 1B to Figure 1C Human albumin (HSA) binding ELISA showing some of the 4A01 humanized variants. Figure 1D to Figure 1E Cynomolgus monkey albumin (CSA) binding ELISA showing some 4A01 humanized variants. Figure 1F to Figure 1G Murine albumin (MSA) binding ELISA showing some of the 4A01 humanized variants. Figures 1H to 1I Rat albumin (RSA) binding ELISA showing some of the 4A01 humanized variants.

[0008] FIG. 2A to FIG. 2D Human albumin (HSA; hz4A01v51) displayed Figure 2A ), cynomolgus monkey albumin (CSA; Figure 2B ), murine albumin (MSA; Figure 2C ) and rat albumin (RSA; Figure 2D ) combined with ELISA.

[0009] FIG. 3A to FIG. 3B . Figure 3A Schematic diagram showing the experimental design for biolayer interferometry assessing binding to albumin domain 3. Figure 3B Biofilm interferometry traces are shown showing that 4A01-NNT-hFc (SEQ ID NOs: 23 and 68) does not bind to albumin domain 3, while 1C04 binds to albumin domain 3.

[0010] FIG. 4A to FIG. 4B . Figure 4ASchematic diagram showing the experimental design of biomembrane interferometry used to assess B2M-FcRn binding to albumin. Figure 4B is a biofilm interferometry trace showing that 4A01-NNT-hFc (SEQ ID NOs: 23 and 68) and humanized hz4A01v51-NNT-hFc (SEQ ID NO: 102) do not compete with albumin for binding to B2m-FcRn, whereas 1C04 does.

[0011] Figure 5 Certain exemplary formats tested in binding and functional assays are shown. Formats (i) and (iii) are bivalent bispecific polypeptides targeting FcRn (black) and albumin (grey). Format (ii) is a trivalent bispecific polypeptide consisting of two units targeting FcRn and one unit targeting albumin. In formats (i) and (ii) the binding units are linked via a glycine-serine linker, and in format (iii) the binding units are directly linked.

[0012] FIG. 6A to FIG. 6G Multivalent, multispecific polypeptides targeting FcRn and albumin are shown to bind to cell subsets in FcRn-transfected cHO cells and human peripheral blood cells (PBMCs). Fig. 6A and Figure 6B Demonstrated binding to transiently transfected cHO cells at pH 7.4, Figure 6C Demonstration of binding to FcRn at pH 7.4 using ELISA, Fig.6D and 6E Binding to transiently transfected cHO cells at pH 6 is demonstrated. Fig. 6F and Figure 6G Binding to human PBMCs at pH 7.4 and pH 6 is shown, respectively. Fig. 6A , Fig.6D , Fig. 6F and Figure 6G , peptides such as Figure 5 (i) is described in the formalization, for Figure 6B and Fig. 6E , peptides such as Figure 5 (i) formatted as described above, except for cx12032, which is as follows Figure 5 (iii) is described in the specification. Figure 6C , cx11383 Figure 5 (i) and cx11385 as described in Figure 5 (ii) as described above.

[0013] FIG. 7A to FIG. 7DDemonstration of binding of multivalent, multispecific polypeptides targeting FcRn and albumin to FcRn and human albumin using ELISA. Fig. 7A and Figure 7B exhibited binding at pH 7.4, while Figure 7C and Fig.7D Binding is shown at pH 5.5. The constructs in FIG. 7 are Figure 5 (i) as described above, except that cx12032 is Figure 5 (Stylized as described in (iii).

[0014] Figure 8 The multivalent, multispecific polypeptides targeting FcRn and albumin were shown to have a dose-dependent ability to prevent IgG binding to FcRn-transfected 293 cells at pH 6. Figure 5 (i) described in (cx11383) and Figure 5 (ii) as described in (cx11385).

[0015] 9A to 9D The pharmacokinetic (PK) profiles and in vivo clearance of IgG of multivalent, multispecific polypeptides targeting FcRn and albumin are presented in comparison to an in-house generated sequence analog of Nipocalimab. Fig. 9A , Fig. 9B and Fig. 9C Serum PK curves of BALB / c mice are shown ( Fig. 9A and Fig. 9B ) or human FcRn / albumin transgenic C57BL / 6 mice ( Fig. 9C ). Fig.9D Shown are serum levels of human IgG administered to transgenic mouse strains after administration of FcRn blocking test articles. Figure 5 (i) described in (cx11558 and cx11383) and Figure 5 (ii) as described (cx11642 and cx11385).

[0016] FIG. 10A to FIG. 10C In vivo study results are shown in cynomolgus monkeys administered 100 mg / kg and 20 mg / kg of the FcRn blocking test articles cx11558 or cx11642 or 20 mg / kg of the recombinant engineered IgG Fc fragment efgartigimod. Fig. 10A Serum levels of cynomolgus monkey IgG are shown, plotted as % change from baseline (indicated by the dotted line). Fig. 10B Display receptor occupancy. Fig. 10CSerum levels of cx11558, cx11642 and igamod are shown (the lower limit of quantitation for each test article is indicated by a dotted line).

[0017] FIG. 11A to FIG. 11C In vivo results are shown following administration of 100 mg / kg or 20 mg / kg IV or 100 mg / kg SC of the multispecific peptide cx12007 targeting FcRn and albumin in cynomolgus monkeys. Fig.11A Serum levels of cynomolgus monkey IgG are shown, plotted as % change from baseline (indicated by the dotted line). Fig. 11B Display receptor occupancy. Fig. 11C Serum levels of cx12007 are shown (lower limit of quantitation indicated by dotted line).

[0018] FIG. 12A to FIG. 12B In vivo results of a multispecific peptide cx12007 targeting FcRn and albumin were presented in cynomolgus monkeys after administration of 20 mg / kg, 40 mg / kg or 70 mg / kg SC. Fig. 12A Serum levels of cynomolgus monkey IgG are shown, plotted as % change from baseline (indicated by the dotted line). Fig. 12B Serum levels of cx12007 are shown (lower limit of quantitation indicated by dotted line). DETAILED DESCRIPTION

[0019] Embodiments provided herein relate to FcRn-binding polypeptides, including FcRn-binding polypeptides and albumin-binding polypeptides, and uses thereof. Definition and various implementations

[0020] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0021] All references cited herein, including patent applications, patent publications, and Genbank Accession Numbers, are incorporated by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0022] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are routinely employed using conventional methods, such as the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (2003)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology (Academic Press, Inc.); Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, 1998) Academic Press; Animal Cell Culture (RI Freshney), 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) Plenum Press; Cell and TissueCulture Laboratory Procedures (edited by A.Doyle, JBGriffiths and DG Newell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (edited by DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wileyand Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (edited by D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, 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 JD Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., JS Lippincott Company, 1993); and updated versions thereof.

[0023] Unless otherwise defined, scientific and technical terms used in conjunction with the present invention will have the meanings generally understood by those skilled in the art. In addition, unless the context otherwise requires or otherwise clearly indicated, singular terms shall include the plural and plural terms shall include the singular. With respect to any conflicts in definitions between various sources or references, the definitions provided herein shall prevail.

[0024] In general, the residue numbering in immunoglobulin heavy chains is that of the EU index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0025] It should be understood that the embodiments of the invention described herein include "consisting of" and / or "consisting essentially of". As used herein, the singular forms "a", "an", and "the" include plural references unless otherwise indicated. The use of the term "or" herein is not meant to imply that alternatives are mutually exclusive.

[0026] In this application, unless explicitly stated or understood by one skilled in the art, the use of "or" means "and / or." In the context of multiple dependent items, the use of "or" refers back to more than one of the preceding independent items or dependent items.

[0027] The phrase "reference sample", "reference cell" or "reference tissue" means a sample with at least one known characteristic that can be used as a comparison with a sample with at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. A reference sample can be used to determine, for example, the amount of protein and / or mRNA present in a healthy tissue, in contrast to the amount of protein and / or mRNA present in a sample with an unknown characteristic. In some embodiments, the reference sample is from the same subject, but from a different part of the subject than the test sample. In some embodiments, the reference sample is from a tissue region around or near the cancer. In some embodiments, the reference sample is not from the subject being tested, but from a sample of a subject known to have or not have the disorder being discussed. In some embodiments, the reference sample is from the same subject, but from a time point before the subject suffers from cancer. In some embodiments, the reference sample is from a benign cancer sample from the same or different subject. When using a negative reference sample for comparison, the expression or amount of the molecule discussed in the negative reference sample will indicate the amount that a person skilled in the art will understand when assuming that there is no molecule and / or a molecule with a low content in the present invention. When a positive reference sample is used for comparison, the expression level or amount of the molecule in question in the positive reference sample will indicate the amount that one skilled in the art would understand if a certain amount of the molecule in the present invention were present.

[0028] The terms "benefit", "clinical benefit", "response" and "therapeutic response" as used herein in the context of benefiting from or responding to the administration of a therapeutic agent can be measured by assessing various evaluation indicators, such as inhibiting disease progression to some extent, including slowing and complete arrest; reducing the number of disease attacks and / or symptoms; reducing the size of lesions; inhibiting (i.e., reducing, slowing or completely stopping) disease cell infiltration to adjacent peripheral organs and / or tissues; inhibiting (i.e., reducing, slowing or completely stopping) disease spread; alleviating to some extent one or more symptoms associated with the disorder; increasing the length of time that the patient is disease-free (e.g., progression-free survival) after treatment; increasing overall survival; improving the response rate; and / or reducing the mortality rate at a given time point after treatment. "Non-responsive" or "unresponsive" subjects or cancers are subjects or cancers that fail to meet the above-mentioned restrictions on "response".

[0029] The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably and refer to nucleotide polymers. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0030] 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 may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers composed of amino acid residues. Full-length proteins and fragments thereof are encompassed in this definition. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation and similar modifications. In addition, for the purposes of the present invention, "polypeptide" refers to a protein, which includes modifications to the native sequence, such as deletions, additions and substitutions (which are usually conservative in practice), as long as the protein maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis; or may be accidental, such as by mutations in the host producing the protein or by errors caused by PCR amplification.

[0031] As used herein, "albumin" refers to any naturally occurring mature albumin produced by processing albumin precursors in cells. Unless otherwise specified, the term includes albumin from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring variants of albumin, such as splice variants or allelic variants. A non-limiting exemplary human albumin amino acid sequence is shown, for example, in UniProt Accession No. P02768.2. See SEQ ID NO.1. Non-limiting exemplary murine, cynomolgus and rat albumin amino acid sequences are shown in SEQ ID NOs: 2-4.

[0032] As used herein, "FcRn" refers to any naturally occurring mature Fc receptor produced by processing FcRn precursors in cells. Unless otherwise specified, the term includes FcRn from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring FcRn variants, such as splice variants or allelic variants. Non-limiting exemplary human FcRn amino acid sequences are shown, for example, at UniProt accession number P55899. See SEQ ID NO.92. Non-limiting exemplary murine, cynomolgus and rat FcRn amino acid sequences are shown in SEQ ID NOs: 93-95.

