Fusion protein capable of blocking action of BAFF and april on receptor thereof

Abstract

A fusion protein capable of inhibiting BAFF and APRIL signaling, a pharmaceutical composition containing the fusion protein, and a related use. In addition, also provided are a single-domain antibody specifically binding to serum albumin or an antigen-binding fragment thereof, a fusion protein containing the single-domain antibody or the antigen-binding fragment thereof, and a use for prolonging the drug half-life.

Description

A fusion protein that blocks the action of BAFF, APRIL on their receptors

[0001] This application claims the benefit of Chinese application No. 202411109747.0, filed on August 13, 2024, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, and more particularly, to a fusion protein capable of inhibiting BAFF and APRIL signaling, a pharmaceutical composition containing the fusion protein, and related uses. In addition, the present application also relates to a single-domain antibody specifically binding to serum albumin, a fusion protein containing the single-domain antibody, and its use for extending the half-life of a drug. BACKGROUND

[0003] B cells are associated with autoimmune diseases, such as systemic lupus erythematous (SLE), etc. This is because they can present antigens to autoreactive T cells, secrete inflammatory cytokines, and differentiate into antibody-secreting cells (ASCs), i.e., plasma blasts and plasma cells, which are responsible for producing pathogenic autoantibodies. Therefore, autoimmune diseases can be treated by depleting or inhibiting B cells.

[0004] Key factors for B cell development, differentiation, and survival include B cell activating factor (BAFF, also known as Blys) and a proliferating inducing ligand (APRIL). BAFF binds to B cell-expressed receptors with different affinities, including BAFF receptor (BAFF-R), transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), and B-cell maturation antigen (BCMA). APRIL binds to TACI, BCMA, and heparin sulfate proteoglycan (HSPG). These cytokines have been shown to be elevated in various autoantibody-related diseases, including SLE.

[0005] Due to the role of BAFF / APRIL system in autoimmune diseases, it has become a therapeutic target for many new biological agents. Blocking of BAFF or APRIL has also shown clinical prospects. Belimumab is an anti-BAFF antibody drug on the market, approved for the treatment of SLE and SLE-related lupus nephritis (LN), but only a small number of patients achieve clinical remission, such as lupus low disease activity state (LLDAS) or complete renal response remission rate of 12-14% or 30%, respectively. In addition, anti-APRIL antibody BION-1301 and Sibeprenlimab under development have also shown pharmacodynamic activity in clinical trials.

[0006] Atacicept and Telitacicept are soluble wild-type (WT) TACI extracellular domain Fc fusion proteins. Both Atacicept and Telitacicept have shown clinical efficacy in SLE, however, Atacicept failed to meet its primary endpoint in a key trial in SLE, and is currently in phase III clinical indications for lupus nephritis (LN) and IgA nephropathy (Berger's disease); Telitacicept was approved in China in 2021 for the treatment of SLE.

[0007] Due to the limitations of the above antibody drugs or Fc fusion protein drugs in clinical application, there is still a need to develop more effective drugs to meet the huge clinical demand. SUMMARY

[0008] The present inventors have obtained a trimeric fusion protein molecule that can efficiently block the binding of ligands to TNF receptors (e.g., efficiently block the binding of Blys and / or APRIL to their receptors) through extensive research, thereby inhibiting the signaling mediated by the binding of the ligands to their TNF receptors and treating related diseases. (e.g., inhibiting the signaling mediated by the binding of Blys and / or APRIL to their receptors, thereby reducing the amount of autoantibody production, which is expected to be used for the treatment of B cell-related diseases). Further, the trimeric fusion protein of the present application also has improved drugability, such as better stability and / or reduced amino acid modification (e.g., glycosylation modification, hydroxylation modification), low production cost, and can be better applied to large-scale industrial production.

[0009] Trimeric fusion protein molecule

[0010] Accordingly, in a first aspect, the present application provides a polypeptide comprising a first peptide segment and a second peptide segment; wherein the first peptide segment comprises or consists of an extracellular domain of a member of the tumor necrosis factor (TNF) receptor family or a fragment thereof; and the second peptide segment comprises or consists of a trimerization domain derived from cartilage matrix protein or a trimerization domain derived from procollagen.

[0011] In certain embodiments, the first peptide segment comprises a ligand binding domain of the TNF receptor family member.

