Albumin-binding polypeptides and uses thereof

Abstract

The present invention provides VHH-containing polypeptides that bind to albumin. Uses of the VHH-containing polypeptides are also provided.

Description

[0001] Cross-references to related applications

[0002] This application claims the rights of U.S. Provisional Application No. 63 / 338,629, filed May 5, 2022, and U.S. Provisional Application No. 63 / 351,362, filed June 11, 2022, the full text of which is incorporated herein by reference for any purpose.

[0003] The sequence list is incorporated by reference.

[0004] This application is incorporated by reference in the serial list submitted with this application, which is in electronic format named 01202-0020-00PCT_ST26, created on April 27, 2023, and is 133,294 bytes in size. Technical Field

[0005] This invention relates to albumin-binding peptides and methods of using albumin-binding peptides, such as methods of improving the half-life of other molecules. Background Technology

[0006] Plasma proteins are eliminated through recirculation via two main mechanisms: renal filtration of molecules below 60 kDa and micropinocytosis by endothelial cells. Proteins below the renal threshold are rapidly cleared through recirculation, resulting in a half-life of 1 day or less, while proteins above the renal threshold are primarily cleared via micropinocytosis, with a half-life of approximately 3 to 5 days. Albumin and immunoglobulin G (IgG) are proteins with long plasma half-lives of approximately 15 to 30 days due to their large size (66 and 150 kDa respectively) and their ability to recirculate via micropinocytosis through pH-dependent binding to nascent Fc receptors (FcRn).

[0007] Long plasma half-lives can be used in therapeutic agents to accurately and precisely control plasma drug concentrations, optimize efficacy while limiting toxicity, and reduce the frequency and amount of drug required. Therapeutic proteins with short plasma half-lives, due to their small size or lack of ability to recycle via FcRn binding, can fuse with albumin-binding entities to prolong their plasma half-life. The VHH domain of single-domain antibodies is an ideal albumin-binding entity because it is a small domain of approximately 12 to 15 kDa, a single domain composed of a single polypeptide that can be easily fused to another protein or peptide via recombinant methods, has the potential for easy humanization to reduce immunogenicity, and contains many naturally occurring proteins A for affinity purification.

[0008] Therefore, there is a need for the VHH domain that binds to albumin. Summary of the Invention

[0009] Implementation Scheme 1. A polypeptide comprising at least one albumin-binding VHH domain, wherein the at least one albumin-binding VHH domain comprises a CDR1 sequence selected from SEQ ID NO:5-8, a CDR2 sequence selected from SEQ ID NO:9-21, and a CDR3 sequence selected from SEQ ID NO:22.

[0010] Implementation Scheme 2. The polypeptide of Implementation Scheme 1, wherein each albumin-binding VHH domain independently comprises a CDR1 sequence selected from SEQ ID NO: 5-8, a CDR2 sequence selected from SEQ ID NO: 9-21, and a CDR3 sequence selected from SEQ ID NO: 22.

[0011] Implementation Scheme 3. The polypeptide of Implementation Scheme 1 or Implementation Scheme 2, wherein at least one VHH domain binding albumin comprises a sequence selected from the following CDR1, CDR2 and CDR3 sequences: SEQ ID NO: 5, 9 and 22; SEQ ID NO: 5, 10 and 22; SEQ ID NO: 5, 11 and 22; SEQ ID NO: 5, 12 and 22; SEQ ID NO: 5, 13 and 22; SEQ ID NO: 5, 14 and 22; SEQ ID NO: 5, 15 and 22; SEQ ID NO: 6, 15 and 22; SEQ ID NO: 7, 15 and 22; SEQ ID NO: 8, 15 and 22; SEQ ID NO: 6, 16 and 22; SEQ ID NO: 6, 17 and 22; SEQ ID NO: 6, 18 and 22; SEQ ID NO: 6, 19 and 22; SEQ ID NO: 6, 20 and 22; and SEQ ID NO: 6, 21 and 22.

[0012] Implementation Scheme 4. The polypeptide of Implementation Scheme 3, wherein each VHH domain binding albumin independently comprises a sequence selected from the following CDR1, CDR2 and CDR3 sequences: SEQ ID NO: 5, 9 and 22; SEQ ID NO: 5, 10 and 22; SEQ ID NO: 5, 11 and 22; SEQ ID NO: 5, 12 and 22; SEQ ID NO: 5, 13 and 22; SEQ ID NO: 5, 14 and 22; SEQ ID NO: 5, 15 and 22; SEQ ID NO: 6, 15 and 22; SEQ ID NO: 7, 15 and 22; SEQ ID NO: 8, 15 and 22; SEQ ID NO: 6, 16 and 22; SEQ ID NO: 6, 17 and 22; SEQ ID NO: 6, 18 and 22; SEQ ID NO: 6, 19 and 22; SEQ ID NO: 6, 20 and 22; and SEQ ID NO: 6, 21 and 22.

[0013] Implementation Scheme 5. The polypeptide of any one of Implementation Schemes 1 to 4, wherein at least one VHH domain of albumin is humanized.

[0014] Implementation Scheme 6. The polypeptide of Implementation Scheme 5, wherein each VHH domain that binds albumin is humanized.

[0015] Implementation Scheme 7. The polypeptide of any one of Implementation Schemes 1 to 6, wherein at least one VHH domain of albumin-binding protein comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequences to those selected from SEQ ID NO:23-43 and 71-74.

[0016] Implementation Scheme 8. The polypeptide of Implementation Scheme 7, wherein each VHH domain binding albumin contains at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequences to those selected from SEQ ID NO: 23-43 and 71-74.

[0017] Implementation Scheme 9. The polypeptide of any one of Implementation Schemes 1 to 7, wherein at least one VHH domain that binds albumin comprises a sequence selected from SEQ ID NO:23-43 and 71-74.

[0018] Implementation Scheme 10. The polypeptide of any one of Implementation Schemes 1 to 9, wherein each VHH domain binding albumin comprises a sequence selected from SEQ ID NO:23-43 and 71-74.

[0019] Implementation Scheme 11. A polypeptide of any one of Implementation Schemes 1 to 10, wherein at least one VHH domain of albumin binds human albumin and at least one albumin selected from cynomolgus monkey, mouse and rat albumin.

[0020] Implementation Scheme 12. The polypeptide of any one of Implementation Schemes 1 to 11, wherein each VHH domain of the albumin-binding protein binds human albumin and at least one albumin selected from cynomolgus monkey, mouse and rat albumin.

[0021] Implementation Scheme 13. A polypeptide of any one of Implementation Schemes 1 to 12, wherein at least one VHH domain of albumin binds to human, cynomolgus monkey, mouse and rat albumin.

[0022] Implementation Scheme 14. The polypeptide of any one of Implementation Schemes 1 to 13, wherein each VHH domain of the albumin-binding polypeptide binds to human, cynomolgus monkey, mouse and rat albumin.

[0023] Implementation Scheme 15. A polypeptide of any one of Implementation Schemes 1 to 14, wherein at least one VHH domain that binds albumin binds human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM.

[0024] Implementation Scheme 16. A polypeptide of any one of Implementation Schemes 1 to 15, wherein at least one VHH domain that binds albumin binds to 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.

[0025] Implementation Scheme 17. The polypeptide of any one of Implementation Schemes 1 to 16, wherein each albumin-binding VHH domain binds human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM.

[0026] Implementation Scheme 18. The polypeptide of any one of Implementation Schemes 1 to 17, wherein each albumin-binding VHH domain binds to 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.

[0027] Implementation Scheme 19. The polypeptide of any one of Implementation Schemes 1 to 18, wherein each albumin-binding VHH domain does not bind to the albumin domain 3.

[0028] Implementation Scheme 20. The polypeptide of any one of Implementation Schemes 1 to 19, wherein each VHH domain that binds albumin does not interfere with the binding of albumin to FcRn.

[0029] Implementation Scheme 21. A polypeptide of any one of Implementation Schemes 1 to 20, wherein the polypeptide comprises at least one binding domain for binding a non-albumin protein.

[0030] Implementation Scheme 22. The polypeptide of Implementation Scheme 21, wherein at least one binding domain of a non-albumin protein is VHH.

[0031] Implementation Scheme 23. The polypeptide of Implementation Scheme 22, wherein the binding domain of each protein that binds to non-albumin is VHH.

[0032] Implementation Scheme 24. The polypeptide of Implementation Scheme 21, wherein at least one binding domain of a non-albumin protein comprises a heavy chain variable region and a light chain variable region.

[0033] Implementation Scheme 25. The polypeptide of Implementation Scheme 24, wherein each binding domain of the non-albumin-binding protein includes a heavy chain variable region and a light chain variable region.

[0034] Implementation Scheme 26. The polypeptide of any one of Implementation Schemes 21 to 25, wherein at least one binding domain of a non-albumin protein is a binding domain of a therapeutic antibody.

[0035] Implementation Scheme 27. The polypeptide of Implementation Scheme 26, wherein the binding domain of each protein that binds to non-albumin is the binding domain of a therapeutic antibody.

[0036] Implementation Scheme 28. The polypeptide of Implementation Scheme 26 or Implementation Scheme 27, wherein the therapeutic antibody can be used to treat diseases or conditions selected from autoimmune diseases or conditions, inflammatory diseases or conditions, infections and cancers.

[0037] Implementation Scheme 29. A polypeptide of any one of Implementation Schemes 1 to 28, wherein the polypeptide comprises the amino acid sequence of a therapeutic protein.

[0038] Implementation Scheme 30. The polypeptide of Implementation Scheme 29, wherein the therapeutic protein can be used to treat diseases or conditions selected from autoimmune diseases or conditions, inflammatory diseases or conditions, infections, and cancers.

[0039] Implementation Scheme 31. A polypeptide as described in any one of Implementation Schemes 1 to 30, wherein the polypeptide contains an Fc region.

[0040] Implementation scheme 32. The polypeptide of implementation scheme 31, wherein the Fc region binds FcRn.

[0041] Implementation scheme 33. The polypeptide of implementation scheme 31 or implementation scheme 32, wherein the Fc region is the IgG1 Fc region.

[0042] Implementation Scheme 34. A polypeptide of any one of Implementation Schemes 31 to 33, wherein the Fc region comprises one or more substitutions that enhance half-life.

[0043] Implementation Scheme 35. The polypeptide of Implementation Scheme 34, wherein the Fc region comprises one or more substitutions that enhance FcRn binding and / or reduce the dissociation rate of Fc and FcRn at at least one pH.

[0044] Implementation Scheme 36. A polypeptide of any one of Implementation Schemes 31 to 35, wherein the Fc region comprises a substitution at one or more amino acid positions selected from 252, 254, 256, 428 or 434.

[0045] Implementation Scheme 37. The polypeptide of Implementation Scheme 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.

[0046] Implementation Scheme 38. The polypeptide of Implementation Scheme 37, wherein the Fc region comprises substitutions for M252Y, S254T and T256E; M252Y and M428V; or M428L and N434S.

[0047] Implementation Scheme 39. The polypeptide of any one of Implementation Schemes 31 to 38, wherein the Fc region comprises a sequence selected from SEQ ID NO: 47-68 and 85-87.

[0048] Implementation Scheme 40. A polypeptide of any one of Implementation Schemes 1 to 39, wherein the half-life of the polypeptide is greater than the half-life of the same polypeptide lacking the VHH domain that binds albumin.

[0049] Implementation Scheme 41. A pharmaceutical composition comprising a polypeptide as described in any one of Implementation Schemes 1 to 40 and a pharmaceutically acceptable carrier.

[0050] Implementation Scheme 42. An isolated nucleic acid encoding a polypeptide as described in any one of Implementation Schemes 1 to 40.

[0051] Implementation Scheme 43. A vector comprising nucleic acid as described in Implementation Scheme 42.

[0052] Implementation Scheme 44. A host cell comprising a nucleic acid as described in Implementation Scheme 42 or a vector as described in Implementation Scheme 43.

