Albumin-binding polypeptide and its use

Abstract

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

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 338,629, filed May 5, 2022, and U.S. Provisional Application No. 63 / 351,362, filed Jun. 11, 2022, the entire contents of which are incorporated herein by reference for all purposes.

[0002] [Incorporation by Reference of Sequence Listing] This application incorporates by reference a Sequence Listing of 133,294 bytes in size, titled 01202 - 0020 - 00PCT_ST26, created on Apr. 27, 2023, and submitted in electronic format together with this application.

[0003] The present invention relates to albumin - binding polypeptides and methods of using albumin - binding polypeptides to, for example, improve the half - life of other molecules.

Background Art

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

[0005] A long plasma half-life is useful for a therapeutic agent to precisely and accurately control plasma drug concentration, optimize efficacy while limiting toxicity, and reduce the frequency and amount of drug required. Therapeutic proteins with a short plasma half-life due to their small size or lacking the ability to be recycled via FcRn binding can have their plasma half-life extended by fusion with an albumin conjugate. The single-domain antibody VHH domain is a small domain with a size of about 12 kDa to 15 kDa, a single domain composed of a single polypeptide that can be easily fused to another protein or peptide by recombinant means, is easily humanized to reduce the potential for immunogenicity, and many naturally bind to protein A for affinity purification, making it an ideal albumin conjugate.

Summary of the Invention

[0006] Therefore, a VHH domain that binds to albumin is required.

Means for Solving the Problems

[0007] Embodiment 1. A polypeptide comprising at least one VHH domain that binds to albumin, wherein the at least one VHH domain that binds to albumin comprises a CDR1 sequence selected from SEQ ID NO: 5 to SEQ ID NO: 8, a CDR2 sequence selected from SEQ ID NO: 9 to SEQ ID NO: 21, and a CDR3 sequence of SEQ ID NO: 22.

[0008] Embodiment 2. Each VHH domain that binds to albumin independently comprises a CDR1 sequence selected from SEQ ID NO: 5 to SEQ ID NO: 8, a CDR2 sequence selected from SEQ ID NO: 9 to SEQ ID NO: 21, and a CDR3 sequence of SEQ ID NO: 22, the polypeptide according to Embodiment 1.

[0009] Embodiment 3. At least one VHH domain that binds to albumin comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO:5, SEQ ID NO:9, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:10, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:11, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:12, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:13, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:14, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:7, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:8, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:16, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:17, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:18, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:19, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:20, and SEQ ID NO:22; and SEQ ID NO:6, SEQ ID NO:21, and SEQ ID NO:22, and is a polypeptide according to Embodiment 1 or Embodiment 2.

[0010] Embodiment 4. Each VHH domain that binds to albumin independently comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO:5, SEQ ID NO:9, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:10, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:11, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:12, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:13, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:14, and SEQ ID NO:22; SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:7, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:8, SEQ ID NO:15, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:16, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:17, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:18, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:19, and SEQ ID NO:22; SEQ ID NO:6, SEQ ID NO:20, and SEQ ID NO:22; and SEQ ID NO:6, SEQ ID NO:21, and SEQ ID NO:22, and is a polypeptide according to Embodiment 3.

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

[0012] Embodiment 6. Each VHH domain that binds to albumin is a humanized polypeptide according to Embodiment 5.

[0013] Embodiment 7. The at least one 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 to 43 and SEQ ID NOs: 71 to 74, and is a polypeptide according to any one of Embodiments 1 to 6.

[0014] Embodiment 8. 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 to 43 and SEQ ID NOs: 71 to 74, and is a polypeptide according to Embodiment 7.

[0015] Embodiment 9. The at least one VHH domain that binds to albumin comprises a sequence selected from SEQ ID NOs: 23 to 43 and SEQ ID NOs: 71 to 74, and is a polypeptide according to any one of Embodiments 1 to 7.

[0016] Embodiment 10. Each VHH domain that binds to albumin comprises a sequence selected from SEQ ID NOs: 23 to 43 and SEQ ID NOs: 71 to 74, and is a polypeptide according to any one of Embodiments 1 to 9.

[0017] Embodiment 11. The at least one VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 1 to 10, which binds to human albumin and binds to at least one albumin selected from cynomolgus monkey, mouse, and rat albumin.

[0018] Embodiment 12. Each VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 1 to 11, which binds to human albumin and binds to at least one albumin selected from cynomolgus monkey, mouse, and rat albumin.

[0019] Embodiment 13. The at least one VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 1 to 12, which binds to human, cynomolgus monkey, mouse, and rat albumin.

[0020] Embodiment 14. Each VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 1 to 13, which binds to human, cynomolgus monkey, mouse, and rat albumin.

[0021] Embodiment 15. The at least one VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 1 to 14, which binds to human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

[0022] Embodiment 16. The at least one VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 1 to 15, which 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.

[0023] Embodiment 17. Each VHH domain that binds to albumin is the polypeptide according to any one of Embodiments 1 to 16, which binds to human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

[0024] Embodiment 18. The polypeptide according to any one of Embodiments 1 to 17, wherein each VHH domain that binds to albumin binds to each of human, cynomolgus monkey, mouse, and rat albumins with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

[0025] Embodiment 19. The polypeptide according to any one of Embodiments 1 to 18, wherein each VHH domain that binds to albumin does not bind to albumin domain 3.

[0026] Embodiment 20. The polypeptide according to any one of Embodiments 1 to 19, wherein each VHH domain that binds to albumin does not interfere with the binding of albumin to FcRn.

[0027] Embodiment 21. The polypeptide according to any one of Embodiments 1 to 20, wherein the polypeptide comprises at least one binding domain that binds to a protein other than albumin.

[0028] Embodiment 22. The polypeptide according to Embodiment 21, wherein at least one binding domain that binds to a protein other than albumin is a VHH.

[0029] Embodiment 23. The polypeptide according to Embodiment 22, wherein each binding domain that binds to a protein other than albumin is a VHH.

[0030] Embodiment 24. The polypeptide according to Embodiment 21, wherein at least one binding domain that binds to a protein other than albumin comprises a heavy chain variable region and a light chain variable region.

[0031] Embodiment 25. The polypeptide according to Embodiment 24, wherein each binding domain that binds to a protein other than albumin comprises a heavy chain variable region and a light chain variable region.

[0032] Embodiment 26. The polypeptide according to any one of Embodiments 21 to 25, wherein at least one binding domain that binds to a protein other than albumin is a binding domain of a therapeutic antibody.

[0033] Embodiment 27. The polypeptide according to Embodiment 26, wherein each binding domain that binds to a protein other than albumin is a binding domain of a therapeutic antibody.

[0034] Embodiment 28. The polypeptide according to Embodiment 26 or 27, wherein the therapeutic antibody is useful for the treatment of a disease or disorder selected from autoimmune diseases or disorders, inflammatory diseases or disorders, infectious diseases, and cancers.

[0035] Embodiment 29. The polypeptide according to any one of Embodiments 1 to 28, wherein the polypeptide comprises the amino acid sequence of a therapeutic protein.

[0036] Embodiment 30. The polypeptide according to Embodiment 29, wherein the therapeutic protein is useful for the treatment of a disease or disorder selected from autoimmune diseases or disorders, inflammatory diseases or disorders, infectious diseases, and cancers.

[0037] Embodiment 31. The polypeptide according to any one of Embodiments 1 to 30, wherein the polypeptide comprises an Fc region.

[0038] Embodiment 32. The polypeptide according to Embodiment 31, wherein the Fc region binds to FcRn.

[0039] Embodiment 33. The polypeptide according to Embodiment 31 or 32, wherein the Fc region is an IgG1 Fc region.

[0040] Embodiment 34. The polypeptide according to any one of Embodiments 31 to 33, wherein the Fc region comprises one or more substitutions that enhance the half-life.

[0041] Embodiment 35. The polypeptide according to embodiment 34, wherein the Fc region comprises one or more substitutions that enhance FcRn binding at at least one pH and / or decrease the dissociation rate between Fc and FcRn.

[0042] Embodiment 36. The polypeptide according to any one of embodiments 31 - 35, wherein the Fc region comprises substitutions at one or more amino acid positions selected from 252, 254, 256, 428, or 434.

[0043] Embodiment 37. The polypeptide according to embodiment 36, wherein the Fc region comprises substitutions at amino acid positions 252, 254, and 256; or amino acid positions 252 and 428; or amino acid positions 428 and 434.

[0044] Embodiment 38. The polypeptide according to embodiment 37, wherein the Fc region comprises substitutions M252Y, S254T, and T256E; M252Y and M428V; or M428L and N434S.

[0045] Embodiment 39. The polypeptide according to any one of embodiments 31 - 38, wherein the Fc region comprises a sequence selected from SEQ ID NOs: 47 - SEQ ID NOs: 68 and SEQ ID NOs: 85 - SEQ ID NOs: 87.

[0046] Embodiment 40. The polypeptide according to any one of embodiments 1 - 39, wherein the half-life of the polypeptide is longer than the half-life of the same polypeptide lacking a VHH domain that binds to albumin.

[0047] Embodiment 41. A pharmaceutical composition comprising the polypeptide according to any one of embodiments 1 - 40 and a pharmaceutically acceptable carrier.

[0048] Embodiment 42. An isolated nucleic acid encoding the polypeptide according to any one of embodiments 1 - 40.

[0049] Embodiment 43. A vector comprising the nucleic acid according to embodiment 42.

[0050] Embodiment 44. A host cell comprising the nucleic acid according to Embodiment 42 or the vector according to Embodiment 43.

[0051] Embodiment 45. A host cell that expresses the polypeptide according to any one of Embodiments 1 to 40.

[0052] Embodiment 46. A method for producing the polypeptide according to any one of Embodiments 1 to 40, the method comprising incubating the host cell according to Embodiment 44 or 45 under conditions suitable for the expression of the polypeptide.

[0053] Embodiment 47. The method according to Embodiment 46, further comprising isolating the polypeptide.

[0054] Embodiment 48. A method comprising administering to a subject the polypeptide according to any one of Embodiments 1 to 40 or the pharmaceutical composition according to Embodiment 41.

[0055] Embodiment 49. A method for treating a disease or disorder, the method comprising administering to a subject suffering from the disease or disorder a pharmaceutically effective amount of the polypeptide according to any one of Embodiments 1 to 40 or the pharmaceutical composition according to Embodiment 41.

[0056] Embodiment 50. The method according to Embodiment 49, wherein the disease or disorder is selected from autoimmune diseases or disorders, inflammatory diseases or disorders, infectious diseases, and cancers.

Brief Description of the Drawings

[0057]

Figure 1A - 1C

Figure 2A - 2B

Figure 3A - 3B

Figure 4A - 4I

Figure 5A - 5D

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Figure 7A - 7B

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Figure 9A - 9D

Figure 10

Figure 11A - 11B

Figure 12A - 12D

Modes for Carrying Out the Invention

[0058] The embodiments shown herein relate to albumin - binding polypeptides and their use.