[0033] The term "specifically binds" an antigen or epitope is a well-understood term 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" if it reacts or binds to a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than it does to alternative cells or substances. A single domain antibody (sdAb) or VHH-containing polypeptide is said to "specifically bind" or "preferentially bind" a target if it binds to it with greater affinity, greater avidity, more readily, and / or for a longer duration than it binds 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 greater affinity, avidity, more readily, and / or for a longer duration than it binds to other FcRn epitopes or non-FcRn epitopes. By reading this definition, it should also be 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. Reference to binding generally but not necessarily means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0034] As used herein, the term "inhibit" with respect to target protein activity refers to a decrease in protein activity. In some embodiments, "inhibit" refers to a decrease in activity compared to the protein in the absence of a modulator.

[0035] As used herein, the term "epitope" refers to the position to which an antigen binding molecule (e.g., sdAb or VHH-containing polypeptide) binds on a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate, or lipid). Epitopes often include a group of molecules with chemical activity on the surface, such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural characteristics and charge-to-mass ratio characteristics. Epitopes can be formed by adjacent and / or juxtaposed non-adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed by adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. Epitopes may include, 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 length of the epitope is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen. In some embodiments, epitopes can be identified according to a certain minimum distance relative to the CDR residues on the antigen binding molecules. In some embodiments, epitopes can be identified according to the above distance, and are further limited to those residues involved in the bond (such as hydrogen bond) between the residues of the antigen binding molecules and the antigen residues. The epitope can also be identified by various scans, such as alanine or arginine scanning can indicate one or more residues that can interact with the antigen binding molecules. Unless explicitly stated, a group of residues as an epitope does not exclude other residues as part of the epitope of a specific antigen binding molecule. In fact, the presence of this group of epitopes represents the smallest epitope series (or species group). Therefore, in some embodiments, the residue set identified as an epitope represents the minimum relevant epitope of the antigen, rather than the exclusive residue list of the epitope on the non-antigen.

[0036] The term "antibody" is used in the broadest sense and encompasses various polypeptides comprising antibody-like antigen binding domains, including but not limited to conventional antibodies (usually comprising at least one heavy chain and at least one light chain), single domain antibodies (sdAbs, which comprise at least one VHH domain and optionally a selected Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and fragments of any of the foregoing, as long as they exhibit the desired antigen binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include but are not limited to heavy chain constant domains (comprising CH1, hinge, CH2, and CH3, wherein CH1 is usually paired with a light chain constant domain CL, and the hinge mediates dimerization) and Fc regions (comprising hinge, CH2, and CH3, wherein the hinge mediates dimerization).

[0037] 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, and the like.

[0038] As used herein, the term "antigen binding domain" refers to a portion of an antibody sufficient to bind to an antigen. In some embodiments, the antigen binding domain of a conventional antibody comprises three heavy chain CDRs and three light chain CDRs. Therefore, 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 an 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 an antigen. In some embodiments, an sdAb or an antigen binding domain containing a VHH polypeptide comprises three CDRs of a VHH domain. Therefore, in some embodiments, an sdAb or an antigen binding domain containing a VHH polypeptide comprises a VHH domain, which comprises CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to an antigen.

[0039] 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 a shark antibody. In some embodiments, a VHH comprises three CDRs and four framework regions, denoted FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N-terminus or C-terminus so that it comprises only part of FR1 and / or FR4, or lacks one or both of those framework regions, as long as the VHH substantially maintains antigen binding and specificity.

[0040] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody comprising at least one light chain-free monomer domain such as a VHH domain and an optionally selected Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each of which comprises 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, such as polypeptides having the structures VHH1-VHH2, VHH1-VHH2-Fc, VHH1-VHH2-VHH3, or VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different.

[0041] 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, wherein 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. In addition, in some such embodiments, the VHH polypeptide may form a dimer. The VHH-containing polypeptide, also an sdAb, has non-limiting structures including VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of such structures, one VHH may be connected to another VHH via a linker, or one VHH may be connected to Fc via a linker. In some such embodiments, the linker comprises 1 to 20 amino acids, preferably 1 to 20 amino acids mainly comprising glycine and optionally comprising serine. In some embodiments, when the VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, if the structure VHH1-VHH2-Fc forms a dimer, it is considered to be tetravalent (i.e., the dimer has four VHH domains). Similarly, if the structure VHH1-VHH2-VHH3-Fc forms a dimer, it is considered to be hexavalent (i.e., the dimer has six VHH domains).

[0042] The term "monoclonal antibody" refers to an antibody (including sdAb or VHH polypeptide) of a substantially homogeneous antibody group, that is, the individual antibodies comprising this group are identical except for the possible presence of a small amount of naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Therefore, a sample of a monoclonal antibody can bind to the same epitope on the antigen. The modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a substantially homogeneous antibody group, and should not be understood as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be produced by the fusion tumor method first described by Kohler and Milstein, 1975, Nature 256:495, or can be produced by a recombinant DNA method such as described in U.S. Patent No. 4,816,567. For example, monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554.

[0043] The term "CDR" means complementarity determining region, which is defined by one skilled in the art by at least one identification method. In some embodiments, CDRs may be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. VHHs comprise three CDRs, referred to as CDR1, CDR2, and CDR3.

[0044] As used herein, the term "heavy chain constant region" refers to a region comprising at least three heavy chain constant domains C H 1. Hinge, C H 2 and C H3. Of course, unless otherwise indicated, non-functional altering deletions and modifications in these domains are included within the scope of the term "heavy chain constant region". 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 an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an α constant region is an IgA antibody. In addition, an antibody comprising a μ constant region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Specific isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising a γ1 constant region), IgG2 (comprising a γ2 constant region), IgG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0045] As used herein, "Fc region" refers to the 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 any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0046] As discussed herein, an "acceptor human framework" as used herein is a framework comprising an amino acid sequence of a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human common framework. An acceptor human framework derived from a human immunoglobulin framework or a human common framework may comprise the same amino acid sequence as that thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes in all human frameworks of a single antigen binding domain such as a VHH 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.

[0047] "Affinity" refers to the strength of the sum 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 can generally be expressed by the dissociation constant (K) and the affinity of a molecule X for its partner Y, respectively. D ) or K D-表观 Affinity can be measured by common methods known in the art (such as ELISA K D , KinExA, flow cytometry and / or surface plasmon resonance devices), including those described herein. These methods include but are not limited to those involving or flow cytometry methods.

[0048] As used herein, the term "K D ” refers to the equilibrium dissociation constant of the antigen binding molecule / antigen interaction. When the term “K D ", it includes K D and K D-表观 .

[0049] In some embodiments, the K of the antigen binding molecule is D The mean fluorescence measured at each antibody concentration is measured by flow cytometry using a cell line expressing the antigen and fitting the nonlinear single-site binding equation (Prism Software graphpad). In some such embodiments, K D K D-表观 .

[0050] The term "biological activity" refers to any one or more biological properties of a molecule (whether found naturally in vivo or provided or achieved by recombinant means). Biological properties include, but are not limited to, binding to a ligand, inducing or increasing cell proliferation, and inducing or increasing cytokine expression.

[0051] "Agonist" or "activating" antibodies are antibodies that increase and / or activate 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.

[0052] "Antagonist", "blocking" or "neutralizing" antibodies are antibodies that inhibit, reduce and / or do not activate 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.

[0053] An "affinity matured" sdAb or VHH-containing polypeptide refers to a sdAb or VHH-containing polypeptide having one or more alterations in one or more CDRs that improve the affinity of the sdAb or VHH-containing polypeptide for an antigen, compared to a parent sdAb or VHH-containing polypeptide that does not have such alterations.

[0054] As used herein, "humanized VHH" refers to a VHH in which one or more framework regions have been substantially replaced by human framework regions. In some cases, certain framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized VHHs may include residues that are neither present in the original VHH nor in the human framework sequence, but include such residues to further improve and optimize the performance of sdAb or VHH-containing polypeptides. In some embodiments, humanized sdAbs or VHH-containing polypeptides include human Fc regions. It will be appreciated that humanized sequences can be identified by their primary sequence and do not necessarily represent the process of producing antibodies.

[0055] 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, etc. Such effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays.

[0056] A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0057] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification. In some embodiments, a "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or compared to the Fc region of a parent polypeptide, for example, about one to about ten amino acid substitutions in the native sequence Fc region or in the Fc region of a parent polypeptide, and preferably about one to about five amino acid substitutions. In some embodiments, the variant Fc region herein has at least about 80% sequence identity with the native sequence Fc region and / or the Fc region of a parent polypeptide, has at least about 90% sequence identity therewith, has at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith.

[0058] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FcγR is a native human FcR. In some embodiments, an FcR is an FcR (gamma receptor) that binds to an IgG antibody and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allele variants and alternative splicing forms of those receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), both of which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain, and the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are reviewed in, e.g., 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). The term "FcR" herein encompasses other FcRs including FcRs to be identified in the future. For example, the term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for 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 regulates the homeostasis of immunoglobulins. 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.)).

[0059] As used herein, the term "substantially similar" or "substantially the same" means that the similarity between two or more values ​​is high enough so that, in the case of a biological characteristic measured by the value, a person skilled in the art would consider the difference between the two or more values ​​to be minimal or not biologically and / or statistically significant. In some embodiments, two or more substantially similar values ​​differ by no more than about any of the following: 5%, 10%, 15%, 20%, 25%, or 50%.

[0060] A polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence identity percentage, and not 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 a polypeptide. In some embodiments, the variant will have at least about 80% amino acid sequence identity. In some embodiments, the variant will have at least about 90% amino acid sequence identity. In some embodiments, the variant will have at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0061] As used herein, "percentage (%) of amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences 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, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved by various means within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. One skilled in the art can determine the parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0062] Amino acid substitutions may 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 may be introduced into the antibody of interest and the product may be screened for desired activity (e.g., maintained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC). Table 1

[0063] Amino acids can be grouped based on common side chain properties: (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0064] Non-conservative substitutions will result in exchanging a member of one of these classes for another class.

[0065] The term "vector" is used to describe a polynucleotide that can be engineered to contain one or more polynucleotides that can be propagated in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences that regulate the expression of a polypeptide of interest (such as a promoter and / or enhancer), and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0066] "Host cell" refers to a cell that can be or has been a recipient of a vector or 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 cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, Host cells include 293 and CHO cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutations. Host cells include cells transfected in vivo with the polynucleotides provided herein.

[0067] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components that are commonly found or produced in nature. For example, a polypeptide is said to be "isolated" when it is separated from at least some of the components of the cell that produced the polypeptide. If the polypeptide is secreted by a cell after expression, then physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be an "isolated" polypeptide. Similarly, a polynucleotide is said to be "isolated" when it is not part of a larger polynucleotide commonly found in nature (such as genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide), or is separated from at least some of the components of the cell that produced it (for example, in the case of an RNA polynucleotide). Thus, a DNA polynucleotide contained in a vector inside a host cell can be said to be "isolated."