[0053] Implementation Scheme 45. A host cell that expresses a polypeptide as described in any one of Implementation Schemes 1 to 40.

[0054] Implementation Scheme 46. A method for producing a polypeptide as described in any one of Implementation Schemes 1 to 40, comprising culturing a host cell as described in Implementation Scheme 44 or Implementation Scheme 45 under conditions suitable for expressing the polypeptide.

[0055] Implementation Scheme 47. The method of Implementation Scheme 46, further comprising isolating the polypeptide.

[0056] Implementation Scheme 48. A method comprising administering to a subject a polypeptide as described in any one of Implementation Schemes 1 to 40 or a pharmaceutical composition as described in Implementation Scheme 41.

[0057] Implementation Scheme 49. A method of treating a disease or condition, comprising administering to a subject suffering from the disease or condition a pharmaceutically effective amount of a polypeptide as described in any one of Implementation Schemes 1 to 40 or a pharmaceutical composition as described in Implementation Scheme 41.

[0058] Implementation scheme 50. The method of implementation scheme 49, wherein the disease or condition is selected from autoimmune diseases or conditions, inflammatory diseases or conditions, infections and cancers. Attached Figure Description

[0059] Figures 1A to 1C . Figure 1A This is a schematic diagram of a monomeric single-domain antibody (sdAb-NNT-Fc) that binds albumin and contains Fc with mutations in S364N, Y407N, and K409T. Figures 1B to 1C The results show that 4A01-NNT-hFc (SEQ ID NO: 23 and 68) binds monovalently to human serum albumin (HAS; 1B) and murine serum albumin (MSA; 1C).

[0060] Figures 2A to 2B . Figure 2A A schematic diagram illustrating the experimental design for assessing biolayer interferometry of binding to albumin domain 3. Figure 2B The biological layer interferometry track showed that 4A01-NNT-hFc (SEQ ID NO: 23 and 68) did not bind to albumin domain 3, while 1CO4 did.

[0061] Figures 3A to 3B . Figure 3A A schematic diagram illustrating the experimental design for evaluating the binding of B2M-FcRn to albumin using a biolayer interferometry method. Figure 3B The track was obtained by biological layer interferometry analysis, which showed that 4A01-NNT-hFc (SEQ ID NO: 23 and 68) and humanized 4A01v51-NNT-hFc did not block B2m-FcRn from binding to albumin, while 1CO4 did.

[0062] Figures 4A to 4I . Figure 4A The comparison shows certain humanized variants of 4A01 (SEQ ID NO:24-43). Figures 4B to 4C ELISA showing human albumin (HSA) binding to certain humanized variants of 4A01. Figures 4D to 4E ELISA showing the binding of certain humanized variants of cynomolgus macaque albumin (CSA). Figures 4F to 4G ELISA showing the binding of murine albumin (MSA) to certain humanized variants of 4A01. Figures 4H to 4I Rat albumin (RSA) binding ELISA for certain humanized variants of 4A01.

[0063] Figures 5A to 5D Human albumin (HAS) showing humanized hz4A01v51; Figure 5A Crab-eating macaque albumin (CSA); Figure 5B ), Murine albumin (MSA; Figure 5C ) and rat albumin (RSA; Figure 5DCombined with ELISA.

[0064] Figure 6 Various monospecific and multispecific single-domain antibody forms are shown. Form (i) contains two albumin-specific VHH domains (black) formalized as VHH-hIgG Fc (hIgG Fc shown in gray). Form (ii) is a monovalent albumin-specific VHH domain (black) formalized as VHH-hIgG-NNT-Fc (monomer Fc, gray). Form (iii) is a bispecific polypeptide consisting of an albumin-binding VHH domain (black) and a non-albumin-targeting VHH domain (gray) linked via a glycine-serine linker. Form (iv) contains two albumin-specific VHH domains (black) formalized as hIgG1 Fc-VHH (hIgG Fc shown in gray). Form (v) is a bispecific polypeptide, bivalent to each target, containing an albumin-specific VHH domain (black) fused to the C-terminus of the heavy chain of a non-albumin antibody (containing both light and heavy chains shown in gray). The heavy chain of this polypeptide can be formalized as VH-CH1-hIgG Fc-VHH. Form (vi) is a bispecific polypeptide, bivalent to each target, containing an albumin-specific VHH domain (black) fused to the N-terminus of the heavy chain of a non-albumin antibody (containing both light and heavy chains shown in gray). The heavy chain of this polypeptide can be formalized as VHH-VH-CH1-hIgG Fc. Form (vii) is a bispecific polypeptide, bivalent to each target, containing an albumin-specific VHH domain (black) fused to the area between the CH1 and Fc regions of a non-albumin antibody (containing both light and heavy chains shown in gray). The heavy chain of this polypeptide can be formalized as VH-CH1-VHH-hIgG Fc. Form (viii) is a bispecific polypeptide, bivalent to each target, containing an albumin-specific VHH domain (black) fused to the N-terminus of a light chain of a non-albumin antibody (including the light and heavy chains shown in gray). The light chain of this polypeptide can be formalized as VHH-VL-CL. Form (ix) is a bispecific polypeptide, bivalent to each target, containing an albumin-specific VHH domain (black) fused to the C-terminus of a light chain of a non-albumin antibody (including the light and heavy chains shown in gray). The light chain of this polypeptide can be formalized as VL-CL-VHH.

[0065] Figures 7A to 7B The ELISA assay shows the binding of albumin-bound and non-albumin-bound sdAb peptides to human albumin. Figure 7A The binding was shown at pH 7.4, while Figure 7B The binding was shown at pH 6. Figures 7A to 7B The constructs in Figure 6 The formalization described in (iii) is as follows.

[0066] Figure 8 The monovalent sdAb peptide (cx5009) was shown to bind to albumin in humans, cynomolgus monkeys, mice, or rats via ELISA at pH 7.4. Figure 8 The constructs in Figure 6 The formalization described in (ii) is as follows.

[0067] Figures 9A to 9D This displays the in vivo pharmacokinetic (PK) profiles of albumin-bound divalent VHH-hIgG1-xELL-Fc peptide (cx11956) compared to the non-targeted divalent VHH-hIgG1-xELL-Fc peptide (cx11851). Figure 9A and Figure 9B The serum PK profile of BALB / c mice administered 30 mg / kg of the test substance is shown, as determined by ELISA. Figure 9C and Figure 9D The serum PK profile of mice administered with 0.3 mg / kg of the test substance is shown. Figure 9A and Figure 9C Displays absolute serum concentration, in μg / mL, while Figure 9B and Figure 9D The data show the concentration (cMax) normalized to 30 minutes after administration. Figures 9A to 9D The constructs in Figure 6 The formalization described in (i) is as follows.

[0068] Figure 10 The binding of human IgG1 and human IgG1-xELL to recombinant biotinylated human FCGRT (FcRn) / B2M heterodimer was demonstrated by biolayer interference metric analysis (BLI). Association and dissociation with FcRn were detected at pH 6 and pH 8.

[0069] Figures 11A to 11B The results showed that different monospecific and bispecific albumin-binding single-domain antibody forms and their binding to recombinant human albumin at neutral (7.4) pH were compared. Figure 11A ) and IL-4R ( Figure 11B ) through ELISA binding. Single-specific molecules (cx12583 and cx12584) such as Figure 6 The formalized bispecific molecules described in (iv) are as follows: Figure 6 (v)(cx12587 and cx12594) or Figure 6 The formalization described in (vii)(cx12595 and cx12596) is as follows.

[0070] Figures 12A to 12D The display shows that it is formalized as a monovalent VHH-hIgG1-NNT-Fc polypeptide ( Figures 11A to 11B) and divalent VHH-hIgG1-xELL-Fc polypeptide ( Figures 12C to 12D Some humanized variants of 4A01 at pH 6 ( Figure 12A and Figure 12C ) and pH 7.4 ( Figure 12B and 12D Human albumin (HSA) binding ELISA. Detailed Implementation

[0071] The implementation schemes provided in this article involve albumin-binding peptides and their uses.

[0072] Definitions and various implementation schemes

[0073] The section headings used in this article are for organizational purposes only and should not be construed as limiting the subject matter.

[0074] All references cited in this article (including patent applications, patent publications, and Genback Accession numbers) are incorporated herein by reference, just as the full text of each individual reference is explicitly and individually incorporated by reference.

[0075] The techniques and procedures described or mentioned herein are generally well known to those skilled in the art and commonly used by them. For example, the following widely used methodologies employ: Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYCURRENT PROTOCOLS IN MOLECULAR BIOLOGY (edited by F.M. Susubel et al. (2003)); Series of Methods in Enzymology (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (edited by M.J. MacPherson, B.D. Hames, and G.G. Taylor (1995)); Harlow and Lane (edited by Harlow and Lane, 1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (edited by R.R. Freshney (1987)); Oligonucleotide Synthesis (edited by M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis editor, 1998) Academic Press; Animal Cell Culture (RI Freshney) editor, 1987); Introduction to Cell and Tissue Culture (JPMather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (edited by A.Doyle, JBGriffiths and DG Newell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (edited by DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos (ed., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., ed., 1994); Current Protocols in Immunology (JEColigan et al., ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA 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, ed., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, ed., Harwood). Academic Publishers, 1995; and Cancer: Principles and Practice of Oncology (edited by VT DeVita et al., JBLippincott Company, 1993); and their latest editions.

[0076] Unless otherwise defined, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires or explicitly specifies otherwise, singular terms shall include plural terms and plural terms shall include singular terms. In the event of any conflict between definitions from various sources or references, the definitions provided herein shall prevail.

[0077] Generally, the residues in the constant region of the immunoglobulin heavy chain are numbered according to the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "The EU index in Kabat's book" refers to the residue numbering of human IgG1 EU antibodies.

[0078] It should be understood that embodiments of the invention described herein include “consisting of the embodiments” and / or “substantially consisting of the embodiments”. As used herein, unless otherwise specified, the singular forms “a / an” and “the” include plural references. The use of the term “or” herein is not intended to imply that substitutes are mutually exclusive.

[0079] In this application, unless expressly specified or understood by those skilled in the art, the word "or" means "and / or". In the context of multiple subordinate technical solutions, "or" is used to indicate more than one previous independent or subordinate technical solution.

[0080] The phrases “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. The reference sample can be used to establish the levels of proteins and / or mRNA present in, for example, healthy tissue, compared to the levels of proteins and / or mRNA present in a sample with unknown characteristics. In some embodiments, the reference sample is from the same subject, but from a different part of the subject being tested. In some embodiments, the reference sample is from a tissue region surrounding or adjacent to cancer. In some embodiments, the reference sample is not from the subject being tested, but is a sample from a subject known to have or not have the condition in question. In some embodiments, the reference sample is from the same subject, but from a time point prior to the development of cancer in the subject. In some embodiments, the reference sample is from a benign cancer sample from the same or different subjects. When a negative reference sample is used for comparison, the expression level or amount of the molecule in question in the negative reference sample will indicate, as those skilled in the art would understand, the absence of the molecule and / or the presence of a molecule at low levels, in light of the invention. When a positive reference sample is used for comparison, the degree or amount of expression of the molecule in question in the positive reference sample will indicate the extent that a person skilled in the art would understand, given the present invention, that a certain amount of the molecule exists.

[0081] As used herein, in the context of benefit from self-administered therapeutics or response to administered therapeutics, the terms “benefit,” “clinical benefit,” “responsiveness,” and “treatment responsiveness” can be measured by assessing various endpoints, such as, to some extent, inhibition of disease progression, including slowing and complete inhibition; reduction in the number of disease onsets and / or symptoms; reduction in lesion size; inhibition (i.e., reduction, slowing, or complete cessation) of disease cell infiltration into adjacent peripheral organs and / or tissues; inhibition (i.e., reduction, slowing, or complete cessation) of disease spread; to some extent, relief of one or more symptoms associated with the disease; increased length of disease-free survival after treatment, such as progression-free survival; increased overall survival; higher response rate; and / or reduced mortality at a given time point after treatment. “Non-responsive” or “unresponsive” subjects or cancers are those who do not meet the eligibility criteria specified above as “responsive.”

