Novel serum albumin-binding single domain antibody

Abstract

The present invention relates to a serum albumin-binding single domain antibody useful for providing a protein therapeutic agent having a sufficient half-life even in the absence of an Fc domain. The antibody can effectively extend the half-life of various protein therapeutic agents by binding the protein therapeutic agents to albumin having a long half-life. The present invention can also be applied to protein therapeutic agents, including anticancer agents, having improved half-lives, and therapeutic methods using same.

Description

Novel serum albumin-binding single-domain antibody

[0001] The present invention relates to a novel serum albumin-binding antibody. Specifically, the antibody according to the present invention is a single-domain antibody. The antibody according to the present invention can extend the half-life of various protein therapeutics by binding them to albumin having a long half-life. In this respect, the present invention also relates to a protein therapeutic with an improved half-life, particularly a drug used to treat cancer, or a treatment method using the same.

[0002] Protein therapeutics exist in various forms, such as antibodies, antibody fragments, growth factors, enzymes, and peptides, and a long serum half-life is essential to maximize efficacy and reduce the frequency of administration. However, small proteins of 40–50 kDa or less have a limitation in that they are rapidly removed from the blood through renal filtration, resulting in short half-lives. Various methods have been developed to overcome this, including fusion with antibody binding domains (Fc domains), PEGylation, PASylation, glycosylation, fusion with XTEN protein polymers, and fusion with serum albumin or albumin-binding proteins and peptides. These methods help increase the in vivo pharmacological availability of small molecules and extend their half-lives.

[0003] In particular, proteins that bind to serum albumin offer several advantages over Fc fusions. While Fc fusion is a powerful strategy that increases serum half-life and provides efficacy, it consequently results in proteins having a size and structure similar to IgG. In contrast, serum albumin-binding proteins can maintain a small size, which is advantageous for enhancing tumor invasion capabilities, reducing side effects, and avoiding non-specific immune responses. Due to these advantages, serum albumin-binding proteins are attracting attention as an attractive strategy to replace Fc fusions.

[0004] Serum albumin is synthesized in the liver and has high concentrations in plasma due to an intracellular recycling pathway via FcRn (neonatal Fc receptor). Albumin binds to FcRn in a weakly acidic environment, and the binding dissociates from FcRn at the cell surface, where the pH is neutral, releasing it into the bloodstream. Through this process, albumin has a long half-life, which can be utilized to fuse albumin-binding proteins to various protein therapeutics to extend their half-life. Design requirements for this include (i) the ability to bind to various types of albumin with moderate to high affinity, (ii) binding characteristics that do not interfere with albumin binding to FcRn, and (iii) the ability to maintain albumin binding ability when fused to the N- or C-terminus of various biological agents.

[0005] The present invention is intended to provide a serum albumin-binding antibody that may be useful for providing a protein (e.g., antibody) therapeutic agent capable of maintaining a sufficient half-life even without an Fc domain. Preferably, the albumin-binding antibody according to the present invention is a microscopic single-domain antibody, e.g., a VHH antibody. Furthermore, the present invention is also intended to provide a use for the serum albumin-binding antibody as described above in a fused form with a heterologous protein (e.g., antibody, CAR, etc.) that specifically binds to a therapeutic protein, e.g., a tumor-associated antigen, a T-cell-associated antigen, or a B-cell-associated antigen.

[0006] One aspect of the present invention provides an albumin-binding protein having a novel sequence.

[0007] The numbered items below exemplify some of the embodiments described in this specification.

[0008] 1. An albumin-binding protein comprising a single variable domain that binds to human albumin protein at both pH 5.5 and pH 7.4 and includes a combination of complementary determining regions selected from the group consisting of combinations of the following complementary determining regions (CDR1, CDR2, and CDR3):

[0009] Sequence No. 1 (CDR1), Sequence No. 2 (CDR2), and Sequence No. 3 (CDR3);

[0010] Sequence No. 4 (CDR1), Sequence No. 5 (CDR2), and Sequence No. 6 (CDR3);

[0011] Sequence No. 7 (CDR1), Sequence No. 8 (CDR2), and Sequence No. 9 (CDR3);

[0012] Sequence No. 10 (CDR1), Sequence No. 11 (CDR2), and Sequence No. 12 (CDR3);

[0013] Sequence No. 13 (CDR1), Sequence No. 14 (CDR2), and Sequence No. 15 (CDR3);

[0014] Sequence No. 16 (CDR1), Sequence No. 17 (CDR2), and Sequence No. 18 (CDR3);

[0015] Sequence No. 19 (CDR1), Sequence No. 20 (CDR2), and Sequence No. 21 (CDR3);

[0016] Sequence No. 22 (CDR1), Sequence No. 23 (CDR2), and Sequence No. 24 (CDR3);

[0017] Sequence No. 25 (CDR1), Sequence No. 26 (CDR2), and Sequence No. 27 (CDR3);

[0018] Sequence No. 28 (CDR1), Sequence No. 29 (CDR2), and Sequence No. 30 (CDR3);

[0019] Sequence No. 31 (CDR1), Sequence No. 32 (CDR2), and Sequence No. 33 (CDR3);

[0020] Sequence No. 34 (CDR1), Sequence No. 35 (CDR2), and Sequence No. 36 (CDR3);

[0021] Sequence No. 37 (CDR1), Sequence No. 38 (CDR2), and Sequence No. 39 (CDR3);

[0022] Sequence No. 40 (CDR1), Sequence No. 41 (CDR2), and Sequence No. 42 (CDR3);

[0023] Sequence No. 43 (CDR1), Sequence No. 44 (CDR2), and Sequence No. 45 (CDR3);

[0024] Sequence No. 46 (CDR1), Sequence No. 47 (CDR2), and Sequence No. 48 (CDR3);

[0025] Sequence No. 49 (CDR1), Sequence No. 50 (CDR2), and Sequence No. 51 (CDR3);

[0026] Sequence No. 52 (CDR1), Sequence No. 53 (CDR2), and Sequence No. 54 (CDR3);

[0027] Sequence No. 55 (CDR1), Sequence No. 56 (CDR2), and Sequence No. 57 (CDR3);

[0028] Sequence No. 58 (CDR1), Sequence No. 59 (CDR2), and Sequence No. 60 (CDR3);

[0029] Sequence No. 61 (CDR1), Sequence No. 62 (CDR2), and Sequence No. 63 (CDR3);

[0030] Sequence No. 64 (CDR1), Sequence No. 65 (CDR2), and Sequence No. 66 (CDR3);

[0031] Sequence No. 67 (CDR1), Sequence No. 68 (CDR2), and Sequence No. 69 (CDR3);

[0032] Sequence No. 70 (CDR1), Sequence No. 71 (CDR2), and Sequence No. 72 (CDR3);

[0033] Sequence No. 73 (CDR1), Sequence No. 74 (CDR2), and Sequence No. 75 (CDR3); and

[0034] Sequence No. 76 (CDR1), Sequence No. 77 (CDR2), and Sequence No. 78 (CDR3).

[0035] 2. An albumin-binding protein in which, in the first embodiment, a single variable domain comprises one of the following combinations of complementarity determining regions (CDR1, CDR2, and CDR3):

[0036] Sequence No. 1 (CDR1), Sequence No. 2 (CDR2), and Sequence No. 3 (CDR3);

[0037] Sequence No. 7 (CDR1), Sequence No. 8 (CDR2), and Sequence No. 9 (CDR3);

[0038] Sequence No. 25 (CDR1), Sequence No. 26 (CDR2), and Sequence No. 27 (CDR3);

[0039] Sequence No. 34 (CDR1), Sequence No. 35 (CDR2), and Sequence No. 36 (CDR3);

[0040] Sequence No. 43 (CDR1), Sequence No. 44 (CDR2), and Sequence No. 45 (CDR3);

[0041] Sequence No. 46 (CDR1), Sequence No. 47 (CDR2), and Sequence No. 48 (CDR3);

[0042] Sequence No. 49 (CDR1), Sequence No. 50 (CDR2), and Sequence No. 51 (CDR3);

[0043] Sequence No. 52 (CDR1), Sequence No. 53 (CDR2), and Sequence No. 54 (CDR3); and

[0044] Sequence No. 76 (CDR1), Sequence No. 77 (CDR2), and Sequence No. 78 (CDR3).

[0045] 3. An albumin-binding protein according to the first or second embodiment, wherein a single variable domain binds to albumin using domain II of the human albumin protein as the major binding site.

[0046] 4. An albumin-binding protein in any one of the first to third embodiments, wherein a single variable domain comprises one of the following combinations of complementarity determining regions (CDR1, CDR2, and CDR3):

[0047] Sequence No. 34 (CDR1), Sequence No. 35 (CDR2), and Sequence No. 36 (CDR3);

[0048] Sequence No. 49 (CDR1), Sequence No. 50 (CDR2), and Sequence No. 51 (CDR3);

[0049] Sequence No. 52 (CDR1), Sequence No. 53 (CDR2), and Sequence No. 54 (CDR3); and

[0050] Sequence No. 76 (CDR1), Sequence No. 77 (CDR2), and Sequence No. 78 (CDR3).

[0051] 5. An albumin-binding protein that competitively binds to Domain II of a human albumin protein with a single variable domain antibody comprising one of the following combinations of complementarity determining regions (CDR1, CDR2, and CDR3) in any one of the first to fourth embodiments:

[0052] Sequence No. 34 (CDR1), Sequence No. 35 (CDR2), and Sequence No. 36 (CDR3);

[0053] Sequence No. 49 (CDR1), Sequence No. 50 (CDR2), and Sequence No. 51 (CDR3);

[0054] Sequence No. 52 (CDR1), Sequence No. 53 (CDR2), and Sequence No. 54 (CDR3); and

[0055] Sequence No. 76 (CDR1), Sequence No. 77 (CDR2), and Sequence No. 78 (CDR3).

[0056] 6. An albumin-binding protein having a single variable domain VHH in any one of the first to fifth embodiments.

[0057] 7. An albumin-binding protein that does not competitively inhibit albumin from binding to FcRn in any one of the first to sixth embodiments.

[0058] 8. An albumin-binding protein in any one of the first to seventh embodiments, wherein the FR1 region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs 79, 80, and 151-174.

[0059] 9. An albumin-binding protein in any one of the first to eighth embodiments, wherein the FR2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 81-84 and 175-183.

[0060] 10. An albumin-binding protein in any one of the first to ninth embodiments, wherein the FR3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 85-100 and 184-202.

[0061] 11. An albumin-binding protein in any one of the first to tenth embodiments, wherein the FR4 region comprises the amino acid sequences of SEQ ID NOs. 101 and 203-206.

[0062] 12. An albumin-binding protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 103 to 135 or an amino acid sequence that is 85% or more identical thereto, in any one of the first to eleventh embodiments.

[0063] 13. An albumin-binding protein in which, in any one of the first to twelfth embodiments, a single variable domain is fused with a first heterogeneous protein.

[0064] 14. In the 13th embodiment, the first heterogeneous protein is an albumin-binding protein that specifically binds to a tumor-associated antigen, a T cell-associated antigen or a B cell-associated antigen, or a fragment thereof.

[0065] 15. In the 14th embodiment, the first heterogeneous protein is an albumin-binding protein that is an antibody or its antigen-binding fragment that specifically binds to a tumor-associated antigen, a T cell-associated antigen or a B cell-associated antigen, or a fragment thereof.

[0066] 16. In the 15th embodiment, the first heterogeneous protein is an albumin-binding protein in the form of Fab, VHH, or scFv.

[0067] 17. In the 13th embodiment, the first heterogeneous protein is an enzyme, cytokine, chemokine, growth factor, hormone, receptor binding protein, ligand protein, proteinase inhibitor, or albumin binding protein that is an active regulatory protein.

[0068] 18. An albumin-binding protein in any one of the 13th to 17th embodiments, wherein the first heterogeneous protein is directly connected to a single variable domain or connected through a linker.

[0069] 19. An albumin-binding protein in any one of the 13th to 18th embodiments, wherein the first heterogeneous protein is connected to a single variable domain through a linker, and the linker is cleavable.

[0070] 20. An albumin-binding protein in which, in any one of the 13th to 19th embodiments, the half-life of the fused protein containing the first heterogeneous protein is improved compared to the case where it is not fused with the albumin-binding protein.

[0071] 21. An albumin-binding protein in which a second heterogeneous protein is additionally fused in any one of the 13th to 20th embodiments.

[0072] 22. In the 21st embodiment, the second heterogeneous protein is an albumin-binding protein that specifically binds to a tumor-associated antigen, a T cell-associated antigen or a B cell-associated antigen, or a fragment thereof.

[0073] 23. An albumin-binding protein in which, in the 21st or 22nd embodiment, the second heterogeneous protein is connected to a single variable domain or the first heterogeneous protein directly or through a linker.

[0074] 24. An albumin-binding protein in which, in any one of the 21st to 23rd embodiments, the half-life of the fused protein containing the second heterogeneous protein is improved compared to the case where it is not fused with the albumin-binding protein.

[0075] One aspect of the present invention provides an isolated nucleic acid molecule for expressing a novel albumin-binding protein described herein, a vector comprising said nucleic acid molecule, and a host cell comprising said vector.

[0076] 25. An isolated nucleic acid molecule encoding an albumin-binding protein as described in any one of the first to 24th embodiments.

[0077] 26. A vector comprising a nucleic acid molecule described in the 25th embodiment.

[0078] 27. A host cell comprising the vector described in the 26th embodiment.

[0079] One aspect of the present invention provides a pharmaceutical composition comprising a novel albumin-binding protein described herein and a medical use thereof.

[0080] 28. A pharmaceutical composition for treating or preventing cancer comprising an albumin-binding protein and a pharmaceutically acceptable carrier as described in any one of the first to 24th embodiments.

[0081] 29. A method for treating or preventing cancer comprising administering the pharmaceutical composition described in the 28th embodiment to a patient who requires treatment or prevention of cancer.

[0082] 30. A pharmaceutical composition for controlling or improving the pharmacokinetic properties of a therapeutic protein or antibody, comprising an albumin-binding protein described in any one of the first to 24th embodiments.

[0083] 31. A method for controlling or improving the pharmacokinetic properties of a therapeutic protein or antibody using an albumin-binding protein described in any one of the first to 24th embodiments.

[0084] The albumin-binding single-domain antibody according to the present invention binds stably to human serum albumin. In particular, by not interfering with FcRn-mediated albumin recycling, it can maintain stable binding to human serum albumin for a long time. Due to these characteristics, when the albumin-binding single-domain antibody according to the present invention is fused with a heterologous protein (e.g., antibody, CAR, etc.) that specifically binds to a therapeutic protein, e.g., a tumor-associated antigen, a T-cell-associated antigen, or a B-cell-associated antigen, it can improve the half-life of the heterologous protein and thereby sustain the therapeutic effect of the heterologous protein for a long time.

[0085] Figure 1 shows the results of measuring whether the anti-albumin VHH antibodies according to the present invention interfere with the binding between FcRn and albumin through epitope competition analysis via the Viacore system.

[0086] Figure 2 is a sensorogram result according to a viacor binding analysis obtained using anti-albumin VHH antibodies and FcRn according to the present invention against human albumin.

[0087] Figure 3 is a schematic diagram showing the full length of human serum albumin (HSA) and each domain used for albumin binding domain analysis.

[0088] Figure 4 shows the EC50 values ​​derived from an ELISA experiment confirming that the anti-albumin VHH antibodies according to the present invention bind to domain II of human serum albumin.

[0089] FIG. 5 is a schematic diagram of a fusion protein structure comprising an anti-albumin VHH antibody according to the present invention together with an anti-CEACAM5 antibody and an anti-CD3 antibody.

[0090] Figure 6 shows the EC50 values ​​derived from an ELISA experiment confirming that the protein structures of Figure 5 bind to human serum albumin.

[0091] Figure 7 shows the results of measuring the half-life of a heterogeneous protein by comparing the case where the anti-albumin VHH antibody (DPVH18) according to the present invention is fused with the anti-CEACAM5 antibody and the anti-CD3 antibody, and the case where the anti-albumin VHH antibody is not fused. Figure 7A is a schematic diagram of the protein structure used in the experiment, and Figure 7B is a half-life graph showing antibody concentration over time.

[0092] FIG. 8 shows the results of measuring the half-life of a heterogeneous protein by comparing the case where the anti-albumin VHH antibody (DPVH42) according to the present invention is fused with the anti-CEACAM5 antibody and the anti-CD3 antibody, and the case where the anti-albumin VHH antibody is not fused. FIG. 8A is a schematic diagram of the protein structure used in the experiment, and FIG. 8B is a half-life graph showing antibody concentration over time.

[0093] Unless otherwise specifically defined, the technical and scientific terms used in this specification have the meaning generally understood by those skilled in the art to which the present invention pertains.

[0094] The embodiments described in this specification and the configurations illustrated in the drawings are merely specific examples to aid in understanding the invention and do not limit the technical scope of the invention. At the time of filing this application, various modifications, equivalents, and applications performing substantially the same functions are possible, and a person skilled in the art will understand that such modifications and applications are also included within the scope of the invention.

[0095] The embodiments, features, components, and technical means described herein may be implemented independently or in any combination, unless otherwise explicitly stated to be exclusive or mutually excluded. In particular, technical features described in different paragraphs, items, or embodiments herein may be combined or optionally combined to the extent that there is no technical contradiction, as understood by a person skilled in the art. Such combinations should be understood to be included within the technical spirit and scope of the invention.