[0068] The terms "individual" and "subject" are used interchangeably herein and refer to an animal, such as a mammal. In some embodiments, methods of treating mammals are provided, including, but not limited to, humans, rodents, monkeys, felines, canines, equines, bovines, porcines, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets. In some embodiments, an "individual" or "subject" refers to a subject in need of treatment for a disease or disorder. In some embodiments, the subject receiving treatment may be a patient, indicating the fact that the subject has been identified as having a disorder associated with the treatment, or is at great risk of contracting the disorder.

[0069] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0070] The term "autoimmune disorder" refers to a disease or disorder generally associated with non-allergic hypersensitivity reactions (Type II, III and / or IV hypersensitivity reactions), which are generally caused by the individual's own humoral and / or cell-mediated immune response to one or more immunogenic substances of endogenous or exogenous origin.

[0071] The term "inflammatory disorder" refers to a disorder associated with inflammation, including but not limited to chronic or acute inflammatory diseases, and specifically includes inflammatory autoimmune diseases and inflammatory allergic conditions.

[0072] The terms "infection" and "infectious disease or condition" refer to a disease or condition caused by an exogenous infectious agent such as, but not limited to, bacteria, viruses, fungi, protozoa, and parasites.

[0073] The terms "cancer" and "tumor" encompass both solid cancers and blood / lymphatic cancers, and also encompass malignant, precancerous, and benign growths, such as dysplasia. 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 and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; hepatocarcinoma; liver cancer; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; Colon cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; lymphoma, including Hodgkin's and non-Hodgkin's lymphomas and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-lytic cell NHL; mass-lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia (Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and other carcinomas and sarcomas; and post-transplantation lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation with macular (cell) disease, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0074] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. As will be apparent to those skilled in the art, "modulation" may also include changes (which may be increases or decreases) in affinity, avidity, specificity and / or selectivity for a ligand, binding partner, one or more of the partners binding to a homopolymer or heteropolymer form or substrate of a target or antigen, compared to the same conditions but without the test agent; changes (which may be increases or decreases) in 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 cell proliferation or cytokine production. Depending on the target involved, this may be determined by any suitable means and / or using any suitable assay known per se or described herein.

[0075] As used herein, "treatment" is a method for obtaining a favorable or desired clinical outcome. "Treatment" as used herein encompasses any administration or application of a therapeutic regimen for a disease in a mammal, including humans. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, any one or more of the following: alleviating one or more symptoms, reducing the extent of the disease, preventing or delaying disease spread (e.g., metastasis, such as metastasis to the lungs or lymph nodes), preventing or delaying disease recurrence, delaying or slowing disease progression, improving disease state, inhibiting disease or disease progression, inhibiting or slowing disease or its progression, blocking its development and alleviating (whether partially or completely). "Treatment" also encompasses mitigation of the pathological consequences of proliferative diseases. The methods provided herein encompass any one or more of these treatment aspects. According to the above, the term treatment does not require 100% removal of all aspects of the disease.

[0076] "Relieve" means that one or more symptoms are alleviated or improved compared to when no therapeutic agent is administered. "Improvement" also includes shortening or reducing the duration of symptoms.

[0077] The term "anticancer agent" is used herein in its broadest sense to refer to an agent used to treat one or more cancers. Exemplary types of these agents include, but are not limited to, chemotherapeutic agents, anticancer biological agents (such as cytokines, receptor extracellular domain-Fc fusions and antibodies), radiation therapy, CAR-T therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNA) and oncolytic viruses.

[0078] The term "biological sample" means a quantity of a substance from a living or formerly living organism. Such substances include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0079] The term "control" or "reference" refers to a composition known to not contain the analyte (a "negative control") or a composition known to contain the analyte (a "positive control"). A positive control may comprise a known concentration of the analyte.

[0080] The term "inhibition" or "inhibit" refers to a reduction or cessation of any phenotypic characteristic, or a reduction or cessation of the incidence, degree or likelihood of the characteristic. "Reduction" or "inhibition" is to reduce, decrease or stagnate the activity, function and / or amount compared to a reference. In some embodiments, "reduction" or "inhibition" means the ability to reduce the overall by 10% or more. In some embodiments, "reduction" or "inhibition" means the ability to reduce the overall by 50% or more. In some embodiments, "reduction" or "inhibition" means the ability to reduce the overall by 75%, 85%, 90%, 95% or more. In some embodiments, the amounts mentioned above are inhibited or reduced over the same period of time relative to a control over a period of time.

[0081] As used herein, "delaying disease progression" means delaying, hindering, slowing, stifling, stabilizing, inhibiting and / or postponing the development of a disease (such as cancer). This delay can be of varying lengths of time, depending on the disease being treated and / or the individual's medical history. As will be apparent to one skilled in the art, a sufficient or significant delay can actually encompass prevention, such that the individual does not develop the disease. For example, the development of advanced cancers, such as cancer metastases, can be delayed.

[0082] As used herein, "prevention" includes providing a preventive effect on the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease. Unless otherwise specified, the terms "reducing," "inhibiting," or "preventing" do not imply or require complete prevention all the time, only over the measured period of time.

[0083] A "therapeutically effective amount" of a substance / molecule (agonist or antagonist) may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule (agonist or antagonist) to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the beneficial effects of the treatment outweigh any toxic or deleterious effects of the substance / molecule (agonist or antagonist). A therapeutically effective amount may be delivered via one or more administrations. A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic and / or preventive result at the necessary dosage and for the necessary time.

[0084] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation that is in a form that permits the biological activity of the active ingredient to be effective, and contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. The preparation may be sterile.

[0085] "Pharmaceutically acceptable carrier" refers to a conventional non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid or carrier in the art, which is used together with the therapeutic agent to constitute a "pharmaceutical composition" for administration to a subject. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosage and concentration used and are compatible with the other ingredients of the formulation. Pharmaceutically acceptable carriers are suitable for the formulation used.

[0086] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0087] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents wherein at least some of the administration overlaps in time, or wherein the administration of one therapeutic agent falls within a relatively short period of time relative to the administration of another therapeutic agent, or wherein the therapeutic effects of the two agents overlap for at least a period of time.

[0088] The term "sequentially" is used herein to refer to administration of two or more therapeutic agents that do not overlap in time, or where the therapeutic effects of the agents do not overlap.

[0089] As used herein, "in conjunction with" refers to administering another treatment modality in addition to another treatment modality. Thus, "in conjunction with" refers to administering another treatment modality before, during, or after one treatment modality is administered to a subject.

[0090] As used herein, the term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0091] An "article of manufacture" is any article (e.g., a package or container) or kit comprising at least one agent, such as a drug for treating a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In some embodiments, the article of manufacture or kit is marketed, distributed, or sold in unit form for performing the methods described herein.

[0092] The terms "label" and "detectable label" mean a moiety attached to, for example, an antibody or antigen so that a reaction (e.g., binding) between members of a specific binding pair can be detected. A labeled member of a specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein into which a label is incorporated to facilitate identification of the binding protein. In some embodiments, the label is a detectable marker that produces a signal that can be detected visually or instrumentally, such as a polypeptide that incorporates a radiolabeled amino acid or is linked to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of polypeptide labels include, but are not limited to, the following: 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); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent labels; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelators. Representative examples of labels commonly used in immunoassays include light-generating moieties, such as acridinium compounds, and fluorescence-generating moieties, such as fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable after reaction with another moiety. Exemplary FcRn-binding polypeptides

[0093] Provided herein are single domain antibodies (sdAbs) comprising a VHH domain that binds to FcRn. In some embodiments, the VHH domain that binds to FcRn has an affinity (K) of between 0.01 nM and 5 nM, or between 0.01 nM and 2 nM, or between 0.01 nM and 1 nM, between 0.01 nM and 0.5 nM, between 0.05 nM and 5 nM, or between 0.05 nM and 2 nM, or between 0.05 nM and 1 nM, or between 0.05 nM and 0.5 nM. D )combined.

[0094] In various embodiments, polypeptides are provided that include at least one VHH domain that binds to FcRn. In some embodiments, polypeptides are provided that include one, two, three, four, five, six, seven, or eight VHH domains that bind to FcRn. In some embodiments, the polypeptides provided herein include one, two, three, or four VHH domains that bind to FcRn. Such polypeptides may include one or more additional VHH domains that bind to one or more target proteins other than FcRn.

[0095] In various embodiments, polypeptides comprising one or more VHH domains that bind to FcRn also comprise therapeutic antigen binding domains and / or therapeutic polypeptides. Such therapeutic antigen binding domains include, but are not limited to, traditional antibody antigen binding domains comprising heavy chain variable regions and light chain variable regions, and single domain antibody antigen binding domains, 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 in vivo half-life 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 times, at least 2 times, at least 3 times, at least 4 times, or at least 5 times longer than the half-life of a polypeptide that does not have a VHH domain that binds to albumin.

[0096] In some embodiments, the polypeptide comprising at least one VHH domain that binds to FcRn comprises an Fc region. In some embodiments, the polypeptide provided herein comprises 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.

[0097] In various embodiments, the FcRn-binding VHH domain comprises a CDR1 sequence selected from SEQ ID NOs: 80-81, a CDR2 sequence selected from SEQ ID NOs: 83-84, and a CDR3 sequence of SEQ ID NO: 85. In various embodiments, the FcRn-binding VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the group consisting of SEQ ID NOs: 80, 83, and 85; SEQ ID NOs: 81, 83, and 85; and SEQ ID NOs: 81, 84, and 85.

[0098] In some embodiments, the FcRn-binding VHH domain 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 a sequence selected from SEQ ID NOs: 86-93. In some embodiments, the FcRn-binding VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 86-93.

[0099] In some embodiments, a VHH domain that binds to FcRn is provided that competes for FcRn binding with a VHH domain comprising an amino acid sequence selected from SEQ ID NOs: 86-93. Exemplary Albumin-Binding Polypeptides

[0100] Provided herein are polypeptides comprising a VHH domain that binds to albumin. 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 to domain 3 with an affinity (K) of between 0.01 nM and 5 nM, or between 0.01 nM and 2 nM, or between 0.01 nM and 1 nM, between 0.01 nM and 0.5 nM, between 0.05 nM and 5 nM, or between 0.05 nM and 2 nM, or between 0.05 nM and 1 nM, or between 0.05 nM and 0.5 nM. D )combined.

[0101] In various embodiments, polypeptides are provided that include at least one VHH domain that binds to albumin. In some embodiments, polypeptides are provided that include one, two, three, four, five, six, seven, or eight VHH domains that bind to albumin. In some embodiments, the polypeptides provided herein include one, two, three, or four VHH domains that bind to albumin. Such polypeptides may include one or more additional VHH domains that bind to one or more target proteins other than albumin.

[0102] In various embodiments, polypeptides comprising one or more VHH domains that bind to albumin also comprise therapeutic antigen binding domains and / or therapeutic polypeptides. Such therapeutic antigen binding domains include, but are not limited to, traditional antibody antigen binding domains comprising heavy chain variable regions and light chain variable regions, and single domain antibody antigen binding domains, such as VHH domains. Non-limiting exemplary therapeutic polypeptides include, for example, receptor extracellular domains, enzymes, and ligands. In various embodiments, polypeptides comprising at least one VHH domain that binds to albumin have a longer in vivo half-life than the same polypeptide without at least one VHH domain that binds to albumin. In some embodiments, the half-life is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, or at least 5 times longer than the half-life of a polypeptide without a VHH domain that binds to albumin.