[0082] The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably and refer to a polymer of nucleotides. This polymer of nucleotides may contain natural and / or non-natural nucleotides, including (but 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.

[0083] The terms "peptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues, not limited to a minimum length. This polymer of amino acid residues may contain native or non-native amino acid residues, and includes (but is not limited to) peptides, oligopeptides, dimers, trimers, and polymers containing amino acid residues. Both full-length proteins and fragments thereof are included in this definition. The term also includes post-expression modifications of peptides, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of this invention, "peptide" refers to a protein containing modifications to its native sequence, such as deletions, additions, and substitutions (generally inherently conservative), provided that the protein maintains its desired activity. This modification may be intentional, such as through site-directed mutagenesis, or accidental, such as through mutations in the host producing the protein or due to errors in PCR amplification.

[0084] As used herein, “albumin” means any initially mature albumin derived from the processing of albumin precursors in cells. Unless otherwise specified, the term includes albumin from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys or rhesus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring variants of albumin, such as splice variants or paired gene variants. A non-limiting exemplary amino acid sequence of mature human albumin is shown in, for example, UniProt registration number P02768.2. See SEQ ID NO. 1. Non-limiting exemplary amino acid sequences of murine, cynomolgus monkey, and rat albumin are shown in SEQ ID NO: 2-4.

[0085] The term "specific binding" to an antigen or epitope is well known in the art, as are methods for measuring such specific binding. A molecule is considered to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently or rapidly with a specific cell or substance than with a substitute cell or substance, with a longer duration and / or greater affinity. A single-domain antibody (sdAb) or VHH-containing peptide binds to a target with greater affinity, antibody affinity, faster binding, and / or longer duration than binding to other substances, and is therefore considered to exhibit "specific binding" or "preferential binding" to the target. For example, an sdAb or VHH-containing peptide that specifically or preferentially binds to an albumin epitope has greater affinity, antibody affinity, faster binding, and / or longer duration than an sdAb or VHH-containing peptide that binds to that epitope. This definition also implies 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. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it may include) specific binding. Generally, but not necessarily, the mention of binding refers to preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0086] As used herein, the term "inhibition" in relation to the activity of a target protein refers to a reduction in the activity of the protein. In some embodiments, "inhibition" refers to a reduction in the activity of the protein compared to the absence of a regulator.

[0087] 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) that an antigen-binding molecule (e.g., an sdAb or a VHH-containing polypeptide) binds to. Epitopes typically comprise chemically active surface groups of molecules (e.g., amino acids, polypeptides, or sugar side chains) and have specific three-dimensional structural features and specific charge features. Epitopes can be formed from both adjacent and / or juxtaposed non-adjacent residues (e.g., amino acid, nucleotide, sugar, lipid moieties) of the target molecule. Epitopes formed from consecutive residues (e.g., amino acid, nucleotide, sugar, lipid moieties) are generally retained upon exposure to denaturing solvents; however, epitopes formed from tertiary folding are generally lost upon treatment with denaturing solvents. Epitopes may contain (but are not limited to) at least 3, at least 5, or 8 to 10 residues (e.g., amino acids or nucleotides). In some embodiments, the epitope length is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within that antigen. In some embodiments, epitopes can be identified by a minimum distance from CDR residues on an antigen-binding molecule. In some embodiments, epitopes can be identified by the above distance, and further limited to those residues involved in the bond (e.g., hydrogen bond) between residues of the antigen-binding molecule and antigen residues. Epitopes can also be identified by various scans, such as alanine or arginine scans, which can indicate one or more residues that the antigen-binding molecule can interact with. Unless explicitly specified, a set of residues that is an epitope does not exclude other residues that are part of an epitope of a particular antigen-binding molecule. Rather, the presence of this set specifies a minimal series (or set of species) of epitopes. Thus, in some embodiments, a set of residues identified as an epitope specifies a minimum number of epitopes associated with the antigen, rather than an exclusive list of epitope residues on the antigen.

[0088] A “nonlinear epitope” or “configurational epitope” comprises a non-adjacent polypeptide, amino acid, and / or sugar within an antigen protein to which an antigen-binding molecule specifically binds. In some embodiments, at least one of the residues will be non-adjacent to other designated residues of the epitope; however, one or more of the residues may be adjacent to other residues.

[0089] A “linear epitope” comprises an adjacent polypeptide, amino acid, and / or sugar within an antigen protein to which an antigen-binding molecule specifically binds. It should be noted that in some embodiments, not every residue within the linear epitope needs to be directly bound (or involved in a bond) by an antigen-binding molecule. In some embodiments, the linear epitope may originate from an immunization with a peptide whose sequence is effectively composed of the linear epitope, or from a structural portion of a protein relatively separate from the rest of the protein (such that the antigen-binding molecule can be intact, at least primarily), having only that sequence portion.

[0090] The term "antibody" is used in the broadest sense and includes a variety of polypeptides containing an antibody-like antigen-binding domain, including (but not limited to) well-known antibodies (typically comprising at least one heavy chain and at least one light chain) and fragments thereof (e.g., scFv, Fab), single-domain antibodies (sdAbs, comprising at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and fragments of any of the above, provided they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerizing domain. This dimerizing domain includes (but is not limited to) a heavy chain constant domain (comprising CH1, a hinge, CH2, and CH3, wherein CH1 typically pairs with the light chain constant domain CL, and the hinge mediates dimerization) and an Fc region (comprising a hinge, CH2, and CH3, wherein the hinge mediates dimerization).

[0091] The term antibody also includes (but is not limited to) chimeric antibodies, humanized antibodies, and antibodies from various species, such as camels (including rheas), sharks, mice, humans, cynomolgus monkeys, etc.

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

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

[0094] In this document, the terms "single-domain antibody" and "sdAb" are used interchangeably to refer to an antibody comprising at least one monomeric domain (such as a VHH domain without a light chain) and an Fc region. In some embodiments, the sdAb is a dimer of two peptides, each peptide comprising at least one VHH domain and an Fc region. As used herein, the terms "single-domain antibody" and "sdAb" include peptides comprising multiple VHH domains, such as peptides having the structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different.

[0095] The term "VHH-containing polypeptide" refers to a polypeptide containing at least one VHH domain. In some embodiments, the VHH polypeptide contains two, three, four, or more VHH domains, wherein the VHH domains may be the same or different. In some embodiments, the VHH-containing polypeptide contains an Fc region. In some of these embodiments, the VHH-containing polypeptide may be referred to as an sdAb. Additionally, in some of these embodiments, the VHH polypeptide may form a dimer. Non-limiting structures of VHH-containing polypeptides (which are also sdAbs) include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of this structure, one VHH may be linked to another VHH via a linker, or one VHH may be linked to an Fc via a linker. In some embodiments, the linker comprises 1 to 20 amino acids, preferably 1 to 20 amino acids mainly composed of glycine and optionally serine. Non-limiting linkers are shown in SEQ ID NO: 88-93. In some embodiments, when the VHH-containing polypeptide contains Fc, it forms a dimer. Therefore, if the structure VHH1-VHH2-Fc forms a dimer, it is considered tetravalent (i.e., the dimer has four VHH domains). Similarly, if the structure VHH1-VHH2-VHH3-Fc forms a dimer, it is considered hexavalent (i.e., the dimer has six VHH domains).

[0096] The term "monoclonal antibody" refers to an antibody (including sdAbs or VHH-containing peptides) that is substantially homologous to a population of antibodies, i.e., the population of individual antibodies is identical, except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, unlike multi-strain antibody formulations that typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. Therefore, a sample of monoclonal antibodies can bind to the same epitope on the antigen. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homologous 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 prepared by the hybridoma method first described in Kohler and Milstein, 1975, Nature 256:495, or by a recombinant DNA method such as that described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries produced using techniques such as those described in McCafferty et al., 1990, Nature 348:552-554.

[0097] The term "CDR" stands for Complementary Decision Region, as defined by at least one identification method known to those skilled in the art. In some embodiments, the CDR may be defined according to any of the following: Chothia numbering scheme, Kabat numbering scheme, combination of Kabat and Chothia, AbM definition, and / or contact definition. The VHH comprises three CDRs, designated as CDR1, CDR2, and CDR3.

[0098] As used in this article, the term "heavy-chain constant region" refers to a region containing at least three heavy-chain constant regions C. H 1. Hinges, C H 2 and C H Region 3. Of course, unless otherwise specified, any non-functional changes or modifications within these regions are included within the scope of the term "heavy chain constant region". A non-limiting exemplary heavy chain constant region includes γ, δ, and α. A non-limiting exemplary heavy chain constant region also includes ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. Additionally, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. Some isotypes may be further subdivided into subtypes. For example, IgG antibodies include (but are not limited to) IgG1 (containing the γ1 constant region), IgG2 (containing the γ2 constant region), IgG3 (containing the γ3 constant region) and IgG4 (containing the γ4 constant region) antibodies; IgA antibodies include (but are not limited to) IgA1 (containing the α1 constant region) and IgA2 (containing the α2 constant region) antibodies; and IgM antibodies include (but are not limited to) IgM1 and IgM2.

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

[0100] As used herein, “receptor human framework” refers to a heavy chain variable domain (V) containing a component derived from the human immunoglobulin framework or the human common framework as discussed herein. H The amino acid sequence of the frame. Receptor human frames derived from the human immunoglobulin frame or the human common frame may contain the same amino acid sequence, or may contain amino acid sequence variations. In some embodiments, the number of amino acid variations across all human frames across a single antigen-binding domain (such as VHH) is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3.

[0101] "Affinity" refers to the sum of the strengths of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody, such as an sdAb or a VHH-containing peptide) and its binding partner (e.g., an antigen). The affinity, or apparent affinity, of molecule X for its partner Y can generally be expressed as a dissociation constant (K0). D ) or K D-表观 It is indicated. Affinity can be expressed through common methods known in the art (such as, for example, ELISA K). D (e.g., KinExA, flow cell counting, and / or surface plasma resonance devices), including those measurements described herein. This method includes (but is not limited to) methods involving... Alternatively, flow cytometry can be used.

[0102] As used in this article, the term "K" D "K" refers to the equilibrium dissociation constant of antigen-binding molecules / antigen interactions. When the term "K" is used in this article... D When “, it contains K D and K D-表观 .

[0103] In some implementations, the K of the antigen-binding moleculeD The antigen-expressing cell lines were used for flow cell counting, and the average fluorescence measured at various antibody concentrations was fitted to a nonlinear unit point binding equation (Prism software graphpad). In some of these embodiments, K... D For K D-表观 .

[0104] The term "bioactivity" refers to any one or more biological properties of a molecule (whether naturally present when found in living organisms or provided or realized through recombinant methods). Biological properties include (but are not limited to) binding ligands, inducing or increasing cell proliferation, and inducing or increasing the expression of cytokines.

[0105] "Agonist" or "activating" antibodies are those that increase and / or activate the biological activity of the 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.

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

[0107] "Affinity-matured" sdAb or VHH-containing peptide refers to an sdAb or VHH-containing peptide with one or more modified CDRs compared to a parental sdAb or VHH-containing peptide without one or more modifications, which results in improved affinity of the sdAb or VHH-containing peptide for the antigen.

[0108] As used herein, “humanized VHH” refers to a VHH in which one or more frame regions have been substantially replaced by human frame regions. In some embodiments, certain frame region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. Furthermore, humanized VHHs may contain residues not found in the original VHH or in the human frame sequence, but included to further improve and optimize the performance of the VHH-containing peptide in the sdAb. In some embodiments, the humanized sdAb or VHH-containing peptide contains a human Fc region. It should be understood that the humanized sequence can be identified by its primary sequence and does not necessarily indicate a method for producing the antibody.