[0059] Definitions and Various Embodiments The section headings used herein are for the purpose of organization only and are not to be construed as limiting the subject matter described.

[0060] All references cited herein, including patent applications, patent publications, and Genbank accession numbers, are hereby incorporated by reference as if each individual reference were specifically and individually indicated as being incorporated by reference in its entirety to form a part of this specification.

[0061] The techniques and procedures described or referenced in this specification are generally well understood and commonly used, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)), the series METHODS IN ENZYMOLOGY (Academic Press, Inc.), PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)), Oligonucleotide Synthesis (M. J. Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press, Animal Cell Culture (R. I. Freshney, ed., 1987), Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press, Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell eds., 1993-8) J. Wiley and Sons, Handbook of Experimental Immunology (D. M. Weir and C.C. Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (J. E. Coligan et al. eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C. A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988 - 1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company, 1993) and the commonly used methodologies described in their latest editions, such as those described by those skilled in the art, are used using conventional methodologies by those skilled in the art.

[0062] Unless otherwise specified, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those skilled in the art. Further, unless the context requires otherwise or there is an express specific indication to the contrary, singular nouns shall include the plural and plural nouns shall include the singular. In case of any conflict in definitions between various sources or references, the definitions set forth in this specification shall prevail.

[0063] Generally, the numbering of residues in the immunoglobulin heavy chain constant regions is by the EU index numbering as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibody.

[0064] Embodiments of the invention described herein are understood to include "consisting" embodiments and / or "consisting essentially of" embodiments. As used herein, the singular forms ("a", "an", and "the") include plural references unless otherwise specifically indicated. The use of the term "or" herein is not to be construed as meaning that the alternatives are mutually exclusive.

[0065] In this application, the use of "or" means "and / or" unless expressly specified otherwise or not understood by those skilled in the art. In the context of multiple dependent claims, the use of "or" refers to the citation of two or more preceding independent or dependent claims.

[0066] The terms "reference sample", "reference cell", or "reference tissue" refer to a sample having at least one known characteristic that can be used for comparison with a sample having at least one unknown characteristic. In some embodiments, the reference sample can be used as a positive or negative indicator. By using a reference sample, it is possible to establish, for example, the levels of proteins and / or mRNAs present in a sample having unknown characteristics relative to the levels of proteins and / or mRNAs present in healthy tissue. In some embodiments, the reference sample is a sample that is from the same subject but from a different part of the subject than the part being tested. In some embodiments, the reference sample is a sample from a tissue region surrounding or adjacent to cancer. In some embodiments, the reference sample is not from the subject being tested but is from a subject known to have or not have the disorder of interest. In some embodiments, the reference sample is from the same subject but at a time point before the subject develops cancer. In some embodiments, the reference sample is a sample from a benign cancer sample from the same or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule of interest in the negative reference sample indicates a level at which one of ordinary skill in the art, considering the present disclosure, would recognize that the molecule is absent and / or that a low level of the molecule is present. When a positive reference sample is used for comparison, the expression level or amount of the molecule of interest in the positive reference sample indicates a level at which one of ordinary skill in the art, considering the present disclosure, would recognize that a certain level of the molecule is present.

[0067] As used herein in the context of a subject receiving a benefit from or responding to administration of a therapeutic agent, the terms "benefit", "clinical benefit", "responsiveness", and "therapeutic responsiveness" can be gauged by assessing various endpoints, such as inhibition of disease progression, including deceleration and complete cessation; reduction in the number of disease episodes and / or symptoms; reduction in lesion size; inhibition (i.e., reduction, deceleration, or complete cessation) of infiltration of diseased cells into adjacent peripheral organs and / or peripheral tissues; inhibition (i.e., reduction, deceleration, or complete cessation) of disease spread; some alleviation of one or more symptoms associated with the disorder; disease-free presentation after treatment, such as an increase in the length of the progression-free survival period, an increase in the overall survival period, a higher response rate, and / or a decrease in the mortality rate at a given time point after treatment. A "non-responsive" or "non-responding" subject or cancer is one that does not meet the above conditions for "responding".

[0068] The terms "nucleic acid molecule", "nucleic acid", and "polynucleotide" are used interchangeably and can refer to a polymer of nucleotides. Such a polymer of nucleotides can include natural and / or non-natural nucleotides and can include, but is not limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0069] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues include natural or unnatural amino acid residues and, without limitation, may include peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition includes both full-length proteins and fragments thereof. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Further, for the purposes of the present disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative with respect to properties) to the native sequence, so long as the protein maintains the desired activity. These modifications can be intentional, such as by site-directed mutagenesis, or can be accidental, such as by mutations in the host producing the protein or errors during PCR amplification.

[0070] As used herein, "albumin" refers to any natural mature albumin resulting from the processing of albumin precursors within cells. This term includes albumin from any vertebrate origin, including, without limitation, mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. This term also includes naturally occurring variants of albumin, such as splice variants or allelic variants. A non-limiting exemplary mature human albumin amino acid sequence is shown, for example, in UniProt accession number P02768.2. See SEQ ID NO: 1. Non-limiting exemplary mouse, cynomolgus monkey, and rat albumin amino acid sequences are shown in SEQ ID NOs: 2-4.

[0071] The term "specifically binds to" an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" when it reacts or associates more frequently, more rapidly, for a longer duration, and / or with a higher affinity with a particular cell or substance than with another cell or substance. A single domain antibody (sdAb) or VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target when it binds with a higher affinity, 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 albumin epitope is an sdAb or VHH-containing polypeptide that binds to this epitope with a higher affinity, avidity, more readily, and / or for a longer duration than it binds to other albumin epitopes or non-albumin epitopes. Also, by interpreting this definition, it is understood that, for example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, although not always, a reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0072] As used herein, the term "inhibits" with respect to the activity of a target protein refers to a decrease in the activity of the protein. In some embodiments, "inhibits" refers to a decrease in activity as compared to the protein in the absence of the modulator.

[0073] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an sdAb or a VHH-containing polypeptide) binds. Epitopes often include chemically active surface compilations of molecules such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural features and specific charge features. Epitopes can be formed from both contiguous residues and / or juxtaposed non-contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are usually retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes can 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 epitope has a length of less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues. Two antibodies can bind to the same epitope within an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope can be specified by a certain minimum distance from CDR residues on the antigen-binding molecule. In some embodiments, an epitope can be specified by such a distance and can be further limited to those residues involved in a bond (e.g., a hydrogen bond) between a residue of the antigen-binding molecule and an antigen residue. Epitopes can also be specified by various scans. For example, an alanine scan or an arginine scan can indicate one or more residues with which an antigen-binding molecule can interact. Unless explicitly indicated otherwise, a set of residues as an epitope does not exclude other residues since it is part of the epitope for a particular antigen-binding molecule. Rather, the presence of such a set indicates a minimal epitope sequence (or set of types). Thus, in some embodiments, a set of residues specified as an epitope does not represent an exclusive list of residues for the epitope on the antigen, but rather indicates the minimal epitope associated with the antigen.

[0074] A "non-linear epitope" or "conformational epitope" comprises discontinuous polypeptides, amino acids and / or sugars within an antigenic protein to which an antigen-binding molecule specific for the epitope binds. In some embodiments, at least one residue is discontinuous with other indicated residues of the epitope, although one or more residues may be continuous with other residues.

[0075] A "linear epitope" comprises continuous polypeptides, amino acids and / or sugars within an antigenic protein to which an antigen-binding molecule specific for the epitope binds. It should be noted that in some embodiments, not all of the residues within a linear epitope need to be directly bound (or involved in binding) by the antigen-binding molecule. In some embodiments, a linear epitope may be derived from immunization with a peptide consisting essentially of the sequence of the linear epitope, or may be derived from a structural region of a protein that is relatively separated from the remainder of the protein (such that the antigen-binding molecule can interact with at least primarily just that sequence region).

[0076] The term "antibody" is used in the broadest sense and includes, without limitation, conventional antibodies (typically comprising at least one heavy chain and at least one light chain) and fragments thereof (e.g., scFv, Fab), single domain antibodies (sdAb, comprising at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and various polypeptides comprising an antibody-like antigen-binding domain including any of the above fragments so long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include, without limitation, heavy chain constant domains (including CH1, hinge, CH2, and CH3, where CH1 typically pairs with the light chain constant domain CL while the hinge mediates dimerization) and Fc regions (including hinge, CH2, and CH3, where the hinge mediates dimerization).

[0077] The term "antibody" includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as camel (including llama), shark, mouse, human, cynomolgus monkey, etc.

[0078] As used herein, the term "antigen-binding domain" refers to the portion of an antibody sufficient to bind an antigen. In some embodiments, the antigen-binding domain of a conventional antibody comprises three heavy-chain CDRs and three light-chain CDRs. Thus, in some embodiments, the antigen-binding domain comprises a heavy-chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 necessary to maintain binding to the antigen, and a light-chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 necessary to maintain binding to the antigen. In some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises the three CDRs of the VHH domain. Thus, in some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises a VHH domain comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 necessary to maintain binding to the antigen.

[0079] 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 camel antibody or a shark antibody. In some embodiments, a VHH comprises three CDRs and four framework regions designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N-terminus and / or C-terminus such that it comprises only partial FR1 and / or FR4 or lacks one or both of those framework regions, so long as the VHH substantially maintains antigen binding and specificity.

[0080] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody comprising at least one monomeric domain such as a VHH domain that lacks a light chain and an Fc region. In some embodiments, the sdAb is a dimer of two polypeptides, each polypeptide comprising at least one VHH domain and an Fc region. As used herein, the terms "single domain antibody" and "sdAb" refer to polypeptides comprising multiple VHH domains, e.g., structured VHH 1 -VHH 2 -Fc or VHH 1 -VHH 2 -VHH 3 -Fc, where VHH 1 , VHH 2 , and VHH 3 may be the same or different and include polypeptides.

[0081] The term "VHH-containing polypeptide" refers to a polypeptide comprising at least one VHH domain. In some embodiments, the VHH polypeptide comprises two, three, or four or more VHH domains, where each VHH domain may be the same or different. In some embodiments, the VHH-containing polypeptide comprises an Fc region. In some such embodiments, the VHH-containing polypeptide may be referred to as an sdAb. Further, in some such embodiments, the VHH polypeptide may form a dimer. Non-limiting structures of VHH-containing polypeptides, also referred to as sdAbs, include VHH 1 -Fc, VHH 1 -VHH 2 -Fc, and VHH 1 -VHH 2 -VHH 3 -Fc, where VHH 1 , VHH 2 , and VHH 3may be the same or different. In some embodiments of such a structure, one VHH may be linked to another VHH by a linker, or one VHH may be linked to an Fc by a linker. In some such embodiments, the linker comprises from 1 to 20 amino acids, preferably from 1 to 20 amino acids consisting mainly of glycine and optionally serine. Non-limiting linkers are shown in SEQ ID NOs: 88 to 93. In some embodiments, when the VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, the structure VHH 1 -VHH 2 -Fc is considered to be tetravalent when it forms a dimer (i.e., the dimer has 4 VHH domains). Similarly, the structure VHH 1 -VHH 2 -VHH 3 -Fc is considered to be hexavalent when it forms a dimer (i.e., the dimer has 6 VHH domains).