[0103] In some embodiments, the polypeptide comprising 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.

[0104] In various embodiments, the albumin binding VHH domain comprises a CDR1 sequence selected from SEQ ID NOs: 5-8, a CDR2 sequence selected from SEQ ID NOs: 9-21, and a CDR3 sequence of SEQ ID NO:22. In various embodiments, the albumin binding VHH domain comprises a CDR1, CDR2 and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 5, 9 and 22; SEQ ID NOs: 5, 10 and 22; SEQ ID NOs: 5, 11 and 22; SEQ ID NOs: 5, 12 and 22; SEQ ID NOs: 5, 13 and 22; SEQ ID NOs: 5, 14 and 22; SEQ ID NOs: 5, 15 and 22; SEQ ID NOs: 6, 15 and 22; SEQ ID NOs: 7, 15 and 22; SEQ ID NOs: 8, 15 and 22; SEQ ID NOs: 6, 16 and 22; SEQ ID NOs: 6, 17 and 22; SEQ ID NOs: 6, 18 and 22; SEQ ID NOs: 6, 19 and 22; SEQ ID NOs: 6, 20 and 22; and SEQ ID NOs: 6, 21 and 22. In some embodiments, the albumin binding VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6, 21, and 22.

[0105] In some embodiments, the albumin binding VHH domain 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 a sequence selected from SEQ ID NOs: 23-43 and 97-100. In some embodiments, the albumin binding VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 23-43 and 97-100. In some embodiments, the albumin binding VHH domain 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 albumin binding VHH domain comprises the amino acid sequence SEQ ID NO: 99.

[0106] In some embodiments, a VHH domain that binds albumin is provided that competes for albumin binding with a VHH domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-43 and 97-100. Exemplary FcRn-binding polypeptides and albumin-binding polypeptides

[0107] In some embodiments, a polypeptide is provided 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.

[0108] In some embodiments, a polypeptide is provided that comprises 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. Non-limiting exemplary polypeptides comprising at least one VHH domain that binds to FcRn and at least one FcRn that binds to albumin are shown in Figure 5 middle.

[0109] In various embodiments, the polypeptides provided herein may include one or more additional antigen binding domains that bind to one or more target antigens other than albumin or FcRn. In various embodiments, a polypeptide comprising one or more VHH domains that bind to albumin and 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, traditional antibody antigen binding domains comprising heavy chain variable regions and light chain variable regions, and single domain antibody antigen binding domains, such as VHH domains. Non-limiting exemplary therapeutic polypeptides include, for example, receptor extracellular domains, enzymes, and ligands. The VHH domains that bind to FcRn and / or the VHH domains that bind to albumin may be connected to the antigen binding domain or therapeutic polypeptide at the N-terminus or at the C-terminus of the antigen binding domain or therapeutic polypeptide (e.g., VHH-VHH-antigen binding domain or antigen binding domain-VHH-VHH), or they may each be located at different ends (e.g., VHH-antigen binding domain-VHH).

[0110] In some embodiments of the polypeptide, adjacent portions of the polypeptide, such as the first VHH and the second VHH or the VHH and the antigen binding domain, may be connected to each other by a joint. In some such embodiments, the joint comprises 1 to 20, 1 to 12 or 1 to 8 amino acids, preferably mainly composed of glycine and optionally selected serine. A non-limiting example of such a joint 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.

[0111] In some embodiments, the polypeptide comprising at least one VHH domain that binds to albumin and one VHH domain that binds to FcRn does not comprise an Fc region. In some embodiments, the polypeptide comprising at least one VHH domain that binds to albumin and one VHH domain that binds to FcRn comprises an Fc region. In some embodiments, the polypeptide provided herein comprises 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.

[0112] 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 without at least one VHH domain that binds to albumin. In some embodiments, the half-life is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, or at least 5 times longer than the half-life of a polypeptide without a VHH domain that binds to albumin.

[0113] In some embodiments, the 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 NOs: 69-77. In some embodiments, the 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 NOs: 69-77.

[0114] In some embodiments, the polypeptide comprising at least one VHH domain that binds albumin and one VHH domain that binds 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: 69-74 and 77. In some embodiments, the polypeptide comprising at least one VHH domain that binds albumin and one VHH domain that binds FcRn comprises a sequence selected from SEQ ID NOs: 69-74 and 77. Humanized VHH domain

[0115] In some embodiments, the VHH domain is humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) are suitable for use as therapeutic molecules because humanized antibodies reduce or eliminate human immune responses to non-human antibodies, which may cause immune responses to antibody therapeutics and reduce the effectiveness of therapeutics. In general, humanized VHHs include CDRs (or portions thereof) derived from non-human antibodies and FRs (or portions thereof) derived from human antibody sequences. In some embodiments, some FR residues in humanized VHHs are replaced with corresponding residues from non-human antibodies (e.g., VHHs from which CDR residues are derived), thereby, for example, restoring or improving antibody specificity or affinity.

[0116] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633, and are 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. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 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 the "guided selection" method of FR shuffling).

[0117] 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 the consensus sequence of human antibodies having a particular subgroup of heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:2296). 1:2623); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633); and framework regions derived from screening 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, the FR regions of a VHH are replaced with human FR regions to generate a humanized VHH. In some embodiments, certain FR residues of a human FR are replaced in order to improve one or more properties of a humanized VHH. VHH domains with such replaced residues are still referred to herein as "humanized". Fc domain

[0118] In various embodiments, the Fc region included in the polypeptide is a human Fc region, or is derived from a human Fc region.

[0119] In some embodiments, the Fc region included in the polypeptide is derived from a human Fc region and comprises mutations designed for heterodimerization, referred to herein as "knob" and "hole". In some embodiments, the "knob" Fc region comprises the mutation T366W. In some embodiments, the "hole" Fc region comprises the mutations T366S, L368A, and Y407V. In some embodiments, the Fc region used for heterodimerization comprises additional mutations, such as the mutation S354C located on the first member of the heterodimeric Fc pair that forms an asymmetric disulfide bond and the corresponding mutation Y349C located on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair comprises the modification H435R or H435K to prevent protein A binding while maintaining FcRn binding. In some embodiments, one member of the heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc region comprises the modification H435R or H435K (in some cases, when the modification is H435R, referred to as "hole-R"), and the knob Fc region does not comprise a modification. In some cases, the hole-R mutation improves the purification of heterodimers relative to the homodimeric hole Fc region that may be present.

[0120] Non-limiting exemplary Fc regions that can be used in the polypeptide include Fc regions comprising the amino acid sequence of SEQ ID NOs: 47-68. Peptide expression and production

[0121] A nucleic acid molecule is provided, which comprises a polynucleotide encoding a polypeptide provided herein. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs secretion of the polypeptide, which leader sequence is usually cleaved so that it is not present in the secreted polypeptide. The leader sequence may be a native heavy chain (or VHH) leader sequence, or may be another heterologous leader sequence.

[0122] Nucleic acid molecules can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0123] Provide a vector comprising a nucleic acid encoding a polypeptide provided herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector optimized to express the polypeptide in a desired cell type such as a CHO or CHO-derived cell or NSO cell is selected. Exemplary such vectors are described in, for example, Running Deer et al., Biotechnol. Prog. 20: 880-889 (2004).

[0124] In some embodiments, the polypeptides provided herein can be expressed in prokaryotic cells, such as bacterial cells; or eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13 CHO cells, and FUT8 CHO cells; cells (Crucell); and NSO cells. In some embodiments, the polypeptides can be expressed in yeast. See, for example, U.S. Publication No. US2006 / 0270045 A1. In some embodiments, a particular eukaryotic host cell line is selected based on its ability to perform a desired post-translational modification on a polypeptide. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptides produced in 293 cells.

[0125] The introduction of one or more nucleic acids (such as vectors) into the desired host cells can be accomplished by any method, including but not limited to calcium phosphate transfection, DEAE-polydextrose mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into the desired host cells according to any suitable method.

[0126] A host cell comprising any of the nucleic acids or vectors described herein is also provided. In some embodiments, a host cell expressing the polypeptides described herein is provided. The polypeptides expressed in the host cell can be purified by any suitable method. Such methods include, but are not limited to, using affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and reagents that bind to the Fc region. For example, protein A, protein G, protein A / G or antibody affinity columns can be used to bind to the Fc region and purify polypeptides comprising the Fc region. Hydrophobic interaction chromatography, such as butyl or phenyl columns, can also be used to purify some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) can also be used to purify 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.) is also suitable for purifying some polypeptides, such as antibodies. A variety of methods for purifying polypeptides are known in the art.

[0127] In some embodiments, the polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described in, e.g., 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).

[0128] In some embodiments, polypeptides prepared by the methods described above are provided. In some embodiments, polypeptides are prepared in host cells. In some embodiments, polypeptides are prepared in cell-free systems. In some embodiments, polypeptides are purified. In some embodiments, cell culture media comprising polypeptides are provided.

[0129] In some embodiments, compositions comprising antibodies prepared by the methods described above are provided. In some embodiments, compositions comprise polypeptides provided herein prepared in host cells. In some embodiments, compositions comprise polypeptides prepared in cell-free systems. In some embodiments, compositions comprise purified polypeptides. Exemplary methods for treating diseases using FcRn-binding polypeptides

[0130] In some embodiments, a method for treating a disease in an individual is provided, comprising administering a therapeutic polypeptide comprising an FcRn-binding domain as provided herein. Such diseases include any disease that would benefit from treatment with a therapeutic polypeptide comprising an FcRn-binding domain. Non-limiting exemplary diseases that can be treated with a therapeutic polypeptide comprising an FcRn-binding domain as provided herein include infectious diseases, immune diseases or disorders (e.g., autoimmune diseases or disorders), inflammatory diseases or disorders, and cancer. In some embodiments, the disease or disorder is an autoantibody-mediated disease or disorder. In some embodiments, the disease or condition is pemphigus vulgaris, lupus nephritis, myasthenia gravis, Guillain-Barre syndrome, antibody-mediated rejection, antiphospholipid antibody syndrome, chronic inflammatory demyelinating polyneuropathy, immune complex-mediated vasculitis, glomerulitis, ion channel lesions, neuromyelitis optica, autoimmune encephalitis, autoimmune Graves' disease, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, immune neutropenia, dilated cardiomyopathy or serum sickness. The method comprises administering to the subject an effective amount of a therapeutic polypeptide comprising an FcRn-binding domain as provided herein. In some embodiments, the treatment method promotes the clearance of autoantibodies in the subject. In some embodiments, the treatment method blocks an immune response in the subject (e.g., blocking immune complex-based activation of the immune response). Such treatment methods may be directed to humans or animals. In some embodiments, methods for treating humans are provided.