[0012] As used herein, the term "ligand binding domain of a TNF receptor family member" has the meaning as understood by one of skill in the art, and generally refers to a portion of or the entire extracellular region of a TNF receptor that is responsible for binding to a ligand, typically a member of the TNF ligand family, such as TNF-a, TNF-b, LT-a, CD40L, etc. These domains typically contain repeated "tumor necrosis factor receptor (TNFR) domains" of about 40 amino acids in length.

[0013] For example, when the TNF receptor family member is BCMA, the ligand binding domain comprised therein generally refers to a portion of the extracellular region of the BCMA extracellular region that is responsible for binding to APRIL, BLys ligand.

[0014] For example, when the TNF receptor family member is TACI, the ligand binding domain comprised therein generally refers to a portion of the extracellular region of the TACI extracellular region that is responsible for binding to APRIL, BLys ligand.

[0015] In certain embodiments, the TNF receptor family member is selected from one or more of the following group: TNFR1, TNFR2, BAFFR, BCMA, TACI, OX40, GITR, CD27, 4-1BB, CD40, DcR1, DcR2, DR3, HVEM, LTbR, RANK, Fn14, Fas, TRAILR1, TRAILR2.

[0016] In certain embodiments, the TNF receptor family member is a human TNF receptor family member.

[0017] In certain embodiments, the TNF receptor family member is BCMA or TACI.

[0018] As used herein, "BCMA", i.e., B-cell maturation antigen, has the meaning commonly understood by one of skill in the art. BCMA is a transmembrane protein mainly expressed on the surface of mature B lymphocytes and plasma cells, and can play an important role in the development, survival and proliferation of B cells, etc. The specific amino acid sequence of the wild-type BCMA protein can be obtained from public databases (e.g., GenBank database). One of skill in the art understands that BCMA can include BCMA of various species of origin, and there can be differences between the amino acid sequences of BCMA of different species of origin. In the present application, when BCMA is described, it not only includes BCMA having an extracellular region as shown in SEQ ID NO: 173, but also includes BCMA of various species of origin having an extracellular region different from that shown in SEQ ID NO: 173.

[0019] As used herein, "TACI", i.e., transmembrane activator and calcium-modulator and cyclophilin ligand interactor, has the meaning commonly understood by one of skill in the art, and is of great significance in the activation, proliferation and immune regulation of B cells, etc. The specific amino acid sequence of the wild-type TACI protein can be obtained from public databases (e.g., GenBank database). One of skill in the art understands that TACI can include TACI of various species of origin, and there can be differences between the amino acid sequences of TACI of different species of origin. In the present application, when TACI is described, it not only includes TACI having an extracellular region as shown in SEQ ID NO: 170, but also includes TACI of various species of origin having an extracellular region different from that shown in SEQ ID NO: 170.

[0020] In certain embodiments, the first peptide segment comprises or consists of an extracellular domain of BCMA, or a fragment thereof.

[0021] In certain embodiments, the first peptide segment comprises or consists of a ligand binding domain of the BCMA.

[0022] In certain embodiments, the BCMA extracellular domain is selected from a wild-type BCMA extracellular domain and a variant thereof; wherein the BCMA extracellular domain variant has the ligand binding activity of the wild-type BCMA extracellular domain from which it is derived.

[0023] In certain embodiments, the BCMA ectodomain variant has reduced glycosylation sites (e.g., N-glycosylation sites) or does not contain glycosylation sites (e.g., N-glycosylation sites) as compared to the wild-type BCMA ectodomain. In certain embodiments, the BCMA ectodomain variant does not contain any signature sequence N-X-(S or T) as compared to the wild-type BCMA ectodomain, wherein N represents an asparagine residue, X represents any one of amino acid residues except proline residue, S represents a serine residue, and T represents a threonine residue. In certain embodiments, the BCMA ectodomain variant has the amino acid residue at the position corresponding to position 42 of SEQ ID NO: 173 replaced with an amino acid residue other than an asparagine residue as compared to the wild-type BCMA ectodomain. In certain embodiments, the BCMA ectodomain variant has the amino acid residue at the position corresponding to position 42 of SEQ ID NO: 173 replaced with a glutamine residue as compared to the wild-type BCMA ectodomain.