[0082] The term "monoclonal antibody" refers to antibodies (including sdAb or VHH-containing polypeptides) of a substantially homogeneous antibody population. That is, the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Further, typically, in contrast to polyclonal antibody preparations that contain different antibodies against different determinants (epitopes), each monoclonal antibody is an antibody against a single determinant on the antigen. Thus, a sample of monoclonal antibodies can bind to the same epitope on the antigen. The modifying phrase "monoclonal" indicates the nature of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies can be produced by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods as described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries produced using techniques described, for example, in McCafferty et al., 1990, Nature 348:552-554.

[0083] The term "CDR" refers to complementarity-determining regions defined by at least one particular scheme for one of ordinary skill in the art. In some embodiments, the CDRs can 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. VHH contains three CDRs designated CDR1, CDR2, and CDR3.

[0084] As used herein, the term "heavy chain constant region" refers to at least three heavy chain constant domains, namely, C H 1, the hinge, C H 2, and C HRefers to the region containing 3. Of course, deletions and modifications that do not change the function within the domain are included within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to one antibody isotype. For example, an antibody containing the γ constant region is an IgG antibody, an antibody containing the δ constant region is an IgD antibody, and an antibody containing the α constant region is an IgA antibody. Furthermore, an antibody containing the μ constant region is an IgM antibody, and an antibody containing the ε constant region is an IgE antibody. A particular isotype can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 antibody (containing the γ 1 constant region), IgG2 antibody (containing the γ 2 constant region), IgG3 antibody (containing the γ 3 constant region), and IgG4 antibody (containing the γ 4 constant region); IgA antibodies include, but are not limited to, IgA1 antibody (containing the α 1 constant region) and IgA2 antibody (containing the α 2 constant region); IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0085] As used herein, "Fc region" refers to a portion of the heavy chain constant region that includes CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In various embodiments, when the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is derived from a human Fc region and lacks a C-terminal lysine residue. In some embodiments, the Fc region is derived from a human Fc region and includes a C-terminal lysine residue. In some embodiments, the C-terminal amino acid of the Fc region is an amino acid other than lysine. In some embodiments, the Fc region is derived from a human Fc region and lacks C-terminal glycine and lysine residues.

[0086] As used herein, "acceptor human framework" is a framework that includes the amino acid sequence of the heavy chain variable domain (V H ) framework derived from a human immunoglobulin framework or a human consensus framework, as discussed herein. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can include the same amino acid sequence thereof, or can include changes in the amino acid sequence. In some embodiments, the number of amino acid changes is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3 across all human frameworks within a single antigen-binding domain such as a VHH.

[0087] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody such as an sdAb or a VHH-containing polypeptide) and its binding partner (e.g., an antigen). The affinity or apparent affinity of a molecule X for its partner Y is generally the dissociation constant (K D ) or K D(見かけ)It can be represented by. Affinity can be measured by conventional methods known in the art, including the methods described herein (e.g., ELISA K D , KinExA, flow cytometry, and / or surface plasmon resonance devices, etc.). Such methods include, but are not limited to, methods requiring BIAcore (registered trademark), Octet (registered trademark), or flow cytometry.

[0088] 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 " is used herein, it includes K D and K D(見かけ) .

[0089] In some embodiments, the K D of the antigen-binding molecule is measured by flow cytometry using an antigen-expressing cell line and fitting the mean fluorescence measured at each antibody concentration to a non-linear one-site binding equation (graphpad's Prism Software). In some such embodiments, K D is K D(見かけ) .

[0090] The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as seen in vivo, or provided or made possible by recombinant means). Biological properties include, but are not limited to, ligand binding, induction or increase of cell proliferation, and induction or increase of cytokine expression.

[0091] An "agonist" antibody or "activating" antibody is an antibody that increases and / or activates the biological activity of a target antigen. In some embodiments, the agonist antibody binds to the antigen and increases its biological activity by at least about 20%, 40%, 60%, 80%, 85% or more.

[0092] An "antagonist" antibody, "blocking" antibody, or "neutralizing" antibody is an antibody that inhibits, reduces, and / or inactivates the biological activity of a target antigen. In some embodiments, a neutralizing antibody binds to an antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99% or more.

[0093] An "affinity matured" sdAb or VHH-containing polypeptide refers to an sdAb or VHH-containing polypeptide that has one or more such modifications in one or more CDRs compared to a parental sdAb or VHH-containing polypeptide that has no modifications that confer an improvement in the affinity of the sdAb or VHH-containing polypeptide for an antigen.

[0094] As used herein, a "humanized VHH" refers to a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some cases, certain framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Further, a humanized VHH may contain residues that are not found in either the original VHH or the human framework sequence, but are included to further improve and optimize the performance of the sdAb or VHH-containing polypeptide. In some embodiments, a humanized sdAb or VHH-containing polypeptide includes a human Fc region. As will be understood, a humanized sequence can be identified by its primary sequence and does not necessarily indicate the process by which the antibody was made.

[0095] 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, among others. Such effector functions generally require the combination of an Fc region with a binding domain (e.g., an antibody variable domain) and can be evaluated using a variety of assays.

[0096] The "Fc region of a native sequence" includes an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. Examples of the Fc region of a native sequence of a human include the Fc region of a native sequence of human IgG1 (non-A allotype and A allotype), the Fc region of a native sequence of human IgG2, the Fc region of a native sequence of human IgG3, and the Fc region of a native sequence of human IgG4, as well as naturally occurring variants thereof.

[0097] The "mutant Fc region" includes an amino acid sequence different from the amino acid sequence of the Fc region of a native sequence due to at least one amino acid modification. In some embodiments, the "mutant Fc region" includes an amino acid sequence different from the amino acid sequence of the Fc region of a native sequence due to at least one amino acid modification, but retains at least one effector function of the Fc region of a native sequence. In some embodiments, the mutant Fc region has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the Fc region of a native sequence or the Fc region of a parent polypeptide as compared to the Fc region of a native sequence or the Fc region of a parent polypeptide. In some embodiments, the mutant Fc region of the present specification has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the Fc region of a native sequence and / or the Fc region of a parent polypeptide.

[0098] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR binds to IgG antibodies (gamma receptors) and is a receptor of the FcγRI subclass, FcγRII subclass, and FcγRIII subclass, including allelic variants and alternatively spliced forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences but mainly differ in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which plays a role in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin isotype.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.)).

[0099] As used herein, the terms "substantially similar" or "substantially the same" indicate a sufficiently high degree of similarity between two or more numerical values such that one of ordinary skill in the art would consider the difference between the two or more values to have little or no biological significance and / or statistical significance within the context of the biological characteristics measured by the above values. In some embodiments, two or more substantially similar values differ by no more than one of the approximate values of 5%, 10%, 15%, 20%, 25%, or 50%.

[0100] A polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Such variants include, for example, polypeptides having one or more amino acid residues added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, the variant has at least about 80% amino acid sequence identity. In some embodiments, the variant has at least about 90% amino acid sequence identity. In some embodiments, the variant has at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0101] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide sequence, polypeptide sequence, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide sequence or polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignments for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve the maximum alignment over the full length of the sequences being compared.

[0102] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into the antibody of interest and the product screened for retention / improvement of a desired activity, such as antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC.

[0103] [Table 1]

[0104] Amino acids can be grouped according to common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that influence chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0105] Non-conservative substitutions involve exchanging one member of these classes for another.

[0106] The term "vector" is used to describe a polynucleotide that can be manipulated to contain a cloned polynucleotide(s) that can be propagated within a host cell. A vector can contain one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., a promoter and / or enhancer, etc.) that regulate the expression of the polypeptide of interest, and / or one or more selectable marker genes (e.g., an antibiotic resistance gene and genes that can be used in a colorimetric assay, e.g., β-galactosidase, etc.). The term "expression vector" refers to a vector that is used to express the polypeptide of interest in a host cell.

[0107] "Host cell" refers to a cell that can be or has been a recipient of a vector or an isolated polynucleotide. A host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate animal cells, fungal cells such as yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6™ cells (Crucell), as well as 293 cells and CHO cells, and their derivatives, such as 293-6E cells, CHO-DG44 cells, CHO-K1 cells, CHO-S cells, and CHO-DS cells. Host cells include the progeny of a single host cell, but due to natural mutations, accidental mutations, or intentional mutations, the progeny are not necessarily identical (in morphology or genomic DNA complement) to the original parent cell. Host cells also include cells that have been transfected in vivo with the polynucleotide(s) provided herein.

[0108] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components that are typically found together or produced together in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least a portion of the components of the cell that produced it. When a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered "isolating" the polypeptide. Similarly, a polynucleotide is "isolated" when it is not part of a larger polynucleotide (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) in which it is typically found in nature, or when, for example, in the case of an RNA polynucleotide, it is separated from at least a portion of the components of the cell that produced it. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

[0109] The terms "individual" and "subject" are used interchangeably herein to refer to an animal, such as a mammal. In some embodiments, but not limited to, methods of treating mammals including humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets are provided. In some examples, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject to be treated can be a patient who has been identified as having or being at sufficient risk of having a disorder associated with the treatment.

[0110] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0111] The term "autoimmune disorder" typically refers to a disease or disorder associated with a non-anaphylactic hypersensitivity reaction (type II, III, and / or type IV hypersensitivity reactions) caused by the subject's own humoral and / or cellular immune response to one or more immunogenic substances of generally endogenous / exogenous origin.

[0112] The term "inflammatory disorder" refers to a disorder associated with inflammation, including but not limited to chronic or acute inflammatory diseases, and explicitly includes inflammatory autoimmune diseases and inflammatory allergic conditions.

[0113] The terms "infection" and "infectious disease or infectious disorder" refer to a disease or disorder caused by exogenous infectious agents such as, but not limited to, bacteria, viruses, fungi, protozoa, and parasites.

[0114] The terms "cancer" and "tumor" include solid cancers and hematological cancers / lymphadenocarcinomas, and also include malignant tumors such as dysplasia, premalignant tumors, and benign tumors. Exemplary cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal carcinoma, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), melanoma, multiple myeloma, neuroblastoma, oral cancer (lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and other carcinomas and sarcomas, as well as post-transplant lymphoproliferative disorder (PTLD), and abnormalities in angiogenesis associated with nevus flammeus, edema (such as edema associated with brain tumors), and the Meigs syndrome.

[0115] In some embodiments, "increase" or "decrease" each refers to a statistically significant increase or decrease. As will be apparent to those skilled in the art, "modulation" also includes a change (which can be either an increase or a decrease) in the affinity, avidity, specificity, and / or selectivity of a target or antigen for one or more of its ligands, binding partners, homomultimers or heteromultimers, partners or substrates that associate thereto, as compared to the same conditions except for the presence of the test agent, a change (which can be either an increase or a decrease) in the sensitivity of the target or antigen to one or more conditions (such as pH, ionic strength, presence of cofactors, etc.) in the medium or environment in which the target or antigen is present, and / or may include cell proliferation or cytokine production. This can be determined in any suitable manner known per se or described herein and / or using any suitable assay, depending on the target involved.