[0109] The "effector-positive Fc region" possesses the "effector function" of the native 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. This effector function generally requires the combination of the Fc region with a binding domain (e.g., antibody variable domain) and can be assessed using various analyses.

[0110] The “natural sequence Fc region” contains the same amino acid sequence as the Fc region found in nature. The natural sequence human Fc region includes the natural sequence human IgG1 Fc region (non-A and A allotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, as well as its naturally occurring variants.

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

[0112] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, FcγR is the original human FcR. In some embodiments, FcR is an IgG antibody-binding receptor (γ receptor) and a receptor comprising the FcγRI, FcγRII, and FcγRIII subclasses, including paired variants and alternative spliced ​​forms of those receptors. The FcγRII receptor comprises FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor-based tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor-based tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). For example, FcRs are discussed 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 included in the term “FcR” in this document. For example, the term “Fc receptor” or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward, Immunol. Today 18(12):592-598(1997); Ghetie et al., Nature Biotechnology, 15(7):637-640(1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216(2004); WO 2004 / 92219(Hinton et al.)).

[0113] As used herein, the terms "substantially similar" or "substantially identical" mean that two or more values ​​are similar enough that a person skilled in the art would consider the difference between the two or more values ​​to be negligible or non-biologically and / or statistically significant in the context of the biological characteristic measured by the value. In some embodiments, the difference between two or more substantially similar values ​​does not exceed any one of about 5%, 10%, 15%, 20%, 25%, or 50%.

[0114] A peptide “variant” means a bioactive peptide that, after sequence alignment and the introduction of gaps (if necessary) to achieve maximum sequence identity and without considering any retained substitutions as part of sequence identity, has at least about 80% amino acid sequence identity with the native peptide sequence. This variant includes, for example, peptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus. In some embodiments, the variant will have at least about 80% amino acid sequence identity. In some embodiments, the variant will have at least about 90% amino acid sequence identity. In some embodiments, the variant will have at least about 95% amino acid sequence identity with the native peptide sequence.

[0115] As used herein, “percentage of amino acid sequence identity (%)” and “homology” for peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence after alignment and the introduction of gaps (if necessary) to achieve maximum sequence identity % and without considering any retaining substitutions as part of sequence identity. Alignments for the purpose of determining the percentage of amino acid sequence identity can be performed in various ways within the capabilities of those skilled in the art (e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software). Those skilled in the art can determine suitable parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0116] Amino acid substitutions may include (but are not limited to) the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions may be introduced into the antibody of interest and for desired activities, such as preserving / improving antigen binding, reducing immunogenicity, or improving ADCC or CDC screening products.

[0117] Table 1

[0118]

[0119] Amino acids can be grouped according to common side chain properties:

[0120] (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

[0121] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0122] (3) Acidity: Asp, Glu;

[0123] (4) Alkaline: His, Lys, Arg;

[0124] (5) Residues that affect chain orientation: Gly, Pro;

[0125] (6) Fang tribe: Trp, Tyr, Phe.

[0126] Non-retentional substitution involves exchanging members of one category for members of another.

[0127] The term "vector" is used to describe a polynucleotide that can be engineered to contain selected polynucleotides capable of replicating in a host cell. A vector may contain one or more of the following components: a replication origin, one or more regulatory sequences (such as, for example, promoters and / or enhancers) regulating the expression of the polypeptide of interest, and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric analysis, such as β-galactosidase). The term "expression vector" refers to a vector used to express the polypeptide of interest in a host cell.

[0128] "Host cell" refers to a cell that can be, or has been, a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Suitable eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include (but are not limited to) NSO cells. Crucells and 293 and CHO cells and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Host cells include the progeny of a single host cell, and these progeny may not be identical to the original parent cell (morphologically or in terms of genomic DNA complement) due to natural, accidental, or intentional mutations. Host cells include cells transfected in vivo with the polynucleotides described herein.

[0129] As used herein, the term "isolated" refers to a molecule that has been isolated from at least some of its components, using which the molecule is normally found or produced in nature. For example, a polypeptide is called "isolated" when it is isolated from at least some of the components of the cell that produced it. In the case where a polypeptide is secreted by a cell after expression, the physical isolation of the supernatant containing the polypeptide from the cell that produced it is considered "isolated" polypeptide. Similarly, a polynucleotide is called "isolated" when it is not a part of a larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA in the case of DNA polynucleotides), in which the larger polynucleotide is normally found in nature, or isolated from at least some of the components of the cell that produced it, for example, in the case of RNA polynucleotides. Thus, DNA polynucleotides contained in a vector within a host cell can be called "isolated".

[0130] In this document, the terms "subject" and "subject" are used interchangeably to refer to an animal; for example, a mammal. In some embodiments, methods are provided for treating mammals, including (but not limited to) humans, rodents, apes, cats, dogs, horses, cattle, pigs, sheep, goats, laboratory mammals, farm mammals, competitive mammals, and pet mammals. In some embodiments, "subject" or "subject" refers to a subject or person who requires treatment for a disease or condition. In some embodiments, a subject receiving treatment may be a patient who has been identified as having the relevant condition to be treated or is at appropriate risk of exposure to the condition.

[0131] As used in this article, "disease" or "symptom" refers to a condition that requires and / or expects treatment.

[0132] The term "autoimmune disease" refers to a disease or condition that is typically associated with non-allergic hypersensitivity reactions (Class II, III, and / or IV), which generally arise from an immune response mediated by the subject's own body fluids and / or cells to one or more immunogenic substances of endogenous or exogenous origin.

[0133] The term "inflammatory condition" refers to a condition associated with inflammation, including (but not limited to) chronic or acute inflammatory diseases and specifically including inflammatory autoimmune diseases and inflammatory allergic symptoms.

[0134] The terms “infection” and “infectious disease or condition” refer to a disease or condition caused by exogenous infectious agents such as (but not limited to) bacteria, viruses, fungi, protozoa and parasites.

[0135] The terms "cancer" and "tumor" encompass solid cancers and hematologic / lymphomas, as well as malignant, pre-malignant, and benign growths such as dysplasia. Exemplary cancers include (but are not limited to): basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancers, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancers, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, leukemia, liver cancer, and lung cancer (e.g., small cell lung cancer, non-small cell lung cancer). Lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung; melanoma; 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; gastric cancer; testicular cancer; thyroid cancer; uterine cancer or endometrial cancer; urinary system cancer; vulvar cancer; lymphoma (including Hodgkin's and...). Non-Hodgkin's lymphoma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade spreading NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-lytic cell NHL, large-volume disease NHL, mantle cell lymphoma, AIDS-related lymphoma and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic medulloblastic leukemia, and other cancers and sarcomas; and post-transplant lymphoproliferative disorders (PTLD) and abnormal angiogenesis, edema (such as those associated with brain tumors) associated with leukoblastic leukemia and Meigs' syndrome.

[0136] In some implementations, “increase” or “decrease” each refers to a statistically significant increase or decrease. As will be apparent to those skilled in the art, “modulation” may also involve changes in the affinity, antibody affinity, specificity, and / or selectivity of the target or antigen for one or more of its ligands, binding partners, partners or substrates in the form of homopolymers or heteropolymers (which may be increases or decreases); changes in the sensitivity of the target or antigen to one or more conditions in the medium or to the environment in which the target or antigen is present (such as pH, ionic strength, presence of cofactors, etc.) (which may be increases or decreases); and / or cell proliferation or cytokine production compared to the same conditions but without the assay reagent. This may be done in any suitable manner and / or using any suitable analytical assay known per se or described herein, depending on the target involved.

[0137] As used herein, “treatment” refers to a method of achieving a beneficial or desired clinical outcome. As used herein, “treatment” encompasses any administration or application of a therapeutic agent for a disease in mammals, including humans. For the purposes of this invention, a beneficial or desired clinical outcome includes (but is not limited to) any one or more of the following: relief of one or more symptoms, reduction of disease severity, prevention or delay of disease spread (e.g., metastasis, such as to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of disease state, inhibition of disease or disease progression, inhibition or slowing of disease or its progression, cessation of its development, and remission (whether partial or complete). “Treatment” also encompasses a reduction in the pathological outcome of proliferative diseases. The methods provided herein consider any one or more of these aspects of treatment. Consistent with the foregoing, the term “treatment” does not require 100% removal of all aspects of the disease.

[0138] "Improvement" means the reduction or improvement of one or more symptoms compared to not receiving a treatment. "Improvement" also includes a reduction or decrease in the duration of symptoms.

[0139] In this document, the term "anticancer agent" is used in its broadest sense to refer to an agent used to treat one or more types of cancer. Exemplary categories of such agents include (but are not limited to) chemotherapeutic agents, anticancer biological agents (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiotherapy, CAR-T therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNA), and oncolytic viruses.

[0140] The term "biological sample" refers to a quantity of material derived from a living organism or pre-organism. This material includes (but is 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.

[0141] The terms "control" or "reference" refer to a composition known to contain either no analyte ("negative control") or an analyte ("positive control"). A positive control may contain a known concentration of the analyte.

[0142] The term "inhibition" or "inhibit" refers to a reduction or cessation of any expressed characteristic, or a reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Reduction" or "inhibition" means a reduction, decrease, or inhibition of activity, function, and / or amount compared to a reference. In some embodiments, "reduction" or "inhibit" means the ability to cause an overall reduction of 10% or greater. In some embodiments, "reduction" or "inhibit" means the ability to cause an overall reduction of 50% or greater. In some embodiments, "reduction" or "inhibit" means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or greater. In some embodiments, the amounts specified above represent inhibition or reduction over a period of time relative to a control over the same period of time.

[0143] As used herein, “delaying the development of a disease” means postponing, hindering, slowing, delaying, stabilizing, inhibiting, and / or postponing the development of a disease (such as cancer). This delay can be of varying lengths, depending on the patient’s medical history and / or the patient’s current treatment. As will be apparent to those skilled in the art, a substantial or significant delay can effectively include prevention, in that the patient does not develop the disease. For example, the development of advanced cancer, such as metastasis, can be delayed.

[0144] As used herein, “prevention” includes providing prevention related to the occurrence or recurrence of a disease in subjects who are predisposed to the disease but have not yet been diagnosed with it. Unless otherwise specified, the terms “reduction,” “suppression,” or “prevention” do not imply or require complete prevention at all times, but only during the period of positive measurement.

[0145] The "therapeuticly effective amount" of a substance / molecule, agonist, or antagonist can vary depending on factors such as the subject's disease state, age, sex, and weight, and the ability of the substance / molecule, agonist, or antagonist to elicit the desired response in the subject. Therapeuticly effective amount is also the amount by which any toxic or adverse effect of the substance / molecule, agonist, or antagonist outweighs the therapeutically beneficial effect. Therapeuticly effective amount can be delivered in one or more doses. Therapeuticly effective amount refers to the amount at which the desired therapeutic and / or preventative outcome is effectively achieved at the required dose and for the required duration.

[0146] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a formulation in such a form as to allow the bioactivity of the active ingredient(s) to be effective and without any other components that would have unacceptable toxicity to the subject to whom the formulation will be administered. This formulation may be sterile.

[0147] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier well known in the art and used in conjunction with a therapeutic agent, comprising a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used.

[0148] "Combined with one or more other therapeutic agents" includes simultaneous (combined) and sequential administration in any order.

[0149] As used herein, the term “combination” refers to the administration of two or more therapeutic agents, wherein at least part of the administration overlaps in time, or the administration of one therapeutic agent falls within a short time period relative to the administration of another therapeutic agent, or the therapeutic effects of two agents overlap for at least a period of time.

[0150] As used in this article, the term "sequentially" refers to the administration of two or more therapeutic agents that do not overlap in time or in which the therapeutic effects of the agents do not overlap.

[0151] As used herein, “in combination with” refers to administration of one therapeutic modality in addition to another. Therefore, “in combination with” means administration of one therapeutic modality before, during, or after administration of another therapeutic modality to the subject.