[0024] As will be readily understood by those skilled in the art, the signature sequence N-X-(S or T) in the wild-type BCMA ectodomain can be mutated by one or more mutations selected from the group consisting of:

[0025] (1) the N residue is deleted or replaced with one or more other amino acid residues (e.g., one non-N amino acid residue);

[0026] (2) the (S or T) residue is deleted or replaced with one or more other amino acid residues (e.g., one non-S and non-T amino acid residue);

[0027] (3) the X residue is deleted or replaced with a proline residue;

[0028] (4) one or more amino acid residues (e.g., one non-N amino acid residue) is added between the N residue and the X residue; and

[0029] (5) one or more amino acid residues (e.g., one non-S and non-T amino acid residue) is added between the X residue and the (S or T) residue;

[0030] Thus, the resulting BCMA ectodomain variant does not contain any signature sequence N-X-(S or T).

[0031] In certain embodiments, in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 173 (e.g., a methionine residue) is replaced with an amino acid residue other than a methionine residue (e.g., a leucine residue, a valine residue, a glycine residue, a proline residue, or a serine residue) as compared to the wild-type BCMA ectodomain. In certain embodiments, in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 173 is replaced with a glycine residue, a proline residue, or a serine residue as compared to the wild-type BCMA ectodomain.

[0032] In certain embodiments, in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 173 (e.g., a lysine residue) is replaced with an amino acid residue other than a lysine residue (e.g., an arginine residue, a serine residue, a glutamine residue, a glutamic acid residue, a glycine residue, or an alanine residue) as compared to the wild-type BCMA ectodomain.

[0033] In certain embodiments, in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 173 (e.g., a methionine residue) is replaced with an amino acid residue other than a methionine residue (e.g., a leucine residue, a valine residue, a glycine residue, a proline residue, or a serine residue) as compared to the wild-type BCMA ectodomain. In certain embodiments, in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 173 is replaced with a glycine residue, a proline residue, or a serine residue as compared to the wild-type BCMA ectodomain.

[0034] In certain embodiments, the BCMA ectodomain variant comprises a mutation selected from the group consisting of: (i) the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 173 (e.g., a methionine residue) is replaced with a leucine residue, a valine residue, a glycine residue, a proline residue, or a serine residue; (ii) the amino acid residue at the position corresponding to position 42 of SEQ ID NO: 173 (e.g., an asparagine residue) is replaced with a glutamine residue, and (iii) a combination of (i) and (ii). In certain embodiments, the BCMA ectodomain variant comprises a mutation selected from the group consisting of: (i) the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 173 (e.g., a methionine residue) is replaced with a glycine residue, a proline residue, or a serine residue; (ii) the amino acid residue at the position corresponding to position 42 of SEQ ID NO: 173 (e.g., an asparagine residue) is replaced with a glutamine residue, and (iii) a combination of (i) and (ii), as compared to the wild-type BCMA ectodomain.

[0035] In certain embodiments, the BCMA ectodomain variant comprises a mutation selected from the group consisting of: (i) the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 173 (e.g., a lysine residue) is replaced with an arginine residue, a serine residue, a glutamine residue, a glutamic acid residue, a glycine residue, or an alanine residue; (ii) the amino acid residue at the position corresponding to position 42 of SEQ ID NO: 173 (e.g., an asparagine residue) is replaced with a glutamine residue, and (iii) a combination of (i) and (ii), as compared to the wild-type BCMA ectodomain.

[0036] In certain embodiments, the BCMA ectodomain variant comprises a mutation selected from the group consisting of: (i) substitution of the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 173 (e.g., a methionine residue) with a leucine residue, a valine residue, a glycine residue, a proline residue, or a serine residue; (ii) substitution of the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 173 (e.g., a lysine residue) with an arginine residue, a serine residue, a glutamine residue, a glutamic acid residue, a glycine residue, or an alanine residue; (iii) substitution of the amino acid residue at the position corresponding to position 42 of SEQ ID NO: 173 (e.g., an asparagine residue) with a glutamine residue, and (iv) combinations of (i), (ii), and (iii). In certain embodiments, the BCMA ectodomain variant comprises a mutation selected from the group consisting of: (i) substitution of the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 173 (e.g., a methionine residue) with a glycine residue, a proline residue, or a serine residue; (ii) substitution of the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 173 (e.g., a lysine residue) with an arginine residue, a serine residue, a glutamine residue, a glutamic acid residue, a glycine residue, or an alanine residue; (iii) substitution of the amino acid residue at the position corresponding to position 42 of SEQ ID NO: 173 (e.g., an asparagine residue) with a glutamine residue, and (iv) combinations of (i), (ii), and (iii).