[0096] Accordingly, even if a component or feature described in a specific embodiment or paragraph is not explicitly described together with another embodiment or paragraph, it should be interpreted as being directly and clearly derivable from the disclosure of the present invention where it is recognized as technically reasonable by a person skilled in the art.

[0097] definition

[0098] Expressions used in the singular form in this specification are used to include the plural unless the context clearly indicates otherwise. Additionally, the expression “or” is interpreted to include the meaning of “and / or” unless otherwise specified in the context.

[0099] As used herein, the term “comprising” should be understood as an open expression that essentially includes the described components, components, steps, etc., unless specifically stated otherwise, without excluding the existence of additional components, components, steps, etc. Accordingly, the term “comprising” is interpreted as a concept that includes the more restrictive “consisting of” or “consisting essentially of.”

[0100] In this specification, the term “sequence” may be interpreted, depending on the context, as a nucleic acid (or polynucleotide) molecule or a protein (or polypeptide) molecule having a given sequence.

[0101] As used herein, the terms “sequence identity” or “sequence homology” refer to a value expressed as a percentage of the total length of the sequences, representing the number of residues present at the same position when two amino acid sequences or nucleic acid sequences are aligned. When a specific sequence is described in this specification as having “at least X% sequence identity,” X may be, for example, 85%, 90%, 95%, 98%, or 99%. Sequence identity is typically calculated using BLAST (Basic Local Alignment Search Tool), ClustalW, EMBOSS, or other known sequence alignment algorithms, based on default parameters. For example, when aligning amino acid sequences using BLASTP, the identity value calculated using a BLOSUM62 matrix and a gap penalty as default values ​​may be used as the basis. Additionally, in this specification, “sequence identity” or “sequence homology” may include values ​​calculated according to global alignment or local alignment optimized by considering insertions, deletions, substitutions, etc., during sequence alignment, and is also used as a criterion to describe the scope of functional equivalents capable of maintaining the technical effects of the invention.

[0102] As used herein, the term “conservative amino acid substitution” refers to a substitution between amino acids with similar physicochemical properties (e.g., charge, size, hydrophobicity, polarity, etc.), and includes a substitution in which the structural stability or biological function of the protein can be substantially maintained.

[0103] For example, the following substitutions within an amino acid group may be considered conservative substitutions:

[0104] Hydrophobic amino acid group: Ala, Val, Leu, Ile, Met

[0105] Polar non-charged amino acid groups: Ser, Thr, Gln, Asn

[0106] Acidic amino acid group: Asp, Glu

[0107] Basic amino acid group: Lys, Arg, His

[0108] Aromatic amino acid group: Phe, Tyr, Trp

[0109] In addition, exemplary amino acid substitutions that can be considered as conservative substitutions for each amino acid are as follows.

[0110] Ala (A): Val, Leu, Ile

[0111] Arg (R): Lys, Gln, Asn

[0112] Asn (N): Gln, His, Asp, Lys, Arg

[0113] Asp (D): Glu, Asn

[0114] Cys (C): Ser, Ala

[0115] Gln (Q): Asn, Glu

[0116] Glu (E): Asp, Gln

[0117] Gly (G): Ala

[0118] His (H): Asn, Gln, Lys, Arg

[0119] Ile (I): Leu, Val, Met, Ala, Phe

[0120] Leu (L): Ile, Val, Met, Ala

[0121] Lys (K): Arg, Gln, Asn

[0122] Met (M): Leu, Phe, Ile

[0123] Phe (F): Leu, Val, Ile, Ala, Tyr

[0124] Pro (P): Ala

[0125] Ser (S): Thr

[0126] Thr (T): Ser

[0127] Trp (W): Tyr, Phe

[0128] Tyr (Y): Trp, Phe, Thr, Ser

[0129] Val (V): Ile, Leu, Met, Phe, Ala

[0130] In this specification, the expression “having one or more conservative amino acid substitutions” includes the fact that the function of the protein (e.g., antigen binding ability) is substantially maintained even when the original amino acid is substituted with a physicochemically similar amino acid.

[0131] To determine whether a conservative amino acid substitution maintains protein function, methods such as a binding affinity assay or a cell-based functional assay may generally be used. For example, if a variant antibody containing the substituted amino acid substantially maintains its binding ability to albumin compared to an antibody containing the original sequence (e.g., maintaining more than 80% of the binding ability relative to the original), or if biological activities such as internalization within cells or the ability to kill targeted cancer cells are substantially maintained, such substitution may be considered a conservative substitution. Such conservative substitutions correspond to examples of variants that can be derived by a person skilled in the art through ordinary experiments based on the core CDR sequence or variable region sequence specified in the embodiments of the invention, and are recognized as substantial variations of the technical concept of the embodiments described in the specification. Therefore, where it is specified in this specification that it includes “conservative amino acid substitution,” this is interpreted to include substitutions to amino acids that are structurally similar and functionally equivalent.

[0132] As used herein, the term “antibody” refers to a protein of the immunoglobulin family comprising a binding site capable of binding to a specific antigen, and includes all forms that are naturally occurring or produced by recombinant technology. This includes full-length antibodies (e.g., IgG, IgA, IgM, IgD, IgE), as well as all fragments that retain the functional characteristics of antibodies (Fab, Fab′, F(ab′)₂, scFv, VHH, single-domain antibodies, etc.) and variants derived from humanized, chimeric, fully human, or synthetic libraries. Additionally, it includes variants or fusion protein forms modified to improve binding affinity, stability, half-life, heterogeneous function, etc., while maintaining the ability to specifically bind to an antigen.

[0133] As used herein, the term “specifically bound” means a binding that has significantly higher binding affinity and selectivity than non-specific interactions with respect to a binding target (e.g., antigen, receptor, ligand, etc.). Such specificity can be determined through known analytical methods that can be easily performed by a person skilled in the art (e.g., enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), biolayer interferometry (BLI), fluorescence or radiolabeled binding assay, etc.), and typically includes cases showing a difference in binding affinity of at least 10 times, preferably 100 times or more, compared to non-specific binding. Whether specific binding has occurred can be determined based on results that ensure reproducibility by repeatedly measuring using the said analytical method.

[0134] As used herein, the term “albumin” refers to a protein that is abundant in the plasma of mammals, including humans, that can reversibly bind to various endogenous or exogenous substances, and has a relatively long blood half-life through an FcRn-mediated recycling mechanism. In this specification, “albumin” specifically includes human serum albumin, which is used to mean a protein that includes a known amino acid sequence (e.g., the sequence described in UniProt Accession No. P02768) or a variant, derivative, or functional equivalent substantially homologous thereto.

[0135] As used herein, the term “albumin-binding protein” refers to a protein comprising a binding site capable of specifically binding to human albumin, wherein stability, circulation time, or pharmacokinetic properties in the body can be regulated through binding to albumin. The albumin-binding protein includes not only a solitary form but also a fusion protein form fused with a heterologous protein, wherein the binding ability to albumin is substantially maintained even in such cases. In this specification, “human albumin” is used interchangeably with “human serum albumin.”

[0136] As used in this specification, the term “Complementarity-Determining Region (CDR)” refers to a functional region within a variable region of a protein that is an amino acid sequence region that contributes to direct binding with a target molecule and substantially determines binding specificity and affinity.

[0137] As used in this specification, the term “single variable domain” refers to a protein domain composed of a single variable region that can independently bind to albumin or antigen, and may exist alone or in a fused form with another protein.

[0138] As used herein, the term “variable region” refers to a region within an antibody or antigen-binding protein that has relatively high variation in amino acid sequence and is a protein region that primarily contributes to binding specificity and affinity with a target molecule. A variable region typically includes a complementarity determining region (CDR) and a framework region that supports them to provide structural stability, and may exist as part of the entire antibody or independently as a single variable domain.

[0139] As used herein, the term “Complementary Determining Region (CDR)” refers to a segment of amino acid sequences within the variable region of an antibody that exhibits relatively high sequence diversity and is primarily involved in direct contact and binding with an antigen. A single variable region typically includes three CDRs (CDR1, CDR2, and CDR3) and exists independently in the heavy chain variable region (VH) and the light chain variable region (VL), respectively. The boundaries of the CDRs may be defined according to a system of definitions known in the art, including but not limited to Kabat, Chothia, and IMGT, and unless a specific system of definitions is specified in this specification, they may be interpreted according to standards commonly accepted in the art.

[0140] As used herein, the term “framework region (FR)” refers to a region within a variable region that is the amino acid sequence segment excluding the CDR, which serves to support the spatial arrangement of the CDR and maintain the overall three-dimensional structural stability of the antibody. A single variable region typically comprises four framework regions (FR1, FR2, FR3, and FR4), wherein FR1 refers to the region located at the N-terminus of CDR1, FR2 refers to the region located between CDR1 and CDR2, FR3 refers to the region located between CDR2 and CDR3, and FR4 refers to the region adjacent to the C-terminus of CDR3. The precise boundaries of FR1 through FR4 may vary depending on the selected CDR definition system, but their relative positional relationships are as described above. Unless otherwise noted, the abbreviation “FR” in this specification refers to the “framework region.”

[0141] As used herein, the term “VHH” refers to a single variable domain derived from a heavy-chain antibody, which is a functional protein domain capable of forming a stable binding structure without a light chain and specifically binding to a target molecule.

[0142] As used herein, the term “Fab” refers to an antigen-binding unit derived from an antibody, meaning a protein portion that does not include an Fc region while maintaining the function of recognizing and binding to an antigen.

[0143] The term “scFv” as used in this specification refers to a single-chain antigen-binding protein in which the heavy chain variable region and the light chain variable region of an antibody are connected through a peptide linker.

[0144] As used herein, the term “antigen-binding fragment” refers to a portion of a protein that is part of the whole antibody and retains the ability to bind to an antigen.

[0145] As used herein, the term “FcRn” refers to a receptor that binds to albumin or IgG to evade their lysosomal degradation and mediate their recirculation within the body, thereby extending their half-life.

[0146] As used herein, the term “competitive inhibition” refers to a phenomenon in which two or more molecules bind exclusively to the same or overlapping binding sites, thereby reducing or hindering the binding of one molecule.

[0147] As used in this specification, the term “heterogeneous protein” refers to a protein having a biological function distinct from the original function of an albumin-binding protein, which may exist in a fused form to an albumin-binding protein.

[0148] In this specification, the terms “first” and “second,” used to modify, for example, “heterogeneous protein,” are merely identifying terms used to distinguish components of the same or similar kind from one another, and are not to be interpreted as limiting temporal order, superiority or inferiority of importance, or structural or functional differences.

[0149] The term “tumor-associated antigen” as used in this specification means an antigen that is selectively expressed or overexpressed in tumor tissue compared to normal tissue and can be used as a target for the diagnosis or treatment of cancer.

[0150] As used herein, the term “T cell-associated antigen” refers to a protein or part thereof that is present on the surface of a T cell or is involved in regulating the activation, proliferation, or function of a T cell, and means a target molecule that can be specifically recognized and bound by an antibody or antigen-binding fragment that may be included in the heterologous protein of the present invention.

[0151] As used herein, the term “B cell-associated antigen” means a target molecule that is present on the surface of B cells or is a protein or part thereof associated with the activation, differentiation, or antibody production of B cells, and which can be specifically recognized and bound by an antibody or antigen-binding fragment that may be included in the heterologous protein of the present invention.

[0152] As used herein, the term “enzyme” refers to a protein or a functional fragment thereof having a catalytic function that increases the rate of a chemical reaction with respect to one or more substrates. Enzymes are not chemically consumed before or after a reaction and can catalyze various biochemical reactions, including oxidation-reduction, hydrolysis, condensation, isomerization, and transfer reactions. In this specification, enzymes include not only natural enzymes but also enzymes in the form of modified forms, mutants, and fusion proteins.

[0153] As used herein, the term “cytokine” refers to a protein or polypeptide involved in intercellular signaling and may be involved in immune responses, inflammatory responses, cell proliferation, differentiation, or the regulation of survival. Cytokines may be secreted proteins and may activate or inhibit signaling pathways through binding to receptors. This definition includes interleukins, interferons, tumor necrosis factor family proteins, and functional variants thereof.

[0154] As used herein, the term “chemokine” refers to a protein of the sub-concept of cytokines that has the function of inducing or regulating cell migration (chemotaxis). Chemokines can bind to specific receptors and regulate the localization, tissue infiltration, or distribution of immune cells or other cells. This definition includes C, CC, CXC, and CX3C series chemokines and their variants.

[0155] As used herein, the term “growth factor” means a protein or polypeptide that influences the proliferation, differentiation, survival, or functional activity of cells. Growth factors generally bind to cell surface receptors to induce signal transduction and may be involved in tissue growth, regeneration, or the maintenance of homeostasis. This definition includes vascular, neuronal, epithelial, or connective tissue-related growth factors and their functional variants.

[0156] As used herein, the term “hormone” means a proteinaceous signaling substance produced in a specific tissue or cell that acts on other tissues or cells in the body to regulate physiological functions. Hormones may be delivered via blood or body fluids and may bind to receptors on target cells to induce or inhibit responses related to metabolism, growth, immunity, or homeostasis. This definition includes proteinaceous or peptide hormones and their variants.

[0157] As used herein, the term “receptor-binding protein” means a protein or an antigen-binding fragment thereof capable of specifically binding to a receptor on a cell surface or within a cell. Such proteins may have receptor-activating, inhibitory, or signal transduction-regulating functions and are not necessarily physiological ligands. This definition includes antibodies, single-domain antibodies, ligand-like proteins, and their variants.

[0158] As used herein, the term “ligand protein” means a protein or polypeptide that binds to one or more receptors to induce or regulate biological signal transduction. Ligand proteins can activate or block receptors and include natural ligands as well as artificially designed binding proteins. In this definition, ligand proteins may have a single function or multiple functions.

[0159] As used herein, the term “protein inhibitor” means a protein or a functional fragment thereof capable of reducing or blocking the function of a specific enzyme, receptor, ligand, or signaling protein. Such inhibition may occur in a competitive or non-competitive manner and includes forms that interfere with binding, catalytic activity, or interaction. This definition includes antibodies, single-domain antibodies, and inhibitory protein variants.

[0160] As used herein, the term “active regulatory protein” means a protein or polypeptide having the function of increasing or decreasing the activity of a target protein, receptor, or signaling pathway. Activity regulation may be achieved through direct binding, induction of structural changes, or indirect interactions, and includes both activating factors and inhibitors.

[0161] As used in this specification, the term “linker” means a linking sequence or binding part that structurally connects two or more proteins or domains to maintain or regulate the function of each component.

[0162] As used herein, the expression “linker cleavable” means a characteristic in which fused components can be separated by selective cleavage under an in vivo environment, enzymatic action, or specific conditions.

[0163] As used herein, the term “fusion protein” means that two or more proteins or domains are linked by a single polypeptide chain to be expressed as a single protein.

[0164] As used in this specification, the term “isolated” means that it is separated from its natural state or artificially manufactured and exists in a form distinct from its original biological environment.

[0165] As used herein, the term “nucleic acid” means genetic material that exists in the form of DNA or RNA and can encode proteins.

[0166] As used in this specification, the term “vector” means a carrier used to deliver or express nucleic acids into a cell.

[0167] As used in this specification, the term “host cell” means a cell capable of receiving a vector and maintaining or expressing foreign nucleic acids.

[0168] As used in this specification, the term “cancer” means a disease characterized by abnormal cell proliferation and invasion or metastasis.

[0169] As used herein, the term “pharmaceuticalally acceptable carrier” means a substance that enables a pharmaceutical composition containing an active ingredient to be safely administered in vivo and provides physical and chemical stability, solubility, or suitability for administration of the active ingredient. Such a carrier may include water-soluble or water-insoluble media depending on the route of administration and may be selected within a range that does not substantially affect the pharmacological activity of the active ingredient.

[0170] As used herein, the term “pharmaceuticalally acceptable diluent” refers to a substance used to adjust the concentration of an active ingredient or to increase the volume or weight of a formulation, and which serves to improve the ease of administration, quantification, or uniformity of a pharmaceutical composition. The diluent may be selected within a range that is chemically stable with respect to the active ingredient and does not adversely affect the safety and efficacy of the pharmaceutical composition.

[0171] As used herein, the term “pharmaceuticalally acceptable excipient” means an auxiliary component used to improve the stability, release characteristics, bioavailability, or physical properties of an active ingredient during the preparation, storage, or administration of a pharmaceutical composition. Such excipients may include, but are not limited to, stabilizers, buffers, isotonic agents, preservatives, antioxidants, binders, disintegrants, or coating agents, for example.

[0172] As used in this specification, the term “treatment” means alleviating, improving, or eliminating the symptoms of a disease, or inhibiting the progression of a disease.

[0173] As used in this specification, the term “prevention” means suppressing the occurrence of a disease or reducing the risk of its occurrence.

[0174] As used herein, the term “pharmacokinetic properties” refers to properties related to the processes of absorption, distribution, metabolism, and excretion of a protein, peptide, or complex thereof administered in vivo. These pharmacokinetic properties include, but are not limited to, patterns of change in blood concentration, residence time in the body, circulation time, elimination rate, systemic exposure, volume of distribution, clearance rate, and half-life. Pharmacokinetic properties in this specification may be modified by the structure, size, binding partners, fusion, or interactions such as binding with albumin of the protein, and are not necessarily limited to specific numerical values. “Improvement” or “modification” of pharmacokinetic properties involves a relative change or adjustment of one or more of these properties, which may include, for example, an increase in blood residence time, a decrease in elimination rate, or an increase in systemic exposure.