[0152] The term "instructions for use of a drug" refers to the information commonly included in the commercially available packaging of a therapeutic product, which contains information about indications, uses, dosage, administration, combination therapies, contraindications, and / or warnings regarding the use of the therapeutic product.

[0153] "Article" refers to any manufacture (e.g., packaging or container) or kit that contains at least one reagent, such as a pharmaceutical agent for treating a disease or condition (e.g., cancer), or a probe for specifically detecting the biomarkers described herein. In some embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0154] The terms "tag" and "detectable tag" refer to, for example, a portion linked to an antibody or antigen such that the reaction (e.g., binding) between members of a specific binding pair is detectable. The labeled member of a specific binding pair is referred to as "detectably labeled." Therefore, the term "labeled binding protein" refers to a protein with an incorporated tag that provides identification of the binding protein. In some embodiments, the tag is a detectable biomarker that can generate a signal detectable by visual or instrumental methods (e.g., incorporating or linking a radiolabeled amino acid to a biotinylated moiety detectable by labeled avidin (e.g., a polypeptide containing a fluorescent marker or enzymatically active streptavidin detectable by optical or colorimetric methods)). Examples of polypeptide tags include (but are not limited to) the following: radioisotopes or radionuclides (e.g., 3 H, 14 C 35 S, 90 Y、 99 Tc, 111 In、 125 I, 131 I, 177 Lu、 166 Ho or 153 Sm); chromogens, fluorescent tags (e.g., FITC, rhodamine, lanthanide fluorescent powders); enzyme tags (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporter sequences (e.g., leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, epitope tags); and magnetic agents, such as gadolinium chelates. Representative examples of tags commonly used in immunoassays include a light-generating portion, such as an acridine compound, and a fluorescence-generating portion, such as luciferin. In this respect, the portion itself is not detectable, but may become detectable after reacting with another portion.

[0155] Exemplary albumin-binding peptide

[0156] This document provides single-domain antibodies (sdAbs) comprising a VHH domain that binds albumin. In some embodiments, the VHH domain that binds albumin does not interfere with albumin binding to FcRn. In some embodiments, the VHH domain that binds albumin does not bind to domain 3 of albumin. In some embodiments, the VHH domain that binds albumin has an affinity (K0) between 0.01 nM and 5 nM, or between 0.01 nM and 2 nM, or between 0.01 nM and 1 nM, or between 0.01 nM and 0.5 nM, or between 0.05 nM and 5 nM, or between 0.05 nM and 2 nM, or between 0.05 nM and 1 nM, or between 0.05 nM and 0.5 nM. D(Combined)

[0157] In various embodiments, a polypeptide comprising at least one albumin-binding VHH domain is provided. In some embodiments, a polypeptide comprising one, two, three, four, five, six, seven, or eight albumin-binding VHH domains is provided. In some embodiments, the polypeptide provided herein comprises one, two, three, or four albumin-binding VHH domains. This polypeptide may comprise one or more additional VHH domains binding one or more non-albumin target proteins.

[0158] In various embodiments, the polypeptide comprising one or more albumin-binding VHH domains further comprises a therapeutic antigen-binding domain and / or a therapeutic polypeptide. This therapeutic antigen-binding domain includes (but is not limited to) conventional antibody antigen-binding domains comprising heavy chain variable regions and light chain variable regions, and single-domain antibody antigen-binding domains (such as VHH domains). Figure 6 (v) through (ix) provide non-limiting forms of peptides comprising one or more albumin-binding VHH domains and one or more conventional antibody domains. Other non-limiting exemplary therapeutic peptides include, for example, receptor extracellular domains, enzymes, and ligands. In various embodiments, peptides comprising at least one albumin-binding VHH domain have a longer in vivo half-life than the same peptide not having at least one albumin-binding VHH domain. In some embodiments, the half-life is at least 1.5x, at least 2x, at least 3x, at least 4x, or at least 5x longer than the half-life of peptides not having an albumin-binding VHH domain.

[0159] In some embodiments, a polypeptide comprising at least one albumin-binding VHH domain includes an Fc region. In some embodiments, the polypeptides provided herein comprise one, two, three, or four albumin-binding VHH domains and Fc regions. In some embodiments, the Fc region mediates polypeptide dimerization under physiological conditions.

[0160] In various embodiments, the VHH domain binding albumin includes a CDR1 sequence selected from SEQ ID NO:5-8, a CDR2 sequence selected from SEQ ID NO:9-21, and a CDR3 sequence selected from SEQ ID NO:22. In various embodiments, the VHH domain binding albumin comprises a sequence selected from the following CDR1, CDR2, and CDR3 sequences: SEQ ID NO: 5, 9, and 22; SEQ ID NO: 5, 10, and 22; SEQ ID NO: 5, 11, and 22; SEQ ID NO: 5, 12, and 22; SEQ ID NO: 5, 13, and 22; SEQ ID NO: 5, 14, and 22; SEQ ID NO: 5, 15, and 22; SEQ ID NO: 6, 15, and 22; SEQ ID NO: 7, 15, and 22; SEQ ID NO: 8, 15, and 22; SEQ ID NO: 6, 16, and 22; SEQ ID NO: 6, 17, and 22; SEQ ID NO: 6, 18, and 22; SEQ ID NO: 6, 19, and 22; SEQ ID NO: 6, 20, and 22; and SEQ ID NO: 6, 21, and 22.

[0161] In some embodiments, the VHH domain binding to albumin comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequences selected from SEQ ID NO:23-43 and 71-74. In some embodiments, the VHH domain binding to albumin comprises an amino acid sequence selected from SEQ ID NO:23-43 and 71-74.

[0162] In some embodiments, a VHH domain for binding albumin is provided, which competes with a VHH domain comprising an amino acid sequence selected from SEQ ID NO:23-43 and 71-74 for binding to albumin.

[0163] In some embodiments, the VHH domain binding to albumin may be humanized. Humanized antibodies (such as sdAbs or VHH-containing peptides) can be used as therapeutic molecules because they reduce or eliminate human immune responses to non-human antibodies, which can lead to reduced immune responses to antibody therapeutics and reduced efficacy of the therapeutics. Generally, humanized antibodies comprise one or more variable domains, wherein 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. Optionally, humanized antibodies may also comprise 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., an antibody derived from CDR residues) to, for example, restore or improve antibody specificity or affinity.

[0164] Humanized antibodies and their preparation methods are described, 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. Patents Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; and Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol. Immunol. 28:489-498 (description of “Surface Rework”); Dall'Acqua et al., (2005) Methods 36:43-60 (description of “FR Remodeling”); and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (description of the “Guided Selection” approach to FR remodeling).

[0165] Human frame regions that can be used for humanization include (but are not limited to): frame regions selected using a “best fit” method (see, for example, Sims et al. (1993) J. Immunol. 151:2296); frame regions of the common sequence of human antibodies derived from the heavy chain variable region of a specific subgroup (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:262). 3) Human mature (somatic mutation) framework regions or human germline framework regions (see, for example, Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633); and framework regions derived from screening FR libraries (see, for example, Baca et al., (1997) J. Biol. Chem. 272:10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, the FR regions of VHHs are replaced with human FR regions to prepare humanized VHHs. In some embodiments, certain FR residues of human FRs are replaced to improve one or more properties of the humanized VHH. The VHH domain having these replaced residues is still referred to herein as “humanized”.

[0166] As provided herein, sdAbs containing the VHH domain of albumin-binding peptides can slow the clearance of molecules linked thereto, including molecules containing human Fc regions. In various embodiments, the Fc region contained in the albumin-binding peptide is a human Fc region or is derived from a human Fc region. Non-limiting sdAb forms containing Fc regions are shown in… Figure 6 middle.

[0167] In some embodiments, the Fc region contained in the albumin-binding peptide is derived from the human Fc region and includes the deletion of three amino acids in the low-carbon hinge corresponding to IgG1, E233, L234, and L235, referred to herein as "Fc xELL". The FcxELL peptide does not bind to FcγR and is therefore referred to as "effecton silencing" or "effecton zero". However, in some embodiments, the xELL Fc region binds to FcRn and thus has an extended half-life and transcytosis associated with FcRn-mediated recycling.

[0168] In some embodiments, the Fc region contained in the albumin-binding peptide is derived from the human Fc region and includes mutations M252Y and M428V, which may be referred to as "YV". In some embodiments, this mutation enhances binding to FcRn at an acidic pH (around 6.5) in the endosome, while losing detectable binding at a neutral pH (around 7.2), allowing enhanced FcRn-mediated recycling and an extended half-life. In some embodiments, the Fc region contained in the albumin-binding peptide is derived from the human Fc region and includes mutations M252Y, S254T, and T256E, which may be referred to as "YTE". In some embodiments, this mutation prolongs the human serum half-life by significantly reducing the dissociation rate of Fc and FcRn. In some embodiments, the Fc region contained in the albumin-binding peptide is derived from the human Fc region and includes mutations M428L and N434S, which may be referred to as "LS". In some implementations, this mutation prolongs the serum half-life in humans by increasing the binding affinity of Fc to FcRn at pH 6 and decreasing the dissociation rate. Various Fc mutations that can enhance the circulating half-life are described, for example, in Saunders, Front.Immunol.doi.org / 10.3389 / fimmu.2019.01296 (2019).

[0169] In some embodiments, the Fc region contained in the albumin-binding polypeptide is derived from the human Fc region and includes mutations designed for heterodimerization, referred to herein as "knobs" and "holes". 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 for heterodimerization contains additional mutations, such as the mutation S354C on the first member of the heterodimer Fc pair, which forms an asymmetric disulfide with the corresponding mutation Y349C on the second member of the heterodimer Fc pair. In some embodiments, one member of the heterodimer Fc pair contains the mutation H435R or H435K to prevent protein A binding while maintaining FcRn binding. In some embodiments, one member of the heterodimer Fc pair contains the mutation H435R or H435K, while the second member of the heterodimer Fc pair is not modified at H435. In various embodiments, the mortar Fc region contains a modification of H435R or H435K (when modified to H435R, it is referred to as "mortar-R" in some embodiments), while the pestle Fc region does not contain it. In some embodiments, the mortar-R mutation improves the purification of heterodimers on the mortar Fc region where heterodimers may exist.

[0170] Non-limiting exemplary Fc regions that can be used in albumin-binding peptides include Fc regions containing the amino acid sequences of SEQ ID NO:47-68 and 85-87.

[0171] Exemplary activity of albumin-binding peptides

[0172] In various embodiments, the albumin-binding peptides provided herein bind to albumin epitopes outside domain 3. In some embodiments, the albumin-binding peptides provided herein do not interfere with (i.e., do not inhibit) albumin binding to FcRn. Methods for determining whether the albumin-binding peptides interfere with albumin binding to FcRn are known in the art; non-limiting exemplary methods are also provided herein.

[0173] In some embodiments, the albumin-binding domain-containing peptides provided herein have a longer in vivo half-life than peptides lacking an albumin-binding domain. In various embodiments, the albumin-binding domain-containing peptides provided herein have a half-life at least 1.5x, at least 2x, at least 3x, at least 4x, or at least 5x longer than that of peptides lacking an albumin-binding domain.

[0174] Peptide expression and production

[0175] A nucleic acid molecule comprising a polynucleotide encoding a polypeptide containing an albumin-binding domain is provided. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs the secretion of the albumin-binding domain-containing polypeptide, the leader sequence being typically cleaved so that it is not present in the secreted polypeptide. The leader sequence may be an initial heavy chain (or VHH) leader sequence or another heterologous leader sequence.

[0176] Nucleic acid molecules can be constructed using recombinant DNA techniques well known in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in selected host cells.