[0037] In certain embodiments, the wild-type BCMA ectodomain has: (a) an amino acid sequence as set forth in SEQ ID NO: 173; (b) an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 173; or, (c) a sequence that has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence set forth in SEQ ID NO: 173.

[0038] In certain embodiments, the BCMA ectodomain variant has a sequence as set forth in any one of SEQ ID NOs: 175, 181, 183, 185, 187, 189, 235-240, 255-260, 275-280.

[0039] In certain embodiments, the BCMA is human BCMA.

[0040] In certain embodiments, the first peptide stretch comprises the amino acid residues at positions corresponding to positions 8-41 of SEQ ID NO: 173 in the wild-type BCMA ectodomain or in the BCMA ectodomain variant.

[0041] As used herein, the expression "the amino acid residues at positions corresponding to positions 8-41 of SEQ ID NO: 173 in the wild-type BCMA ectodomain or in the BCMA ectodomain variant" means the amino acid positions / residues in the sequence of the wild-type BCMA ectodomain or of the BCMA ectodomain variant under comparison which are in equivalent positions to the amino acid residues at positions 8-41 of SEQ ID NO: 173, when the sequence of the wild-type BCMA ectodomain or of the BCMA ectodomain variant is optimally aligned with SEQ ID NO: 173, i.e. when the sequence of the wild-type BCMA ectodomain or of the BCMA ectodomain variant is aligned with SEQ ID NO: 173 to obtain the highest percentage identity.

[0042] Unless specifically indicated otherwise or apparently contradicted by context, the meaning of the remaining similar expressions herein is defined in a manner analogous to the above.

[0043] In certain embodiments, the first peptide segment comprises or consists of the amino acid residues at positions 8-41 (or 8-42, or 8-43, or 8-44, or 8-45, or 8-46, or 8-47, or 8-48, or 8-49, or 8-51, or 8-54, or 7-41, or 7-42, or 7-43, or 7-44, or 7-45, or 7-46, or 7-47, or 7-48, or 7-49, or 7-51, or 7-54, or 4-41, or 4-42, or 4-43, or 4-44, or 4-45, or 4-46, or 4-47, or 4-48, or 4-49, or 4-51, or 4-54, or 3-41, or 3-42, or 3-43, or 3-44, or 3-45, or 3-46, or 3-47, or 3-48, or 3-49, or 3-51, or 3-54, or 2-41, or 2-42, or 2-43, or 2-44, or 2-45, or 2-46, or 2-47, or 2-48, or 2-49, or 2-51, or 2-54, or 1-41, or 1-42, or 1-43, or 1-44, or 1-45, or 1-46, or 1-47, or 1-48, or 1-49, or 1-51, or 1-54) of SEQ ID NO: 173 in the wild-type BCMA ectodomain or the BCMA ectodomain variant.

[0044] In certain embodiments, the first peptide segment comprises or consists of the amino acid residues at positions 7-41 (or 7-42, or 7-43, or 7-44, or 7-45, or 7-46, or 7-48, or 7-49, or 7-51, or 7-54, or 4-41, or 4-42, or 4-43, or 4-44, or 4-45, or 4-46, or 4-48, or 4-49, or 4-51, or 4-54, or 3-41, or 3-42, or 3-43, or 3-44, or 3-45, or 3-46, or 3-48, or 3-49, or 3-51, or 3-54, or 2-41, or 2-42, or 2-43, or 2-44, or 2-45, or 2-46, or 2-48, or 2-49, or 2-51, or 2-54, or 1-41, or 1-42, or 1-43, or 1-44, or 1-45, or 1-46, or 1-48, or 1-49, or 1-51, or 1-54) of SEQ ID NO: 173 in the wild-type BCMA ectodomain or the BCMA ectodomain variant.

[0045] In certain embodiments, the first peptide segment has a sequence as set forth in any one of SEQ ID NOs: 173-204, 235-280.

[0046] In certain embodiments, the first peptide segment comprises or consists of an ectodomain of TACI, or a fragment thereof.

[0047] In certain embodiments, the first peptide segment comprises or consists of a ligand binding domain of the TACI.