[0175] 1. Albumin-binding antibody

[0176] One aspect of the present invention provides a novel albumin-binding protein that binds to human albumin protein.

[0177] Human serum albumin (HSA) is the most abundant protein in human plasma, accounting for about 50–60% of total plasma proteins, and plays an important role in maintaining osmotic pressure and transporting various endogenous and exogenous substances.

[0178] HSA is synthesized in the liver and is translated into a precursor form containing signal peptides and propeptides, then processed to exist as a mature single polypeptide consisting of 585 amino acids, with a molecular weight of approximately 66.5 kDa. Structurally, HSA consists of three homologous domains (domain I, domain II, and domain III). The canonical amino acid sequence of HSA is known (see, for example, UniProt accession number P02768), and the amino acid sequences of each domain are also known in the art.

[0179] HSA has a long blood half-life, which is maintained by intracellular recycling pathways. Specifically, HSA binds to the neonatal Fc receptor (FcRn) in the slightly acidic environment within endosomes and is released to the cell surface under neutral pH conditions. Due to these characteristics, HSA is utilized as a useful platform in drug delivery and the development of protein therapeutics for extending half-lives.

[0180] The albumin-binding protein according to the present invention preferably comprises an antibody or antigen-binding fragment comprising three complementarity determining regions (CDR1, CDR2, and CDR3) as follows. The CDR1, CDR2, and CDR3 sequences, denoted by the same clone name (e.g., DVH-30 CDR1, DVH-30 CDR2, and DVH-30 CDR3), constitute a set of CDRs corresponding to one antibody or antigen-binding fragment.

[0181] DVH-30 CDR1: DYTMY(Sequence No. 1)

[0182] DVH-30 CDR2: SITSGDSAYYAGSVKG(Sequence No. 2)

[0183] DVH-30 CDR3: NRWLTNRDY(Sequence No. 3)

[0184] DVH-31 CDR1: INAIG(Sequence No. 4)

[0185] DVH-31 CDR2: TISMGGSTDYADSVKG(Sequence No. 5)

[0186] DVH-31 CDR3: RWGWDYSGPER(Sequence No. 6)

[0187] DVH-39 CDR1: NYAMR(Sequence No. 7)

[0188] DVH-39 CDR2: TITSTGGSTNYANSVKG(Sequence No. 8)

[0189] DVH-39 CDR3: GNSWSSRYDY(Sequence No. 9)

[0190] DVH-40 CDR1: VYAMR(Sequence No. 10)

[0191] DVH-40 CDR2: AISSAGGSTNYADSVKG(Sequence No. 11)

[0192] DVH-40 CDR3: GNSWSSRYDY(Sequence No. 12)

[0193] DVH-42 CDR1: RYAVG (Sequence No. 13)

[0194] DVH-42 CDR2: AINWSGGSTNTADSVKG(Sequence No. 14)

[0195] DVH-42 CDR3: VFRIVPPTTTNYHY(Sequence No. 15)

[0196] DVH-51 CDR1: RYAMG (Sequence No. 16)

[0197] DVH-51 CDR2: TISWSGASTYYGDSVKG(Sequence No. 17)

[0198] DVH-51 CDR3: AWDLGSTLYGATGYKYDY (Sequence No. 18)

[0199] DVH-52 CDR1: RYAMG (Sequence No. 19)

[0200] DVH-52 CDR2: TISWSASSTYYADSVKG(Sequence No. 20)

[0201] DVH-52 CDR3: AWDLGSTLYGASGYKYDY (Sequence No. 21)

[0202] DVH-53 CDR1: WYAMG (Sequence No. 22)

[0203] DVH-53 CDR2: AIRRRGVSTNYADSVKG(Sequence No. 23)

[0204] DVH-53 CDR3: AWDLDSDSRLKYDY(Sequence No. 24)

[0205] DPVH-04 CDR1: FNPMG(Sequence No. 25)

[0206] DPVH-04 CDR2: AISWSGSNTLYSDSVKG(Sequence No. 26)

[0207] DPVH-04 CDR3: RSEYGVTSIEGEYDY(Sequence No. 27)

[0208] DPVH-07 CDR1: SYDMS (Sequence No. 28)

[0209] DPVH-07 CDR2: AIMSAGGSTNYADSVKG(Sequence No. 29)

[0210] DPVH-07 CDR3: HIDWPLEDYDY(Sequence No. 30)

[0211] DPVH-10 CDR1: LRSMY(Sequence No. 31)

[0212] DPVH-10 CDR2: AISGSGGDTRYVDSVRG(Sequence No. 32)

[0213] DPVH-10 CDR3: PQALRLFIDLEDRNLY(Sequence No. 33)

[0214] DPVH-11 CDR1: AMG (Sequence No. 34)

[0215] DPVH-11 CDR2: AISWTGGSTYFEDSVKG(Sequence No. 35)

[0216] DPVH-11 CDR3: RRPPFLAIATAGYEYDY(Sequence No. 36)

[0217] DPVH-13 CDR1: FNPMG(Sequence No. 37)

[0218] DPVH-13 CDR2: AISWSGSNTLYSDSVKG(Sequence No. 38)

[0219] DPVH-13 CDR3: RSEYGLTSIEGEYDY (Sequence No. 39)

[0220] DPVH-14 CDR1: FNPMG(Sequence No. 40)

[0221] DPVH-14 CDR2: AISWSGSNTLYSDSVKG(Sequence No. 41)

[0222] DPVH-14 CDR3: RSEYGLTSIEGEYDY (Sequence No. 42)

[0223] DPVH-15 CDR1: FNPMG (Sequence No. 43)

[0224] DPVH-15 CDR2: AISWSGGYTLYSDSVKG(Sequence No. 44)

[0225] DPVH-15 CDR3: RSEYGLTSIEGGYDY (Sequence No. 45)

[0226] DPVH-16 CDR1: FNPMG (Sequence No. 46)

[0227] DPVH-16 CDR2: AISWSGSITLYSDSVKG(Sequence No. 47)

[0228] DPVH-16 CDR3: RSEYGVTSIEGEYDY (Sequence No. 48)

[0229] DPVH-17 CDR1: FNPMG (Sequence No. 49)

[0230] DPVH-17 CDR2: AISWSGSNTLYSDSVKG(Sequence No. 50)

[0231] DPVH-17 CDR3: RSGYGVTSIEGEYDY(Sequence No. 51)

[0232] DPVH-18 CDR1: SYAMA (Sequence No. 52)

[0233] DPVH-18 CDR2: AITWSGESTYYASSVKG(Sequence No. 53)

[0234] DPVH-18 CDR3: NRSPQTSLFSRVVFSRDSNDYNY(Sequence No. 54)

[0235] DPVH-19 CDR1: AYAMA (Sequence No. 55)

[0236] DPVH-19 CDR2: AITWSGESTYYASSVKG(Sequence No. 56)

[0237] DPVH-19 CDR3: NRSPQTSLFSRVVFSRDSNDYNY(Sequence No. 57)

[0238] DPVH-24 CDR1: GYAMS (Sequence No. 58)

[0239] DPVH-24 CDR2: AIAEAGGSTNYADSVKG(Sequence No. 59)

[0240] DPVH-24 CDR3: HITWPLEDYDY (Sequence No. 60)

[0241] DPVH-27 CDR1: TYAMG (Sequence No. 61)

[0242] DPVH-27 CDR2: GISWGYGSTYYADSVRG(Sequence No. 62)

[0243] DPVH-27 CDR3: HREWVLEDYDY (Sequence No. 63)

[0244] DPVH-32 CDR1: SYAMG (Sequence No. 64)

[0245] DPVH-32 CDR2: TISWSGGSTYYADSVKG(Sequence No. 65)

[0246] DPVH-32 CDR3: HTVWPLEEYDY (Sequence No. 66)

[0247] DPVH-33 CDR1: IYAMG (Sequence No. 67)

[0248] DPVH-33 CDR2: TISSGGSTKYLDSVKG (Sequence No. 68)

[0249] DPVH-33 CDR3: LPGSWPIRDFGS(Sequence No. 69)

[0250] DPVH-37 CDR1: NYAMH (Sequence No. 70)

[0251] DPVH-37 CDR2: GITSTARAINVADSVQG(Sequence No. 71)

[0252] DPVH-37 CDR3: YRVRGDGKLMY(Sequence No. 72)

[0253] DPVH-39 CDR1: SYIMA (Sequence No. 73)

[0254] DPVH-39 CDR2: RITSGGHTNYADSVKG (Sequence No. 74)

[0255] DPVH-39 CDR3: RTVQRDY (Sequence No. 75)

[0256] DPVH-42 CDR1: DYAMS (Sequence No. 76)

[0257] DPVH-42 CDR2: GISIAGSKTDYGDSVKG(Sequence No. 77)

[0258] DPVH-42 CDR3: GWGKAYF (Sequence No. 78)

[0259] The CDR sequences listed in SEQ ID NOs 1 to 78 are defined according to the Kabat amino acid numbering system.

[0260] The albumin-binding protein according to the present invention preferably comprises three complementarity determining regions of an albumin-binding protein selected from the group consisting of DVH-30, DVH-39, DPVH-04, DPVH-11, DPVH-15, DPVH-16, DPVH-17, DPVH-18, and DPVH-42 described herein.

[0261] In one embodiment, the albumin-binding protein according to the present invention may bind to albumin using domain II of the human albumin protein as the major binding site. Here, “major binding site” means a case where the binding characteristics of the antibody toward the entire antigen are primarily determined by binding to a specific antigen domain, and includes, for example, cases where the EC50 value for said domain is similar to or lower than the EC50 value for the entire antigen. A protein that binds to albumin using domain II of the human albumin protein as the major binding site can bind to albumin without substantially interfering with the FcRn-mediated albumin recycling pathway.

[0262] The albumin-binding protein according to the above embodiment preferably comprises three complementarity determining regions of an albumin-binding protein selected from the group consisting of DPVH-11, DPVH-17, DPVH-18, and DPVH-42 described herein.

[0263] In one embodiment, the albumin-binding protein according to the present invention is

[0264] A CDR1 comprising an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, and 76,

[0265] A CDR2 comprising an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, and 77, and / or

[0266] A CDR3 may have an amino acid sequence that is 85% or more identical to an amino acid sequence selected from the group consisting of sequence numbers 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, and 78.

[0267] As described above, the albumin-binding protein according to the present invention may include an amino acid sequence that is identical to, or at least 85% identical to, an amino acid sequence selected from the group consisting of specific sequence numbers for each of CDR1, CDR2, and CDR3. Here, “identity of amino acid sequence” refers to sequence homology calculated according to a conventional sequence alignment method, and the range of identity described in the present invention is intended to allow for modifications that maintain albumin-binding ability when considering the length, structural characteristics, and functional requirements of the CDR. Such sequence identity may be at least 85% in one embodiment, at least 90% in another embodiment, at least 95% in yet another embodiment, at least 98% in yet another embodiment, and at least 99% in yet another embodiment.

[0268] Generally, the CDR is a region primarily involved in direct binding with a target molecule in an antibody or a single variable domain, but it is widely known in the art that some amino acid residues within the CDR may not be directly involved in binding, or that the overall stereostructure and binding specificity can be maintained through conservative amino acid substitutions. Accordingly, the sequence identity range described in the present invention can be understood to include modifications that substantially maintain binding specificity and affinity for albumin, including conservative amino acid substitutions, substitutions, deletions, or insertions of some residues.

[0269] Furthermore, the above range of sequence identity is intended to encompass sequence variations that may occur in different expression systems, manufacturing conditions, or during the optimization process for improving protein stability, and includes variations in which the binding function with albumin, pH-dependent binding characteristics, or FcRn-mediated half-life extension effect are substantially maintained despite such modifications.

[0270] In this specification, “albumin binding ability is maintained” means that the binding affinity or binding signal to human albumin is maintained at a substantial level when evaluated by commonly used assays (e.g., ELISA, SPR, or BLI, etc.) compared to an albumin-binding protein containing the original CDR sequence. For example, such binding ability may be described as being maintained at a level of about 70% or more, preferably about 80% or more, more preferably about 90% or more compared to a reference protein, but is not limited thereto.

[0271] In one embodiment, the albumin-binding protein according to the present invention is

[0272] A CDR1 having 1 to 3 amino acid substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, and 76,

[0273] A CDR2 having 1 to 3 amino acid substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, and 77, and / or

[0274] A CDR3 having 1 to 5 amino acid substitutions compared to an amino acid sequence selected from the group consisting of sequence numbers 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75 and 78 may have

[0275] As described above, the albumin-binding protein according to the present invention may include a limited number of amino acid substitutions for each of CDR1, CDR2, and CDR3 compared to a reference amino acid sequence selected from the group consisting of specific sequence numbers. Here, “amino acid substitution” means that one amino acid residue is replaced by another amino acid residue, and such substitution is permitted within a range that substantially maintains the overall length, structural stability, and binding function with albumin of the CDR.

[0276] Generally, the CDR is a key region directly involved in binding to a target molecule in an antibody or a single variable domain, but not all amino acid residues within the CDR are essential for binding, and some residues are known to serve as structural support or have a limited effect on binding affinity. Accordingly, the albumin-binding protein according to the present invention may include 1 to 3 amino acid substitutions compared to the reference sequence for CDR1 and CDR2, and may include 1 to 5 amino acid substitutions compared to the reference sequence for CDR3, which is relatively longer and has greater structural and functional flexibility. Variants including this range of amino acid substitutions can substantially maintain binding specificity and affinity for albumin.

[0277] The above amino acid substitution may include conservative amino acid substitution, in which case the overall stereostructure and binding characteristics can be maintained through substitution between amino acids with similar charge, polarity, hydrophobicity, or stereochemical properties. Additionally, in some embodiments, the above amino acid substitution may be performed for the purpose of improving the expression efficiency, stability, or manufacturing process characteristics of the protein.

[0278] In one embodiment, the albumin-binding protein according to the present invention may include an FR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 79 to 80 and 151 to 174.

[0279] The above FR1 region is a framework region located at the N-terminus of the variable region, which supports the structural arrangement of CDR1 and can contribute to the stable folding of the variable domain and the maintenance of the three-dimensional structure. In one embodiment, the FR1 region may include the entire region of the corresponding amino acid sequence selected from the group consisting of SEQ ID NOs 79 to 80 and 151 to 174, and in another embodiment, may include a part thereof.

[0280] In another embodiment, the FR1 region may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs 79 to 80 and 151 to 174. The sequence identity may be determined by a sequence alignment method commonly used in the art.

[0281] In another embodiment, the FR1 region may include one or more, for example, one to five or one to ten amino acid substitutions for an amino acid sequence selected from the group consisting of SEQ ID NOs 79 to 80 and 151 to 174, and said substitutions may be conservative substitutions. Notwithstanding such modifications, the FR1 region may substantially maintain albumin binding function while maintaining the structural framework of the variable region.

[0282] In another embodiment, the FR1 region may be modified for the purpose of humanization, improved stability, improved expression efficiency, or reduced immunogenicity.

[0283] Preferably, the albumin-binding protein according to the present invention is a humanized antibody, and the FR1 region is the FR1 region of the humanized antibody.

[0284] Preferably, the FR1 region comprises the amino acid sequence of SEQ ID NO. 79 or 80.

[0285] In one embodiment, the albumin-binding protein according to the present invention may include an FR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 81 to 84 and 175 to 183.

[0286] The above FR2 region is a framework region located between CDR1 and CDR2, and can contribute to stabilizing the relative arrangement of CDR1 and CDR2 and maintaining the structural integrity of the variable region. In one embodiment, the FR2 region may include the entire or part of the corresponding region of the above sequence number.

[0287] In another embodiment, the FR2 region may include an amino acid sequence having 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs 81 to 84 and 175 to 183.

[0288] In another embodiment, the FR2 region may include one or more, for example, one to five or one to ten amino acid substitutions, and said substitutions may be conservative substitutions. Notwithstanding such variations, the FR2 region may substantially maintain the three-dimensional structure of the variable region.

[0289] In another embodiment, the FR2 region may be partially modified for the purpose of humanization, affinity regulation, improved stability, improved expression efficiency, or reduced immunogenicity.

[0290] Preferably, the albumin-binding protein according to the present invention is a humanized antibody, and the FR2 region is the FR2 region of the humanized antibody.

[0291] Preferably, the FR2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 81 to 84.

[0292] In one embodiment, the albumin-binding protein according to the present invention may include an FR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 85 to 100 and 184 to 202.

[0293] The above FR3 region is a framework region located between CDR2 and CDR3, and can support the spatial arrangement of CDR2 and CDR3 and contribute to maintaining the overall structural stability of the variable domain and the proper formation of antigen binding sites. FR3 typically forms a relatively long segment within the variable domain and can affect the orientation and structural flexibility of CDR3. In one embodiment, the FR3 region may include the entire or a part of the corresponding region of the above sequence number.

[0294] In another embodiment, the FR3 region may include an amino acid sequence having 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs 85 to 100 and 184 to 202. The sequence identity may be determined by a sequence alignment method commonly used in the art.

[0295] In another embodiment, the FR3 region may include one or more, for example, one to five or one to ten amino acid substitutions for an amino acid sequence selected from the group consisting of SEQ ID NOs 85 to 100 and 184 to 202, and said substitutions may be conservative substitutions. Notwithstanding such modifications, the FR3 region may maintain albumin binding function while substantially maintaining the structural framework of the variable region.

[0296] In another embodiment, the FR3 region may be partially modified for the purpose of humanization, affinity regulation, improved stability, improved expression efficiency, or reduced immunogenicity.