[0177] A vector comprising a nucleic acid encoding a polypeptide containing an albumin-binding domain is provided. This vector includes (but is not limited to) DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector optimized for expression of the polypeptide in a desired cell type (such as CHO or CHO-derived cells or NSO cells) is selected. An exemplary vector is described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0178] In some embodiments, peptides containing an albumin-binding domain may be expressed in prokaryotic cells (such as bacterial cells) or eukaryotic cells (such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells). This expression may be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express peptides include (but are not limited to) COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells; Crucells; and NSO cells. In some embodiments, the peptide may be expressed in yeast. See, for example, U.S. Publication No. US2006 / 0270045 A1. In some embodiments, a specific eukaryotic host cell is selected based on its ability to perform the desired post-translational modifications on the peptide. For example, in some embodiments, CHO cells produce peptides with a higher degree of sialylation than the same peptide produced in 293 cells.

[0179] The introduction of one or more nucleic acids (such as vectors) into desired host cells can be achieved by any method, including (but not limited to) calcium phosphate transfection, DEAE-glucan-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, 3rd edition, Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into desired host cells using any suitable method.

[0180] Host cells comprising any of the nucleic acids or vectors described herein are also provided. In some embodiments, host cells expressing polypeptides comprising albumin-binding domains described herein are provided. The polypeptide expressed in the host cells can be purified by any suitable method. This method includes (but is not limited to) the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and agents that bind to the Fc region. For example, protein A, protein G, protein A / G, or antibody affinity columns can be used to bind to the Fc region and purify polypeptides containing the Fc region. Hydrophobic interaction chromatography (e.g., butyl or phenyl columns) is also suitable for purifying some polypeptides (such as antibodies). Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) is also 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.) is also suitable for purifying some polypeptides (such as antibodies). Many methods for purifying polypeptides are known in the art.

[0181] In some embodiments, the peptide is generated 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).

[0182] In some embodiments, a polypeptide comprising an albumin-binding domain prepared by the above-described method is provided. In some embodiments, the polypeptide is prepared in host cells. In some embodiments, the polypeptide is prepared in a cell-free system. In some embodiments, the polypeptide is purified. In some embodiments, a cell culture medium comprising the polypeptide is provided.

[0183] In some embodiments, an antibody-containing composition prepared by the above-described method is provided. In some embodiments, the composition comprises a polypeptide containing an albumin-binding domain prepared in host cells. In some embodiments, the composition comprises a polypeptide prepared in a cell-free system. In some embodiments, the composition comprises a purified polypeptide.

[0184] An exemplary method of treating diseases using albumin-binding peptides

[0185] In some embodiments, a method of treating a subject for a disease is provided, comprising administering a therapeutic polypeptide comprising an albumin-binding domain provided herein. This disease includes any disease from which treatment with the therapeutic polypeptide would benefit. 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 conditions, inflammatory diseases or conditions, and cancer. The method comprises administering to a subject an effective amount of the therapeutic polypeptide comprising an albumin-binding domain provided herein. This treatment may be performed in humans or animals. In some embodiments, a method of treating a human is provided.

[0186] The therapeutic peptides containing an albumin-binding domain provided herein may be administered to subjects as needed. The determination of the dosing frequency may be made by those skilled in the art, such as by an attending physician based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health status of the subject being treated, and the like. In some embodiments, an effective dose of the therapeutic peptide is administered to the subject once or multiple times. In some embodiments, an effective dose of the therapeutic peptide is administered to the subject daily, bi-weekly, weekly, bi-weekly, monthly, etc. An effective dose of the therapeutic peptide is administered to the subject at least once. In some embodiments, an effective dose of the therapeutic peptide may be administered multiple times, including multiple times over a period of at least one month, at least six months, or at least one year.

[0187] In some embodiments, the pharmaceutical composition is administered in an amount that is effective in treating the disease. Therapeuticly effective amounts typically depend on the weight of the subject being treated, their physical or health condition, the extent of the condition to be treated, or the age of the subject being treated. Generally, the antibody can be administered in amounts ranging from 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 amounts ranging from about 10 μg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the antibody can be administered in amounts ranging from about 50 μg / kg body weight to about 5 mg / kg body weight per dose. In some embodiments, the antibody can be administered in amounts ranging from about 100 μg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody can be administered in amounts ranging from about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody can be administered in amounts ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.5 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.05 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.05 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 5 mg / kg body weight or less, for example, less than 4, less than 3, less than 2, or less than 1 mg / kg of antibody.

[0188] In some implementations, the therapeutic peptide can be administered in vivo via various routes, including (but not limited to) intravenous, intra-arterial, non-enteral, intraperitoneal, or subcutaneous administration. Appropriate formulations and routes of administration can be selected based on the intended application.

[0189] Pharmaceutical Composition

[0190] In some embodiments, compositions comprising polypeptides containing albumin-binding domains are provided in formulations having a wide variety of pharmaceutically acceptable carriers (see, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th edition, (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd edition, Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers (including mediators, adjuvants, and diluents) are available. In addition, a variety of pharmaceutically acceptable excipients (such as pH adjusters and buffers, tonicants, stabilizers, wetting agents, and the like) are also available. Non-limiting exemplary carriers include saline, buffered saline, dextran, water, glycerol, ethanol, and combinations thereof.

[0191] In some embodiments, the pharmaceutical composition comprises a polypeptide containing 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.

[0192] Non-limiting exemplary diagnostic and treatment methods

[0193] In some embodiments, the methods described herein can be used to evaluate subjects and / or samples from subjects (e.g., cancer patients). In some embodiments, the evaluation is one or more of diagnosis, prognosis, and / or response to treatment.

[0194] In some embodiments, the methods described herein include assessing the presence, absence, or quantity of a protein. In some embodiments, the methods described herein include assessing the presence, absence, or expression level of a nucleic acid. The compositions described herein can be used for this measurement. In some embodiments, the assessment can guide treatment (including treatment with the peptides described herein).

[0195] Reagent test kit

[0196] Articles and kits are also provided comprising any of the polypeptides containing an albumin-binding domain as described herein, and suitable packaging. In some embodiments, the invention comprises a kit having: (i) a polypeptide containing an albumin-binding domain, and (ii) instructions for administering the polypeptide to a subject using the kit.

[0197] Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, cans, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. This product may be further sterilized and / or sealed. Unit dosage forms comprising the compositions described herein are also provided. These unit dosage forms may be stored in suitable packaging as single or multiple unit doses and may be further sterilized and / or sealed. Instructions supplied with the kits of the present invention are typically written instructions as labels or pharmaceutical instructions (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a disk or optical storage disc) are also acceptable. Instructions related to antibody use generally include information on dosage, dosing schedule, and route of administration for intended therapeutic or industrial use. The kit may further include descriptions of suitable subjects or treatments.

[0198] The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. For example, kits may also be provided containing sufficient doses of the molecules disclosed herein to provide an effective duration of treatment for subjects, such as approximately 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 longer. The kit may also contain multiple unit doses of the molecules, instructions for use, and in quantities sufficient for storage and use in pharmacies (e.g., hospital pharmacies and dispensing pharmacies). In some embodiments, the kit comprises a dry (e.g., lyophilized) composition that can be rehydrated, resuspended, or rehydrated to typically form an antibody suspension.

[0199] Example

[0200] The embodiments discussed below are intended purely as examples of the invention and should not be considered as limiting the invention in any way. These embodiments are not intended to represent all or only experiments conducted. Efforts have been made to ensure accuracy regarding quantities used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise specified, parts are by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0201] Example 1: Development of anti-albumin single-domain antibody (sdAb)

[0202] The single-domain antibody targeting human albumin was generated via immunization with a recombinant form of rhea and alpaca serum albumin (SEQ ID NO:1).

[0203] After developing the specific anti-albumin antibody titer, peripheral blood mononuclear cells (PBMCs) of rheas / alpas were separated from 500 mL of blood from immunized animals, and total mRNA was isolated using the Qiagen RNeasy Maxi kit and subsequently converted into first-strand cDNA using Thermo Superscript IV reverse transcriptase and oligo-dT initiation. The VHH sequence was specifically amplified by PCR using cDNA as a template and selected for colonization into a yeast surface presentation vector as the VHH-Fc-AGA2 fusion protein. Fc is human IgG1 Fc or, in some cases, a variant of IgG1 Fc with reduced effector function.

[0204] A yeast library containing the VHH-Fc-AGA2 fusion protein was separated using a recombinant form of human albumin via magnetic beads and then concentrated using fluorescence activated cell sorting (FACS). Sorted yeast plates were inoculated, and isolated pure lines were selected into 96-well blocks for induction of VHH-Fc-AGA2 fusion protein expression on the yeast cell surface. Biotinylated recombinant human albumin or unrelated biotinylated protein (albumin-negative) was directly applied to the induced yeast, washed, treated with fluorophore-labeled streptavidin, and analyzed by 96-well flow cytometry.

[0205] The nucleic acid sequences encoding VHH, which bind to biotinylated recombinant human albumin and do not bind to unrelated biotinylated proteins, were selected within the framework of the human Fc xELL coding region and colonized into a mammalian expression vector. Expression was then achieved via transient transfection with polyethyleneimine in HEK293 free-form cells (293F cells) or CHO cells. Supernatants were collected after 3 to 7 days, and the secreted recombinant protein was purified by protein A chromatography. Concentration was calculated based on absorbance and extinction coefficient at 280 nm.

[0206] Anti-albumin sdAb 4A01 was selected for humanization.

[0207] Example 2: Monovalent binding of anti-albumin sdAb 4A01 to human and mouse albumin

[0208] Monomeric anti-albumin sdAb 4A01 is produced by fusing 4A01 VHH into a human Fc containing S364N, Y407N and K409T mutations (4A01-NNT-hFc; SEQ ID NO: 23 and 68). Figure 1AThe monomeric anti-albumin sdAb 4A01 was prepared by ELISA by titrating the monomeric sdAb onto immobilized albumin from a specified species (Medisorp plate) and by using an anti-human Fc (HRP) assay, as follows.

[0209] The plate was coated with albumin of the specified species (Sigma) at 2 μg / ml, 50 μl / well, and incubated overnight at 4°C. 1x fish glue (blocker, Biotium) was added to the coated wells and incubated at RT for 1 hour. Titration with 4A01-NNT-hFc was added (starting at 100 nM, diluted 1:3, with a final well blank), and the plate was incubated at RT for 1 hour. The plate was then washed three times with 0.1% D-PBST, followed by the addition of anti-human Fc HRP antibody (1:2000 in 0.1% D-PBST, Jackson). The plate was incubated at RT for 30 minutes and washed three times with 0.1% D-PBST. TMB substrate was then added, and the absorbance was read at 650 nm using a Molecular Devices reader. The data were plotted using the equation unit point-total binding (Y = Bmax*X / (Kd+X) + NS*X + background, GraphPad Prism).

[0210] like Figure 1B and Figure 1C As shown, anti-albumin sdAb 4A01 binds to both human and mouse albumin with comparable affinity. Human albumin's K... D The K of 0.23 nM and mouse albumin D It is 0.20 nM.

[0211] Example 3: Anti-albumin sdAb 4A01 does not bind to albumin domain 3

[0212] Albumin primarily binds to the β-2 microglobulin FcRn complex via domain 3, and this binding is believed to involve an increased half-life of proteins fused to anti-albumin antibodies or to albumin itself. To determine whether anti-albumin sdAb 4A01 binds to albumin domain 3, the binding of 4A01-NNT-hFc was assessed using a biolayer interferometry assay, as follows.

[0213] Albumin domain 3 (mouse Fc-labeled) was immobilized on an anti-mouse IgG Fc capture biosensor. All buffer / protein formulations were prepared in MBST5 (50 nM MES pH 5, 150 mM NaCl, 0.025% Tween). A baseline was established using buffer-only. Mouse Fc-labeled human albumin domain III (10 μg / ml) was loaded onto an anti-mouse IgG Fc capture sensor (ForteBio). Anti-albumin sdAbs 4A01 (4A01-NNT-hFc) and 1CO4 (similar form) were then loaded and allowed to associate with the captured biotinylate domain 3, followed by dissociation using MBST5. sdAb 1CO4 is known to bind to albumin domain 3 and was used as a positive control. See also Figure 2A .