[0048] In certain embodiments, the ectodomain of TACI has: (a) an amino acid sequence as set forth in SEQ ID NO: 170; (b) an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 170; or (c) a sequence that has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence set forth in SEQ ID NO: 170.

[0049] In certain embodiments, the TACI is human TACI.

[0050] In certain embodiments, the TACI extracellular domain is selected from the group consisting of wild-type TACI extracellular domain and variants thereof, wherein the TACI extracellular domain variant has the ligand binding activity of the wild-type TACI extracellular domain from which it is derived.

[0051] In certain embodiments, the first peptide segment comprises (or consists of) amino acid residues in the TACI (e.g., the wild-type TACI extracellular domain or the TACI extracellular domain variant) at positions corresponding to positions 71-104 of SEQ ID NO: 170.

[0052] In certain embodiments, the first peptide segment comprises (or consists of) amino acid residues in the TACI (e.g., the wild-type TACI extracellular domain or the TACI extracellular domain variant) at positions corresponding to positions 71-104 (or 71-110, or 71-118, or 71-130, or 71-165, or 68-104, or 68-110, or 68-118, or 68-130, or 68-165, or 50-104, or 50-110, or 50-118, or 50-130, or 50-165, or 30-104, or 30-110, or 30-118, or 30-130, or 30-165, or 13-104, or 13-110, or 13-118, or 13-130, or 13-165, or 1-104, or 1-110, or 1-118, or 1-130, or 1-165) of SEQ ID NO: 170.

[0053] In certain embodiments, the first peptide segment comprises (or consists of) amino acid residues in the TACI (e.g., the wild-type TACI extracellular domain or the TACI extracellular domain variant) at positions corresponding to positions 68-110 (or 68-118, or 13-110, or 13-118) of SEQ ID NO: 170.

[0054] In certain embodiments, the first peptide segment has a sequence as set forth in any one of SEQ ID NOs: 170-172.

[0055] In certain embodiments, the second peptide segment comprises (or consists of) a trimerization domain derived from cartilage matrix protein.

[0056] In certain embodiments, the cartilage matrix protein is selected from the group consisting of wild-type cartilage matrix protein and variants thereof; wherein the cartilage matrix protein variant has a trimerization activity (e.g., an activity to associate with two other identical or similar polypeptides containing a cartilage matrix protein trimerization domain to form a trimer (e.g., a homotrimer or a heterotrimer)).

[0057] In certain embodiments, the cartilage matrix protein has: (a) an amino acid sequence as set forth in SEQ ID NO: 229; (b) an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 229; or (c) a sequence that has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence set forth in SEQ ID NO: 229.

[0058] In certain embodiments, the cartilage matrix protein is a human cartilage matrix protein.

[0059] In certain embodiments, the second peptide segment comprises or consists of amino acid residues in the cartilage matrix protein (e.g., the wild-type cartilage matrix protein or a variant thereof) at positions corresponding to positions 458-466 (or 458-480, or 458-496, or 454-466, or 454-480, or 454-496, or 445-466, or 445-480, or 445-496) of SEQ ID NO: 229.

[0060] In certain embodiments, the second peptide segment comprises or consists of amino acid residues in the cartilage matrix protein (e.g., the wild-type cartilage matrix protein or a variant thereof) at positions corresponding to positions 454-496 of SEQ ID NO: 229.

[0061] In certain embodiments, the second peptide segment has a sequence as set forth in SEQ ID NO: 230.

[0062] In certain embodiments, the second peptide segment comprises a trimerization domain derived from a procollagen protein.

[0063] In certain embodiments, the second peptide segment comprises a trimerization domain derived from a C-propeptide of a procollagen protein.

[0064] In certain embodiments, the second peptide segment comprises a trimerization domain derived from the C-propeptide of pro.α.1 (II), pro.α.1 (I), pro.α.2 (I), pro.α.1 (III), pro.α.1 (V), pro.α.2 (V), pro.α.1 (XI), pro.α.2 (XI), or pro.α.3 (XI).

[0065] As is generally understood by those skilled in the art, "pro.α.1 (II)" means the α-1 chain of type II procollagen, "pro.α.1 (I)" means the α-1 chain of type I procollagen, "pro.α.2 (I)" means the α-2 chain of type I procollagen, "pro.α.1 (III)" means the α-1 chain of type III procollagen, "pro.α.1 (V)" means the α-1 chain of type V procollagen, "pro.α.2 (V)" means the α-2 chain of type V procollagen, "pro.α.1 (XI)" means the α-1 chain of type XI procollagen, "pro.α.2 (XI)" means the α-2 chain of type XI procollagen, and "pro.α.3 (XI)" means the α-3 chain of type XI procollagen.