[0297] Preferably, the albumin-binding protein according to the present invention is a humanized antibody, and the FR3 region is the FR3 region of the humanized antibody.

[0298] Preferably, the FR3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 85 to 100.

[0299] In one embodiment, the albumin-binding protein according to the present invention may include an FR4 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 101 and 203 to 206.

[0300] The above FR4 region is a framework region adjacent to the C-terminus of CDR3 that forms the C-terminus of the variable region, and can contribute to maintaining the structural stability of the variable domain and to proper connection with the constant region or subsequent domain. FR4 plays a role in completing the overall three-dimensional structure of the variable region and can contribute to stabilizing the spatial orientation of CDR3.

[0301] In one embodiment, the FR4 region may include all or part of an amino acid sequence selected from the group consisting of SEQ ID NOs 101 and 203 to 206.

[0302] In another embodiment, the FR4 region may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs 101 and 203 to 206. The sequence identity may be determined by a sequence alignment method commonly used in the art.

[0303] In another embodiment, the FR4 region may include one or more, for example, one to five or one to ten amino acid substitutions for an amino acid sequence selected from the group consisting of SEQ ID NOs 101 and 203 to 206, and said substitutions may be conservative substitutions. Notwithstanding such modifications, the FR4 region may maintain albumin binding function while substantially maintaining the structural framework of the variable region.

[0304] In another embodiment, the FR4 region may be partially modified for the purpose of humanization, improved stability, improved expression efficiency, or reduced immunogenicity.

[0305] Preferably, the albumin-binding protein according to the present invention is a humanized antibody, and the FR4 region is the FR4 region of the humanized antibody.

[0306] Preferably, the FR4 region comprises the amino acid sequence of SEQ ID NO. 101 or 206.

[0307] In one embodiment, the albumin-binding protein according to the present invention is

[0308] FR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 79 to 80 and 151 to 174,

[0309] FR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 81 to 84 and 175 to 183,

[0310] FR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 85 to 100 and 184 to 202, and / or

[0311] FR4 may have an amino acid sequence selected from the group consisting of SEQ ID NOs 101 and 203 to 206.

[0312] In one embodiment, the albumin-binding protein according to the present invention may include the amino acid sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and at this time

[0313] (i) FR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 79 to 80 and 151 to 174, and

[0314] (ii) CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73 and 76, and

[0315] (iii) FR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 81 to 84 and 175 to 183, and

[0316] (iv) CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, and 77, and

[0317] (v) FR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 85 to 100 and 184 to 202, and

[0318] (vi) CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75 and 78, and

[0319] (vii) FR4 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 101 and 203 to 206.

[0320] In one embodiment, the albumin-binding protein according to the present invention may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs 103 to 135 or an amino acid sequence that is 85% or more identical thereto.

[0321] DVH-30:

[0322] QVQLVESGGGLVQPGGSLRLSCAASEIIIDDYTMYWYRQAPGEQRELVASITSGDSAYYAGSVKGRFTISRDNGKNMVYLQMNSLKPEDTAVYFCNANRWLTNRDYWGQGTQVTVSS (SEQ ID NO: 103)

[0323] DVH-31:

[0324] QVQLVESGGGLVQAGGSLRLSCATSGSTFSINAIGWYRQAPGKQRELVATISMGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNARWGWDYSGPERWGQGTQVTVSS (서열번호 104)

[0325] DVH-39:

[0326] QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMRWYRQAPGKERELVATITSTGGSTNYANSVKGRFTISRDNAGNTVYLQMNSLKPDDTAVYYCSAGNSWSSRYDYWGQGTQVTVSS (서열번호 105)

[0327] DVH-40:

[0328] QVQLVESGGGWVQPGGSLRLSCLASGFTFSVYAMRWYRQAPGKERELVAAISSAGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAGNSWSSRYDYWGQGTQVTVSS (서열번호 106)

[0329] DVH-42:

[0330] QVQLVESGGGLVQAGGSLRLSCAASGFFSFSRYAVGWFRQSPGKEREFVAAINWSGGSTNTADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAVFRIPPTTTNYHYWGQGAQVTVSS (서열번호 107)

[0331] DVH-51:

[0332] QVQLVESGGGLVQAGGSLRLSCAASGRTLSRYAMGWFRQAPGKEREFVATISWSGASTYYGDSVKGRFTISRENAKNAVYLQMNSLKPEDTAVYCAAAWDLGSTLYGATGYKYDYWGQGTQVTVSS (서열번호 108)

[0333] DVH-52:

[0334] QVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREFVATISWSASSTYYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAAAWDLGSTLYGASGYKYDYWGQGTQVTVSS (서열번호 109)

[0335] DVH-53:

[0336] QVQLVESGGGLVQAAGSLRLSCAASGGTFTWYAMGWFRQAPGKEREFVAAIRRRGVSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAWDLDSDSRLKYDYWGQGTQVTVSS (서열번호 110)

[0337] DPVH-04:

[0338] QVQLVESGGGLVQAGGSLRLSCAASGRTSRFNPMGWFRQAPGKEREFVAAISWSGSNTLYSDSVKGRFTISRDIGKTSAYLQMISLKPEDTAVYYCAARSEYGVTSIEGEYDYWGQGTQVTVSS (서열번호 111)

[0339] DPVH-07:

[0340] EVQLVESGGGSVQPGGSLRLSCAASGFTFSSYDMSWYRQAPGKERELVAAIMSAGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSAHIDWPLEDYDYWGQGTQVTVSS (서열번호 112)

[0341] DPVH-10:

[0342] QVQLVESGGGLVQAGGSLRLSCEASGRTFSLRSMYWFRQDPGKEREFVAAISGSGGDTRYVDSVRGRFTISREYLRNTVYLQMSNLLPEDTAVYYCAAPQALRLFIDLEDRNLYWGQGTQVTVSS (서열번호 113)

[0343] DPVH-11:

[0344] QVKLEESGGGLVQAGGSLRLSCAASGRNTYAMGWFRQAPGKEREFVAAISWTGGSTYFEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARRPPFLAIATAGYEYDYWGQGTQVTVSS (서열번호 114)

[0345] DPVH-13:

[0346] AVQLVESGGGLVQAGGSLRLSCAASGHTSRFNPMGWFRQAPGKEREFVAAISWSGSNTLYSDSVKGRFTISRDIGKISAYLQMNSLKPEDTAVYYCAARSEYGLTSIEGEYDYWGQGTQVTVSS (서열번호 115)

[0347] DPVH-14:

[0348] QVQLVESGGGLVQAGGSLRLSCAASGRTSRFNPMGWFRQAPGKEREFVAAISWSGSNTLYSDSVKGRFTISRDIGKISAYLQMNSLKPEDTAVYYCAARSEYGLTSIEGEYDYWGQGTQVTVSS (서열번호 116)

[0349] DPVH-15:

[0350] QVQLVESGGELVQAGGSLRLSCAASGGTGRFNPMGWFRQAPGKEREFVAAISWSGGYTLYSDSVKGRFTISRDIGKIAAYLQMNNLKPEDTAVYYCAARSEYGLTSIEGGYDYWGQGTQVTVSS (서열번호 117)

[0351] DPVH-16:

[0352] QVQLVESGGGLVQAGGSLRLSCAASGRTSRFNPMGWFRQAPGKEREFVAAISWSGSITLYSDSVKGRFTISRDIGKTSAYLQMISLKPEDTAVYYCAARSEYGVTSIEGEYDYWGQGTQVTVSS (서열번호 118)

[0353] DPVH-17:

[0354] AVQLVESGGGLVQAGGSLRLSCAASGRTSRFNPMGWFRQAPGKEREFVAAISWSGSNTLYSDSVKGRFTISRDIGKTSAYLQMISLKPEDTAVYYCAARSGYGVTSIEGEYDYWGQGTQVTVSS (서열번호 119)

[0355] DPVH-18:

[0356] QVQLVESGGGLVQAGGSLGLSCAASGGTFSSYAMAWFRQAPGKEREFVAAITWSGESTYYASSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYSCAGNRSPQTSLFSRVVFSRDSNDYNYWGQGTQVTVAS (서열번호 120)

[0357] DPVH-19:

[0358] QVKLEESGGGLVQAGASLRLSCAASGRTFNAYAMAWFRQAPGKEREFVAAITWSGESTYYASSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYSCAGNRSPQTSLFSRVVFSRDSNDYNYWGQGTQVTVAS (서열번호 121)

[0359] DPVH-24:

[0360] EVQLVESGGGLVQAGGSLGLSCAASGFTFSGYAMSWYRQAPGKERELVAAIAEAGGSTNYADSVKGRFTIFRDNAQNTVYLQMNSLKPEDTGVYYCNAHITWPLEDYDYWGQGTQVTVSS (서열번호 122)

[0361] DPVH-27:

[0362] DVQLVESGGGLVQAGGSLTLSCAASGLTFSTYAMGWFRQAPGKEREFVAGISWGYGSTYYADSVRGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCNAHREWVLEDYDYWGQGTQVTVSS (서열번호 123)

[0363] DPVH-32:

[0364] QVQLVESGGGLVQAGGSLRLSCAASGFTFSSYAMGWFRQAPGKEREFVATISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAHTVWPLEEYDYWGQGTQVTVSS (서열번호 124)

[0365] DPVH-33:

[0366] QVKLEESGGGLVQPGGSLRLSCAASGSTFSIYAMGWYRQAPGKQRELVATISSGGSTKYLDSVKGRLTISRDNAKNMVYLQMNSLEPEDTAVYYCNALPGSWPIRDFGSWGQGTQVTVSS (서열번호 125)

[0367] DPVH-37:

[0368] DVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMHWYRQAPGGERELVAGITSTARAINVADSVQGRFTISRDNAKNTVSLQMNGLKPEDTAVYYCNAYRVRGDGKLMYWGQGTQVTVSS (서열번호 126)

[0369] DPVH-39:

[0370] EVQLVESGGGLVQPGGSLRLSCVVSGNILSSYIMAWYRQTPGNERELVARITSGGHTNYADSVKGRFTISGETFKNTVYLQMMSLKPEDTAVYICNQRTVQRDYWGQGTQVTVSS (서열번호 127)

[0371] DPVH-42:

[0372] AVQLVESGGGLAQPGGSLRLSCTASGFTFTDYAMSWYRQVPGKERELVAGISIAGSKTDYGDSVKGRFTISRDNAKNMVYLQMNSLKPEDTAMYYCTRGWGKAYFWGQGTQVTVSS (서열번호 128)

[0373] DP-686:

[0374] EVQLVESGGGLVQPGGSLRLSCTASGFTFTDYAMSWVRQVPGKGLEWVSGISIAGSKTDYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAMYYCTRGWGKAYFWGQGTLVTVSS (서열번호 129)

[0375] DP-687:

[0376] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYAMSWVRQAPGKGLEWVSGISIAGSKTDYGDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCTRGWGKAYFWGQGTLVTVSS (서열번호 130)

[0377] DP-688:

[0378] EVQLVESGGGLVQPGNSLRLSCAASGFTFTDYAMSWVRQAPGKGLEWVSGISIAGSKTDYGDSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTRGWGKAYFWGQGTLVTVSS (서열번호 131)

[0379] DP-689:

[0380] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYAMSWVRQAPGKGLEWVSGISIAGSKTDYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGWGKAYFWGQGTLVTVSS (서열번호 132)

[0381] DP-724:

[0382] EVQLVESGGGVVQPGGSLRLSCAASGFTFTDYAMSWYRQAPGKGLELVAGISIAGSKTDYGDSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCTRGWGKAYFWGQGTLVTVSS (서열번호 133)

[0383] hDPVH18-1:

[0384] EVQLLESGGGLVQPGGSLRLSCAASGGTFSSYAMAWFRQAPGKGLEFVSAITWSGESTYYASSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGNRSPQTSLFSRVVFSRDSNDYNYWGQGTLVTVAS (SEQ ID NO: 134)

[0385] hDPVH18-11:

[0386] EVQLLESGGGLVQPGGSLRLSCAASGGTFSSYAMAWFRQAPGKGLEFVSAITWSGESTYYASSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGNRSPQTSLFSRVVFSRDSNDYNYWGQGTLVTVSS (SEQ ID NO: 135)

[0387] The sequence identity with the above reference sequence may be 90% or more in one embodiment and 95% or more in another embodiment.

[0388] The albumin-binding protein according to the present invention is preferably an antibody or antigen-binding fragment.

[0389] Albumin-binding proteins in the form of antibodies or antigen-binding fragments can function as molecules that provide specific binding ability to human albumin while simultaneously possessing high binding affinity, structural stability, and biocompatibility. These albumin-binding proteins may exist alone or in a fused form with other proteins or functional domains.

[0390] In one embodiment, the antibody or antigen-binding fragment may include, but is not limited to, a full-length antibody, Fab, scFv, VHH, etc. Additionally, the albumin-binding protein according to the present invention may be implemented in various antibody-derived structures or modified antigen-binding fragment forms other than these exemplary forms, as long as the antigen-binding function is maintained. Depending on these molecular forms, there may be differences in molecular weight, structural stability, binding characteristics, or tissue penetration.

[0391] Additionally, albumin-binding proteins in the form of antibodies or antigen-binding fragments may or may not include an Fc region. If an Fc region is included, it may affect circulating characteristics or half-life in the body through interaction with FcRn; even in the case of antigen-binding fragments that do not include an Fc region, a half-life extension effect may be provided through binding with albumin.

[0392] In one embodiment, the antigen-binding fragment may be a single variable domain antibody.

[0393] Single variable domain antibodies are antibody fragments composed solely of the variable region responsible for the antibody's antigen-binding function; they possess a relatively small molecular size and simple structure while providing specific binding ability to target molecules. Since these single variable domain antibodies do not require a pair of heavy and light chains, they offer the advantages of easy expression and manufacturing, as well as excellent structural stability.

[0394] In one embodiment, the single variable domain antibody may include, but is not limited to, a single domain antibody such as a single domain derived from a heavy chain variable region (VH), a single domain derived from a light chain variable region (VL), or a VHH derived from a camelid antibody. Additionally, a humanized single variable domain antibody, a chimeric form of a single variable domain antibody, or a single variable domain antibody with a modified amino acid sequence may also be included as long as they maintain albumin binding function.

[0395] Albumin-binding proteins in the form of single variable domain antibodies can have enhanced tissue penetration due to their small molecular size, and their circulation time or half-life in the body can be regulated through binding to albumin. Accordingly, single variable domain antibodies, as one embodiment of the albumin-binding protein according to the present invention, can be suitably utilized for various therapeutic or prophylactic applications.

[0396] In one embodiment, the albumin-binding protein according to the present invention is a VHH antibody.

[0397] VHH antibodies are single-variable domain antibodies composed of the variable region of heavy-chain antibodies derived from camelids, and are also referred to as nanobodies or single-domain antibodies. VHH antibodies possess a structural characteristic that allows them to perform antigen-binding functions solely through their single variable domain, without containing a light chain.

[0398] The crystal structure of VHH antibodies is elliptical, measuring approximately 4 nm × 2.5 nm × 3 nm, and has a molecular weight of approximately 12-14 kDa, which is significantly smaller than that of conventional IgG antibodies (approximately 150 kDa). Due to this ultra-small structure, VHH antibodies exhibit rapid distribution within the body and, through high tissue penetration, can effectively recognize hidden epitopes within tumor antigens that are difficult for conventional antibodies to access.

[0399] In addition, VHH antibodies can maintain high binding specificity and affinity for target molecules despite their simple single-domain structure, and they have excellent structural stability and are easy to express and manufacture. Due to these characteristics, VHH antibodies can be utilized as binding modules suitable for designing various protein fusions or multifunctional proteins.

[0400] In one embodiment, an albumin-binding protein composed of a VHH antibody can have its circulation time, stability, or pharmacokinetic properties modified through binding to albumin, thereby exhibiting in vivo behavior suitable for therapeutic or prophylactic purposes. Additionally, the VHH antibody may have enhanced tissue penetration due to its relatively small size, and these physical properties can be complemented by its albumin-binding characteristics.

[0401] In one embodiment, the albumin-binding protein according to the present invention has the characteristic of being able to bind to human albumin protein under weakly acidic to neutral pH conditions. In one embodiment, the albumin-binding protein can exhibit binding ability to albumin under both pH 5.5 and pH 7.4 conditions.

[0402] The characteristic that albumin-binding proteins can bind to albumin under both weakly acidic and neutral pH conditions implies that binding to albumin can be maintained even under various pH conditions in the body's internal environment. For example, in a neutral pH environment such as plasma, binding to albumin allows it to exist in a stable complex form during circulation, and binding characteristics can be maintained even in a relatively weakly acidic environment such as intracellular organelles.

[0403] In one embodiment, the albumin-binding protein can exhibit binding ability to albumin even under conditions of approximately pH 5.5, so that interaction with albumin can be maintained even in a weakly acidic environment such as an endosome. Additionally, by exhibiting binding ability under conditions of approximately pH 7.4, it can exist in a state bound to albumin in plasma. These binding characteristics across the entire pH range are technical features indicating that the albumin-binding protein according to the present invention can bind to albumin in an in vivo environment.

[0404] Additionally, albumin binding ability under the above pH conditions can be evaluated through conventional binding analysis methods such as ELISA, surface plasmon resonance (SPR), or bio-layer interferometry (BLI). In this specification, maintaining albumin binding ability may include cases where the binding signal or binding affinity does not significantly decrease based on the said analysis method.