[0116] As used herein, "treatment" is a procedure for obtaining a beneficial or desired clinical outcome. "Treatment" as used herein is directed to any administration or application of a therapeutic agent for a disease in a mammal, including a human. For the purposes of the present disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the degree of the disease, prevention or delay of the progression of the disease (e.g., metastasis, e.g., to the lung or lymph nodes), prevention or delay of the recurrence of the disease, delay or deceleration of the progression of the disease, improvement of the disease state, inhibition of the disease or the progression thereof, inhibition or deceleration of the disease or its progression, prevention of its development, and remission (partial or total). "Treatment" also includes reduction of the pathological consequences of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. Accordingly, the term treatment does not require the complete elimination of all aspects of the disorder by 100%.

[0117] "Improvement" means that one or more symptoms are alleviated or improved as compared to the case where no therapeutic agent is administered. "Improvement" also includes shortening or reduction of the duration of the symptoms.

[0118] The term "anticancer agent" is used herein in its broadest sense to refer to an agent used in the treatment of one or more cancers. Exemplary classes of such agents include, but are not limited to, chemotherapeutic agents, anticancer biologics (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiation therapy agents, CAR-T therapy agents, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNA), and oncolytic viruses.

[0119] The term "biological sample" means an amount of 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, white blood cells, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0120] The term "control" or "reference" refers to a composition known to be free of the analyte ("negative control") or a composition known to contain the analyte ("positive control"). A positive control may contain a known concentration of the analyte.

[0121] The term "inhibit" or "inhibiting" refers to a decrease or cessation of any phenotypic characteristic, or a decrease or cessation in the incidence, degree, or likelihood of that characteristic. "Reducing" or "inhibiting" means decreasing, reducing, or stopping the activity, function, and / or amount as compared to a reference. In some embodiments, "reducing" or "inhibiting" means the ability to cause an overall decrease of 10% or more. In some embodiments, "reducing" or "inhibiting" means the ability to cause an overall decrease of 50% or more. In some embodiments, "reducing" or "inhibiting" means the ability to cause an overall decrease of 75%, 85%, 90%, 95% or more. In some embodiments, the above amounts are inhibited or decreased over a period of time relative to a control over the same period.

[0122] As used herein, "delaying the onset of a disease" means delaying, preventing, decelerating, retarding, stabilizing, suppressing, and / or extending the onset of a disease (such as cancer). This delay can be of various lengths depending on the medical history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or substantial delay can in fact encompass prevention in the sense that the individual does not develop the disease. For example, it is possible to delay advanced cancer such as the occurrence of metastasis.

[0123] "Prevention" as used herein includes providing prevention against the occurrence or recurrence of a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed with the disease. Unless otherwise indicated, the terms "reduce," "inhibit," or "prevent" do not indicate or require complete prevention over the entire period, but rather indicate or require prevention only over the period measured.

[0124] The "therapeutically effective amount" of a substance / molecule, agonist, or antagonist can vary depending on factors such as the individual's disease state, age, gender, and weight, as well as the ability of the substance / molecule, agonist, or antagonist to induce the desired response in the individual. The therapeutically effective amount is also an amount in which the therapeutically beneficial effect exceeds any toxic or harmful effects of the substance / molecule, agonist, or antagonist. The therapeutically effective amount can be delivered in one or more administrations. The therapeutically effective amount refers to an amount effective to achieve the desired therapeutic and / or prophylactic results over the required dosage and over the required time.

[0125] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation in a form that enables the biological activity of the active ingredient(s) (if any) to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is administered. Such formulations can be sterilized.

[0126] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art used with a therapeutic agent that together with the "pharmaceutical composition" is for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation used.

[0127] Administration "in combination" with one or more additional therapeutic agents includes co-administration (simultaneous administration) and sequential administration in any order.

[0128] As used herein, the term "in combination" refers to administration of two or more therapeutic agents where at least a portion of the administrations overlap in time, or where the administration of one therapeutic agent is included in a short period relative to the administration of the other therapeutic agent, or where the therapeutic effects of both therapeutic agents overlap for at least some period of time.

[0129] As used herein, the term "sequentially" refers to administration of two or more therapeutic agents where the administrations do not overlap in time, or where the therapeutic effects of the therapeutic agents do not overlap.

[0130] As used herein, "in combination with" refers to adding another therapy to the administration of a particular therapy. Thus, "in combination with" refers to administering a particular therapy before, during, or after another therapy is administered to an individual.

[0131] The term "package insert" is used to refer to the instructions customarily included in the commercial package of a therapeutic product that contain information regarding the indications, usage, dosage, administration, combination therapies, contraindications and / or warnings regarding the use of such a therapeutic product.

[0132] "Manufactured article" is any manufactured product (e.g., a package or container) or kit that includes at least one reagent, such as a medicament for treating a disease or disorder (e.g., cancer), or a probe that specifically detects a biomarker described herein. In some embodiments, the manufactured product or kit is advertised, distributed, or sold as a unit for performing the methods described herein.

[0133] The terms "label" and "detectable label" mean a moiety that, for example, binds to an antibody or antigen and enables the detection of a reaction (e.g., binding) between members of a specific binding pair. The labeled member of a specific binding pair is referred to as "detectably labeled." Thus, the term "labeled binding protein" refers to a protein into which a label that provides for the identification of the binding protein has been incorporated. In some embodiments, the label is a detectable marker that can generate a signal detectable visually or by instrumental means, such as the incorporation of a radioactively labeled amino acid or the binding of a biotinylated moiety to a polypeptide that can be detected by a labeled avidin (e.g., streptavidin that includes an enzyme activity detectable by a fluorescent marker or by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153(Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include a light-emitting moiety, e.g., an acridinium compound, and a fluorescence-emitting moiety, e.g., fluorescein. In this regard, although the moiety itself may not be detectably labeled, it may become detectable upon reaction with yet another moiety.

[0134] Exemplary albumin-binding polypeptide Single-domain antibodies (sdAbs) comprising a VHH domain that binds to albumin are provided herein. In some embodiments, the VHH domain that binds to albumin does not interfere with the binding of albumin to FcRn. In some embodiments, the VHH domain that binds to albumin does not bind to domain 3 of albumin. In some embodiments, the VHH domain that binds to albumin has an affinity (K D ) for binding of 0.01 nM to 5 nM, or 0.01 nM to 2 nM, or 0.01 nM to 1 nM, 0.01 nM to 0.5 nM, 0.05 nM to 5 nM, or 0.05 nM to 2 nM, or 0.05 nM to 1 nM, or 0.05 nM to 0.5 nM.

[0135] 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.

[0136] In various embodiments, polypeptides that include one or more VHH domains that bind to albumin also include a therapeutic antigen-binding domain and / or a therapeutic polypeptide. Such therapeutic antigen-binding domains include, but are not limited to, the antigen-binding domains of conventional antibodies that include a heavy chain variable region and a light chain variable region, and the antigen-binding domains of single domain antibodies such as VHH domains. Non-limiting formats of polypeptides that include one or more VHH domains that bind to albumin and one or more conventional antibody domains are presented in FIGS. 6(v)-(ix). Other non-limiting exemplary therapeutic polypeptides include, for example, receptor extracellular domains, enzymes, and ligands. In various embodiments, polypeptides that include at least one VHH domain that binds to albumin have a longer half-life in vivo than the same polypeptides that do not include at least one VHH domain that binds to albumin. In some embodiments, the half-life is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold longer than the half-life of a polypeptide that does not include a VHH domain that binds to albumin.

[0137] In some embodiments, a 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.

[0138] In various embodiments, the VHH domain that binds to albumin comprises a CDR1 sequence selected from SEQ ID NO: 5 to SEQ ID NO: 8, a CDR2 sequence selected from SEQ ID NO: 9 to SEQ ID NO: 21, and a CDR3 sequence of SEQ ID NO: 22. In various embodiments, each VHH domain that binds to albumin comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 12, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 14, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 7, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 8, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 16, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 17, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 18, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 19, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 20, and SEQ ID NO: 22; and SEQ ID NO: 6, SEQ ID NO: 21, and SEQ ID NO: 22.

[0139] In some embodiments, the VHH domain that binds to albumin comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence selected from SEQ ID NO: 23 to SEQ ID NO: 43 and SEQ ID NO: 71 to SEQ ID NO: 74. In some embodiments, the VHH domain that binds to albumin comprises an amino acid sequence selected from SEQ ID NO: 23 to SEQ ID NO: 43 and SEQ ID NO: 71 to SEQ ID NO: 74.

[0140] In some embodiments, a VHH domain that binds to albumin is provided, which competes for binding to albumin with a VHH domain comprising an amino acid sequence selected from SEQ ID NO: 23 to SEQ ID NO: 43 and SEQ ID NO: 71 to SEQ ID NO: 74.

[0141] In some embodiments, the VHH domain that binds to albumin can be humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides, etc.) are useful as therapeutic molecules. This is because humanized antibodies reduce or eliminate the human immune response against non-human antibodies that can cause an immune response against the antibody therapeutic agent and reduce the effectiveness of the therapeutic agent. Generally, a humanized antibody comprises one or more variable domains in which the CDR (or a portion thereof) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. A humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve, for example, the specificity or affinity of the antibody.

[0142] Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and further described, for example, in Riechmann et al., (1988) Nature 332:323-329, Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033, U.S. Patent No. 5,821,337, U.S. Patent No. 7,527,791, U.S. Patent No. 6,982,321, and U.S. Patent No. 7,087,409, Kashmiri et al., (2005) Methods 36:25-34, Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"), Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling"), and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0143] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296), framework regions derived from consensus sequences of human antibodies of certain subgroups of 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, 151:2623), human mature (somatic hypermutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633), and framework regions obtained from screening of FR libraries (see, e.g., Baca et al., (1997) J. Biol. Chem. 272: 10678-10684, and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, humanized VHHs are generated by replacing the FR regions of VHHs with human FR regions. In some embodiments, replacing certain FR residues of the human FR improves one or more properties of the humanized VHH. VHH domains having such replaced residues are also further referred to herein as "humanized."

[0144] As provided herein, sdAbs comprising VHH domains that bind albumin can delay the clearance of the molecules to which they are linked, including molecules comprising a human Fc region. In various embodiments, the Fc region comprised in the albumin-binding polypeptide is a human Fc region or is derived from a human Fc region. A non-limiting sdAb format comprising an Fc region is shown in FIG. 6.

[0145] In some embodiments, the Fc region contained in the albumin-binding polypeptide is derived from a human Fc region and contains three amino acid deletions corresponding to IgG1 E233, L234, and L235 in the lower hinge, herein referred to as "Fc xELL". Since the Fc xELL polypeptide does not bind to FcγR, it is referred to as "effector silent" or "effector null", but in some embodiments, the xELL Fc region binds to FcRn, resulting in an extended half-life and transcytosis associated with FcRn-mediated recycling.