[0131] The therapeutic polypeptide comprising an FcRn-binding domain provided herein can be administered to a subject as needed. The frequency of administration can be determined by a person skilled in the art, such as an attending physician, based on consideration of the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like. In some embodiments, an effective dose of a therapeutic polypeptide is administered to a subject one or more times. In some embodiments, an effective dose of a therapeutic polypeptide comprising an FcRn-binding domain is administered to a subject every day, every half week, every week, every two weeks, once a month, etc. An effective dose of a therapeutic polypeptide comprising an FcRn-binding domain is administered to a subject at least once. In some embodiments, an effective dose of a therapeutic polypeptide can be administered multiple times, including multiple administrations over a time course of at least one month, at least six months, or at least one year.

[0132] In some embodiments, the pharmaceutical composition is administered in an amount effective to treat the disease. The therapeutically effective amount generally depends on the body weight of the subject being treated, its physical or health condition, the extent of the condition being treated, or the age of the subject being treated.

[0133] 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 application. Pharmaceutical composition

[0134] In some embodiments, the composition comprising the polypeptide is provided in a formulation with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th Edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers, including vehicles, adjuvants, and diluents are available. In addition, various pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, and the like, are also available. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Non-Limiting Exemplary Diagnostic and Treatment Methods

[0135] In some embodiments, the methods described herein are applicable to evaluating subjects and / or samples from subjects (e.g., patients with autoimmune diseases or cancer patients). In some embodiments, the evaluation is one or more of a diagnosis, a prognosis, and / or a response to treatment.

[0136] In some embodiments, the methods described herein comprise assessing the presence, absence, or amount of a protein. In some embodiments, the methods described herein comprise assessing the presence, absence, or expression of a nucleic acid. The compositions described herein can be used for such measurements. In some embodiments, the assessment can guide treatment (including treatment with a polypeptide described herein). Reagent test kit

[0137] Also provided are articles of manufacture and kits comprising any of the polypeptides as described herein and suitable packaging.In some embodiments, the invention includes a kit having (i) a polypeptide provided herein and (ii) instructions for using the kit to administer the polypeptide to a subject.

[0138] Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, cans, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Such articles may be further sterilized and / or sealed. Also provided are unit dosage forms comprising the compositions described herein. Such unit dosage forms may be stored in suitable packaging in single or multiple unit doses and may also be further sterilized and sealed. The instructions supplied in the kits of the present invention are written instructions typically on a label or drug instructions (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions for use of antibodies generally include information on dosage, dosing schedule, and route of administration for intended treatment or industrial use. The kit may further include a description of selecting a suitable individual or treatment.

[0139] The container may be a unit dose, bulk (e.g., multi-dose packaging), or a sub-unit dose. For example, a kit containing a sufficient dose of the molecules disclosed herein to provide an extended period of effective treatment to an individual, such as any of about 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 months, may also be provided. The kit may also include multiple unit doses of the molecule and instructions for use and be packaged in sufficient quantities for storage and use in a pharmacy (e.g., a hospital pharmacy and a dispensing pharmacy). In some embodiments, the kit includes a dried (e.g., lyophilized) composition that can be reconstituted, resuspended, or rehydrated to form a generally stable aqueous suspension of the antibody. Example

[0140] The embodiments discussed below are intended only to illustrate the present invention and should not be considered to limit the present invention in any way. These embodiments are not intended to represent that the experiments below are all experiments or the only experiment performed. Every effort has been made to ensure the accuracy of the numbers used (such as amount, temperature, etc.). However, certain experimental errors and deviations should be considered. Unless otherwise specified, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure. Example 1: Development of anti-albumin single domain antibodies (sdAbs)

[0141] Single domain antibodies targeting human albumin are generated by immunizing llamas and / or alpacas with recombinant human serum albumin (SEQ ID NO: 1).

[0142] After the specific anti-albumin antibody titers were generated, llama / alpaca peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of blood from the immunized animals and 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. The Fc was a human IgG1 Fc, or in some cases, a variant IgG1 Fc with reduced effector function.

[0143] Yeast pools displaying VHH-Fc-AGA2 fusion proteins were enriched using a recombinant form of human albumin via magnetic bead separation followed by fluorescence activated cell sorting (FACS). Sorted yeast were pelleted and isolated colonies were picked into 96-well plates and induced for yeast cell surface expression of VHH-Fc-AGA2 fusion proteins. Biotinylated recombinant human albumin or an irrelevant biotinylated protein (albumin negative) was applied directly to the induced yeast, washed, treated with fluorophore-labeled streptavidin, and analyzed by 96-well flow cytometry.

[0144] 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 using polyethyleneimine in HEK293 Freestyle cells (293F cells) or CHO cells. The supernatant was collected after 3 to 7 days, the secreted recombinant protein was purified by protein A chromatography, and the concentration was calculated by absorbance at 280 nm and extinction coefficient.

[0145] The anti-albumin sdAb 4A01 was selected for humanization. Example 2: Humanization and species cross-reactivity of anti-albumin sdAb 4A01

[0146] Various humanized versions of sdAb 4A01 were made based on the human heavy chain framework VH3-23*04. Certain amino acids were backmutated to donor amino acids and certain mutations, such as mutations in CDR2, were tested. Figure 1A An alignment of the human heavy chain receptor sequence with the humanized form of 4A01 is shown.

[0147] The binding of monomeric anti-albumin sdAb 4A01 ("lm4A01") and its humanized version to human serum albumin, cynomolgus monkey serum albumin, murine serum albumin and rat serum albumin was determined by ELISA as follows. Medisorp plates were coated with albumin at 2 μg / ml, 50 μl / well overnight at 4°C (human, murine and rat albumin - Sigma, cynomolgus monkey albumin - Abcam). 1X fish gelatin (blocking agent, Bethyl Laboratories) was added to the albumin coated wells and then incubated for 1 hour at RT. A titration of sdAb fusion protein was added (starting at 100 nM, across 1:3 or down to 1:4) and incubated for 1 hour at room temperature (RT). The plates were washed 3 times with 0.1% D-PBST and then anti-human Fc HRP antibody (1:2000 in 0.1% D-PBST, Jackson) was added and incubated for 30 minutes at RT. Plates were washed 3 times with 0.1% DPBST and then TMB substrate was added. The absorbance at 650 nm was read on a plate reader (Molecular Devices) and the data were plotted using the one site-total binding equation (model: Y=Bmax*X / (Kd+X)+NS*X+background, GraphPad Prism).

[0148] Binding of 4A01 and humanized forms of 4A01 to human albumin is shown in Figure 1B to Figure 1C All sdAbs were expressed with K values ​​between 0.10 and 0.43 nM. D Binding to human albumin. Binding of 4A01 and humanized forms of 4A01 to cynomolgus monkey albumin is shown in Figure 1D to Figure 1E All sdAbs were expressed with K values ​​between 0.11 and 0.34 nM. D Binding to cynomolgus monkey albumin. Binding of 4A01 and humanized forms of 4A01 to murine albumin is shown in Figure 1F to Figure 1G All sdAbs have a K between 0.11 and about 0.25 nM. D Binding to murine albumin. Binding of 4A01 and the humanized form of 4A01 to rat albumin is shown in Figures 1H to 1I All sdAbs had K values ​​between 0.14 and about 0.33 nM. D Binds to rat albumin.

[0149] FIG. 2A to FIG. 2DBinding of 4A01 and humanized hz4A01v51 to human (2A), cynomolgus monkey (2B), murine (2C), and rat (2D) albumin is shown. 4A01 and all humanized variants tested bind to all four albumin species with an affinity of less than 1 nM. Humanized hz4A01v51 binds to all four albumin species with an affinity of less than 0.3 nM and achieves greater than 90% maximal binding.

[0150] The framework regions of hz4A01v51 were further modified, including by back-mutating certain residues to donor amino acids and / or introducing alternative charged residues. The modified monomeric anti-albumin antibodies (comprising hz4A01v51.9, hz4A01v51.11, hz4A01v51.12 or hz4A01v51.13 VHH domains) exhibited similar binding curves at pH 6 and pH 7.4 and all exhibited improved binding over that observed for hz4A01v51, particularly at pH 6 (data not shown). Example 3: Anti-albumin sdAb 4A01 does not bind to albumin domain 3

[0151] Albumin binds to the beta-2 microglobulin FcRn complex primarily via domain 3, and this binding is believed to be involved in improving the half-life of proteins fused to anti-albumin antibodies or to albumin itself. To determine whether anti-albumin sdAb 4A01 binds to albumin domain 3, binding of 4A01-NNT-hFc was analyzed by biofilm interferometry as follows.

[0152] Albumin domain 3 (mouse Fc tagged) was immobilized on an anti-mouse IgG Fc capture biosensor. All buffers / protein preparations were in MBST5 (50 nM MES pH5, 150 mM NaCl, 0.025% Tween). Buffer only was used to establish a baseline. Human albumin domain III (10 μg / ml) with mouse Fc tag was loaded on an anti-mouse IgG Fc capture biosensor (ForteBio). Anti-albumin sdAbs 4A01 (4A01-NNT-hFc) and 1C04 (similar format) were then loaded and allowed to bind to the captured biotin domain 3 and subsequently dissociate from MBST5. sdAb 1C04 is known to bind to albumin domain 3 and was used as a positive control. See Figure 3A .

[0153] like Figure 3B As shown, 1C04, but not 4A01, binds to immobilized albumin domain 3.

[0154] The interference of anti-albumin sdAbs 4A01 (4A01-NNT-hFc), hz4A01v51 and 1C04 on albumin-FcRn binding was then tested as follows. Binding was assessed by biofilm interferometry using biotinylated recombinant FcRn-B2M immobilized on a streptavidin biosensor. The immobilized FcRn-B2M was then complexed with recombinant human albumin. All buffer / protein preparations were in MBST5 (50mM MES pH5, 150mM NaCl, 0.025% Tween). Only buffer was used to establish a baseline. Biotinylated FcRn-B2M (10μg / ml, Acro Biosystems) was loaded onto a streptavidin biosensor (ForteBio) and another baseline determined. 50μM recombinant human albumin (Sigma) was then added and allowed to bind to the immobilized FcRn-B2M. Anti-albumin sdAbs 4A01 and 4A01v51 and sdAb 1C04 were then loaded and allowed to bind to the captured biotin domain 3 and subsequently dissociate from MBST5. Figure 4A .

[0155] like Figure 3B As shown, both 4A01 and hx4A01v51 bound to albumin bound to FcRn-B2M, but 1C04 did not. Example 4: Development of FcRn single domain antibodies (sdAbs)

[0156] Single domain antibodies targeting human FcRn were generated by immunizing llama with polypeptides comprising human B2m (SEQ ID NO: 79) and the extracellular domain of human FcRn (SEQ ID NO: 78), which were joined to a flexible linker and fused to a polyhistidine tag for purification. After the generation of specific anti-FcRn antibody titers, llama peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of blood from the immunized animals, and 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 sequence was 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.