[0066] In certain embodiments, the second peptide segment comprises a trimerization domain derived from the C-propeptide of pro.α.1 (I).

[0067] In certain embodiments, the second peptide segment comprises a trimerization domain derived from the C-propeptide of pro.α.1 (I), or consists of the same.

[0068] In certain embodiments, the second peptide segment comprises a trimerization domain derived from the C-propeptide of pro.α.1 (I), and optionally other amino acid residues (e.g., other than the trimerization domain in the C-propeptide).

[0069] In certain embodiments, the second peptide segment comprises a trimerization domain derived from the C-propeptide of pro.α.1 (I), or consists of the same.

[0070] In certain embodiments, the pro.a.1 (I) has: (a) an amino acid sequence as set forth in SEQ ID NO: 231; (b) an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 231; or, (c) a sequence that has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence set forth in SEQ ID NO: 231.

[0071] In certain embodiments, the second peptide segment comprises or consists of amino acid residues in the pro.a.1 (I) corresponding to positions 1220-1282 (or 1220-1300, or 1220-1350, or 1220-1400, or 1220-1464, or 1218-1282, or 1218-1300, or 1218-1350, or 1218-1400, or 1218-1464) of SEQ ID NO: 231.

[0072] In certain embodiments, the second peptide segment comprises or consists of amino acid residues in the pro.a.1 (I) corresponding to positions 1220-1464 of SEQ ID NO: 231.

[0073] In certain embodiments, the procollagen protein is a human procollagen protein.

[0074] In certain embodiments, the second peptide segment is inert to the action of a protease.

[0075] In certain embodiments, the second peptide segment is inert to the action of a BMP-1 (Bone Morphogenetic Protein 1) enzyme (e.g., the second peptide segment cannot be cleaved / enzymatically digested by a BMP-1 enzyme).

[0076] In certain embodiments, the second peptide segment further comprises additional amino acid residues R and S. In certain embodiments, the second peptide segment comprises amino acid residues R and S at the N-terminus of the trimerization domain of the C-propeptide derived from pro.a.1 (I). In certain embodiments, the second peptide segment comprises an amino acid residue R and an amino acid residue S, in that order, from the N-terminus to the C-terminus at the N-terminus of the trimerization domain of the C-propeptide derived from pro.a.1 (I).

[0077] In some embodiments, the second peptide segment has a sequence as set forth in SEQ ID NO: 232.

[0078] In some embodiments, the second peptide segment is directly linked or optionally linked through a first linker (e.g., a first peptide linker) to the N- or C-terminus (e.g., C-terminus) of the first peptide segment.

[0079] In some embodiments, the first linker is a peptide linker consisting of 3-35 (e.g., 5-30, 10-30, 15-30) amino acid residues.

[0080] In some embodiments, the first linker comprises a sequence as set forth in any one of SEQ ID NOs: 205-219.

[0081] In some embodiments, the polypeptide further comprises a single-domain antibody or an antigen-binding fragment thereof that specifically binds to serum albumin.

[0082] In some embodiments, the single-domain antibody or the antigen-binding fragment thereof comprises:

[0083] a CDR1 or a variant thereof, a CDR2 or a variant thereof, a CDR3 or a variant thereof, as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21, 220-225;

[0084] The variant has one or several (e.g., 1, 2, or 3) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived.

[0085] In some embodiments, the CDRs are defined by the Kabat, IMGT, Chothia, or AbM numbering system.