[0405] The ability of the albumin-binding protein according to the present invention to bind to albumin under different pH conditions may include the possibility that the interaction with albumin is maintained even in an environment associated with the FcRn-mediated albumin recycling pathway. This characteristic may contribute to the extension of the residence time or half-life in the blood.

[0406] In one embodiment, the albumin-binding protein according to the present invention does not competitively inhibit albumin from binding to FcRn. The phrase “does not competitively inhibit” includes cases where the interaction between albumin and FcRn is not substantially hindered even when the albumin-binding protein is bound to albumin.

[0407] In one embodiment, the albumin-binding protein may interact with albumin by binding to a site that does not overlap with the FcRn binding site of albumin, or by not directly participating in binding with FcRn. Accordingly, even if the albumin-binding protein binds to albumin, the ability of FcRn to recognize and bind to albumin can be maintained.

[0408] In addition, the process of albumin binding to FcRn is known to occur primarily under weakly acidic pH conditions and to dissociate under neutral pH conditions. In one embodiment, the albumin-binding protein according to the present invention may bind to albumin in a manner compatible with these FcRn-mediated binding characteristics, and as a result, may include cases where the intracellular recycling pathway of albumin is not substantially disrupted.

[0409] In this specification, the statement that albumin does not competitively inhibit binding to FcRn may include cases where, based on FcRn binding analysis, cell-based binding analysis, or conventional competitive binding analysis, the FcRn binding signal or binding affinity is not significantly reduced compared to albumin alone.

[0410] In one embodiment, the albumin-binding protein according to the present invention binds to domain II of albumin.

[0411] In one embodiment, the albumin-binding protein according to the present invention comprises (i) a CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, and 76, and (ii) an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, and 77. A single-domain antibody comprising a CDR2 including, and (iii) a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75 and 78 may be an antibody that competitively binds to domain II of albumin. Such competitive binding characteristics may be evaluated, for example, through ELISA, surface plasmon resonance (SPR), or other conventional binding analysis methods.

[0412] Albumin is structurally composed of three major domains: Domain I, Domain II, and Domain III. It is known in the art that the neonatal Fc receptor (FcRn) binds to Domain I and Domain III of albumin. Considering this, when an albumin-binding protein binds to Domain II of albumin, this may involve binding to a region that does not overlap with the albumin-binding site of FcRn. Accordingly, in one embodiment, the anti-albumin antibody according to the present invention may bind to a region spatially distinct from the interaction between FcRn and albumin, thereby maintaining binding to albumin without substantially hindering FcRn-mediated albumin recycling.

[0413] Whether the albumin-binding protein binds to the albumin domain II, or whether there is competitive inhibition of the interaction between FcRn and albumin, can be determined by methods commonly known in the art, such as binding analysis using domain-specific recombinant albumin proteins, competitive binding analysis, surface plasmon resonance (SPR), biolayer interference (BLI), or cell-based analysis. For example, if the binding between albumin and FcRn is not significantly reduced in the presence of the albumin-binding antibody according to the present invention, it can be determined that the antibody does not substantially interfere with FcRn binding.

[0414] In one embodiment, the anti-albumin VHH antibody according to the present invention exhibits more selective or stronger binding characteristics to domain II of albumin compared to conventionally known anti-albumin VHH antibodies. These binding characteristics reflect improved selective binding to domain II of albumin and can be advantageous in extending the blood residence time or half-life while preserving the FcRn-mediated albumin recycling pathway. Accordingly, the albumin-binding antibody of the present invention can be applied in the form of a fusion with a therapeutic protein or antibody.

[0415] The albumin-binding protein according to the present invention can also bind to albumin of several species with sufficient affinity (e.g., medium to high affinity). For example, the albumin-binding antibody according to the present invention can bind to human albumin as well as albumin of cynomolgus monkeys, mice, and / or rats.

[0416] 2. Fusion protein

[0417] One aspect of the present invention provides a fusion protein in which the albumin-binding protein described herein is fused with one or more heterogeneous proteins.

[0418] The above heterogeneous protein refers to a protein having a different origin, structure, or biological function from the albumin-binding protein according to the present invention, and includes a protein in which the in vivo stability, blood retention time, half-life, or pharmacological properties of the said protein can be controlled or improved by being fused to the albumin-binding protein. Such heterogeneous proteins are not limited to antibodies and may include various proteins that can be used for therapeutic, diagnostic, or drug delivery purposes.

[0419] In one embodiment, the heterogeneous protein may be an antibody or an antibody fragment. For example, the heterogeneous protein may include a full-length antibody (IgG), Fab, F(ab′)₂, scFv, VHH, a single-domain antibody, or a variant thereof, and may include humanized, chimeric, or fully human antibodies. However, the heterogeneous protein according to the present invention is not limited to these forms.

[0420] In one embodiment, the heterogeneous protein may be a protein that specifically binds to a tumor-associated antigen, a T cell-associated antigen, or a B cell-associated antigen. Non-limiting examples include tumor-associated antigens such as CEACAM5, HER2, EGFR, EpCAM, PSMA, MSLN, EGFRvIII, GD2, Claudin18.2, Trop-2, DLL3, NY-ESO-1, or MAGE family antigens. T cell-associated antigens may include CD3, CD28, 4-1BB (CD137), OX40 (CD134), CD2, CD5, or CD7, and B cell-associated antigens may include CD19, CD20, CD22, CD79a, CD79b, CD37, or BCMA. The above antigens are merely exemplary, and a person skilled in the art will understand that various antigens, including immune cell surface proteins, tumor-specific or tumor-associated markers, and disease-specifically expressed target molecules, can be selected as binding targets for heterologous proteins.

[0421] Proteins that bind to such antigens may be antibodies or antibody fragments, but are not limited thereto, and may include non-antibody binding proteins, single variable domain proteins, or modifications thereof that have antigen-binding ability.

[0422] In one embodiment, the heterogeneous protein fused with the albumin-binding protein according to the present invention is not limited to antibodies or antibody fragments and may include various proteins that are not antibodies.

[0423] For example, the heterogeneous protein may be an enzyme, cytokine, chemokine, growth factor, hormone, receptor-binding protein, ligand protein, proteinaceous inhibitor, or active regulatory protein. As a non-limiting example, the heterogeneous protein may be an interleukin (e.g., IL-2, IL-7, IL-10, IL-15, IL-21), an interferon (e.g., IFN- , IFN- It may include ), tumor necrosis factor family proteins, or functional variants thereof. Additionally, the heterogeneous protein may be an angiogenesis regulator, an immune regulator, a protein involved in cell signaling, or a protein containing a binding domain of a cell surface receptor.

[0424] In another embodiment, the heterogeneous protein may include a proteinaceous toxin, an enzyme-active domain, a prodrug-activating enzyme, or a therapeutic peptide, and the in vivo stability or duration of action of the said protein may be controlled through fusion with an albumin-binding protein.

[0425] In addition, the above heterogeneous protein may include a diagnostic protein, an imaging protein, or a labeling protein, and in such cases, fusion with an albumin-binding protein may be utilized to control the distribution characteristics in the body or the persistence of the detection signal.

[0426] A person skilled in the art will understand that, considering the characteristics of the albumin-binding protein according to the present invention, various proteins suitable for therapeutic, diagnostic, or drug delivery purposes can be selected as heterologous proteins.

[0427] In one embodiment, the fusion protein according to the present invention has a structure in which one heterogeneous protein is fused with an albumin-binding protein.

[0428] In another embodiment, the fusion protein according to the present invention has a multi-specific structure in which two or more heterogeneous proteins are fused to an albumin-binding protein. For example, a structure may be implemented in which one heterogeneous protein binds to a tumor-associated antigen and another heterogeneous protein binds to a T cell-associated antigen or a B cell-associated antigen, and such a configuration may be implemented in the form of a bispecific antibody or a variant thereof. As used herein, the terms “first heterogeneous protein” and “second heterogeneous protein” are used as identifying terms to describe such a configuration in which two or more heterogeneous proteins are fused to a single albumin-binding protein.

[0429] When the fusion protein according to the present invention comprises two or more heterogeneous proteins and an albumin-binding protein, the arrangement of these protein components may vary. For example, when comprising a heterogeneous protein (A) that specifically binds to a tumor-associated antigen, an albumin-binding protein (B), and a heterogeneous protein (C) that specifically binds to a T cell or B cell-associated antigen, the fusion protein may comprise an arrangement of ABC, BAC, ACB, CBA, BCA, or CAB. Such arrangements may be implemented linearly within a single polypeptide chain, and each component may be directly connected or connected through one or more linkers.

[0430] Fusion between an albumin-binding protein and a heterogeneous protein according to the present invention can be achieved through direct peptide bonding or can be connected through one or more linkers. The fusion site may be any one of the N-terminus, C-terminus, or internal region of the albumin-binding protein or the heterogeneous protein, and may be selected to maintain the binding function and structural stability of each component.

[0431] When a linker is used, the linker may consist of an amino acid sequence that does not substantially affect the expression, folding, or functional characteristics of the fusion protein, and may be a flexible linker, a cleavable linker, or a non-cleavable linker. When two or more linkers are used in the fusion protein according to the present invention, each linker may independently be composed of a flexible linker, a cleavable linker, or a non-cleavable linker. In one embodiment, any linker included in the fusion protein according to the present invention may be configured to be enzymatically cleaved in the body or in an intracellular environment, thereby allowing the albumin-binding protein and the heterologous protein to function in a functionally separated form.

[0432] A heterogeneous protein existing in a fused form with an albumin-binding protein according to the present invention may have an improved blood retention time or half-life compared to the same heterogeneous protein without the albumin-binding protein bound.

[0433] The fusion protein according to the present invention can exist in a state bound to albumin in the body due to the albumin binding characteristics of the albumin-binding protein, and accordingly, the fused heterogeneous protein can also maintain stability during circulation in the body in a form linked to albumin. This albumin-linked structure can be involved in mitigating non-specific removal or degradation in the body, and as a result, the blood residence time or half-life of the fused protein containing the heterogeneous protein can be improved compared to the case where it is not fused with the albumin-binding protein.

[0434] In one embodiment, these effects can be quantitatively evaluated through pharmacokinetic (PK) analysis. For example, compared to a protein not fused with albumin-bound VHH, a protein fused with albumin-bound VHH may show an increase in AUC_last and AUC_inf values, which represent blood exposure levels, and a tendency for the maximum blood concentration (C_max) to also increase. Additionally, the fusion protein may exhibit characteristics such as a decrease in clearance (CL) or an increase in half-life (t_1 / 2) or mean residence time (MRT_inf). These changes in PK parameters reflect that the fusion protein can circulate in the body for a longer period through binding with albumin.

[0435] As such, the fusion protein according to the present invention can have its in vivo stability and circulation characteristics regulated through the albumin binding properties of the albumin binding protein, and as a result, the half-life of the fused protein containing the heterologous protein can be improved compared to the case where it is not fused with the albumin binding protein.

[0436] Furthermore, even in the case of fusion proteins having multiple specific structures, each heterogeneous protein can independently maintain binding functions to different targets, and albumin-binding proteins can contribute to the improvement of in vivo circulation characteristics by integrating these functions within a single protein platform. Accordingly, the fusion protein according to the present invention can be utilized in various therapeutic applications requiring an extended duration of action.

[0437] 3. Nucleic Acids, Vectors, and Host Cells

[0438] One aspect of the present invention provides an isolated nucleic acid molecule encoding an albumin-binding protein or a fusion protein comprising the same according to the present specification, a vector comprising said isolated nucleic acid molecule, and a host cell comprising said vector.

[0439] In one embodiment, the isolated nucleic acid molecule may be DNA or RNA and may include a base sequence encoding an amino acid sequence of an albumin-binding protein or fusion protein according to the present invention. The nucleic acid molecule may be a naturally derived sequence or may include a codon-optimized sequence considering expression efficiency, stability, or host cell compatibility.

[0440] The above vector may be an expression vector capable of containing the isolated nucleic acid molecule and may include regulatory elements such as a promoter, a transcription initiation site, a termination sequence, a selection marker, or a replication origin. These regulatory elements may be selected depending on the type of host cell used, and the vector may include a plasmid, a viral vector, or a variant thereof. Such a vector may be appropriately selected by a person skilled in the art depending on the desired expression characteristics, the type of host cell, or the purpose of use.

[0441] In one embodiment, the host cell may be a prokaryotic or eukaryotic cell and may include, for example, a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. The host cell may stably maintain or transiently receive the vector to express the albumin-binding protein or fusion protein according to the present invention. In some embodiments, the host cell may be selected to facilitate protein expression, folding, post-translational modification, or secretion, and a person skilled in the art may select an appropriate host cell system according to the characteristics of the albumin-binding protein or fusion protein according to the present invention.

[0442] The nucleic acid molecule, vector, and host cell according to the present invention may be used to produce an albumin-binding protein or a fusion protein containing the same according to the present specification through recombinant technology, and such production can be easily performed by a person skilled in the art without excessive experimentation through standard molecular biological and cell engineering techniques known in the art.

[0443] 4. Medical Uses

[0444] One aspect of the present invention provides using the albumin-binding protein or the fusion protein containing the same described herein for medical purposes for the treatment or prevention of a disease.

[0445] In one embodiment, the albumin-binding protein or fusion protein may be used in a form bound to a therapeutic protein or antibody acting on a biological pathway associated with a target disease, thereby controlling or improving the in vivo stability, blood retention time, or half-life of said therapeutic protein or antibody. These characteristics may contribute to extending the duration of therapeutic efficacy, reducing the frequency of administration, or achieving a more stable manifestation of the therapeutic effect.

[0446] In one embodiment, the medical use may include the treatment or prevention of various diseases, including cancer, inflammatory diseases, immune diseases, autoimmune diseases, infectious diseases, or metabolic diseases. For example, an albumin-binding protein fused with a heterologous protein that binds to a tumor-associated antigen, a T-cell-associated antigen, or a B-cell-associated antigen may exhibit therapeutic effects through the regulation of the immune response or targeted action within the tumor microenvironment.

[0447] In one embodiment, the medical use is the treatment or prevention of cancer.

[0448] In one embodiment, the albumin-binding protein according to the present invention or the fusion protein containing it may be used in a form combined with a heterologous protein that specifically binds to a tumor-associated antigen, thereby enabling targeted binding to tumor cells or selective action within the tumor microenvironment. These binding characteristics may provide action on cancer-associated cells or tissues while mitigating non-specific action on normal tissues.

[0449] In one embodiment, the fusion protein according to the present invention may include an antibody or antibody fragment that binds to a tumor-associated antigen and may exhibit more stable circulation characteristics in the body through fusion with an albumin-binding protein. As a result, the blood retention time or half-life of the anticancer active protein or antibody may be controlled or improved, which may contribute to the sustainability of anticancer treatment or the flexibility of the administration strategy.

[0450] In addition, in one embodiment, the albumin-binding protein according to the present invention or the fusion protein containing the same may be used to regulate the interaction between immune cells and tumor cells. For example, when including a heterologous protein that binds to a T cell-associated antigen or a B cell-associated antigen, effects associated with the induction or regulation of an immune response within the tumor microenvironment may occur.

[0451] In one embodiment, the cancer may include a solid tumor or a blood cancer, and the type of cancer is not limited to a specific tissue, organ, or cell type. Additionally, the medical use according to the present invention may be performed as monotherapy or in combination with other anticancer agents, immunotherapies, radiation therapy, or surgery, and the in vivo stability and functional characteristics of the albumin-binding protein or fusion protein may be maintained even under such combination therapy.

[0452] In another aspect, the albumin-binding protein according to the present invention can be used as a platform protein for improving the pharmacokinetic properties of heterologous proteins, particularly therapeutic proteins or antibodies.

[0453] In one embodiment, the albumin-binding protein may be provided in a form fused or bound to a therapeutic protein or antibody, and through such binding, the in vivo stability, blood retention time, or half-life of the heterologous protein may be controlled or improved. Changes in these pharmacokinetic properties may be associated with the duration of therapeutic effect, control of administration intervals, or flexibility of administration strategies.

[0454] In one embodiment, the albumin-binding protein according to the present invention may exist in a state associated with albumin in the blood through its binding properties with human albumin, and accordingly, the fused or bound heterologous protein may also maintain stability in circulation in the body in an albumin-associated form. These properties may contribute to mitigating loss through pathways such as non-specific removal, renal excretion, or protein degradation.

[0455] In addition, in one embodiment, the albumin-binding protein according to the present invention can maintain its binding ability to albumin in a weakly acidic to neutral pH range, so that interaction with albumin can be maintained not only in the blood environment but also in the in vivo environment associated with the FcRn-mediated albumin recirculation pathway. These characteristics may be associated with pharmacokinetic properties that reduce the clearance rate (CL) of the fusion protein containing the heterologous protein or increase the half-life (t₁ / ₂) or mean retention time (MRT).

[0456] In one embodiment, the use as a platform protein is not limited by the type, mechanism of action, or target of a specific therapeutic protein or antibody, and can be applied to various biopharmaceuticals including protein therapeutics, antibody therapeutics, enzyme proteins, cytokines, or immunomodulatory proteins. Accordingly, the albumin-binding protein according to the present invention can be utilized as a universal platform protein for controlling the pharmacokinetic properties of heterologous proteins.

[0457] 5. Pharmaceutical composition

[0458] One aspect of the present invention provides a pharmaceutical composition comprising an albumin-binding protein or a fusion protein containing the same as described herein as an active ingredient.