[0157] Yeast pools displaying VHH-Fc-AGA2 fusion proteins were enriched using a recombinant form of the FcRn ECD via magnetic bead separation followed by fluorescence activated cell sorting (FACS). Sorted yeast were precipitated and isolated colonies were picked and placed in 96-well plates and grown in culture medium to convert surface displayed VHH-Fc expression to secretion into the culture medium. Supernatants from 96-well yeast secretion cultures were 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.

[0158] One VHH domain that binds FcRn (clone B10a) was humanized essentially as described in Example 2. Humanized B10a VHH domains (hzB10a.v1-v2.5) were used to generate bispecific polypeptides targeting human FcRn and albumin with Figure 5 (i) (CX11383, CX11558, CX12003, CX12006, CX12007, CX12008 and CX12032) or Figure 5 (ii) The structures shown in (cx11385 and cx11642). Example 5: Binding of anti-FcRn VHH polypeptides to human FcRn

[0159] Binding of anti-FcRn VHH polypeptides to human FcRn was tested by flow cytometry at neutral (7.4) or endosomal (6) pH using a CHO-based overexpression system or isolated human peripheral blood mononuclear cells (PBMCs). CHO cells were transiently transfected to overexpress the human FcRn extracellular domain (ECD) (SEQ ID NO: 78) fused to the transmembrane domain and intracellular citrine and beta-2 microglobulin (SEQ ID NO: 79).

[0160] CHO cells were plated at 50,000 cells / well in 96-well plates and resuspended in PBS, pH 7.4 or a buffer containing 20 mM His, 150 mM NaCl, pH 6. Serial dilutions of the test article were prepared at 2 times the final concentration in the buffer described above (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 in the respective buffer and then incubated at 4°C for 30 minutes with a fluorescently labeled (AF647) antibody detecting the VHH polypeptide. After the final wash using iQue Screener (Sartorius), the binding of the detection antibody to FcRn on the transfected cells was measured (citrin positive).

[0161] Isolated human PBMCs were plated at 120,000 cells / well in 96-well plates and washed with PBS, pH 7.4. Cells were stained with Zombie Aqua to exclude dead cells and with specific fluorescently labeled antibodies to surface lineage markers (CD14, CD16, CD3, CD56, CD19, and HLA-DR) to allow gating of different PBMC subsets. After surface marker staining, the cells were fixed and permeabilized using the Foxp3 / Transcription Factor Fixation / Permeabilization Kit (eBioscience TM ) The cells were fixed and permeabilized according to the manufacturer's recommended protocol. The cells were then resuspended in the buffer described above to produce pH 7.4 or pH 6 conditions and test article staining was performed as described for CHO cells. After the final wash, binding to FcRn was measured on a Novocyte-Quanteon flow cytometer (Agilent) on PBMC subsets (monocytes, B cells or T cells).

[0162] Some anti-FcRn VHH polypeptides were also tested by ELISA for binding to human FcRn at neutral (7.4) pH. 96-well ELISA plates were coated with PBS containing human FcRn / B2m heterodimer protein at 4°C overnight, washed with PBST and then blocked with PBST containing 3% BSA for 1 hour at room temperature. Serial dilutions of the test article 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 cells were washed with PBST and then incubated with horseradish peroxidase (HRP)-conjugated antibodies to the detection polypeptide for 1 hour at room temperature. The plate was washed with PBS-T, then TMB substrate was added and developed. The TMB substrate was stopped with an equal volume of stop solution (1M H2SO4). The absorbance at 450nm was measured with a 96-well plate reader (Emax, Molecular Devices). All data were plotted and analyzed using GraphPad Prism analysis software.

[0163] like Fig. 6A To Figure B and Fig.6D As shown in Figure E, all tested FcRn-targeting peptides bind to human FcRn overexpressed on CHO cells with apparent affinities as low as sub-nanomolar. Binding is independent of pH and is comparable at pH 7.4 and pH 6. Figure 6C As shown, a polypeptide having a bivalent FcRn binding domain (such as Figure 5 (ii)) binds FcRn with higher affinity than its monovalent counterpart (e.g. Figure 5 (i)). By using a VHH unit specific for FcRn (as a monovalent specific unit for albumin, Figure 5 (i) mediated binding, and the non-mammalian targeting VHH units did not bind to human FcRn with appreciable affinity. Fig. 6F and Figure 6G pH-independent binding to FcRn was shown to be retained on primary human cells. Figure 5 (i) The formatted FcRn and albumin-targeting polypeptide (cx11558) binds with low nanomolar affinity to FcRn-expressing human monocytes at pH 7.4 and pH 6, but not to FcRn-negative B cells or T cells. Example 6: Binding of polypeptides to human albumin

[0164] The binding of humanized VHH-containing polypeptides to human albumin at neutral (7.4) or late endosomal (5.5) pH was tested by ELISA. 96-well ELISA plates were coated with albumin in PBS at 4°C overnight, washed with PBS-T and then blocked with 5% milk powder in PBS-T for 1 hour at room temperature. Serial dilutions of the test article were prepared in PBS pH 7.4 or a buffer containing 20mM His, 150mM NaCl, pH 5.5 and added to each plate. Each plate was incubated for 1 hour at room temperature. After incubation, the cells were washed in the respective buffer and then incubated with an HRP-conjugated antibody that detects the VHH polypeptide for 30 minutes at room temperature. Each plate was washed in the respective buffer and TMB substrate was added and developed. The TMB substrate was stopped with an equal volume of stop solution (1M H2SO4). The absorbance at 450nm was measured with a 96-well plate reader. The data were plotted and analyzed using GraphPad Prism analysis software. The results are shown in FIG. 7A to FIG. 7D middle.

[0165] like FIG. 7A to FIG. 7D As shown in Figure 2, all bispecific anti-FcRn / anti-albumin polypeptides tested bound human albumin with apparent affinities as low as subnanomolar. Binding was independent of pH and was comparable at pH 7.4 and pH 5.5. Figure 5 (i) mediated binding, and the non-mammalian targeting VHH units did not bind to human albumin with appreciable affinity. Example 7: Blocking IgG binding to human FcRn by VHH-containing polypeptides

[0166] Blockade of human IgG binding to human FcRn expressed in HEK 293 cells by humanized VHH polypeptides targeting human FcRn was measured by flow cytometry. HEK 293 cells were transiently transfected to overexpress the human FcRn extracellular domain (ECD) (SEQ ID NO: 78) fused to the transmembrane domain and intracellular citrin and β-2 microglobulin (SEQ ID NO: 79). 50,000 transiently transfected HEK 293 cells were plated in a 96-well plate and resuspended in 20 mM His, 150 mM NaCl, pH 6. Serial dilutions of the test article were prepared at 2 times the final concentration in the buffer described above 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 another 60 minutes. The cells were then washed in the buffer described above and then incubated with fluorescently labeled (AF647) streptavidin for 30 minutes at 4°C to detect binding of IgG to FcRn. After a final wash using iQueScreener (Sartorius), binding of streptavidin to cells expressing FcRn (citrin positive) was measured. Data were plotted and analyzed using GraphPad Prism analysis software. Results are shown in Figure 8 middle.

[0167] like Figure 8 As shown, increasing concentrations of the peptide test article reduced the binding of human IgG to FcRn expressed on HEK 293 cells, as measured by a decrease in the mean fluorescence intensity (MFI) of the AF647-conjugated streptavidin reagent used to detect binding of biotinylated human IgG. The inhibitory activity (IC50) was Figure 5 (i) The described formatted construct (cx11383) and Figure 5 (ii) were similar in the formatted construct described (cx11385) and fell within the low millimolar concentration range for both articles tested. Example 8: In vivo pharmacokinetic profile and IgG clearance of peptides targeting human FcRn and albumin

[0168] The pharmacokinetic (PK) profiles of humanized polypeptides targeting human FcRn and albumin were tested in conventional BALB / c mice and transgenic animals expressing human FcRn and human albumin on a C57BL / 6 background. None of the VHH sequences targeting FcRn used had cross-reactivity with mouse FcRn, but the VHH sequences targeting albumin bound to mouse albumin (data not shown). Therefore, the ability to clear IgG in vivo after injection of human IgG was analyzed only in the human FcRn / albumin transgenic mouse system.

[0169] To assess serum exposure in BALB / c mice, a single dose of 30 mg / kg or 0.3 mg / kg of the polypeptide test article was injected intravenously into the animals. Serum samples were collected at 30 minutes, 6 hours, 24 hours, 96 hours, and 168 hours after the injection of the test article. Human FcRn / albumin transgenic mice were first injected with 500 mg / kg of human IgG, and then 24 h later with a single dose of 20 mg / kg of the polypeptide test article. Serum samples were collected at 30 minutes, 24 hours, 96 hours, and 168 hours after the administration of the test article. The test article concentration and human IgG content in mouse serum were determined by ELISA. For the test article PK ELISA, the human FcRn / B2M heterodimeric protein (His-tag, Acro Biosystem) was immobilized on a 96-well ELISA plate by incubating a solution of 4 μg / mL protein in PBS at 4 °C for 12 hours. The next day, the plates were blocked with 3% BSA TBS-T buffer for 2 hours, and then serum samples were incubated on these plates for 2 hours. A horseradish peroxidase (HRP)-conjugated secondary detection antibody that could bind to the polypeptide was used to detect the binding of the test article in the serum sample to the FcRn immobilized on the ELISA plate. The secondary antibody was incubated on the plate for 1 hour and the binding was observed using a TMB substrate solution. Then, a stop solution (1 <H2SO4) was added and the absorbance was measured at 450 nm on an Emax spectrophotometer (Molecular Devices).

[0170] The human IgG content in mouse serum was also determined by ELISA. A polyclonal goat capture antibody specific for human IgG was coated on a 96-well ELISA plate at 1 μg / mL in PBS at 4 °C for 12 hours. The next day, the plates were blocked with 1% BSA buffer for 2 hours, and then serum samples were incubated on these plates for 2 hours. In addition to the capture antibody, a second goat anti-human IgG horseradish peroxidase (HRP)-conjugated detection antibody that could bind to IgG was used to detect the binding of human IgG in the serum. The secondary antibody was incubated on the plate for 1 hour as described above and the binding was observed. Using a standard curve from proteins with known concentrations analyzed in parallel with each assay, the absorbance values measured for each ELISA were converted to test article concentrations in SoftMax Pro. A 4-parameter logistic regression was used to fit the standard curve. GraphPad Prism analysis software was used to output and plot the data.