[0146] In some embodiments, the Fc region contained in the albumin-binding polypeptide is derived from a human Fc region and contains mutations M252Y and M428V that can be referred to as "YV". In some embodiments, such mutations enhance binding to FcRn at the acidic pH (near 6.5) of the endosome, while detectable binding is lost at neutral pH (about 7.2), thus enabling enhanced FcRn-mediated recycling and an extended half-life. In some embodiments, the Fc region contained in the albumin-binding polypeptide is derived from a human Fc region and contains mutations M252Y, S254T, and T256E that can be referred to as "YTE". In some embodiments, such mutations extend the serum half-life in humans by significantly reducing the dissociation rate between Fc and FcRn. In some embodiments, the Fc region contained in the albumin-binding polypeptide is derived from a human Fc region and contains mutations M428L and N434S that can be referred to as "LS". In some embodiments, such mutations increase the binding affinity of Fc for FcRn at pH 6 and reduce the dissociation rate, thereby extending the serum half-life in humans. Various Fc mutations that enhance the circulatory half-life are described, for example, in Saunders, Front. Immunol. doi.org / 10.3389 / fimmu.2019.01296 (2019).

[0147] In some embodiments, the Fc region included in the albumin-binding polypeptide is derived from a human Fc region and contains mutations designed for heterodimerization, referred to herein as "knob" and "hole". In some embodiments, the "knob" Fc region contains the mutation T366W. In some embodiments, the "hole" Fc region contains the mutations T366S, L368A, and Y407V. In some embodiments, the Fc region used for heterodimerization contains additional mutations such as the mutation S354C on the first member of the heterodimeric Fc pair, which forms an asymmetric disulfide with the corresponding mutation Y349C on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair contains the modification H435R or H435K to prevent binding of protein A while maintaining FcRn binding. In some embodiments, one member of the heterodimeric Fc pair contains 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 contains the modification H435R or H435K (when the modification is H435R, sometimes referred to as "hole-R"), while the knob Fc region does not contain it. In some cases, the hole-R mutation improves the purification of the heterodimer relative to the homodimeric hole Fc region that may be present.

[0148] Non-limiting exemplary Fc regions that can be used in the albumin-binding polypeptide include Fc regions containing the amino acid sequences of SEQ ID NOs: 47 to 68 and SEQ ID NOs: 85 to 87.

[0149] Exemplary activities of the albumin-binding polypeptide In various embodiments, the albumin-binding polypeptides provided herein bind to epitopes of albumin outside of domain 3. In some embodiments, the albumin-binding polypeptides provided herein do not (i.e., do not inhibit) interfere with the binding of albumin to FcRn. Methods for determining whether an albumin-binding polypeptide interferes with the binding of albumin to FcRn are known in the art, and exemplary non-limiting methods are also provided herein.

[0150] In some embodiments, polypeptides comprising an albumin-binding domain provided herein have a longer half-life in vivo than polypeptides lacking the albumin-binding domain. In various embodiments, polypeptides comprising an albumin-binding domain provided herein have a half-life that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold longer than the half-life of a polypeptide that does not contain the albumin-binding domain.

[0151] Expression and production of polypeptides Nucleic acid molecules are provided that comprise a polynucleotide encoding a polypeptide comprising an albumin-binding domain. In some embodiments, the nucleic acid molecule can also encode a leader sequence that directs the secretion of the polypeptide comprising the albumin-binding domain, which leader sequence is typically cleaved so that it is not present in the secreted polypeptide. The leader sequence can be a native heavy chain (or VHH) leader sequence or another heterologous leader sequence.

[0152] The nucleic acid molecule can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0153] A vector comprising a nucleic acid encoding a polypeptide comprising an albumin binding domain is provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, a vector optimized for expression of the polypeptide in a desired cell type, such as a CHO cell or a CHO-derived cell, or an NSO cell, is selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0154] In some embodiments, the polypeptide comprising an albumin binding domain can be expressed in prokaryotic cells such as bacterial cells, or in eukaryotic cells such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used for expression of the polypeptide include, but are not limited to, COS cells including COS7 cells, 293 cells including 293-6E cells, CHO cells including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells, PER.C6™ cells (Crucell), and NSO cells. In some embodiments, the polypeptide can be expressed in yeast. See, for example, U.S. Patent Application Publication No. 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform desired post-translational modifications on the polypeptide. For example, in some embodiments, CHO cells produce a polypeptide having a higher level of sialylation than the same polypeptide produced in 293 cells.

[0155] Introduction of one or more nucleic acids (such as vectors) into the desired host cell can be achieved by any method including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press (2001). The nucleic acid can be transiently or stably transfected into the desired host cell according to any suitable method.

[0156] Also provided are host cells comprising any of the nucleic acids or vectors described herein. In some embodiments, host cells are provided that express a polypeptide comprising an albumin binding domain described herein. The polypeptide expressed in the host cell can be purified by any suitable method. Such methods include, but are not limited to, the use of an affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include agents that bind to the ROR1 ECD and the Fc region. For example, Protein A, Protein G, Protein A / G, or an antibody affinity column can be used to bind to the Fc region to purify a polypeptide containing the Fc region. Hydrophobic interaction chromatography, such as a butyl column or a phenyl column, may also be suitable for purifying some polypeptides such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be suitable for purifying some polypeptides such as antibodies. Mixed mode chromatography (e.g., reverse phase / anion exchange, reverse phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for purifying some polypeptides such as antibodies. Many methods for purifying polypeptides are known in the art.

[0157] In some embodiments, the polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009), Spirin, Trends Biotechnol. 22: 538-45 (2004), Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0158] In some embodiments, polypeptides comprising an albumin-binding domain produced by the above methods are provided. In some embodiments, the polypeptide is produced in a host cell. In some embodiments, the polypeptide is produced in a cell-free system. In some embodiments, the polypeptide is purified. In some embodiments, a cell culture medium comprising the polypeptide is provided.

[0159] In some embodiments, compositions comprising an antibody produced by the above methods are provided. In some embodiments, the composition comprises a polypeptide comprising an albumin-binding domain produced in a host cell. In some embodiments, the composition comprises a polypeptide produced in a cell-free system. In some embodiments, the composition comprises a purified polypeptide.

[0160] Exemplary methods of treating a disease using an albumin-binding polypeptide In some embodiments, provided is a method of treating a disease in an individual, comprising administering a therapeutic polypeptide comprising an albumin binding domain provided herein. Such diseases include any disease that would benefit from treatment with the therapeutic polypeptide. Non-limiting exemplary diseases that can be treated with the therapeutic polypeptide comprising an albumin binding domain provided herein include infectious diseases, autoimmune diseases or disorders, inflammatory diseases or disorders, and cancer. The method comprises administering to the individual an effective amount of the therapeutic polypeptide comprising an albumin binding domain provided herein. Such a method of treatment can be a method of treatment in a human or an animal. In some embodiments, provided is a method of treating a human.

[0161] The therapeutic polypeptide comprising an albumin binding domain provided herein can be administered to a subject as needed. The determination of the frequency of administration can be made by one of ordinary skill in the art, such as a treating physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, etc. In some embodiments, an effective dose of the therapeutic polypeptide is administered to the subject one or more times. In some embodiments, an effective dose of the therapeutic polypeptide is administered to the subject daily, twice a week, weekly, every two weeks, once a month, etc. An effective dose of the therapeutic polypeptide is administered to the subject at least once. In some embodiments, an effective dose of the therapeutic polypeptide can be administered in multiple doses over at least one month, at least six months, or at least one year.

[0162] In some embodiments, the pharmaceutical composition is administered in an amount effective to treat a disease. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of the subject, the extent of the medical condition being treated, or the age of the subject being treated. Generally, the antibody can be administered in an amount in the range of about 0.05 mg / kg (body weight) to about 100 mg / kg (body weight) per dose. In some embodiments, the antibody can be administered in an amount in the range of about 10 μg / kg (body weight) to about 100 mg / kg (body weight) per dose. In some embodiments, the antibody can be administered in an amount in the range of about 50 μg / kg (body weight) to about 5 mg / kg (body weight) per dose. In some embodiments, the antibody can be administered in an amount in the range of about 100 μg / kg (body weight) to about 10 mg / kg (body weight) per dose. In some embodiments, the antibody can be administered in an amount in the range of about 100 μg / kg (body weight) to about 20 mg / kg (body weight) per dose. In some embodiments, the antibody can be administered in an amount in the range of about 0.5 mg / kg (body weight) to about 20 mg / kg (body weight) per dose. In some embodiments, the antibody can be administered in an amount in the range of about 0.5 mg / kg (body weight) to about 10 mg / kg (body weight) per dose. In some embodiments, the antibody can be administered in an amount in the range of about 0.05 mg / kg (body weight) to about 20 mg / kg (body weight) per dose. In some embodiments, the antibody can be administered in an amount in the range of about 0.05 mg / kg (body weight) to about 10 mg / kg (body weight) per dose. In some embodiments, the antibody can be administered in an amount in the range of about 5 mg / kg (body weight) or less, such as less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, or less than 1 mg / kg of antibody per dose.

[0163] 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 depending on the intended use.

[0164] Pharmaceutical composition In some embodiments, a composition comprising a polypeptide comprising an albumin binding domain is provided as a formulation comprising a variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed., Lippencott Williams and Wilkins (2004), Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd ed., Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers are available, including vehicles, adjuvants, and diluents. In addition, a variety of pharmaceutically acceptable auxiliary substances are available, such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, etc. Non-limiting exemplary carriers include physiological saline, buffered physiological saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0165] In some embodiments, the pharmaceutical composition comprises a polypeptide comprising an albumin binding domain at a concentration of at least 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 225 mg / mL, or 250 mg / mL.

[0166] Non-limiting exemplary methods of diagnosis and treatment In some embodiments, the methods described herein are useful for evaluating a subject and / or a sample from a subject (e.g., a cancer patient). In some embodiments, the evaluation is one or more of diagnosis, prognosis, and / or response to treatment.

[0167] In some embodiments, the methods described herein include evaluating the presence, absence, or level of a protein. In some embodiments, the methods described herein include evaluating the presence, absence, or level of expression of a nucleic acid. The compositions described herein can be used for these measurements. In some embodiments, the evaluation can indicate treatment (including treatment with the polypeptides described herein).

[0168] Kit Also provided are articles of manufacture and kits comprising any of the polypeptides comprising an albumin binding domain described herein and appropriate packaging. In some embodiments, the invention includes kits comprising (i) a polypeptide comprising an albumin binding domain and (ii) instructions for using the kit to administer the polypeptide to an individual.