[0086] In some embodiments, the single-domain antibody or the antigen-binding fragment thereof comprises:

[0087] (1) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 7; and, a CDR3 as set forth in SEQ ID NO: 8;

[0088] (2) a CDR1 as set forth in SEQ ID NO: 10; a CDR2 as set forth in SEQ ID NO: 11; and, a CDR3 as set forth in SEQ ID NO: 12;

[0089] (3) a CDR1 as set forth in SEQ ID NO: 14; a CDR2 as set forth in SEQ ID NO: 15; and, a CDR3 as set forth in SEQ ID NO: 16;

[0090] (4) a CDR1 as depicted in SEQ ID NO: 14; a CDR2 as depicted in SEQ ID NO: 233; and, a CDR3 as depicted in SEQ ID NO: 16;

[0091] (5) a CDR1 as depicted in SEQ ID NO: 18; a CDR2 as depicted in SEQ ID NO: 19; and, a CDR3 as depicted in SEQ ID NO: 20;

[0092] (6) a CDR1 as depicted in SEQ ID NO: 18; a CDR2 as depicted in SEQ ID NO: 234; and, a CDR3 as depicted in SEQ ID NO: 20;

[0093] (7) a CDR1 as depicted in SEQ ID NO: 22; a CDR2 as depicted in SEQ ID NO: 23; and, a CDR3 as depicted in SEQ ID NO: 24; or,

[0094] (8) a CDR1 as depicted in SEQ ID NO: 226; a CDR2 as depicted in SEQ ID NO: 227; and, a CDR3 as depicted in SEQ ID NO: 228.

[0095] In certain embodiments, the CDRs are defined by the Kabat numbering system.

[0096] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region derived from an immunoglobulin.

[0097] As will be readily understood by those skilled in the art, the heavy chain framework region derived from an immunoglobulin includes both a heavy chain framework region directly derived from an immunoglobulin, as well as a heavy chain framework region that has been engineered or modified from a heavy chain framework region derived from an immunoglobulin. Without being limited by theory, the immunoglobulin can be derived from any species, for example, a mammal (e.g., a human or a camelid).

[0098] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises:

[0099] (i) a sequence as depicted in any one of SEQ ID NOs: 5, 9, 13, 17, 21;

[0100] (ii) a sequence having one or several (e.g., 1, 2, 3, 4 or 5) amino acid substitutions, deletions or additions compared to the sequence as depicted in any one of SEQ ID NOs: 5, 9, 13, 17, 21; or

[0101] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any one of SEQ ID NOs: 5, 9, 13, 17, 21.

[0102] In certain embodiments, the substitution is a conservative substitution.

[0103] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21.

[0104] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof is humanized.

[0105] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline antibody gene).

[0106] As will be readily understood by those skilled in the art, the heavy chain framework region derived from a human immunoglobulin includes both a heavy chain framework region directly derived from a human immunoglobulin and a heavy chain framework region obtained by modifying or otherwise manipulating a heavy chain framework region derived from a human immunoglobulin.

[0107] In certain embodiments, the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from a human-derived residue to a camelid-derived residue.

[0108] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises:

[0109] (i) a sequence as set forth in any one of SEQ ID NOs: 220-225;

[0110] (ii) a sequence comprising one or several (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions compared to the sequence set forth in any one of SEQ ID NOs: 220-225; or

[0111] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 220-225.

[0112] In certain embodiments, the substitution is a conservative substitution.

[0113] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NOs: 220-225.

[0114] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof possesses cross-binding activity to human, murine (e.g., mouse, rat), monkey serum albumin.

[0115] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof is directly linked or optionally linked via a second linker (e.g., a second peptide linker) to the N-terminus or C-terminus of the polypeptide.

[0116] In certain embodiments, the second linker is a peptide linker consisting of 3-35 (e.g., 5-30, 10-25, 10-30) amino acid residues.

[0117] In certain embodiments, the second linker comprises a sequence as set forth in any one of SEQ ID NOs: 205-219.

[0118] In certain embodiments, the second peptide segment is directly linked or optionally linked via a first linker to the C-terminus of the first peptide segment, and the single-domain antibody or antigen-binding fragment thereof is directly linked or optionally linked via a second linker to the C-terminus of the second peptide segment.

[0119] In certain embodiments, the polypeptide has a sequence as set forth in any one of SEQ ID NOs: 34-60, 62-110, 112, 114-169.

[0120] In certain embodiments, the first peptide segment and the second peptide segment are derived from the same or different species.

[0121] In certain embodiments, the polypeptide further comprises a signal peptide and / or a detectable label (e.g., a tag protein).

[0122] In certain embodiments, the polypeptide comprises a signal peptide and / or a detectable label (e.g., a tag protein) at its N-terminus and / or C-terminus.

[0123] In certain embodiments, the polypeptide is capable of blocking the binding of human BLys and / or APRIL to its ligand.

[0124] In certain embodiments, the polypeptide is capable of blocking the binding of murine BLys and / or APRIL to its ligand.