[0171] As Fig. 9A and Fig. 9B shown, as Figure 5 (i)(cx11558) and Figure 5(ii) The multivalent, multispecific polypeptide targeting FcRn and albumin formatted as described (cx11642) had significantly higher serum cMax levels and total serum exposure at 30 minutes than the non-albumin-binding polypeptide (cx11915). Both bispecific formats showed similar PK profiles at the dose levels tested. Fig. 9C Show as Figure 5 (i)(cx11383) and Figure 5 (ii) PK profiles of two similar constructs stylized as described in (cx11385) and highlighting their serum exposure compared to a nikalimab (anti-FcRn antibody) sequence analog generated based on publicly available information. All three test articles started at similar cMax serum concentrations. However, the exposure time was significantly longer for the multivalent multispecific single polypeptide compared to the conventional antibody nikalimab. Between the two polypeptides, as Figure 5 (i) The described formatted construct (cx11383) has a longer serum half-life. The extended serum exposure also translates into a preferential clearance of pre-injected human IgG in a human FcRn / albumin transgenic mouse system. Compared to the nikalimab sequence analog, both complexes cx11383 and cx11385 induced accelerated clearance of human IgG and resulted in lower levels of residual serum IgG at the end of the study (168 hours). See Fig.9D . Example 9: In vivo IgG depletion mediated by peptides targeting human FcRn and albumin in cynomolgus monkeys

[0172] The pharmacodynamic (PD) and pharmacokinetic (PK) profiles of humanized peptides targeting human FcRn and albumin were evaluated in several studies in cynomolgus monkeys. In one study, untreated male cynomolgus monkeys aged 4 to 5 years were divided into 5 treatment groups, each containing 2 monkeys. Groups 1 and 2 were treated with 100 mg / kg and 20 mg / kg cx11558 (monovalent), respectively, groups 3 and 4 were treated with 100 mg / kg and 20 mg / kg cx11642 (bivalent for FcRn binding), respectively, and group 5 was treated with 20 mg / kg agamod (NDC73475-3041-5, batch number AHUC01A, recombinant engineered IgG Fc fragment). All treatments were administered via IV using a 60 min infusion at 10 mL / kg. Serum samples were collected from approximately 2 mL of whole blood from all animals and all groups at the end of the 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.

[0173] The concentration of cynomolgus monkey IgG was analyzed by the cynomolgus monkey IgG ELISA kit from Molecular Innovations (IMNCYIGGKT) according to the manufacturer's protocol. In short, the cynomolgus monkey IgG standard dilution was added to the pre-coated ELISA plate in duplicate, and the serum samples prepared at a final dilution of 2×10^6 to 3×10^6 were added to the plate in triplicate and the plate was incubated for 30 minutes. The plate was then washed and incubated with biotinylated anti-cynomolgus monkey IgG primary antibody for 30 minutes, washed and incubated with horseradish peroxide-conjugated streptavidin antibody for 30 minutes. The plate was then washed and TMB substrate solution was added to all wells for 2 to 10 minutes, and the reaction was quenched by adding 1NH2SO4 to all wells. The absorbance at 450nM was measured using a plate spectrophotometer, the background was subtracted from all standards and samples, and A was plotted relative to the amount of IgG in all standards. 450 , and the standard curve was analyzed using a four-parameter logistic (4PL) curve fit in the Prism software. The standard curve was used to determine the amount of IgG in all samples and this value was multiplied by the dilution factor for each serum sample. Over time, the IgG concentrations of the cx11558 and agamod treatment groups (as a percentage change from baseline) were plotted on Fig. 10A middle.

[0174] The FcRn occupancy of 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 3 separate wells for staining. All cells were stained with ZombieAqua (Biolegend), incubated with cyno IgG (diluted to 0.2 mg / mL) in FACS buffer for 10 minutes, and the following antibody panels were added: CD14-BV421; CD16-BV605; CD8-BV785; CD20-PE / Cy7; NKG2A-PE; CD3-PE-Dazzle 594; HLA-DR-APC-Cy7 (from Biolegend or BD) and incubated in FACS buffer for another 30 minutes at 4°C. After washing, cells were treated with eBioscience fix / perm for 30 minutes at 4°C, washed twice with cold permeabilization buffer, and then incubated with saturating concentrations of B10a.v2.4 VHH-Avl-Hi (in-house reagent to detect free FcRn), saturating concentrations of unlabeled test article (cx11558 or cx11642; to detect total FcRn) or buffer (to detect occupied FcRn) for 30 minutes at 4°C. Cells were then washed and incubated with streptavidin AF647 (to detect B10av2.4VHH-Avi-His) or anti-d-AF647 (in-house reagent to detect cx11558 and cx11642) for 30 minutes at 4°C. After washing, cells were resuspended in FACS buffer and analyzed using a Novocyte Quanteon flow cytometer. The FcRn occupancy of cx11558 and cx11642 is shown in Fig. 10B middle.

[0175] Serum levels of cx11558, cx11642, and igamod were measured over time by ELISA. Biotinylated human TM Streptavidin-coated high-capacity plates (Pierce TM Streptavidin Coated High Capacity Plate) and the standards, controls and serum samples were diluted and added to the plate in duplicate. After 90 to 120 minutes of incubation, the plate was washed and incubated with anti-ID-HRP conjugate (in-house reagent for detection of cx11558 or cx11642) or mouse anti-human IgG Fc HRP conjugate (for detection of igamod) for 60 minutes. The plate was then washed and TMB substrate was added to all wells for 2 to 10 minutes, and the plate was incubated with 1NH2S The reaction was quenched with O4 and the absorbance was measured at 450 nM. The background (650 nM) was subtracted from all standards and samples on a SpectraMax M5e plate reader and expressed as OD 450-650 The absorbance and concentration of the standards were plotted and curve fitting was performed using 4PL regression in Softmax Prov. 5.4.1 or higher. The sample concentrations were then interpolated based on the standard curve regression and reported in ng / mL mass units. The serum concentrations over time were plotted on Fig. 10C middle.

[0176] like Fig. 10A As shown, compared with igamod, cx11558 administered at any dose induced accelerated clearance of cynomolgus monkey IgG and resulted in lower residual serum IgG levels at least through day 27. cx11642 administered at any dose also induced accelerated clearance of cynomolgus monkey IgG to levels below those observed with igamod, however, serum IgG levels appeared to return to normal levels more quickly than observed with cx11558 at a matched dose of 100 mg / kg (data not shown). Fig. 10B As shown, cx11558 maintained full FcRn occupancy on monocytes for about 9 days and about 18 days at 20 mg / kg and 100 mg / kg, respectively, while cx11642 maintained full FcRn occupancy for about half that time. Fig. 10C As shown, the serum concentration of cx11558 administered at a dose of 20 mg / kg and 100 mg / kg remained above 10 μg / mL (which is associated with approximately 100% receptor occupancy) for approximately 9 days and approximately 18 days, respectively, while the serum concentration of cx11642 at 20 mg / kg and 100 mg / kg dropped to below 10 μg / mL on days 6 and 8, respectively, and the serum concentration of igamod dropped to below 10 μg / mL on day 6.

[0177] In another study, the PD and PK profiles of humanized polypeptides targeting human FcRn and albumin administered by intravenous infusion (IV) or subcutaneous injection (SC) were evaluated. For this study, untreated male cynomolgus monkeys were divided into 3 treatment groups, each containing 2 monkeys. Groups 1 and 2 were treated by IV administration of a single dose of 20 mg / kg and 100 mg / kg of cx12007 (infused at 10 mL / kg for 70 minutes), respectively, and group 3 was treated by SC administration of a single dose of 100 mg / kg of cx12007 (1 mL / kg). Serum samples were collected from whole blood of 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, 504, 552, 600, 648, 696, 744, 792, and 840 hours after dosing. Cynomolgus monkey IgG concentrations were analyzed by the NHP Isotyping Kit from Meso Scale Discovery (K15203D) according to the manufacturer's protocol. Briefly, calibration standards and serum samples prepared at a final dilution of 2×10^5 were added to the MSD plate in duplicate, and the plate was incubated for 2 hours. The plate was then washed and incubated in the detection antibody solution for 2 hours. After further washing, read buffer was added and the plate was immediately read on the MSD imager. Use 1 / Y with a calibrated value 2 The standard curve was analyzed using a 4-parameter logistic curve fitting model with a weighting function. The standard curve was used to determine the amount of IgG in all samples and this value was multiplied by the dilution factor used for each serum sample. The IgG concentration of each subject in the treatment group over time (as a percentage change from baseline) is plotted on Fig.11A FcRn occupancy on monocytes was examined by flow cytometry essentially as described above, except that saturating concentrations of unlabeled cx12007 were used (to detect total FcRn). FcRn occupancy of cx12007 is shown in Fig. 11B Serum concentrations of cx12007 were determined by ELISA essentially as described above and Fig. 11C Plotted over time.

[0178] like Fig.11A As shown in Figure 2, all treatment groups showed comparable trough serum IgG clearance, with a maximum clearance of approximately 77% and comparable time to maximum clearance. The fastest time to 40% clearance was approximately 4 days, with the 20 mg / kg and 100 mg / kg doses maintaining low residual serum IgG levels at least until Day 21 and Day 36, respectively. Fig. 11BAs shown, cx12007 maintained full FcRn occupancy on monocytes for 10 and 19 days at 20 mg / kg and 100 mg / kg doses, respectively. Fig. 11C As shown, serum concentrations of cx12007 at 20 mg / kg and 100 mg / kg doses remained above 10 μg / mL for 10 and 19 days, respectively. The curves for the 100 mg / kg dose were similar for both IV and SC routes of administration.

[0179] In another study, the PK and PD curves of the humanized polypeptide cx12007 administered subcutaneously (SC) to cynomolgus monkeys at different doses were evaluated. For this study, untreated male cynomolgus monkeys were divided into 3 treatment groups, each containing 3 monkeys. Groups 1, 2, and 3 were treated with a single dose of 20 mg / kg, 40 mg / kg, and 70 mg / kg of cx12007 administered SC at 1 mL / kg, respectively. Serum samples were collected from whole blood of all animals and all groups before administration, at the end of infusion, and 24, 36, 48, 72, 144, 216, 288, 360, 432, 504, 576, 648, 720, and 792 hours after administration. Cynomolgus monkey IgG concentrations were analyzed as described above via the NHP isotype analysis kit from Meso Scale Discovery (K15203D). IgG concentrations for each treatment group over time (as percent change from baseline) are plotted on Fig. 12A Serum concentrations of cx12007 were determined by ELISA essentially as described above and Fig. 12A Plotted over time.

[0180] like Fig. 12A As shown, all treatment groups showed comparable trough serum IgG clearance, with maximal depletion of approximately >70% and comparable time to maximal depletion. Throughout the study, SC administration of cx12007 at all dose levels maintained low residual serum IgG levels, with IgG levels increasing more rapidly in the 20 mg / kg group. Fig. 12B As shown, serum concentrations of cx12007 administered SC at doses of 20 mg / kg, 40 mg / kg, and 70 mg / kg were maintained at greater than 10 μg / mL for approximately 9 days, at least 12 days, and at least 15 days, respectively.

[0181] In these studies, administration of humanized peptides targeting human FcRn and albumin was well tolerated, and no serious or major adverse events were observed. In summary, these data indicate that peptides targeting human FcRn and albumin are well tolerated, have a longer serum half-life, mediate faster IgG clearance, and maintain lower serum IgG levels for a longer period of time than igamod. These data also indicate that these molecules exhibit the same activity when administered subcutaneously.

[0182] Without departing from the spirit or essential features of the present invention, the present invention may be embodied in other specific forms. The foregoing embodiments are therefore intended to be illustrative rather than limiting of the present invention in all respects. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes within the meaning and scope of the equivalents of the claims are therefore intended to be included herein. Tables of some sequences

Claims

1. A polypeptide comprising at least one FcRn-binding VHH domain, wherein the at least one FcRn-binding VHH domain comprises a CDR1 sequence selected from SEQ ID NOs: 80-81, a CDR2 sequence selected from SEQ ID NOs: 83-84, and a CDR3 sequence of SEQ ID NO:

85.