[0459] The pharmaceutical composition of the present invention may be used for medical purposes as described herein. That is, the pharmaceutical composition may be used for medical purposes for the treatment or prevention of diseases, and may be applied to the treatment or prevention of various diseases, for example, including cancer. In addition, in another embodiment, the pharmaceutical composition may be used for the purpose of controlling or improving the pharmacokinetic properties of a therapeutic protein or antibody.

[0460] The albumin-binding protein according to the present invention can utilize the FcRn-mediated recycling pathway in the body by binding to serum albumin, and as a result, the residence time in the blood and half-life can be significantly increased. Therefore, the pharmaceutical composition of the present invention can provide pharmacological benefits such as increased exposure in the body, extended duration of drug efficacy, and reduced frequency of administration compared to cases where the albumin-binding protein or a fusion protein containing it does not have an albumin-binding function.

[0461] In particular, when the albumin-binding protein according to the present invention is in a form fused with one or more heterogeneous proteins, the in vivo stability and half-life can be improved while maintaining the therapeutic or biological activity of the heterogeneous proteins. Accordingly, the pharmaceutical composition of the present invention can be usefully utilized to maintain an effective concentration of the fusion protein for a long period of time.

[0462] In one embodiment, the pharmaceutical composition may include a pharmaceutically acceptable carrier, diluent, or excipient, and these components may be selected within a range that does not impair the physical and chemical stability, biological activity, or suitability for administration of the albumin-binding protein or fusion protein according to the present invention. The pharmaceutically acceptable carrier, diluent, or excipient may be a material commonly used in the art and may be mixed or formulated with the protein according to the present invention.

[0463] The above pharmaceutical composition may be prepared according to conventional formulation methods and may be provided as an injectable, drip, oral, inhalant, topical, or sustained-release formulation depending on the route of administration. In one embodiment, the pharmaceutical composition may be a liquid or solid formulation suitable for intravenous, subcutaneous, intramuscular, intraperitoneal, or topical administration.

[0464] Additionally, the pharmaceutical composition may include a buffer, an isotonic agent, a stabilizer, a preservative, a surfactant, or an antioxidant as needed, and these components may be selected within the range commonly used in the field of protein formulations. The pharmaceutical composition may be provided in a single dose or multiple dose formulation.

[0465] In one embodiment, the pharmaceutical composition of the present invention may contain an albumin-binding protein as described herein or a fusion protein containing the same in a therapeutically effective amount.

[0466] Herein, “therapeutically effective content” means an amount sufficient to produce one or more biological effects related to the treatment or prevention of a disease when the albumin-binding protein or fusion protein according to the present invention is administered to a subject, and includes an amount within a range that does not cause excessive toxicity or undesirable side effects.

[0467] The above therapeutically effective content may vary depending on the subject's condition, the type and severity of the disease, the route of administration, the frequency of administration, the formulation, or whether combination therapy is used, and such differences or variations are understood to be included within the scope of the technical concept of the present invention.

[0468] In one embodiment, the pharmaceutical composition according to the present invention includes an albumin-binding protein in a fused form with a heterologous protein, in which case the albumin-binding protein may function as a platform component for controlling the in vivo stability, blood retention time, or half-life of the heterologous protein rather than directly exerting a therapeutic effect.

[0469] For such platform applications, the content of the albumin-binding protein can be set to be equal to or proportional to the therapeutically effective content of the heterologous protein, and can be adjusted by considering the effect on pharmacokinetic indicators such as the in vivo exposure time, clearance rate (CL), half-life, or mean retention time (MRT) of the fused heterologous protein, rather than the absolute dose of the albumin-binding protein itself.

[0470] For example, a heterologous protein fused with an albumin-binding protein may exhibit characteristics such as a reduced clearance rate or an increased half-life or average retention time compared to the same heterologous protein without the albumin-binding protein, thereby allowing the same therapeutic effect to be achieved with a lower administration frequency or a different administration schedule.

[0471] The setting of content and the method of administration for such platform applications may vary depending on the characteristics of the heterologous protein, the target disease, the route of administration, and the formulation, and can be implemented in various ways by utilizing the albumin binding characteristics of the albumin-binding protein according to the present invention.

[0472] The pharmaceutical composition according to the present invention can be easily prepared and practiced by a person skilled in the art without requiring excessive experimentation or creative effort, based on the description provided herein and general skills and knowledge known in the art. Pharmaceutically acceptable carriers, excipients, stabilizers, and delivery systems that may be used in the pharmaceutical composition of the present invention include materials and formulation techniques known in the art, and such differences or variations are understood to be included within the scope of the technical concept of the present invention.

[0473] 6. Treatment methods

[0474] One aspect of the present invention provides a method comprising administering an albumin-binding protein or a fusion protein containing the same described herein to an individual requiring medical use as described herein.

[0475] In one embodiment, the medical use includes the treatment or prevention of a disease, and the method may include alleviating symptoms of the disease, inhibiting progression, or reducing the risk of occurrence or recurrence of the disease by administering an albumin-binding protein or a fusion protein containing the same to the individual.

[0476] In one embodiment, the medical use is the treatment or prevention of cancer. The cancer may include solid tumors or blood cancers, and the type of cancer is not limited to a specific tissue, organ, or cell type. The treatment method of the present invention may be performed for the purpose of preventing the occurrence of cancer, alleviating symptoms of cancer that has already occurred, inhibiting tumor growth, delaying progression, or preventing recurrence.

[0477] In one embodiment, the individual may be a human and may include a non-human mammal as needed. The treatment method of the present invention may be performed for the purpose of preventing a disease, or for the purpose of alleviating symptoms of a disease that has already occurred, inhibiting its progression, or restoring tissue function.

[0478] In one embodiment, the treatment method may include administering an albumin-binding protein of the present invention or a fusion protein containing the same in a therapeutically effective amount.

[0479] Herein, “therapeutically effective amount” means an amount sufficient to induce one or more biological effects related to the treatment or prevention of a disease when the albumin-binding protein or fusion protein according to the present invention is administered to an individual, and includes an amount within a range that does not cause excessive toxicity or clinically undesirable side effects.

[0480] The above therapeutically effective amount may vary depending on the type and severity of the disease, the purpose of treatment (treatment or prevention), the individual's condition, the route of administration, the frequency of administration, the form of the protein used (albumin-binding protein alone or fusion protein), and whether combination therapy is used.

[0481] In one embodiment, when an albumin-binding protein is administered in a fused form with a heterologous protein, the therapeutically effective amount may be determined by considering both the therapeutic effect of the heterologous protein and the pharmacokinetic properties regulated by the albumin-binding protein. For example, if the clearance rate decreases or the half-life or mean retention time increases due to fusion with the albumin-binding protein, the administration frequency or schedule required to achieve the same therapeutic effect may be adjusted.

[0482] The albumin-binding protein or fusion protein may be administered alone, or may be administered in combination with other therapeutic agents, such as anticancer agents, immunotherapies, immunomodulators, antibody therapies, cell therapies, or radiation therapy. Combined administration may be performed with the same or different formulations, and may be carried out simultaneously, sequentially, or at regular intervals.

[0483] 7. Methods for Improving Pharmacokinetic Properties

[0484] One aspect of the present invention provides a method for controlling or improving the pharmacokinetic properties of a therapeutic protein or antibody using an albumin-binding protein as described herein.

[0485] In the present method, the albumin-binding protein may be directly bound to a therapeutic protein or antibody, provided in the form of a fusion protein, or form a complex through non-covalent interaction. Through such binding or fusion, the therapeutic protein or antibody acquires or increases its ability to interact with albumin, and as a result, pharmacokinetic properties such as stability, circulation time, elimination rate, or systemic exposure may be modified or improved.

[0486] In this specification, the term “use of albumin-binding protein” includes, for the above purpose, providing the albumin-binding protein together with a therapeutic protein or antibody, binding it thereto in a covalent or non-covalent manner, or preparing and applying it in the form of a fusion protein.

[0487] The control or improvement of pharmacokinetic properties according to the present method may include, but is not limited to, an increase in blood retention time, a decrease in clearance, an extension of half-life, or an increase in mean retention time, and these effects may occur regardless of the structure, molecular weight, target specificity, presence of an Fc domain, or route of administration of the therapeutic protein or antibody.

[0488] The use according to the present invention is not limited to a specific route of administration, time of administration, or therapeutic indication, and can be utilized in the development, manufacture, or provision of various therapeutic proteins or antibodies.

[0489]

[0490] Examples

[0491] Example 1: Production of VHH Antibody

[0492] 1.1. Immunization

[0493] Alpaca was immunized using human albumin protein as an antigen. Specifically, the region from the 25th aspartic acid (Asp) residue to the 609th leucine (Leu) residue of the amino acid sequence of human albumin protein (UniProt Accession No. P02768-1) was used as the antigen.

[0494] A total of five immunizations were performed on alpacas using the above antigens, and the immunization was carried out according to methods commonly used in the relevant technical field. The immunization schedule was as shown in Table 1 below.

[0495]

[0496]

[0497] Peripheral blood samples were collected at each immunization step, and the progress of immunization was evaluated using enzyme-linked immunosorbent assay (ELISA). ELISA analysis was performed using human serum albumin (HSA), cynomolgus monkey serum albumin (CSA), and mouse serum albumin (MSA) as antigens.

[0498] Specifically, each antigen was diluted in coating buffer and then dispensed at a rate of 100 μL into each well of a 96-well plate for incubation. Afterward, the plates were washed with washing buffer, and then 150 μL of blocking buffer was added to each well for incubation. After removing the blocking buffer, 100 μL of either pre-immunization serum or immune serum was added to each well for incubation.

[0499] Subsequently, the mixture was washed three times with washing buffer, incubated with a secondary antibody, and washed three times again. Then, the TMB substrate was reacted at 25°C, the reaction was terminated by adding a stop buffer, and the absorbance was measured at 450 nm.

[0500] The results of the above ELISA analysis are shown in Tables 2 to 4 below.

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507] 1.2. Single B-cell Beacon Screening

[0508] After obtaining partially antigen-specific memory B cells through antigen-specific enrichment, the memory B cells were stimulated and cultured. Analysis of the supernatant collected after 7 days of culture revealed binding activity to anti-camelid VHH and anti-lama IgG(H+L). Based on this, it was determined that most memory B cells were activated, and subsequent screening was performed.

[0509] Activated memory B cells were analyzed using a single-cell beacon screening method on cells acquired after the 4th and 5th immunizations. Specifically, the cells were dispensed onto a chip within the instrument and arranged at the single-cell level, after which reactivity was observed against antibodies secreted from each cell using an anti-camelid VHH-specific secondary antibody. During this process, some B cells were selected that simultaneously showed a response to camelid VHH while binding to human albumin and cynomolgus monkey albumin.

[0510] Selected B cells were transferred to a 96-well plate and PCR was performed to amplify single-cell-derived genes, and plasmid cloning was performed using the amplified genes.

[0511]

[0512] 1.3. Linear Expression Screening

[0513] After transfecting the cloned plasmid into Expi293F cells or ExpiCHO-S cells and culturing them, the supernatant containing VHH was collected. ELISA analysis was performed on the collected supernatant to confirm the expression of VHH. In addition, the relative expression levels of VHH and its binding to human, cynomolgus monkey, and mouse albumin were evaluated via ELISA analysis.

[0514]

[0515] 1.4. Construction of Immunophage Display Library and Specific VHH Screening

[0516] Immunophage display libraries were constructed in accordance with Probio’s Standard Operating Procedures (SOPs), and quality evaluations were performed on the constructed libraries in terms of size, insertion rate, in-frame ratio, and sequence diversity. The SOPs are based on conventional phage display methods and include panning, washing, and elution steps.

[0517] To select VHH that binds to serum albumin, soluble panning using antigen proteins was performed. Specifically, up to three pannings were performed using human albumin and mouse albumin as antigens, and as a result, VHH clones having unique sequences that bind to albumin in ELISA analysis were selected.

[0518]

[0519] 1.5. VHH Production and Refining

[0520] Based on the results of linear expression screening and immune phage display, sequences showing a positive reaction to human and cynomolgus monkey albumin were selected, and VHH antibodies were produced and purified using the selected sequences.

[0521] Selected VHH was expressed in the CHO-S cell line by adding a 6×His-DYKDDDDK tag to the C-terminus of the sequence, and the supernatant containing the expressed VHH was purified using PreDictor RoboColumn IMAC Sepharose 6FF. After purification, the buffer was exchanged with pH 7.2 PBS buffer, and the concentration of VHH was evaluated based on the absorbance at 280 nm.

[0522] In addition, the purity of all VHHs was confirmed to be over 80% through SDS-PAGE and SEC-HPLC analysis. VHH clones derived from beacon screening were named “DVH”, and VHH clones derived from the phage display library were named “DPVH”.

[0523] The amino acid sequences of the produced VHH antibodies are as follows.

[0524] DVH-30:

[0525] QVQLVESGGGLVQPGGSLRLSCAASEIIIDDYTMYWYRQAPGEQRELVASITSGDSAYYAGSVKGRFTISRDNGKNMVYLQMNSLKPEDTAVYFCNANRWLTNRDYWGQGTQVTVSS (SEQ ID NO: 103)

[0526] DVH-31:

[0527] QVQLVESGGGLVQAGGSLRLSCATSGSTFSINAIGWYRQAPGKQRELVATISMGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNARWGWDYSGPERWGQGTQVTVSS (SEQ ID NO: 104)

[0528] DVH-39:

[0529] QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMRWYRQAPGKERELVATITSTGGSTNYANSVKGRFTISRDNAGNTVYLQMNSLKPDDTAVYYSCAGNSWSSSRYDYWGQGTQVTVSS (서열번호 105)

[0530] DVH-40:

[0531] QVQLVESGGGWVQPGGSLRLSCLASGFTFSVYAMRWYRQAPGKERELVAAISSAGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAGNSWSSRYDYWGQGTQVTVSS (서열번호 106)

[0532] DVH-42:

[0533] QVQLVESGGGLVQAGGSLRLSCAASGFFSFSRYAVGWFRQSPGKEREFVAAINWSGGSTNTADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAVFRIVPPTTTNYHYWGQGAQVTVSS (서열번호 107)

[0534] DVH-51:

[0535] QVQLVESGGGLVQAGGSLRLSCAASGRTLSRYAMGWFRQAPGKEREFVATISWSGASTYYGDSVKGRFTISRENAKNAVYLQMNSLKPEDTAVYCAAAWDLGSTLYGATGYKYDYWGQGTQVTVSS (서열번호 108)

[0536] DVH-52:

[0537] QVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREFVATISWSASSTYYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYCAAAWDLGSTLYGASGYKYDYWGQGTQVTVSS (서열번호 109)

[0538] DVH-53:

[0539] QVQLVESGGGLVQAAGSLRLSCAASGGTFTWYAMGWFRQAPGKEREFVAAIRRRGVSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAWDLDSDSRLKYDYWGQGTQVTVSS (서열번호 110)

[0540] DPVH-04:

[0541] QVQLVESGGGLVQAGGSLRLSCAASGRTSRFNPMGWFRQAPGKEREFVAAISWSGSNTLYSDSVKGRFTISRDIGKTSAYLQMISLKPEDTAVYYCAARSEYGVTSIEGEYDYWGQGTQVTVSS (서열번호 111)

[0542] DPVH-07:

[0543] EVQLVESGGGSVQPGGSLRLSCAASGFTFSSYDMSWYRQAPGKERELVAAIMSAGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSAHIDWPLEDYDYWGQGTQVTVSS (서열번호 112)

[0544] DPVH-10:

[0545] QVQLVESGGGLVQAGGSLRLSCEASGRTFSLRSMYWFRQDPGKEREFVAAISGSGGDTRYVDSVRGRFTISREYLRNTVYLQMSNLLPEDTAVYYCAAPQALRLFIDLEDRNLYWGQGTQVTVSS (서열번호 113)

[0546] DPVH-11:

[0547] QVKLEESGGGLVQAGGSLRLSCAASGRNTYAMGWFRQAPGKEREFVAAISWTGGSTYFEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARRPPFLAIATAGYEYDYWGQGTQVTVSS (서열번호 114)

[0548] DPVH-13:

[0549] AVQLVESGGGLVQAGGSLRLSCAASGHTSRFNPMGWFRQAPGKEREFVAAISWSGSNTLYSDSVKGRFTISRDIGKISAYLQMNSLKPEDTAVYYCAARSEYGLTSIEGEYDYWGQGTQVTVSS (서열번호 115)

[0550] DPVH-14:

[0551] QVQLVESGGGLVQAGGSLRLSCAASGRTSRFNPMGWFRQAPGKEREFVAAISWSGSNTLYSDSVKGRFTISRDIGKISAYLQMNSLKPEDTAVYYCAARSEYGLTSIEGEYDYWGQGTQVTVSS (서열번호 116)

[0552] DPVH-15:

[0553] QVQLVESGGELVQAGGSLRLSCAASGGTGRFNPMGWFRQAPGKEREFVAAISWSGGYTLYSDSVKGRFTISRDIGKIAAYLQMNNLKPEDTAVYYCAARSEYGLTSIEGGYDYWGQGTQVTVSS (서열번호 117)

[0554] DPVH-16:

[0555] QVQLVESGGGLVQAGGSLRLSCAASGRTSRFNPMGWFRQAPGKEREFVAAISWSGSITLYSDSVKGRFTISRDIGKTSAYLQMISLKPEDTAVYYCAARSEYGVTSIEGEYDYWGQGTQVTVSS (서열번호 118)

[0556] DPVH-17:

[0557] AVQLVESGGGLVQAGGSLRLSCAASGRTSRFNPMGWFRQAPGKEREFVAAISWSGSNTLYSDSVKGRFTISRDIGKTSAYLQMISLKPEDTAVYYCAARSGYGVTSIEGEYDYWGQGTQVTVSS (서열번호 119)

[0558] DPVH-18:

[0559] QVQLVESGGGLVQAGGSLGLSCAASGGTFSSYAMAWFRQAPGKEREFVAAITWSGESTYYASSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYSCAGNRSPQTSLFSRVVFSRDSNDYNYWGQGTQVTVAS (서열번호 120)

[0560] DPVH-19:

[0561] QVKLEESGGGLVQAGASLRLSCAASGRTFNAYAMAWFRQAPGKEREFVAAITWSGESTYYASSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYSCAGNRSPQTSLFSRVVFSRDSNDYNYWGQGTQVTVAS (서열번호 121)

[0562] DPVH-24:

[0563] EVQLVESGGGLVQAGGSLGLSCAASGFTFSGYAMSWYRQAPGKERELVAAIAEAGGSTNYADSVKGRFTIFRDNAQNTVYLQMNSLKPEDTGVYYCNAHITWPLEDYDYWGQGTQVTVSS (서열번호 122)

[0564] DPVH-27:

[0565] DVQLVESGGGLVQAGGSLTLSCAASGLTFSTYAMGWFRQAPGKEREFVAGISWGYGSTYYADSVRGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCNAHREWVLEDYDYWGQGTQVTVSS (서열번호 123)

[0566] DPVH-32:

[0567] QVQLVESGGGLVQAGGSLRLSCAASGFTFSSYAMGWFRQAPGKEREFVATISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAHTVWPLEEYDYWGQGTQVTVSS (서열번호 124)

[0568] DPVH-33:

[0569] QVKLEESGGGLVQPGGSLRLSCAASGSTFSIYAMGWYRQAPGKQRELVATISSGGSTKYLDSVKGRLTISRDNAKNMVYLQMNSLEPEDTAVYYCNALPGSWPIRDFGSWGQGTQVTVSS (SEQ ID NO: 125)

[0570] DPVH-37:

[0571] DVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMHWYRQAPGGERELVAGITSTARAINVADSVQGRFTISRDNAKNTVSLQMNGLKPEDTAVYYCNAYRVRGDGKLMYWGQGTQVTVSS (SEQ ID NO: 126)

[0572] DPVH-39:

[0573] EVQLVESGGGLVQPGGSLRLSCVVSGNILSSYIMAWYRQTPGNERELVARITSGGHTNYADSVKGRFTISGETFKNTVYLQMMSLKPEDTAVYICNQRTVQRDYWGQGTQVTVSS (SEQ ID NO: 127)

[0574] DPVH-42:

[0575] AVQLVESGGGLAQPGGSLRLSCTASGFTFTDYAMSWYRQVPGKERELVAGISIAGSKTDYGDSVKGRFTISRDNAKNMVYLQMNSLKPEDTAMYYCTRGWGKAYFWGQGTQVTVSS (SEQ ID NO: 128)

[0576] As a positive control, the anti-albumin VHH sequence (ALB8) of ozoralizumab, which is currently commercially available as a treatment for rheumatoid arthritis, was used.

[0577] ALB8 amino acid sequence:

[0578] EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 136)

[0579]

[0580] Example 2: Evaluation of the binding ability of VHH antibodies to albumin antigens

[0581] The binding potency of the synthesized VHH antibodies to albumin was evaluated under conditions ranging from pH 5.0 to 7.4. PBS and PBST (DPBS + 0.05% Tween 20) for each pH condition were custom-made by Dain Bio and used.

[0582] Recombinant human, synomolgus monkey, and mouse albumin proteins (Acrobiosystems) were diluted to a concentration of 0.5 μg / mL, 30 μL was dispensed into each well of a Maxisorp 384-well microplate, and the plates were coated by incubation at 4°C for 16 hours. Subsequently, the plates were washed four times with 100 μL / well of PBST (DPBS + 0.05% Tween 20, Teknova).

[0583] After washing, 80 μL / well of Fish Gelatin Blocking Agent (Biotium) was added to perform a blocking reaction at room temperature for 2.5 hours, followed by four washes with 100 μL / well of PBST. Each VHH antibody was prepared in various concentration ranges by serial dilution in PBS at 3- or 4-fold levels. 30 μL of the prepared VHH antibody solution was added to each well of an albumin-coated plate and incubated at room temperature for 1 hour.

[0584] After washing the plates with PBST, 30 μL of the HRP (horseradish peroxidase)-conjugated anti-FLAG secondary antibody (Sigma) was added to each well diluted to a concentration of 0.05 μg / mL (1:20,000) in binding buffer and incubated at room temperature for 1 hour. Subsequently, after washing in the same manner, 30 μL of TMB (3,3′,5,5′-tetramethylbenzidine, Invitrogen) substrate was added to each well and reacted at room temperature in a dark room for 10 minutes.

[0585] The reaction was terminated by adding 30 μL / well of 0.16 M sulfuric acid (reaction stopping solution, Invitrogen), and absorbance was measured at 450 nm (Synergy H1 multimode reader, Biotek).

[0586] As a result, all VHH antibodies according to the present invention showed significant binding to human albumin under pH 5.5 to 7.4 conditions, and were confirmed to have a sufficient level of binding ability to synomolgus monkey and mouse albumin as well.

[0587] The albumin binding ability of the antibodies used in the test was measured as shown in Tables 5 and 6 below.

[0588]

[0589]

[0590]

[0591]

[0592] Example 3: Antibody Humanization

[0593] In this embodiment, the amino acid sequences of the alpaca-derived VHH antibodies DPVH18 and DPVH42 were humanized.

[0594] Humanization was performed by aligning the VHH antibody sequence with the human VH germline sequence based on the IMGT database and then selecting the human framework sequence exhibiting the highest homology. To maintain antigen binding specificity, the amino acid sequences of the CDR1, CDR2, and CDR3 regions were not altered. On the other hand, amino acid residues included in the framework regions (FR1 to FR4) that are not directly involved in antigen binding were replaced with corresponding human germline amino acids. Additionally, back-mutation was performed on residues predicted to be important for maintaining structural stability.

[0595] The amino acid sequence of the produced humanized VHH variant is as follows.

[0596] DP-686 (Moclone DPVH42):

[0597] EVQLVESGGGLVQPGGSLRLSCTASGFTFTDYAMSWVRQVPGKGLEWVSGISIAGSKTDYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAMYYCTRGWGKAYFWGQGTLVTVSS (SEQ ID NO: 129)

[0598] DP-687 (Moclone DPVH42):

[0599] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYAMSWVRQAPGKGLEWVSGISIAGSKTDYGDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCTRGWGKAYFWGQGTLVTVSS (SEQ ID NO: 130)

[0600] DP-688 (Moclone DPVH42):

[0601] EVQLVESGGGLVQPGNSLRLSCAASGFTFTDYAMSWVRQAPGKGLEWVSGISIAGSKTDYGDSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTRGWGKAYFWGQGTLVTVSS (SEQ ID NO: 131)

[0602] DP-689 (Moclone DPVH42):

[0603] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYAMSWVRQAPGKGLEWVSGISIAGSKTDYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGWGKAYFWGQGTLVTVSS (SEQ ID NO: 132)

[0604] DP-724 (Moclone DPVH42):

[0605] EVQLVESGGGVVQPGGSLRLSCAASGFTFTDYAMSWYRQAPGKGLELVAGISIAGSKTDYGDSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCTRGWGKAYFWGQGTLVTVSS (SEQ ID NO: 133)

[0606] hDPVH18-1 (Moclone DPVH18):

[0607] EVQLLESGGGLVQPGGSLRLSCAASGGTFSSYAMAWFRQAPGKGLEFVSAITWSGESTYYASSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGNRSPQTSLFSRVVFSRDSNDYNYWGQGTLVTVAS (SEQ ID NO: 134)

[0608] hDPVH18-11 (Moclone DPVH18):

[0609] EVQLLESGGGLVQPGGSLRLSCAASGGTFSSYAMAWFRQAPGKGLEFVSAITWSGESTYYASSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGNRSPQTSLFSRVVFSRDSNDYNYWGQGTLVTVAS (SEQ ID NO: 135)

[0610] The binding activity of the humanized VHH variants produced in this manner was evaluated through antigen binding affinity analysis after expression and purification, and the results showed that they exhibited antigen binding activity substantially equivalent to that of the parent VHH.

[0611]

[0612] Example 4: Test to confirm competitive binding of FcRn

[0613] To ensure the effect of extending the half-life, it is desirable that albumin-bound VHH does not competitively inhibit the binding between the neonatal Fc receptor (FcRn) and albumin. Accordingly, to evaluate whether there is competition for the binding between FcRn and albumin, a surface plasmon resonance (SPR)-based epitope competitive assay using the Biacore 8K system was performed.

[0614] HBS-EP+ with a pH of 6.0 was used as the running buffer, and antigen immobilization was performed at 25°C. Specifically, the surface of the sensor chip was activated with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and then the antigen was injected and immobilized; after binding, the remaining active site was blocked with ethanolamine hydrochloride.

[0615] For epitope competition analysis, human serum albumin was immobilized on a CM5 sensor chip, and the test antibody (VHH) was injected first, followed by the sequential injection of human FcRn to observe changes in the binding signal. Competitive binding was evaluated based on whether the binding signal of FcRn significantly decreased in the presence of the test antibody. Data analysis was performed using Biacore 8K Evaluation Software.

[0616] Meanwhile, as a positive control, the anti-albumin VHH sequence (ALB8) of ozoralizumab, which is currently commercially available as a treatment for rheumatoid arthritis, was used.

[0617] Analysis results showed that the tested antibodies exhibited binding characteristics that did not competitively inhibit FcRn from binding to albumin (Figs. 1 and 2). These results suggest that the anti-albumin VHH antibodies according to the present invention can bind to albumin while maintaining the FcRn-mediated albumin recycling pathway, supporting the possibility that they may favorably extend the half-life when applied in vivo.

[0618]

[0619] Example 5: Albumin Domain Binding Analysis

[0620] Domain-specific binding analysis was performed to determine which domain of albumin the selected anti-albumin VHH antibodies bind to.

[0621] Albumin proteins were produced and purified in four forms, respectively: (1) whole albumin (amino acids 25 to 609 of SEQ ID NO. 102), (2) a fragment corresponding to albumin domain II (amino acids 207 to 405 of SEQ ID NO. 102), (3) a fragment corresponding to albumin domain I+II (amino acids 25 to 405 of SEQ ID NO. 102), and (4) a fragment corresponding to albumin domain II+III (amino acids 214 to 609 of SEQ ID NO. 102) (see FIG. 3). After coating the proteins onto plates at equal concentrations, anti-albumin VHH antibodies according to the present invention were added to evaluate binding. This test was performed using the same method as the ELISA conditions described in Example 1.2.

[0622] As a result, the DPVH-11, DPVH-17, DPVH-18, and DPVH-42 antibodies exhibited low EC50 values ​​for whole albumin and albumin domain II proteins and showed strong binding ability. In particular, the antibodies exhibited EC50 values ​​for albumin domain II proteins that were similar to or lower than those for whole albumin proteins, suggesting that the major binding sites of these antibodies are located in albumin domain II (see Fig. 4).

[0623] In contrast, DPVH-27 bound to the entire albumin but did not show binding to Domain II and Domain I+II proteins, exhibiting a binding pattern different from that of the anti-albumin VHH antibodies according to the present invention. While this binding pattern may contribute to extending the residence time in the body to some extent through binding to albumin, it is difficult to consider the FcRn-mediated albumin recycling process as being completely maintained; therefore, it suggests that this may not be considered the optimal binding characteristic for maximizing the half-life extension effect.

[0624]

[0625] Example 6: Preparation of a fusion protein comprising albumin-bound VHH antibody, anti-CEACAM5 antibody, and anti-CD3 antibody

[0626] To evaluate the potential for extending the half-life following the introduction of an albumin-bound VHH antibody, a fusion protein in the form of a T cell engager (TCE) containing the albumin-bound VHH antibody according to the present invention was constructed.

[0627] The above fusion protein was designed by attaching a VHH antibody that specifically binds to anti-human serum albumin (HSA) to a TCE structure composed of an anti-human CD3 antibody domain and an anti-human CEACAM5 antibody domain. To facilitate purification and analysis, a polyhistidine tag (6×His tag) and a FLAG tag (DYKDDDDK) were added to the C-terminus of the fusion protein.

[0628] The above fusion protein was expressed in CHO cells, and the culture supernatant was collected and purified. Primary purification was performed using immobilized metal affinity chromatography (IMAC) with a His tag, and subsequent additional purification was performed using at least one of ion exchange chromatography (IEX) or size exclusion chromatography (SEC).

[0629] The purified proteins were evaluated through SDS-PAGE and liquid chromatography analysis, and it was confirmed that a purity of over 90% was achieved in all samples.

[0630] The constituent protein arrays and amino acid sequences of the constructed fusion proteins are as follows. The underlined parts of the amino acid sequences correspond to the linker sequences.

[0631] DP-345 (containing anti-albumin VHH antibody DPVH-11, anti-CD3 Fab antibody, and anti-CEACAM5 scFv antibody) (see Fig. 5)

[0632] Polypeptide 1: [Anti-albumin VHH antibody DPVH-11] - [linker] - [Anti-CD3 VL-C ] - [linker] - [Hang-CEACAM5 VH] - [scFv linker] - [Hang-CEACAM5 VL]

[0633] QVKLEESGGGLVQAGGSLRLSCAASGRNTYAMGWFRQAPGKEREFVAAISWTGGSTYFEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARRPPPFLAIATAGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFAGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRFVFSLDTSVSTAYLQISSL KAEDTAVYYCAREAGKDYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCSASSSVTFMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISSLQPDDAATYYCQQWNNYPWTFGGGTKVEIK (서열번호 137)

[0634] 제2 POLYPEPIP티드: [항-CD3 VH-CH1] - [linker] - [His-Flag tag]

[0635] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDKATLTVDKSISTAYMELSRLRSDDTAVYYCARSGYYGDSDWYFDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPCGGSHHHHHDYKDDDDK (서열번호138)

[0636] DP-346 (containing anti-albumin VHH antibody DPVH-17, anti-CD3 Fab antibody, and anti-CEACAM5 scFv antibody) (see Fig. 5)

[0637] Polypeptide 1: [Anti-albumin VHH antibody DPVH-17] - [linker] - [Anti-CD3 VL-C ] - [linker] - [Hang-CEACAM5 VH] - [scFv linker] - [Hang-CEACAM5 VL]

[0638] AVQLVESGGGLVQAGGSLRLSCAASGRTSRFNPMGWFRQAPGKEREFVAAIISWSGSNTLYSDSVKGRFTISRDIGKTSAYLQMISLKPEDTAVYYCAARSGYGVTSIEGEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGSDIQMTQS PSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFAGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRFVFSLDTSVSTAYLQISSL KAEDTAVYYCAREAGKDYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCSASSSVTFMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISSLQPDDAATYYCQQWNNYPWTFGGGTKVEIK (SEQ ID NO: 139)

[0639] Polypeptide II: [Anti-CD3 VH-CH1] - [linker] - [His-Flag tag]

[0640] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDKATLTVDKSISTAYMELSRLRSDDTAVYYCARSGYYGDSDWYFDVWGQGTMVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGSHHHHHHDYKDDDDK (SEQ ID NO: 140)

[0641] DP-347 (containing anti-albumin VHH antibody DPVH-18, anti-CD3 Fab antibody, and anti-CEACAM5 scFv antibody) (see Fig. 5)

[0642] Polypeptide 1: [Anti-albumin VHH antibody DPVH-18] - [linker] - [Anti-CD3 VL-C ] - [linker] - [Hang-CEACAM5 VH] - [scFv linker] - [Hang-CEACAM5 VL]

[0643] QVQLVESGGGLVQAGGSLGLSCAASGGTFSSYAMAWFRQAPGKEREFVAAITWSGESTYYASSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYSCAGNRSPQTSLFSRVVFSRDSNDYNYWGQGTQVTVASGGGGSGGGGSGGGGSGGGGSGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGTGDYTLTISSLQPEDFATYFCQQGNTLPWTFAGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRFVFSLDTSVSTAYLQIS SLKAEDTAVYYCAREAGKDYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCSASSSVTFMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISSLQPDDAATYYCQQWNNYPWTFGGGTKVEIK (서열번호 141)

[0644] 제2 POLYPEPIP티드: [항-CD3 VH-CH1] - [linker] - [His-Flag tag]

[0645] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDKATLTVDKSISTAYMELSRLRSDDTAVYYCARSGYYGDSDWYFDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPCGGSHHHHHDYKDDDDK (서열번호142)

[0646] DP-348 (containing anti-albumin VHH antibody DPVH-42, anti-CD3 Fab antibody, and anti-CEACAM5 scFv antibody) (see Fig. 5)

[0647] Polypeptide 1: [Anti-albumin VHH antibody DPVH-42] - [linker] - [Anti-CD3 VL-C ] - [linker] - [Hang-CEACAM5 VH] - [scFv linker] - [Hang-CEACAM5 VL]