[0214] like Figure 2B As shown, 1C04, instead of 4A01, binds to the immobilized albumin domain 3.

[0215] The binding of anti-albumin sdAb 4A01 (4A01-NNT-hFc), hz4A01v51, and 1C04 to albumin-FcRn was then tested, as follows. Binding was assessed using biolayer interferometry with biotinylated recombinant FcRn-B2M immobilized on a strepto-antibiotic biosensor. The immobilized FcRn-B2M was then conjugated with recombinant human albumin. All buffer / protein formulations were prepared in MBST5 (50 nM MES pH 5, 150 mM NaCl, 0.025% Tween). A baseline was established using buffer-only. Biotinylated FcRn-B2M (10 μg / ml, Acro Biosystems) was loaded onto a strepto-antibiotic biosensor (ForteBio), and baseline was further determined. Then, 50 μM recombinant human albumin (Sigma) was added, allowing association with the immobilized FcRn-B2M. Then, antialbumin sdAbs 4A01 and 4A01v51 and sdAb 1C04 were loaded and allowed to associate with the captured biotin domain 3, followed by dissociation using MBST5. See also Figure 3A .

[0216] like Figure 2B As shown, 4A01 and hx4A01v51 both bind to albumin associated with FcRn-B2M, but 1C04 does not bind.

[0217] Example 4: Humanization and Species Cross-Responsiveness of Anti-Albumin sdAb 4A01

[0218] Various humanized forms of sdAb 4A01 were prepared based on the human heavy chain framework VH3-23*04. Certain amino acids were remutated to donor amino acids, and certain mutations in CDR2 were tested (e.g.). Figure 4A This shows a comparison between the human heavy chain receptor sequence and the humanized form of 4A01.

[0219] The binding of monomeric anti-albumin sdAbs 4A01 (“lm4A01”) and its humanized form to human serum albumin, cynomolgus monkey serum albumin, murine serum albumin, and rat serum albumin was determined by ELISA as follows: Medicorp plates were coated with albumin at 2 μg / ml, 50 μl / well and incubated overnight at 4°C (human, murine, and rat albumin – Sigma; cynomolgus monkey albumin – Abcam). 1x fish glue (blocker, Bethyl Laboratories) was added to the albumin-coated wells, followed by incubation at RT for 1 hour. Titration of the sdAb fusion protein was added (starting at 100 nM, 1:3 horizontally or 1:4 downwards) and incubated at RT for 1 hour. The plates were 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 and incubated at RT for 30 minutes. The plate was washed three times with 0.1% D-PBST, and then TMB substrate was added. The absorbance at 650 nm was read on a Molecular Device and the data were plotted using the equation unit point-total combination (Y = Bmax * X / (Kd + X) + NS * X + background, GraphPad Prism).

[0220] The binding of 4A01 and its humanized form to human albumin is shown in... Figures 4B to 4C In the middle. All sdAbs have K values ​​between 0.10 and 0.43 nM. D Binding of human albumin. The binding of 4A01 and its humanized form to cynomolgus monkey albumin is shown in... Figures 4D to 4E In the middle. All sdAb values ​​are between 0.11 and 0.34 nM. D Binding of cynomolgus macaque albumin. The binding of 4A01 and its humanized form to murine albumin is shown in... Figures 4F to 4G In the middle. All sdAbs have K values ​​between 0.11 and approximately 0.25 nM. D Binding of murine albumin. The binding of 4A01 and its humanized form to rat albumin is shown in... Figures 4H to 4I In the middle. All sdAbs have K values ​​between 0.14 and approximately 0.33 nM. D Bind to rat albumin.

[0221] Figures 5A to 5DThe binding of 4A01 and the humanized hz4A01v51 to albumin in humans (5A), cynomolgus monkeys (5B), murines (5C), and rats (5D) is shown. The tested 4A01 and all humanized variants bound to albumin in all four species with an affinity of less than 1 nM. The humanized hz4A01v51 bound to albumin in all four species with an affinity of less than 0.3 nM, achieving a maximum binding maxima of greater than 90%.

[0222] Example 5: Binding of single-domain antibody peptides to human albumin

[0223] The binding of humanized single-domain antibody (sdAb) peptides to human albumin was tested by ELISA at neutral (7.4) or endosome (6) pH. A 96-well ELISA plate was coated with PBS containing 2 μg / mL recombinant albumin and incubated overnight at 4°C. The plate was then washed with PBS / 0.05% Tween-20 (PBS-T) and blocked for 2 hours at room temperature with PBS-T containing 5% milk powder. Serial dilutions of the test sample were prepared in PBS pH 7.4 or buffer pH 6 containing 20 mM His, 150 mM NaCl and added to the plate. The plate was incubated at 4°C for 1 hour. After incubation, cells were washed in their respective buffers and then incubated at room temperature for 30 minutes with horseradish peroxidase (HRP)-conjugated anti-subject genotype antibody for detecting sdAb. The plate was washed in its respective buffer and TMB substrate was added. The HRP-TMB reaction was allowed to develop for 6 minutes and then stopped with an equal volume of HCl-based stop solution. The absorbance at 450 nm was measured using a 96-well plate reader. The data were plotted and analyzed using GraphPad Prism analysis software. The results are shown in Figure 7.

[0224] like Figure 7A and Figure 7B As shown, Figure 6 (iii) The formalized bivalent bispecific sdAb peptide (cx11917) containing the albumin-binding domain and non-mammalian-targeting binding domain of SEQ ID NO:43 (hz4A01v51VHH) binds albumin with low nanomolar to sub-nanomolar affinity. The apparent affinity is only slightly affected by pH, and is similar to that of 2 nM K at pH 6. d In contrast, K+ has 0.7 nM at neutral pH (7.4). d Binding is mediated solely through monovalent targeting of the sdAb subunit of albumin, because... Figure 6 The formalized sdAb peptide (cx11916) described in (iii) containing two non-mammalian-targeting binding domains did not bind albumin with considerable affinity at any of the pH values ​​tested.

[0225] Example 6: Species cross-responsiveness of albumin-bound single-domain antibody peptides

[0226] The binding of a humanized single-domain antibody (sdAb) peptide (cx5009, SEQ ID 69) containing hz4A01v51 VHH (SEQ ID NO: 43) and a monomeric Fc region (with S364N, Y407N, and K409T substituted Fc regions, SEQ ID NO: 68) to recombinant human, cynomolgus monkey, mouse, or rat albumin at neutral (7.4) pH was tested by ELISA. 96-well ELISA plates were coated with PBS containing 2 μg / mL recombinant albumin and incubated overnight at 4°C. The plates were then washed with PBS / 0.05% Tween-20 (PBS-T) and then blocked with 1x fish glue at room temperature for 1 hour. Serial dilutions of the test sample were prepared in PBS-T pH 7.4 and added to the plates. The plates were incubated at room temperature for 1 hour. After incubation, the cells were washed with PBS-T and then incubated at room temperature for 30 minutes with a HRP-bound secondary antibody specifically targeting human IgG1. The plate was then washed, followed by the addition of TMB substrate. HRP-TMB reaction development was allowed, and absorbance at 650 nm was measured using a 96-well plate reader. Data were plotted and analyzed using GraphPad Prism analysis software. Results are shown below. Figure 8 middle.

[0227] like Figure 8 As shown, cx5009, as Figure 6 (ii) The formalized monovalent albumin-specific sdAb, hz4A01v51 VHH-hIgG1-xELL-NNT-Fc (SEQ ID NO: 69), shown in (ii) binds to albumins from humans, cynomolgus monkeys, mice, and rats. It exhibits cross-species similarity in apparent affinity at neutral pH (7.4), with a K+ level in the sub-nanomolar range (approximately 0.2 nM). d .

[0228] Example 7: In vivo pharmacokinetic profile of albumin-bound single-domain antibody peptides

[0229] In healthy mice, the ability of albumin-binding single-domain antibodies (sdAbs) to prolong serum exposure to human IgG was tested. The pharmacokinetic (PK) profiles of a humanized bivalent sdAb peptide (cx11956, SEQ ID NO:70) that is cross-responsive to mouse albumin and formally represented as hz4A01v51VHH-hIgG1-xELL-Fc were compared with those of a non-cross-responsive bivalent sdAb peptide (cx11851) formally represented as the same VHH-hIgG1-xELL-Fc structure. The xELL variant of human IgG1 reduces Fcγ receptor binding but does not affect FcRn binding. This was confirmed in vitro using biolayer interference metric analysis. The cross-responsiveness of human IgG1 to mouse FcRn enables FcRn-mediated recycling of human antibodies in mice.

[0230] To determine the PK profile of the sdAb-hIgG xELL-Fc test item, BALB / c mice were intravenously injected with a single dose of 30 mg / kg or 0.3 mg / kg, and serum samples were collected at 30 min, 6 h, 24 h, 96 h, and 168 h after injection of the test item. The concentration of the test item in mouse serum was determined by ELISA. For the PK ELISA, the human FcRn / B2M heterodimer protein (His tag, AcroBiosystem) was immobilized on a 96-well ELISA plate by incubating a 4 μg / mL protein solution in PBS at 4 °C for 12 h. The next day, the plate was blocked with 3% BSA TBS-T buffer for 2 h, and then the serum samples on the plate were incubated for 2 h. The binding of the test item in the serum samples to the FcRn immobilized on the ELISA plate was detected using an HRP-conjugated secondary anti-subject genotyping antibody that binds to sdAb. The secondary antibody was incubated on a plate for 1 hour and visualized using the TMB substrate solution. Stop solution (1M H₂SO₄) was then added, and the absorbance at 450 nm was measured using an Emax spectrophotometer (Molecular Devices). The absorbance values ​​were converted to test item concentrations using a standard curve with known protein concentrations in SoftMax Pro. The standard curve was fitted using 4-parameter logistic regression. The data were then analyzed and plotted using GraphPad Prism software.

[0231] As shown in Figure 9, the albumin-targeting sdAb peptide slows the clearance of human IgG1 and, when linked to IgG1, prolongs serum exposure. At 30 mg / kg ( Figure 9A ) or 0.3 mg / kg ( Figure 9CFollowing a single dose of the anti-albumin HZ4A01V51VHH-IgG1 xELL-Fc (cx11956), the absolute concentration in serum was significantly higher than that of the non-targeted VHH-IgG1 xELL-Fc (cx11851) of equal size that did not bind albumin. Despite the injection of equal amounts of protein cx11851, the CMax levels were lower in cx11956 at 30 minutes post-injection. Furthermore, the more rapid clearance of the non-targeted construct (cx11851) continued for the first 6 hours post-injection, as shown in the normalized graph (…). Figure 9B and Figure 9D As shown in the figure. Relative to the cMax (30 min) time point, the concentration of non-targeted CX11851 decreased by almost 60% at 6 hours in the 30 mg / kg dose level group, while the concentration of albumin-bound CX11956 decreased by only about 12%. Figure 10 B). Similarly, at low dose levels (0.3 mg / kg), the concentration of albumin-bound cx11956 decreased to only about 87% of CMax, compared to about 72% of CMax for non-albumin-bound cx11851.

[0232] This finding confirms that albumin binding to the VHH domain enhances serum exposure to IgG antibodies. Without intending to limit oneself to any particular theory, enhanced serum exposure can result from the albumin / FcRn recycling channel, independent of IgG / FcRn-mediated recycling.

[0233] To confirm the equivalent binding of human IgG1-xELL and wild-type human IgG to FcRn, the binding of a formalized sdAb peptide of either of the two IgG1 Fc variants to recombinant human FcRn / B2M was measured using a biolayer interferometry assay with an Octet96 Red reader (Sartorius). In short, biotinylated human FcRn / B2m was loaded onto a strepto-antibiotin biosensor. Associativity of the test article was measured for 60 seconds by immersing the sensor in 100 nM of test article dilution. The test article was diluted in a pH 6 buffer containing 50 mM MES, 150 mM NaCl, and 0.025% Tween-20, or a pH 8 buffer containing 50 mM Tris, 150 mM NaCl, and 0.025% Tween-20. Dissociation of the test article was performed for 300 seconds by immersing the sensor in the respective pH buffer without the test article. Use Forte data analysis software to output association and dissociation curves.