2. The polypeptide of claim 1, wherein each VHH domain that binds to FcRn independently comprises a CDR1 sequence selected from SEQ ID NOs: 80-81, a CDR2 sequence selected from SEQ ID NOs: 83-84, and a CDR3 sequence of SEQ ID NO:

85.

3. The polypeptide of claim 1 or 2, wherein at least one FcRn-binding VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 80, 83, and 85; SEQ ID NOs: 81, 83, and 85; and SEQ ID NOs: 81, 84, and 85.

4. The polypeptide of claim 3, wherein each VHH domain that binds FcRn independently comprises a CDR1, CDR2, and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 80, 83, and 85; SEQ ID NOs: 81, 83, and 85; and SEQ ID NOs: 81, 84, and 85.

5. The polypeptide according to any one of claims 1 to 4, wherein at least one FcRn-binding VHH domain is humanized.

6. The polypeptide of claim 5, wherein each VHH domain that binds to FcRn is humanized.

7. The polypeptide of any one of claims 1 to 6, wherein at least one FcRn-binding VHH domain 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: 86-93.

8. The polypeptide of claim 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%, at least 99% identical to a sequence selected from SEQ ID NOs: 86-93.

9. The polypeptide of any one of claims 1 to 7, wherein at least one FcRn-binding VHH domain comprises a sequence selected from the group consisting of SEQ ID NOs: 86-93.

10. The polypeptide of any one of claims 1 to 9, wherein each VHH domain that binds to FcRn comprises a sequence selected from SEQ ID NOs: 86-93.

11. The polypeptide of any one of claims 1 to 10, wherein at least one FcRn-binding VHH domain binds human FcRn with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM.

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

13. The polypeptide of any one of claims 1 to 12, wherein at least one FcRn-binding VHH domain binds human FcRn at pH 6 and pH 7.

4.

14. The polypeptide according to any one of claims 1 to 13, wherein each VHH domain that binds FcRn binds human FcRn at pH 6 and pH 7.

4.

15. The polypeptide of any one of claims 1 to 14, wherein at least one FcRn-binding VHH domain blocks the binding of human IgG to human FcRn.

16. The polypeptide of any one of claims 1 to 15, wherein each VHH domain that binds to FcRn blocks binding of human IgG to human FcRn.

17. The polypeptide according to any one of claims 1 to 16, wherein the polypeptide comprises at least one VHH domain that binds albumin.

18. The polypeptide of claim 17, wherein at least one albumin-binding VHH domain comprises a CDR1 sequence selected from the group consisting of SEQ ID NOs: 5-8, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 9-21, and a CDR3 sequence of SEQ ID NO:

22.

19. The polypeptide of claim 17, wherein each albumin-binding VHH domain independently comprises a CDR1 sequence selected from SEQ ID NOs: 5-8, a CDR2 sequence selected from SEQ ID NOs: 9-21, and a CDR3 sequence of SEQ ID NO:

22.

20. A polypeptide according to claim 17 or 18, wherein at least one albumin binding VHH domain comprises a CDR1, CDR2 and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 5, 9 and 22; SEQ ID NOs: 5, 10 and 22; SEQ ID NOs: 5, 11 and 22; SEQ ID NOs: 5, 12 and 22; SEQ ID NOs: 5, 13 and 22; SEQ ID NOs: 5, 14 and 22; SEQ ID NOs: 5, 15 and 22; SEQ ID NOs: 6, 15 and 22; SEQ ID NOs: 7, 15 and 22; SEQ ID NOs: 8, 15 and 22; SEQ ID NOs: 6, 16 and 22; SEQ ID NOs: 6, 17 and 22; SEQ ID NOs: 6, 18 and 22; SEQ ID NOs: 6, 19 and 22; SEQ ID NOs: 6, 20 and 22; and SEQ ID NOs: 6, 21 and 22.

21. A polypeptide according to claim 17 or 18, wherein each VHH domain that binds albumin independently comprises a CDR1, CDR2 and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 5, 9 and 22; SEQ ID NOs: 5, 10 and 22; SEQ ID NOs: 5, 11 and 22; SEQ ID NOs: 5, 12 and 22; SEQ ID NOs: 5, 13 and 22; SEQ ID NOs: 5, 14 and 22; SEQ ID NOs: 5, 15 and 22; SEQ ID NOs: 6, 15 and 22; SEQ ID NOs: 7, 15 and 22; SEQ ID NOs: 8, 15 and 22; SEQ ID NOs: 6, 16 and 22; SEQ ID NOs: 6, 17 and 22; SEQ ID NOs: 6, 18 and 22; SEQ ID NOs: 6, 19 and 22; SEQ ID NOs: 6, 20 and 22; and SEQ ID NOs: 6, 21 and 22.

22. The polypeptide according to any one of claims 17 to 21, wherein at least one albumin binding VHH domain is humanized.

23. The polypeptide of claim 22, wherein each VHH domain that binds albumin is humanized.

24. A polypeptide according to any one of claims 17 to 23, wherein at least one albumin binding VHH domain 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: 23-43 and 97-100.

25. A polypeptide according to any one of claims 17 to 23, wherein each VHH domain that binds to albumin 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: 23-43 and 97-100.

26. The polypeptide of any one of claims 17 to 23, wherein at least one albumin binding VHH domain comprises a sequence selected from the group consisting of SEQ ID NOs: 23-43 and 97-100.

27. The polypeptide of any one of claims 17 to 23, wherein each VHH domain that binds albumin comprises a sequence selected from SEQ ID NOs: 23-43 and 97-100.

28. The polypeptide of any one of claims 17 to 27, wherein the polypeptide 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: 69-77.

29. The polypeptide according to any one of claims 17 to 27, wherein the polypeptide comprises a sequence selected from SEQ ID NO: 69-77.

30. The polypeptide of any one of claims 17 to 29, wherein at least one albumin binding VHH domain binds human albumin and at least one albumin selected from cynomolgus monkey, mouse and rat albumin.

31. The polypeptide of any one of claims 17 to 29, wherein each VHH domain that binds albumin binds human albumin and at least one albumin selected from cynomolgus monkey, mouse and rat albumin.

32. The polypeptide of any one of claims 17 to 29, wherein at least one albumin binding VHH domain binds human, cynomolgus monkey, mouse and rat albumin.

33. A polypeptide according to any one of claims 17 to 29, wherein each VHH domain that binds albumin binds human, cynomolgus monkey, mouse and rat albumin.

34. The polypeptide of any one of claims 17 to 33, wherein at least one albumin binding VHH domain binds human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM.

35. The polypeptide of any one of claims 17 to 34, wherein at least one albumin binding VHH domain binds to each of human, cynomolgus monkey, mouse and rat albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM.

36. A polypeptide according to any one of claims 17 to 35, wherein each VHH domain that binds albumin binds human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM.

37. A polypeptide according to any one of claims 17 to 36, wherein each VHH domain that binds albumin binds to each of human, cynomolgus monkey, mouse and rat albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM.

38. The polypeptide of any one of claims 17 to 37, wherein each VHH domain that binds albumin does not bind albumin domain 3.

39. The polypeptide of any one of claims 17 to 38, wherein each of the albumin binding VHH domains does not interfere with the binding of albumin to FcRn.

40. The polypeptide of any one of the preceding claims, wherein the polypeptide comprises at least two, at least three or at least four FcRn binding VHH domains and at least one albumin binding VHH domain.

41. The polypeptide of claim 40, wherein the polypeptide comprises two, three or four FcRn binding VHH domains and one albumin binding VHH domain.

42. The polypeptide of any one of claims 1 to 41, wherein the polypeptide comprises at least one binding domain that binds a protein other than albumin or FcRn.

43. The polypeptide of claim 42, wherein at least one binding domain that binds a protein other than albumin or FcRn is a VHH.

44. The polypeptide of claim 43, wherein each binding domain that binds a protein other than albumin or FcRn is a VHH.

45. The polypeptide of claim 42 or 43, wherein at least one binding domain that binds a protein other than albumin or FcRn comprises a heavy chain variable region and a light chain variable region.

46. ​​The polypeptide of claim 45, wherein each binding domain that binds a protein other than albumin or FcRn comprises a heavy chain variable region and a light chain variable region.

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

48. The polypeptide of claim 47, wherein each binding domain that binds a protein other than albumin is a binding domain of a therapeutic antibody.

49. The polypeptide of claim 47 or 48, wherein the therapeutic antibody is useful for treating a disease or condition selected from the group consisting of an autoimmune disease or condition, an inflammatory disease or condition, an infection, and cancer.

50. The polypeptide of any one of claims 1 to 41, wherein the polypeptide comprises the amino acid sequence of a therapeutic protein.

51. The polypeptide of claim 50, wherein the therapeutic protein is useful for treating a disease or condition selected from the group consisting of an autoimmune disease or condition, an inflammatory disease or condition, an infection, and cancer.

52. The polypeptide of any one of the preceding claims, wherein the half-life of the polypeptide is longer than the half-life of the same polypeptide lacking the albumin binding VHH domain.

53. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 52 and a pharmaceutically acceptable carrier.

54. An isolated nucleic acid encoding the polypeptide of any one of claims 1 to 52.

55. A vector comprising the nucleic acid according to claim 54.

56. A host cell comprising the nucleic acid of claim 54 or the vector of claim 55.

57. A host cell expressing the polypeptide according to any one of claims 1 to 52.

58. A method of producing a polypeptide according to any one of claims 1 to 52, comprising cultivating a host cell according to claim 56 or 57 under conditions suitable for expression of the polypeptide.

59. The method of claim 58, further comprising isolating the antibody or polypeptide.

60. A method comprising administering to a subject a polypeptide according to any one of claims 1 to 52 or a pharmaceutical composition according to claim 53.

61. A method for treating a disease or condition, comprising administering a pharmaceutically effective amount of the polypeptide according to any one of claims 1 to 52 or the pharmaceutical composition according to claim 53 to a subject suffering from the disease or condition.

62. The method of claim 61, wherein the disease or disorder is selected from an autoimmune disease or disorder, an inflammatory disease or disorder, an infection, and cancer.

63. The method of claim 61 or 62, wherein the disease or disorder is an autoantibody-mediated disease or disorder.

64. The method of any one of claims 61 to 63, wherein the disease or condition is pemphigus vulgaris, lupus nephritis, myasthenia gravis, Guillain-Barré syndrome, antibody-mediated rejection, antiphospholipid antibody syndrome, chronic inflammatory demyelinating polyneuropathy, immune complex-mediated vasculitis, glomerulitis, ion channelopathy, neuromyelitis optica, autoimmune encephalitis, autoimmune Grave's disease, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, immune neutropenia, dilated cardiomyopathy, or serum sickness.

65. The method of any one of claims 60 to 64, wherein the polypeptide is administered subcutaneously.

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