[0169] Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed mylar or plastic bags), etc. These manufactured products can be further sterilized and / or sealed. Unit dosage forms containing the compositions described herein are also provided. These unit dosage forms can be stored in suitable packaging in single or multiple unit dosages and can be further sterilized and sealed. The instructions provided in the kits of the present invention are typically instructions written on a label or an accompanying document (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions held on a magnetic storage disk or an optical storage disk) are also acceptable. Instructions for use regarding the use of an antibody generally include information regarding dosage, dosing schedule, and route of administration for the intended therapeutic or industrial use. The kit may further include an explanation for selecting an individual appropriate treatment.

[0170] The container can be a unit dose, bulk packaging (e.g., multi-dose packaging), or sub-unit dose. For example, kits can be provided that contain an amount of the molecules disclosed herein sufficient to provide effective treatment to an individual over a long period of time, such as any approximate period of 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more. The kit can also include multiple unit doses of the molecule and instructions for use and can be packaged in an amount sufficient for storage and use in a pharmacy, e.g., a hospital pharmacy and a dispensing pharmacy. In some embodiments, the kit includes a dry (e.g., lyophilized) composition that can generally form a stable aqueous suspension of the antibody upon reconstitution, resuspension, or rehydration.

Examples

[0171] The examples discussed below are intended to purely illustrate the present invention and should in no way be considered as limiting the present invention. These examples are not intended to represent that the following experiments are all or the only experiments conducted. Although efforts have been made to ensure accuracy with respect to the numerical values used (e.g., amounts, temperatures, etc.), some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0172] Example 1: Development of an anti-albumin single-domain antibody (sdAb) Single-domain antibodies targeting human albumin were generated by immunizing llamas and alpacas with recombinant human serum albumin (SEQ ID NO: 1).

[0173] After the expression of specific anti-albumin antibody titers, llama / alpaca peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of the blood of the immunized animals, total mRNA was isolated using the Qiagen RNeasy Maxi Kit, and subsequently converted to first-strand cDNA using Thermo Superscript IV Reverse Transcriptase and oligo dT priming. The VHH sequences were specifically amplified by PCR using the cDNA as a template and cloned into a yeast surface display vector as a VHH-Fc-AGA2 fusion protein. Fc was human IgG1 Fc, or in some cases, a mutant IgG1 Fc with reduced effector function.

[0174] A yeast library presenting the VHH-Fc-AGA2 fusion protein was enriched using recombinant human albumin by fluorescence-activated cell sorting (FACS) following magnetic bead isolation. The sorted yeast were plated out, and the isolated colonies were picked into 96-well blocks, and induction of yeast cell surface expression of the VHH-Fc-AGA2 fusion protein was performed. Biotinylated recombinant human albumin or an irrelevant biotinylated protein (albumin-negative) was directly applied to the induced yeast, washed, treated with fluorophore-labeled streptavidin, and analyzed by 96-well flow cytometry.

[0175] The nucleic acid sequence encoding a VHH that binds to biotinylated recombinant human albumin and does not bind to an irrelevant biotinylated protein was cloned into a mammalian expression vector in-frame with the human Fc xELL coding region and expressed by transient transfection in HEK293 Freestyle cells (293F cells) or CHO cells using polyethyleneimine. After 3 to 7 days, the supernatant was collected, and the secreted recombinant protein was purified by protein A chromatography, and the concentration was calculated from the absorbance at 280 nm and the extinction coefficient.

[0176] The anti-albumin sdAb 4A01 was selected for humanization.

[0177] Example 2: Monovalent binding of anti-albumin sdAb 4A01 to human and mouse albumin The monomeric anti-albumin sdAb 4A01 was generated by fusing the 4A01 VHH to human Fc containing the mutations S364N, Y407N, and K409T (4A01-NNT-hFc; SEQ ID NO: 23 and SEQ ID NO: 68; Figure 1A). The binding of the monomeric anti-albumin sdAb 4A01 was evaluated as follows by ELISA by titrating the monomeric sdAb against immobilized albumin protein of the designated species (Medisorp plate) and detecting with anti-human Fc (HRP).

[0178] Plates were coated overnight at 4°C with the designated species of albumin protein (Sigma) at 2 μg / ml, 50 μl / well. 1× Fish Gelatin (blocking agent, Biotium) was added to the coated wells and incubated for 1 hour at room temperature. After adding the titration of 4A01-NNT-hFc (starting from 100 nM, 1:3 dilution, the last well was blank), it was incubated for 1 hour at room temperature. The plates were then washed three times with 0.1% D-PBST and then anti-human Fc HRP antibody (1:2000 in 0.1% D-PBST, Jackson) was added. After incubating the plates for 30 minutes at room temperature, they were washed three times with 0.1% D-PBST. Then, TMB substrate was added and the absorbance at 650 nm was read using a plate reader (Molecular Devices). Data was plotted using the one-site - total binding equation (Y = Bmax×X / (Kd+X)+NS×X+background, GraphPad Prism).

[0179] As shown in FIGS. 1B and 1C, the anti-albumin sdAb 4A01 bound to both human and mouse albumin with equivalent affinity. The K D for human albumin was 0.23 nM and the K D for mouse albumin was 0.20 nM.

[0180] Example 3: Anti-albumin sdAb 4A01 does not bind to albumin domain 3 Albumin binds mainly via domain 3 to the β2-microglobulin FcRn complex, and this binding is thought to be involved in improving the half-life of proteins fused with anti-albumin antibodies or albumin itself. To determine whether anti-albumin sdAb 4A01 binds to albumin domain 3, the binding of 4A01-NNT-hFc was assayed by biolayer interferometry as follows.

[0181] Albumin domain 3 (with mouse Fc tag) was immobilized on an anti-mouse IgG Fc capture biosensor. All buffer / protein formulations were in MBST5 (50 nM MES (pH 5), 150 mM NaCl, 0.025% Tween®). The baseline was established with buffer only. Human albumin domain III with mouse Fc tag (10 μg / ml) was loaded onto an anti-mouse IgG Fc capture biosensor (ForteBio). Then, anti-albumin sdAb 4A01 (4A01-NNT-hFc) and 1C04 (similar format) were loaded and allowed to associate with the captured biotin domain 3, and then dissociated with MBST5. sdAb 1C04 is known to bind to albumin domain 3 and was used as a positive control. See Figure 2A.

[0182] As shown in Figure 2B, 1C04 bound to the immobilized albumin domain 3, while 4A01 did not bind.

[0183] Next, anti-albumin sdAb 4A01 (4A01-NNT-hFc), hz4A01v51, and 1C04 were tested for interference with albumin-FcRn binding as follows. Binding was evaluated by biolayer interferometry using biotinylated recombinant FcRn-B2M immobilized on a streptavidin biosensor. Then, the immobilized FcRn-B2M was complexed with recombinant human albumin. All buffer / protein formulations were in MBST5 (50 mM MES (pH 5), 150 mM NaCl, 0.025% Tween®). The baseline was established with buffer only. Biotinylated FcRn-B2M (10 μg / ml, Acro Biosystems) was loaded onto a streptavidin biosensor (ForteBio) to determine a further baseline. Then, 50 μM recombinant human albumin (Sigma) was added and allowed to associate with the immobilized FcRn-B2M. Then, anti-albumin sdAb 4A01 and 4A01v51, and sdAb 1C04 were loaded and allowed to associate with the captured biotin domain 3, and then dissociated with MBST5. See Figure 3A.

[0184] As shown in Figure 2B, both 4A01 and hx4A01v51 bound to albumin associated with FcRn-B2M, while 1C04 did not bind.

[0185] Example 4: Humanization and Interspecies Cross-Reactivity of Anti-Albumin sdAb 4A01 Human heavy chain framework VH3-23 * Based on 04, various humanized forms of sdAb 4A01 were generated. Certain amino acids were reverted to donor amino acids by back mutation, and certain mutations were tested, for example, in CDR2. Figure 4A shows the alignment between the human heavy chain acceptor sequence and the humanized form of 4A01.

[0186] The binding of monomeric anti-albumin sdAb 4A01 (「lm4A01」) and its humanized forms to human serum albumin, cynomolgus monkey serum albumin, mouse serum albumin, and rat serum albumin was determined by ELISA as follows. Medisorp plates were coated overnight at 4°C with 2 μg / ml, 50 μl / well of albumin protein (human, mouse, and rat albumin - Sigma, cynomolgus monkey albumin - Abcam). After adding 1-fold Fish Gelatin (blocking agent, Bethyl Laboratories) to the wells coated with albumin, the plates were incubated at room temperature for 1 hour. Titration of the sdAb fusion protein (starting from 100 nM, 1:3 horizontal or 1:4 vertical) was added and the plates were incubated at room temperature for 1 hour. The plates were washed 3 times with 0.1% D-PBST, then anti-human Fc HRP antibody (1:2000 in 0.1% D-PBST, Jackson) was added and the plates were incubated at room temperature for 30 minutes. After washing the plates 3 times with 0.1% D-PBST, TMB substrate was added. The absorbance at 650 nm was read using a plate reader (Molecular Devices), and the data was plotted using the one-site - total binding equation (model: Y = Bmax × X / (Kd + X)+NS × X + background, GraphPad Prism).

[0187] The binding of 4A01 and its humanized forms to human albumin is shown in FIGS. 4B and 4C. All sdAbs bound to human albumin with a K D in the range of 0.10 nM to 0.43 nM. The binding of 4A01 and its humanized forms to cynomolgus monkey albumin is shown in FIGS. 4D and 4E. All sdAbs bound to cynomolgus monkey albumin with a K D in the range of 0.11 nM to 0.34 nM. The binding of 4A01 and its humanized forms to mouse albumin is shown in FIGS. 4F and 4G. All sdAbs bound to mouse albumin with a K D in the range of 0.11 nM to approximately 0.25 nM. The binding of 4A01 and its humanized forms to rat albumin is shown in FIGS. 4H and 4I. All sdAbs bound to rat albumin with a K D in the range of 0.14 nM to approximately 0.33 nM.

[0188] FIGS. 5A-5D show the binding of 4A01 and humanized hz4A01v51 to human (FIG. 5A), cynomolgus monkey (FIG. 5B), mouse (FIG. 5C), and rat (FIG. 5D) albumin. 4A01 and all humanized variants tested bound to all four types of albumin with an affinity of less than 1 nM. Humanized hz4A01v51 bound to all four types of albumin with an affinity of less than 0.3 nM and achieved a maximum binding of over 90%.

[0189] Example 5: Binding of Single-Domain Antibody Polypeptides to Human Albumin The binding of the humanized single-domain antibody (sdAb) polypeptide to human albumin at neutral pH (7.4) or endosomal pH (6) was tested by ELISA. A 96-well ELISA plate was coated overnight at 4 °C with 2 μg / mL recombinant albumin in PBS, washed with PBS / 0.05% Tween®-20 (PBS-T), and then blocked for 2 h at room temperature with 5% nonfat dry milk in PBS-T. Serial dilutions of the test substance were prepared in PBS (pH 7.4) or buffer containing 20 mM His, 150 mM NaCl (pH 6) and added to the plate. The plate was incubated at 4 °C for 1 h. After incubation, the cells were washed with each buffer and then incubated for 30 min at room temperature with a horseradish peroxidase (HRP)-conjugated anti-idiotype antibody to detect the sdAb. The plate was washed with each buffer and TMB substrate was added. The HRP-TMB reaction was allowed to proceed for 6 min and then stopped with an equal volume of HCl-based stop solution. Absorbance at 450 nm was measured with a 96-well plate reader. The data were plotted and analyzed using GraphPad Prism analysis software. The results are shown in Figure 7.