[0234] The results are shown in Figure 10The FcRn binding of IgG1 xELL Fc used in this study was comparable to that of wild-type IgG1 Fc. Both molecules showed similar association rates at pH 6 and exhibited poor affinity for FcRn at pH 8. Therefore, it is expected that albumin binding to the VHH domain will enhance serum exposure to both molecules with the wild-type IgG1 Fc domain and molecules with the IgG1xELL domain.

[0235] Example 8: Binding of various single-domain antibody forms to human albumin

[0236] The ability of different monospecific and bispecific albumin-binding single-domain antibody formulations to bind recombinant human albumin and its second target (IL-4R) at neutral (7.4) pH was evaluated using ELISA. The evaluation included a monospecific antibody containing an albumin-targeting sdAb (hz4A01v51 VHH) linked to the C-terminus of the xELL Fc region via a 6- or 12-residue glycine-serine linker; a bispecific antibody containing the Fab domain (VL-CL (SEQ ID NO:77) and VH-CH1 (SEQ ID NO:76)) of an IL-4R-targeting antibody (dupilumab); a bispecific antibody containing the IgG1 or IgG4 Fc region and an albumin-targeting sdAb (hz4A01v51 VHH) located at different positions; and a monospecific control IL-4R targeting molecule lacking an albumin-targeting sdAb. The names of the test items used in this study and a general structural overview of the peptides are listed in Table 2. For ELISA, 96-well ELISA plates (MaxiSorb, Biolegend) were coated in PBS with human albumin or IL4R at 1 μg / mL (100 μL / well) and incubated overnight at 4°C. The plates were washed 3 times with 0.05% PBST (150 μL / well) and then blocked for 2 hours at RT with 0.05% PBST containing casein (200 μL / well). The plates were washed 3 times with 0.05% PBST, and 100 μL of 0.05% PBST containing the titrated test sample was added to each well (starting at 100 nM, 1:3 dilution, 11-point titration) and incubated at 4°C for 1 hour. After another wash, the plates were incubated at room temperature for 30 minutes with 0.05% PBST (100 μL / well) containing HRP-binding secondary antibody specifically against human IgG1 (Jackson ImmunoResearch). The plate was then washed again, followed by the addition of TMB substrate (100 μL / well), which allowed for RT before addition to the plate. The HRP-TMB reaction was allowed to develop for approximately 10 minutes, after which TMB stop buffer (100 μL / well) was added, and absorbance was measured at 450 nm on a plate reader, subtracting the absorbance at 650 nm (Molecular Devices). The data were plotted and analyzed using GraphPad Prism analysis software. The results are shown below. Figures 11A to 11B middle.

[0237] Table 2

[0238]

[0239]

[0240] like Figure 11AAs shown, all molecules containing anti-albumin VHH exhibit binding to albumin, with peptides containing VHH between the CH1 and Fc regions showing a slightly higher affinity for albumin than molecules with the C-terminus of anti-albumin VHH containing the Fc region. No binding was observed for molecules lacking anti-albumin VHH (cx12585 and cx12590). Figure 11B As shown, all molecules containing the binding domain of the second target (i.e., the Fab domain of dupilumab) exhibited binding to the second target IL-4R with very similar affinity, confirming 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 of the second target (cx12583 and cx12584). This data, along with the data presented in Example 7, shows that albumin-targeting sdAbs can mediate albumin binding when located at the N-terminus, C-terminus, or within the polypeptide (e.g., between domains). Therefore, it is expected that albumin-binding VHH domains will enhance serum exposure to molecules (including those containing Fc), regardless of where the albumin-binding VHH domain is located.

[0241] Example 9: Modification of anti-albumin sdAb Hz4A01v51

[0242] The framework region of Hz4A01v51 was further modified, including by reverting to certain residues of the donor amino acid and / or introducing substituted charged residues. The modified VHHs (Hz4A01v51.9, Hz4A01v51.11, Hz4A01v51.12, and Hz4A01v51.13) were used to generate monovalent (fused to Fc NTT) antialbumin-binding molecules, which possess… Figure 6 The general structure shown in (ii) is also used. Several (Hz4A01v51.9, Hz4A01v51.12, and Hz4A01v51.13) are also used to generate a divalent (VHH fused to Fc xELL) antialbumin-binding molecule, which has Figure 6The general structure is shown in (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 with albumin at 2 μg / mL (100 μL) in PBS and incubated overnight at 4°C, or coated with albumin at 2 μg / mL (100 μL) in 20 mM His-HCl, 150 mM NaCl pH 6 and incubated overnight at 4°C. The plates were washed 3 times with 0.05% PBST or pH 6 buffer and then blocked with 5% milk PBST at RT for 2 hours. Wash the plate 3 times with 0.05% PBST or pH 6 buffer, and add the titrant of the test sample contained in PBST or pH 6 buffer to the plate (starting 100 nM, 1:5 dilution) and incubate at 4°C for 1 hour. After another wash, incubate the plate at room temperature for 30 minutes with an HRP-bound secondary antibody specifically targeting human IgG1. Then wash the plate and add TMB substrate. Allow the HRP-TMB reaction to develop for 6 minutes, add TMB stop buffer, and measure the absorbance at 450 nm using a Molecular Device. Plot and analyze the data using GraphPad Prism analysis software. The 0.16 nM titration point was not tested for negative controls and ELL samples.

[0243] In monovalent form, the modified anti-albumin antibody is displayed at pH 6 ( Figure 12A ) and pH 7.4 ( Figure 12B Similar binding spectra were observed at pH 6, and all showed superior binding compared to Hz4A01v51, particularly at pH 6. In the divalent form, Hz4A01v51.9 and Hz4A01v51.13 showed superior binding compared to Hz4A01v51 at pH 6. Figure 12C ) and pH 7.4 ( Figure 12D Improved similarity binding spectrum under )

[0244] This invention may be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the above embodiments are considered illustrative in all respects and not limiting of the invention. Consequently, the scope of the invention is defined by the appended claims rather than by the foregoing description, and all variations falling within the meaning and equivalents of the claims are intended to be included herein.

[0245] Tables of certain sequences

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

Claims

1. A polypeptide comprising at least one albumin-binding VHH domain, wherein the at least one albumin-binding VHH domain comprises a CDR1 sequence selected from SEQ ID NOs: 5-8, a CDR2 sequence selected from SEQ ID NOs: 9-21, and a CDR3 sequence of SEQ ID NO:

22.

2. The polypeptide of claim 1, wherein each albumin-binding VHH domain independently comprises a CDR1 sequence selected from SEQ ID NOs: 5-8, a CDR2 sequence selected from SEQ ID NOs: 9-21, and a CDR3 sequence of SEQ ID NO:

22.

3. A polypeptide according to claim 1 or claim 2, wherein at least one albumin binding VHH domain comprises a CDR1, CDR2 and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 5, 9 and 22; SEQ ID NOs: 5, 10 and 22; SEQ ID NOs: 5, 11 and 22; SEQ ID NOs: 5, 12 and 22; SEQ ID NOs: 5, 13 and 22; SEQ ID NOs: 5, 14 and 22; SEQ ID NOs: 5, 15 and 22; SEQ ID NOs: 6, 15 and 22; SEQ ID NOs: 7, 15 and 22; SEQ ID NOs: 8, 15 and 22; SEQ ID NOs: 6, 16 and 22; SEQ ID NOs: 6, 17 and 22; SEQ ID NOs: 6, 18 and 22; SEQ ID NOs: 6, 19 and 22; SEQ ID NOs: 6, 20 and 22; and SEQ ID NOs: 6, 21 and 22.

4. The polypeptide of claim 3, wherein each albumin binding VHH domain independently comprises a CDR1, CDR2 and CDR3 sequence selected from the group consisting of: SEQ ID NOs: 5, 9 and 22; SEQ ID NOs: 5, 10 and 22; SEQ ID NOs: 5, 11 and 22; SEQ ID NOs: 5, 12 and 22; SEQ ID NOs: 5, 13 and 22; SEQ ID NOs: 5, 14 and 22; SEQ ID NOs: 5, 15 and 22; SEQ ID NOs: 6, 15 and 22; SEQ ID NOs: 7, 15 and 22; SEQ ID NOs: 8, 15 and 22; SEQ ID NOs: 6, 16 and 22; SEQ ID NOs: 6, 17 and 22; SEQ ID NOs: 6, 18 and 22; SEQ ID NOs: 6, 19 and 22; SEQ ID NOs: 6, 20 and 22; and SEQ ID NOs: 6, 21 and 22.

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

6. The polypeptide of claim 5, wherein each albumin binding VHH domain is humanized.

7. A polypeptide according to any one of claims 1 to 6, wherein at least one albumin binding VHH domain comprises a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence selected from SEQ ID NOs: 23-43 and 71-74.

8. A polypeptide according to claim 7, wherein each albumin binding VHH domain comprises a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence selected from SEQ ID NOs: 23-43 and 71-74.

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

10. The polypeptide of any one of claims 1 to 9, wherein each albumin binding VHH domain comprises a sequence selected from SEQ ID NOs: 23-43 and 71-74.

11. The polypeptide according to any one of claims 1 to 10, wherein at least one albumin binding VHH domain binds human albumin and at least one albumin selected from the group consisting of cynomolgus monkey, mouse and rat albumin.

12. The polypeptide of any one of claims 1 to 11, wherein each albumin binding VHH domain binds human albumin and at least one albumin selected from cynomolgus monkey, mouse and rat albumin.

13. The polypeptide according to any one of claims 1 to 12, wherein at least one albumin binding VHH domain binds human, cynomolgus monkey, mouse and rat albumin.

14. The polypeptide of any one of claims 1 to 13, wherein each albumin binding VHH domain binds human, cynomolgus monkey, mouse and rat albumin.

15. The polypeptide of any one of claims 1 to 14, wherein at least one albumin binding VHH domain binds human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM.

16. The polypeptide of any one of claims 1 to 15, wherein at least one albumin binding VHH domain binds to each of human, cynomolgus monkey, mouse and rat albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM.

17. The polypeptide of any one of claims 1 to 16, wherein each albumin binding VHH domain binds human albumin with an affinity of less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM.

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

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

20. The polypeptide of any one of claims 1 to 19, wherein each albumin binding VHH domain 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 a protein other than albumin.

22. The polypeptide of claim 21, wherein at least one non-albumin binding protein binding domain is a VHH.

23. The polypeptide of claim 22, wherein each non-albumin binding protein binding domain is a VHH.

24. The polypeptide of claim 21, wherein at least one non-albumin binding domain comprises a heavy chain variable region and a light chain variable region.

25. The polypeptide of claim 24, wherein each non-albumin binding protein binding domain comprises a heavy chain variable region and a light chain variable region.

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

27. The polypeptide of claim 26, wherein each binding domain that binds a non-albumin protein is a binding domain of a therapeutic antibody.

28. The polypeptide of claim 26 or claim 27, wherein the therapeutic antibody is useful for treating a disease or condition selected from an autoimmune disease or condition, an inflammatory disease or condition, an infection, and cancer.

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

30. The polypeptide of claim 29, wherein the therapeutic protein is useful for treating a disease or condition selected from the group consisting of an autoimmune disease or condition, an inflammatory disease or condition, an infection, and cancer.

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

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

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

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

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

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

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

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

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

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 of 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 of claim 42 or the vector of claim 43.

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

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

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

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

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

50. The method of claim 49, wherein the disease or disorder is selected from the group consisting of an autoimmune disease or disorder, an inflammatory disease or disorder, an infection, and cancer.

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