[0648] AVQLVESGGGLAQPGGSLRLSCTASGFTFTDYAMSWYRQVPGKERELVAGISIAGSKTDYGDSVKGRFTISRDNAKNMVYLQMNSLKPEDTAMYYCTRGWGKAYFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGSDIQMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFAGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRFVFSLDTSVSTAYLQISSLKA EDTAVYYCAREAGKDYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCSASSSVTFMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISSLQPDDAATYYCQQWNNYPWTFGGGTKVEIK (SEQ ID NO: 143)

[0649] Polypeptide II: [Anti-CD3 VH-CH1] - [linker] - [His-Flag tag]

[0650] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDKATLTVDKSISTAYMELSRLRSDDTAVYYCARSGYYGDSDWYFDVWGQGTMVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGSHHHHHHDYKDDDDK (SEQ ID NO: 144)

[0651] DP-301 (containing anti-CD3 Fab antibody and anti-CEACAM5 scFv antibody; lacking anti-albumin VHH antibody) (see Figures 7A and 8A)

[0652] Polypeptide 1: [Anti-CD3 VH] - [scFv linker] - [Anti-CD3 VL] - [linker] - [Anti-CEACAM5 VL-C ]

[0653] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGNTLPWTFAGGTKVEIKGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDKATLTVDKSISTAYMELSRLRSDDTAVYYCARSGYYGDSDWYFDVWGQ GTMVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCSASSSVTFMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISSLQPDDAATYYCQQWNNYPW TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 145)

[0654] Polypeptide 2: [Anti-CEACAM5 VH-CH1] - [linker] - [His-Flag tag]

[0655] QIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAREAGKDYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGSHHHHHHDYKDDDDK (SEQ ID NO: 146)

[0656] DP-403 (containing anti-albumin VHH antibody DPVH-18, anti-CD3 scFv antibody, and anti-CEACAM5 Fab antibody) (see Fig. 7A)

[0657] Polypeptide 1: [Anti-albumin VHH antibody DPVH-18] - [linker] - [CD3 VH] - [scFv linker] - [Anti-CD3 VL] - [linker] - [Anti-CEACAM5 VL-C ]

[0658] QVQLVESGGGLVQAGGSLGLSCAASGGTFSSYAMAWFRQAPGKEREFVAAITWSGESTYYASSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYSCAGNRSPQTSLFSRVVFSRDSNDYNYWGQGTQVTVASGGGGSGGGGSGGGGSGGGGSGGGGSGGGSGIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGGTDFTLTISSLQPEDVATYYCQQGNTLPWTFAGGTKVEIKGGGGSGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTM NWVRQAPGQGLEWMGLINPYKGVSTYNQKFKDKATLTVDKSISTAYMELSRLRSDDTAVYYCARSGYYGDSDWYFDVWGQGTMVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCSASSSVTFMHWYQQKPGKAPKRLIYDTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDAATYYCQQWNNYPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKWYACEVTHQGLSSPVTKSFNRGEC (서열번호 147)

[0659] 제2 포하티드: [항-CEACAM5 VH-CH1] - [linker] - [His tag] - [linker] - [Flag tag]

[0660] QIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAREAGKDYAMDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGSHHHHHHGGSDYKDDDDK (SEQ ID NO: 148)

[0661] DP-585 (containing anti-albumin VHH antibody DPVH-42, anti-CD3 scFv antibody, and anti-CEACAM5 Fab antibody) (see Fig. 8A)

[0662] Polypeptide 1: [Anti-albumin VHH antibody DPVH-42] - [linker] - [CD3 VH] - [scFv linker] - [Anti-CD3 VL] - [linker] - [Anti-CEACAM5 VL-C ]

[0663] AVQLVESGGGLAQPGGSLRLSCTASGFTFTDYAMSWYRQVPGKERELVAGISIAGSKTDYGDSVKGRFTISRDNAKNMVYLQMNSLKPEDTAMYYCTRGWGKAYFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGSDIQMTQSPSS LSASVGDRVTITCRASQDIRNYLNWYQQKPGKVKPLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGNTLPWTFAGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMNWVRQAPG QGLEWMGLINPYKGVSTYNQKFKDKATLTVDKSISTAYMELSRLRSDDTAVYCARSGYYGDSDWYFDVWGQGTMVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCSASSSVTFMHWYQQKPGKAPKRLIYDTSKLASGVPS RFSGSGSGTEYTLTISSLQPDDAATYYCQQWNNYPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKWYACEVTHQGLSSPVTKSFNRGEC (서열번호 149)

[0664] 제2 포하티드: [항-CEACAM5 VH-CH1] - [linker] - [His tag] - [linker] - [Flag tag]

[0665] QIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAREAGKDYAMDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGSHHHHHHGGSDYKDDDDK (SEQ ID NO: 150)

[0666]

[0667] Example 7: Evaluation of albumin binding ability of a fusion protein containing albumin-binding VHH antibody, anti-CEACAM5 antibody, and anti-CD3 antibody

[0668] In this embodiment, the binding ability to albumin was evaluated when an albumin-binding single-domain antibody (VHH) was fused to a heterologous antibody.

[0669] The albumin binding ability of the synthesized fusion proteins was evaluated using the same ELISA assay as described in Example 2. Specifically, each fusion protein was added to plates coated with recombinant human, synomolgus monkey, and mouse albumin proteins, and the binding was measured.

[0670] As a result, it was confirmed that DPVH-11, DPVH-17, DPVH-18, and DPVH-42 antibodies maintained their ability to bind to albumin regardless of whether they were fused with anti-CEACAM5 antibodies and anti-CD3 antibodies (see Fig. 6). This suggests that the unique albumin binding properties of albumin-binding VHH are preserved even when fused to heterologous antibodies.

[0671]

[0672] Example 8: Evaluation of mouse pharmacokinetic properties of an antibody containing albumin-bound VHH

[0673] In this embodiment, a mouse pharmacokinetic (PK) test was performed to evaluate whether the blood half-life of the antibody in vivo can be improved when the albumin-binding single-domain antibody (VHH) according to the present invention is fused to a heterologous antibody.

[0674] This study consisted of two experiments performed independently of each other, and the analysis of antibody concentrations in serum was performed using the same ELISA method.

[0675] Specifically, a single dose of the test antibody was administered to 9-week-old ICR mice via the tail vein at a dose of 10 mg / kg. After antibody administration, blood was collected at regular intervals to separate the serum, and the antibody concentration in the serum was quantified using the separated serum.

[0676] Blood was collected by orbital blood sampling at 5 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours after antibody administration. In another experiment, blood was collected by abdominal gag sampling at 5 minutes, 1 hour, 3 hours, 6 hours, 9 hours, and 24 hours after antibody administration.

[0677] In each embodiment, the collected blood was placed in a tube that was not treated with an anticoagulant and coagulated at room temperature, and then the serum was separated by centrifuging at 5,000 rpm at 4°C for 10 minutes.

[0678] To analyze serum antibody concentrations, anti-FLAG antibodies were diluted to a concentration of 1 μg / mL in 1× PBS, coated onto 96-well plates, and incubated at 4°C for 16 hours. Subsequently, the plates were washed and blocked with a Fish Gelatin Blocking Agent, after which mouse serum samples diluted with SuperBlock™ Blocking Buffer were added to each well and reacted. The bound antibodies were detected using HRP-conjugated anti-human IgG(H+L) antibodies, and the serum antibody concentration was calculated by measuring the absorbance at 450 nm after the TMB substrate reaction.

[0679] As a result, the antibody containing the albumin-binding VHH domain showed a relatively slower rate of decrease in antibody concentration in mouse blood compared to the control antibody not containing the domain, and a tendency for increased retention time in blood was observed in both blood collection conditions (see Figures 7 and 8).

[0680] Table 7 below summarizes the mouse pharmacokinetic parameters of a protein (DP-403) fused with the DPVH-42 antibody to the anti-CEACAM5 antibody and the anti-CD3 antibody, and a corresponding fusion protein (DP-301) that does not contain albumin-bound VHH.

[0681]

[0682] As shown in the table, DP-403 containing albumin-bound VHH (DPVH-18) has an AUC compared to DP-301, which does not contain VHH. last and AUC inf increased significantly, and C max It also showed an increasing trend. In addition, DP-403 showed a significantly reduced whole-body clearance (CL) compared to DP-301, and the half-life (t1 / 2) and mean residence time (MRT) inf ) increased significantly.

[0683] These results demonstrate that albumin-bound VHH can substantially improve in vivo retention time and pharmacokinetic properties even in a heterogeneous fusion protein format composed of anti-CEACAM5 antibody and anti-CD3 antibody.

Claims

1. An albumin-binding protein comprising a single variable domain that binds to human albumin protein at both pH 5.5 and pH 7.4 and includes a combination of complementary determining regions selected from the group consisting of combinations of the following complementary determining regions (CDR1, CDR2, and CDR3): Sequence No. 1 (CDR1), Sequence No. 2 (CDR2), and Sequence No. 3 (CDR3); Sequence No. 4 (CDR1), Sequence No. 5 (CDR2), and Sequence No. 6 (CDR3); Sequence No. 7 (CDR1), Sequence No. 8 (CDR2), and Sequence No. 9 (CDR3); Sequence No. 10 (CDR1), Sequence No. 11 (CDR2), and Sequence No. 12 (CDR3); Sequence No. 13 (CDR1), Sequence No. 14 (CDR2), and Sequence No. 15 (CDR3); Sequence No. 16 (CDR1), Sequence No. 17 (CDR2), and Sequence No. 18 (CDR3); Sequence No. 19 (CDR1), Sequence No. 20 (CDR2), and Sequence No. 21 (CDR3); Sequence No. 22 (CDR1), Sequence No. 23 (CDR2), and Sequence No. 24 (CDR3); Sequence No. 25 (CDR1), Sequence No. 26 (CDR2), and Sequence No. 27 (CDR3); Sequence No. 28 (CDR1), Sequence No. 29 (CDR2), and Sequence No. 30 (CDR3); Sequence No. 31 (CDR1), Sequence No. 32 (CDR2), and Sequence No. 33 (CDR3); Sequence No. 34 (CDR1), Sequence No. 35 (CDR2), and Sequence No. 36 (CDR3); Sequence No. 37 (CDR1), Sequence No. 38 (CDR2), and Sequence No. 39 (CDR3); Sequence No. 40 (CDR1), Sequence No. 41 (CDR2), and Sequence No. 42 (CDR3); Sequence No. 43 (CDR1), Sequence No. 44 (CDR2), and Sequence No. 45 (CDR3); Sequence No. 46 (CDR1), Sequence No. 47 (CDR2), and Sequence No. 48 (CDR3); Sequence No. 49 (CDR1), Sequence No. 50 (CDR2), and Sequence No. 51 (CDR3); Sequence No. 52 (CDR1), Sequence No. 53 (CDR2), and Sequence No. 54 (CDR3); Sequence No. 55 (CDR1), Sequence No. 56 (CDR2), and Sequence No. 57 (CDR3); Sequence No. 58 (CDR1), Sequence No. 59 (CDR2), and Sequence No. 60 (CDR3); Sequence No. 61 (CDR1), Sequence No. 62 (CDR2), and Sequence No. 63 (CDR3); Sequence No. 64 (CDR1), Sequence No. 65 (CDR2), and Sequence No. 66 (CDR3); Sequence No. 67 (CDR1), Sequence No. 68 (CDR2), and Sequence No. 69 (CDR3); Sequence No. 70 (CDR1), Sequence No. 71 (CDR2), and Sequence No. 72 (CDR3); Sequence No. 73 (CDR1), Sequence No. 74 (CDR2), and Sequence No. 75 (CDR3); and Sequence No. 76 (CDR1), Sequence No. 77 (CDR2), and Sequence No. 78 (CDR3).

2. An albumin-binding protein according to claim 1, wherein a single variable domain comprises one of the following combinations of complementarity determining regions (CDR1, CDR2, and CDR3): Sequence No. 1 (CDR1), Sequence No. 2 (CDR2), and Sequence No. 3 (CDR3); Sequence No. 7 (CDR1), Sequence No. 8 (CDR2), and Sequence No. 9 (CDR3); Sequence No. 25 (CDR1), Sequence No. 26 (CDR2), and Sequence No. 27 (CDR3); Sequence No. 34 (CDR1), Sequence No. 35 (CDR2), and Sequence No. 36 (CDR3); Sequence No. 43 (CDR1), Sequence No. 44 (CDR2), and Sequence No. 45 (CDR3); Sequence No. 46 (CDR1), Sequence No. 47 (CDR2), and Sequence No. 48 (CDR3); Sequence No. 49 (CDR1), Sequence No. 50 (CDR2), and Sequence No. 51 (CDR3); Sequence No. 52 (CDR1), Sequence No. 53 (CDR2), and Sequence No. 54 (CDR3); and Sequence No. 76 (CDR1), Sequence No. 77 (CDR2), and Sequence No. 78 (CDR3).

3. An albumin-binding protein according to claim 1, wherein a single variable domain binds to albumin using domain II of human albumin protein as the major binding site.

4. An albumin-binding protein according to paragraph 3, wherein a single variable domain comprises one of the following combinations of complementarity determining regions (CDR1, CDR2, and CDR3): Sequence No. 34 (CDR1), Sequence No. 35 (CDR2), and Sequence No. 36 (CDR3); Sequence No. 49 (CDR1), Sequence No. 50 (CDR2), and Sequence No. 51 (CDR3); Sequence No. 52 (CDR1), Sequence No. 53 (CDR2), and Sequence No. 54 (CDR3); and Sequence No. 76 (CDR1), Sequence No. 77 (CDR2), and Sequence No. 78 (CDR3).

5. The albumin-binding protein of claim 1, which competitively binds to domain II of human albumin protein and a single variable domain antibody comprising one of the following combinations of complementarity determining regions (CDR1, CDR2, and CDR3): Sequence No. 34 (CDR1), Sequence No. 35 (CDR2), and Sequence No. 36 (CDR3); Sequence No. 49 (CDR1), Sequence No. 50 (CDR2), and Sequence No. 51 (CDR3); Sequence No. 52 (CDR1), Sequence No. 53 (CDR2), and Sequence No. 54 (CDR3); and Sequence No. 76 (CDR1), Sequence No. 77 (CDR2), and Sequence No. 78 (CDR3).

6. An albumin-binding protein according to claim 1, wherein a single variable domain is VHH.

7. An albumin-binding protein according to claim 1 that does not competitively inhibit albumin from binding to FcRn.

8. An albumin-binding protein according to claim 1, wherein the FR1 region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs 79, 80, and 151-174.

9. An albumin-binding protein according to claim 1, wherein the FR2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 81-84 and 175-183.

10. An albumin-binding protein according to claim 1, wherein the FR3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 85-100 and 184-202.

11. An albumin-binding protein according to claim 1, wherein the FR4 region comprises the amino acid sequences of SEQ ID NOs 101 and 203-206.

12. An albumin-binding protein according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 103 to 135 or an amino acid sequence that is 85% or more identical thereto.

13. An albumin-binding protein according to claim 1, wherein a single variable domain is fused with a first heterogeneous protein.

14. In paragraph 13, the first heterogeneous protein is an albumin-binding protein that specifically binds to a tumor-associated antigen, a T cell-associated antigen or a B cell-associated antigen, or a fragment thereof.

15. In paragraph 14, the first heterogeneous protein is an albumin-binding protein that is an antibody or its antigen-binding fragment that specifically binds to a tumor-associated antigen, a T-cell-associated antigen or a B-cell-associated antigen, or a fragment thereof.

16. In paragraph 15, the first heterogeneous protein is an albumin-binding protein of the Fab, VHH, or scFv form.

17. In paragraph 13, the first heterogeneous protein is an enzyme, cytokine, chemokine, growth factor, hormone, receptor binding protein, ligand protein, proteinase inhibitor, or albumin binding protein that is an active regulatory protein.

18. An albumin-binding protein according to claim 13, wherein the first heterogeneous protein is directly connected to a single variable domain or connected via a linker.

19. An albumin-binding protein according to claim 18, wherein the first heterogeneous protein is connected to a single variable domain through a linker, and said linker is cleavable.

20. An albumin-binding protein according to claim 13, wherein the half-life of the fused protein containing the first heterogeneous protein is improved compared to the case where it is not fused with an albumin-binding protein.

21. An albumin-binding protein in which the second heterogeneous protein is additionally fused in paragraph 13.

22. In paragraph 21, the second heterogeneous protein is an albumin-binding protein that specifically binds to a tumor-associated antigen, a T cell-associated antigen or a B cell-associated antigen, or a fragment thereof.

23. An albumin-binding protein according to claim 21, wherein the second heterogeneous protein is connected to a single variable domain or the first heterogeneous protein directly or through a linker.

24. An albumin-binding protein according to claim 21, wherein the half-life of the fused protein containing the second heterogeneous protein is improved compared to the case where it is not fused with an albumin-binding protein.

25. An isolated nucleic acid molecule encoding an albumin-binding protein as described in any one of claims 1 to 24.

26. A vector comprising the nucleic acid molecule described in paragraph 25.

27. A host cell containing the vector described in paragraph 26.

28. A pharmaceutical composition for treating or preventing cancer comprising an albumin-binding protein and a pharmaceutically acceptable carrier as described in any one of claims 1 to 24.

29. A method for treating or preventing cancer comprising administering the pharmaceutical composition described in paragraph 28 to a patient who requires treatment or prevention of cancer.

30. A pharmaceutical composition for controlling or improving the pharmacokinetic properties of a therapeutic protein or antibody, comprising an albumin-binding protein as described in any one of claims 1 to 24.

31. A method for controlling or improving the pharmacokinetic properties of a therapeutic protein or antibody using an albumin-binding protein described in any one of claims 1 to 24.

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