[0190] As shown in FIGS. 7A and 7B, the bivalent bispecific sdAb polypeptide (cx11917) comprising the albumin-binding domain of SEQ ID NO: 43 (hz4A01v51 VHH) and a non-mammalian targeting binding domain, formatted as shown in FIG. 6(iii), binds albumin with low nanomolar to sub-nanomolar affinity. The apparent affinity is only slightly affected by pH, with a K d of 0.7 nM at neutral pH (7.4) compared to 2 nM at pH 6. d The binding is mediated only by the monovalent albumin-targeting sdAb subunit, as the sdAb polypeptide (cx11916) comprising two non-mammalian targeting binding domains, formatted as described in FIG. 6(iii), did not bind albumin with measurable affinity at any of the pHs tested.

[0191] Example 6: Interspecies Cross-Reactivity of Albumin-Binding Single-Domain Antibody Polypeptides The binding of the humanized single-domain antibody (sdAb) polypeptide (cx5009, SEQ ID NO: 69), which contains hz4A01v51 VHH (SEQ ID NO: 43) and a monomeric Fc region (Fc region with substitutions of S364N, Y407N, and K409T, SEQ ID NO: 68), to recombinant human, cynomolgus monkey, mouse, or rat albumin at neutral pH (7.4) was tested by ELISA. A 96-well ELISA plate was coated overnight at 4°C with 2 μg / mL recombinant albumin in PBS, washed with PBS / 0.05% Tween®-20 (PBS-T), and then blocked with 1-fold fish gelatin for 1 hour at room temperature. Serial dilutions of the test substance were prepared in PBS-T (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 PBS-T and then incubated for 30 minutes at room temperature with an HRP-conjugated secondary antibody specific for human IgG1. The plate was then washed and the TMB substrate was added. The HRP-TMB reaction was allowed to proceed, and the absorbance at 650 nm was measured with a 96-well plate reader. The data were plotted and analyzed using GraphPad Prism analysis software. The results are shown in Figure 3.

[0192] As shown in Figure 8, cx5009, a monovalent albumin-specific sdAb, hz4A01v51 VHH-hIgG1-xELL-NNT-Fc (SEQ ID NO: 69), formatted as shown in Figure 6(ii), binds to albumin derived from human, cynomolgus monkey, mouse, and rat. The apparent affinity at neutral pH (7.4) is similar across species in the sub-nanomolar range (about 0.2 nM) of K d D.

[0193] Example 7: In Vivo Pharmacokinetic Profile of Albumin-Binding Single-Domain Antibody Polypeptides The ability of an albumin-binding single domain antibody (sdAb) to extend the serum exposure of human IgG was tested in healthy mice. The pharmacokinetic (PK) profile of a humanized bivalent sdAb polypeptide (cx11956, SEQ ID NO: 70) that is cross-reactive with mouse albumin and is formatted as hz4A01v51 VHH-hIgG1-xELL-Fc was compared to the profile of a bivalent sdAb polypeptide (cx11851) that is not cross-reactive with mice but is formatted with the same VHH-hIgG1-xELL-Fc structure. The xELL mutation of human IgG1 reduces Fcγ receptor binding but does not affect FcRn binding. This was confirmed in vitro using biolayer interferometry. Human IgG1 is cross-reactive with mouse FcRn and enables FcRn-mediated recycling of human antibodies in mice.

[0194] To determine the PK profile of the sdAb-hIgG xELL-Fc test substance, BALB / c mice were injected intravenously with a single dose of either 30 mg / kg or 0.3 mg / kg. Serum samples were collected 30 minutes, 6 hours, 24 hours, 96 hours, and 168 hours after the test substance injection. The concentration of the test substance in mouse serum was determined by ELISA. For the PK ELISA, a human FcRn / B2M heterodimer protein (His-tag, Acro Biosystem) was immobilized on a 96-well ELISA plate by incubating a 4 μg / mL protein solution in PBS at 4°C for 12 hours. The next day, the plates were blocked with 3% BSA TBS-T buffer for 2 hours, and then serum samples were incubated on these plates for 2 hours. Binding of the test substance in the serum samples to FcRn immobilized on the ELISA plate was detected using an HRP-conjugated secondary anti-idiotype detection antibody capable of binding to the sdAb. The secondary antibody was incubated on the plate for 1 hour, and after visualizing the binding using a TMB substrate solution, a stop solution (1M H 2 SO 4) was added, and the absorbance at 450 nm was measured with an Emax spectrophotometer (Molecular Devices). The absorbance values were converted to the test substance concentration using SoftMax Pro with a standard curve generated by a protein of known concentration. The standard curve was fitted using four-parameter logistic regression. The data was exported and graphed using GraphPad Prism analysis software.

[0195] As shown in Figure 9, when the albumin-targeted sdAb polypeptide attaches to IgG1, it can delay the clearance of human IgG1 and extend serum exposure. The absolute concentration of anti-albumin hz4A01v51 VHH-IgG1 xELL-Fc (cx11956) in serum after a single dose of 30 mg / kg (Figure 9A) or 0.3 mg / kg (Figure 9C) is significantly higher than that of an equivalent-sized non-targeted VHH-IgG1 xELL-Fc (cx11851) that does not bind to albumin. Despite injecting the same amount of protein cx11851, the cMax level 30 minutes after injection is already lower than that of cx11956. Furthermore, the more rapid clearance of the non-targeted construct (cx11851) continues over the first 6 hours after injection, as shown in the normalized plots (Figure 9B and Figure 9D). Compared to the cMax (30 minutes) time point, the non-targeted cx11851 concentration decreases by nearly 60% by 6 hours at the 30 mg / kg dose level, while the albumin-binding cx11956 concentration decreases by only about 12% (Figure 10B). Similarly, at the low dose level (0.3 mg / kg), the concentration of albumin-binding cx11956 decreased only to about 87% of Cmax compared to about 72% of the cMax of non-albumin-binding cx11851.

[0196] These findings demonstrate that albumin-binding VHH domains can enhance the serum exposure of IgG antibodies. While not intending to be bound by any particular theory, the enhanced serum exposure may be due to the albumin / FcRn recycling pathway independent of IgG / FcRn-mediated recycling.

[0197] To confirm the equivalent binding of human IgG1-xELL and wild-type human IgG to FcRn, the binding of sdAb polypeptides formatted with either of two IgG1 Fc mutants to recombinant human FcRn / B2M was tested by biolayer interferometry using an Octet96 Red reader (Sartorius). Briefly, biotinylated human FcRn / B2m was loaded onto streptavidin biosensors. The association of the test substance was measured for 60 seconds by immersing the sensors in 100 nM test substance dilutions. The test substances were diluted in either a buffer containing 50 mM MES, 150 mM NaCl and 0.025% Tween®-20 at pH 6 or a buffer containing 50 mM Tris, 150 mM NaCl and 0.025% Tween®-20 at pH 8. The dissociation of the test substance was followed for 300 seconds by immersing the sensors in each pH buffer without the test substance. Association and dissociation curves were exported using Forte data analysis software.

[0198] The results are shown in Figure 10. The FcRn binding of the IgG1 xELL Fc used in these studies is equivalent to the FcRn binding of wild-type IgG1 Fc. Both molecules show similar association rates at pH 6 and have no measurable affinity for FcRn at pH 8. Thus, the albumin-binding VHH domain is expected to enhance the serum exposure of molecules with not only the IgG1 xELL domain but also the wild-type IgG1 Fc domain.

[0199] Example 8: Binding of various single-domain antibody formats to human albumin The ability of different single - specificity and bispecific albumin - binding single - domain antibody formats that bind recombinant human albumin and a second target (IL - 4R) at neutral pH (7.4) was evaluated by ELISA. Single - specificity antibodies containing albumin - targeting sdAb (hz4A01v51 VHH) linked to the C - terminus of the xELL Fc region via a 6 - residue or 12 - residue glycine - serine linker, Fab domains (VL - CL (SEQ ID NO: 77) and VH - CH1 (SEQ ID NO: 76) of an IL - 4R - targeting antibody (dupilumab)), IgG1 or IgG4 Fc regions, and bispecific antibodies containing albumin - targeting sdAb (hz4A01v51 VHH) placed at different positions, as well as single - specificity control IL - 4R - targeting molecules lacking albumin - targeting sdAb were evaluated. The names and general structures of the test substances of the polypeptides used in this study are summarized in Table 2. For ELISA, 96 - well ELISA plates (MaxiSorb, Biolegend) were coated overnight at 4 °C in PBS with 1 μg / mL (100 μL / well) of human albumin or IL4R. After washing the plates three times with 0.05% PBST (150 μL / well), they were blocked for 2 hours at room temperature with casein in 0.05% PBST (200 μL / well). The plates were washed three times with 0.05% PBST, and 100 μL of titrated test substance in 0.05% PBST was added to the wells of the plate (starting at 100 nM, 1:3 dilution, 11 - point titration) and incubated at 4 °C for 1 hour. After washing again, the plates were incubated with an HRP - conjugated secondary antibody (Jackson ImmunoResearch) in 0.05% PBST (100 μL / well) specific for human IgG1 for 30 minutes at room temperature. Then, the plates were washed again and TMB substrate (100 μL / well), which was returned to room temperature before adding to the plate, was added. After allowing the HRP - TMB reaction to proceed for approximately 10 minutes, TMB stop buffer was added (100 μL / well), and the absorbance at 450 nm was measured with a plate reader (Molecular Devices), subtracting the absorbance at 650 nm. The data were plotted and analyzed using GraphPad Prism analysis software. The results are shown in Figures 11A and 11B.

[0200]

Table 2

[0201] As shown in Figure 11A, all molecules containing anti-albumin VHH showed binding to albumin, and the polypeptide containing VHH between the CH1 and Fc regions showed slightly higher affinity for albumin than the molecule containing anti-albumin VHH at the C-terminus of the Fc region. No binding was observed for molecules lacking anti-albumin VHH (cx12585 and cx12590). As shown in Figure 11B, all molecules containing a binding domain for a second target (i.e., the Fab domain of dupilumab) showed binding to the second target, IL-4R, with very similar affinities, demonstrating that the presence of the albumin binding domain does not interfere with binding to the second target. No binding was observed for molecules lacking the binding domain for the second target (cx12583 and cx12584). These data, together with the data presented in Example 7, show that albumin-targeted sdAbs can mediate albumin binding when located at the N-terminus, C-terminus, or within the polypeptide (e.g., between domains) of the molecule. Thus, it is expected that the albumin-binding VHH domain will enhance the serum exposure of molecules containing molecules containing the Fc region, regardless of where the albumin-binding VHH domain is located.

[0202] Example 9: Modification of anti-albumin sdAb Hz4A01v51 The framework region of Hz4A01v51 was further modified, including reverting certain residues to donor amino acids and / or introducing alternative charged residues. Modified VHHs (Hz4A01v51.9, Hz4A01v51.11, Hz4A01v51.12, and Hz4A01v51.13) were used to generate monovalent (VHH fused to Fc NTT) anti-albumin binding molecules with the general structure shown in Figure 6(ii). Also, several of them (Hz4A01v51.9, Hz4A01v51.12, and Hz4A01v51.13) were used to generate divalent (VHH fused to Fc xELL) anti-albumin binding molecules with the general structure shown in Figure 6(i). The binding of monovalent and divalent anti-albumin molecules and monovalent Hz4A01v51-NNT-hFc to human serum albumin at pH 6.0 and pH 7.0 was determined by ELISA as follows. Plates (MaxiSorb, Biolegend) were coated overnight at 4°C in PBS with 2 μg / mL (100 μL) albumin or overnight at 4°C in 20 mM His-HCl, 150 mM NaCl (pH 6) with 2 μg / mL (100 μL) albumin. After washing the plates three times with 0.05% PBST or pH 6 buffer, they were blocked with 5% Milk PBST for 2 hours at room temperature. The plates were washed three times with 0.05% PBST or pH 6 buffer, and titrations of the test substances in PBST or pH 6 buffer were added to the plates (starting at 100 nM, 1:5 dilution) and incubated at 4°C for 1 hour. Then, after washing again, the plates were incubated with an HRP-conjugated secondary antibody specific for human IgG1 for 30 minutes at room temperature. Then, after washing the plates, the TMB substrate was added. After allowing the HRP-TMB reaction to proceed for 6 minutes, the TMB stop buffer was added, and the absorbance at 450 nm was measured with a plate reader (Molecular Devices). The data were plotted and analyzed using GraphPad Prism analysis software. For negative control and ELL samples, the 0.16 nM titration point was not tested.

[0203] In the monovalent format, the modified anti-albumin antibodies showed similar binding profiles at pH 6 (Figure 12A) and pH 7.4 (Figure 12B), and particularly at pH 6, showed improved binding compared to the binding observed with Hz4A01v51 in both cases. In the bivalent format, Hz4A01v51.9 and Hz4A01v51.13 showed similar binding profiles that were improved compared to Hz4A01v51 at both pH 6 (Figure 12C) and pH 7.4 (Figure 12D).

[0204] The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the above-described embodiments should be considered illustrative in all respects and not as limiting the present disclosure. Accordingly, the scope of the present disclosure is indicated by the appended claims rather than the above detailed description, and all modifications within the meaning and equivalent scope of the claims are intended to be included herein.

[0205] [Table 3] TIFF2025517120000005.tif243170TIFF2025517120000006.tif249170TIFF2025517120000007.tif245170TIFF2025517120000008.tif248170TIFF2025517120000009.tif243170TIFF2025517120000010.tif244170TIFF2025517120000011.tif248170TIFF2025517120000012.tif151170

Claims

**Claim 1** A polypeptide comprising at least one VHH domain that binds to albumin, wherein the at least one VHH domain that binds to albumin comprises a CDR1 sequence selected from SEQ ID NO: 5 to SEQ ID NO: 8, a CDR2 sequence selected from SEQ ID NO: 9 to SEQ ID NO: 21, and a CDR3 sequence of SEQ ID NO:

22. **Claim 2** Each VHH domain that binds to albumin independently comprises a CDR1 sequence selected from SEQ ID NO: 5 to SEQ ID NO: 8, a CDR2 sequence selected from SEQ ID NO: 9 to SEQ ID NO: 21, and a CDR3 sequence of SEQ ID NO: 22, the polypeptide according to claim 1. **Claim 3** The at least one VHH domain that binds to albumin comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 12, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 14, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 7, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 8, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 16, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 17, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 18, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 19, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 20, and SEQ ID NO: 22; and SEQ ID NO: 6, SEQ ID NO: 21, and SEQ ID NO: 22, the polypeptide according to claim 1 or 2. **Claim 4** Each VHH domain that binds to albumin independently comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence selected from SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 12, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 14, and SEQ ID NO: 22; SEQ ID NO: 5, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 7, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 8, SEQ ID NO: 15, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 16, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 17, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 18, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 19, and SEQ ID NO: 22; SEQ ID NO: 6, SEQ ID NO: 20, and SEQ ID NO: 22; and SEQ ID NO: 6, SEQ ID NO: 21, and SEQ ID NO:

22. The polypeptide according to claim 3.

5. The polypeptide according to any one of claims 1 to 4, wherein at least one VHH domain that binds to albumin is humanized.

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

7. The polypeptide according to any one of claims 1 to 6, wherein at least one 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%, or at least 99% identical to a sequence selected from SEQ ID NOs: 23 to 43 and SEQ ID NOs: 71 to 74.

8. The polypeptide according to claim 7, 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%, or at least 99% identical to a sequence selected from SEQ ID NOs: 23 to 43 and SEQ ID NOs: 71 to 74.

9. The polypeptide according to any one of claims 1 to 7, wherein at least one VHH domain that binds to albumin comprises a sequence selected from SEQ ID NOs: 23 to 43 and SEQ ID NOs: 71 to 74.

10. Each VHH domain that binds to albumin, the polypeptide according to any one of claims 1 to 9, comprising a sequence selected from SEQ ID NO: 23 to SEQ ID NO: 43 and SEQ ID NO: 71 to SEQ ID NO:

74.

11. At least one VHH domain that binds to albumin, the polypeptide according to any one of claims 1 to 10, which binds to human albumin and binds to at least one albumin selected from cynomolgus monkey, mouse, and rat albumin.

12. Each VHH domain that binds to albumin, the polypeptide according to any one of claims 1 to 11, which binds to human albumin and binds to at least one albumin selected from cynomolgus monkey, mouse, and rat albumin.

13. At least one VHH domain that binds to albumin, the polypeptide according to any one of claims 1 to 12, which binds to human, cynomolgus monkey, mouse, and rat albumin.

14. Each VHH domain that binds to albumin, the polypeptide according to any one of claims 1 to 13, which binds to human, cynomolgus monkey, mouse, and rat albumin.

15. At least one VHH domain that binds to albumin, the polypeptide according to any one of claims 1 to 14, which binds to human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

16. At least one VHH domain that binds to albumin, the polypeptide according to any one of claims 1 to 15, which 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.

17. Each VHH domain that binds to albumin, the polypeptide according to any one of claims 1 to 16, which binds to human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM.

18. Each VHH domain that binds to albumin, the polypeptide according to any one of claims 1 to 17, which 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.

19. The polypeptide according to any one of claims 1 to 18, wherein each VHH domain that binds to albumin does not bind to albumin domain 3.

20. The polypeptide according to any one of claims 1 to 19, wherein each VHH domain that binds to albumin does not interfere with the binding of albumin to FcRn.

21. The polypeptide according to any one of claims 1 to 20, wherein the polypeptide comprises at least one binding domain that binds to a protein other than albumin.

22. The polypeptide according to claim 21, wherein at least one binding domain that binds to a protein other than albumin is a VHH.

23. The polypeptide according to claim 22, wherein each binding domain that binds to a protein other than albumin is a VHH.

24. The polypeptide according to claim 21, wherein at least one binding domain that binds to a protein other than albumin comprises a heavy chain variable region and a light chain variable region.

25. The polypeptide according to claim 24, wherein each binding domain that binds to a protein other than albumin comprises a heavy chain variable region and a light chain variable region.

26. The polypeptide according to any one of claims 21 to 25, wherein at least one binding domain that binds to a protein other than albumin is a binding domain of a therapeutic antibody.

27. The polypeptide according to claim 26, wherein each binding domain that binds to a protein other than albumin is a binding domain of a therapeutic antibody.

28. The polypeptide according to claim 26 or 27, wherein the therapeutic antibody is useful for the treatment of a disease or disorder selected from autoimmune diseases or disorders, inflammatory diseases or disorders, infectious diseases, and cancers.

29. The polypeptide according to any one of claims 1 to 28, wherein the polypeptide comprises the amino acid sequence of a therapeutic protein.

30. The polypeptide according to claim 29, wherein the therapeutic protein is useful for the treatment of a disease or disorder selected from autoimmune diseases or disorders, inflammatory diseases or disorders, infectious diseases, and cancers.

31. The polypeptide according to any one of claims 1 to 30, wherein the polypeptide comprises an Fc region.

32. The polypeptide according to claim 31, wherein the Fc region binds to FcRn.

33. The polypeptide according to claim 31 or 32, wherein the Fc region is an IgG1 Fc region.

34. The polypeptide according to any one of claims 31 to 33, wherein the Fc region comprises one or more substitutions that enhance the half-life.

35. The polypeptide according to claim 34, wherein the Fc region comprises one or more substitutions that enhance FcRn binding at at least one pH and / or decrease the dissociation rate between Fc and FcRn.

36. The polypeptide according to any one of claims 31 to 35, wherein the Fc region comprises substitutions at one or more amino acid positions selected from 252, 254, 256, 428, or 434.

37. The polypeptide according to claim 36, wherein the Fc region comprises substitutions at amino acid positions 252, 254, and 256; or amino acid positions 252 and 428; or amino acid positions 428 and 434.

38. The polypeptide according to claim 37, wherein the Fc region comprises the substitutions M252Y, S254T, and T256E; M252Y and M428V; or M428L and N434S.

39. The polypeptide according to any one of claims 31 to 38, wherein the Fc region comprises a sequence selected from SEQ ID NOs: 47 to 68 and SEQ ID NOs: 85 to 87.

40. The polypeptide according to any one of claims 1 to 39, wherein the half-life of the polypeptide is longer than the half-life of the same polypeptide lacking the VHH domain that binds to albumin.

41. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 40 and a pharmaceutically acceptable carrier.

42. An isolated nucleic acid encoding the polypeptide according to any one of claims 1 to 40.

43. A vector comprising the nucleic acid according to claim 42.

44. A host cell comprising the nucleic acid according to claim 42 or the vector according to claim 43.

45. A host cell expressing the polypeptide according to any one of claims 1 to 40.

46. A method for producing the polypeptide according to any one of claims 1 to 40, comprising incubating the host cell according to claim 44 or 45 under conditions suitable for the expression of the polypeptide.

47. The method according to claim 46, further comprising isolating the polypeptide.

48. A method comprising administering to a subject the polypeptide according to any one of claims 1 to 40, or the pharmaceutical composition according to claim 41.

49. A method for treating a disease or disorder, comprising administering to a subject suffering from the disease or disorder a pharmaceutically effective amount of the polypeptide according to any one of claims 1 to 40, or the pharmaceutical composition according to claim 41.

50. The method according to claim 49, wherein the disease or disorder is selected from autoimmune diseases or disorders, inflammatory diseases or disorders, infectious diseases, and cancers.

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