[0125] In some embodiments, the polypeptide is capable of blocking the binding of monkey BLys and / or APRIL to its ligand.

[0126] In some embodiments, the polypeptide has cross-binding activity with human, monkey, murine BLys.

[0127] In some embodiments, the polypeptide has cross-binding activity with human, monkey, murine APRIL.

[0128] In some embodiments, the polypeptide has good drugability, for example, has good stability (e.g., the peptide bond between one or more amino acid residues at the terminal (N-terminal and / or C-terminal) has good stability) and / or reduced amino acid modification (e.g., glycosylation modification, hydroxylation modification).

[0129] In a second aspect, the present application provides a trimeric protein complex comprising at least one polypeptide of the first aspect.

[0130] In some embodiments, the trimeric protein complex comprises each independently selected from a first polypeptide, a second polypeptide, and a third polypeptide of the first aspect.

[0131] In some embodiments, the first polypeptide, the second polypeptide, and the third polypeptide have the same or different first peptide segment, the same or different second peptide segment, the same or different first linker, and / or the same or different second linker from each other.

[0132] In some embodiments, none of the first polypeptide, the second polypeptide, and the third polypeptide comprises a single-domain antibody or an antigen-binding fragment thereof that specifically binds to serum albumin; or at least one of the first polypeptide, the second polypeptide, and the third polypeptide comprises a single-domain antibody or an antigen-binding fragment thereof that specifically binds to serum albumin.

[0133] In some embodiments, the trimeric protein complex comprises at least two single-domain antibodies or antigen-binding fragments thereof that specifically bind to serum albumin, wherein the at least two single-domain antibodies or antigen-binding fragments thereof that specifically bind to serum albumin are the same or different from each other.

[0134] In some embodiments, the single-domain antibody or antigen-binding fragment thereof that specifically binds to serum albumin is as defined above.

[0135] In some embodiments, the trimeric protein complex is a homotrimeric protein complex.

[0136] In some embodiments, the trimeric protein complex comprises at least one polypeptide of the first aspect, and an additional polypeptide.

[0137] In certain embodiments, the trimeric protein complex comprises a first polypeptide selected from the first aspect, and an additional second polypeptide and a third polypeptide.

[0138] In certain embodiments, the trimeric protein complex comprises each independently a first polypeptide and a second polypeptide selected from the first aspect, and an additional third polypeptide.

[0139] In certain embodiments, the additional polypeptide comprises a trimerization domain, and optionally (i) an extracellular domain of a TNF receptor family member or a fragment thereof, and / or (ii) a single-domain antibody or an antigen-binding fragment thereof that specifically binds serum albumin.

[0140] In certain embodiments, the trimerization domain comprised by the additional polypeptide is the same as or different from the second peptide segment comprised by the polypeptide of the first aspect, the extracellular domain of a TNF receptor family member or a fragment thereof optionally comprised by the additional polypeptide is the same as or different from the first peptide segment comprised by the polypeptide of the first aspect, and / or, the single-domain antibody or an antigen-binding fragment thereof that specifically binds serum albumin optionally comprised by the additional polypeptide is the same as or different from the single-domain antibody or an antigen-binding fragment thereof that specifically binds serum albumin comprised by the polypeptide of the first aspect.

[0141] In certain embodiments, the trimerization domain comprised by the additional polypeptide is the same as the second peptide segment comprised by the polypeptide of the first aspect.

[0142] In certain embodiments, the polypeptides comprised by the trimeric protein complex form covalent or non-covalent linkages between them.

[0143] In certain embodiments, the polypeptides comprised by the trimeric protein complex form disulfide linkages between them.

[0144] In certain embodiments, the second peptide segments of the polypeptides comprised by the trimeric protein complex form disulfide linkages between them.

[0145] In certain embodiments, the trimeric protein complex is capable of blocking the binding of human BLys and / or APRIL to its ligand.

[0146] In certain embodiments, the trimeric protein complex is capable of blocking the binding of murine BLys and / or APRIL to its ligand.

[0147] In certain embodiments, the trimeric protein complex is capable of blocking the binding of monkey BLys and / or APRIL to its ligand.

[0148] In certain embodiments, the trimeric protein complex possesses cross-binding activity to human, monkey, and murine BLys.

[0149] In some embodiments, the trimeric protein complex has cross-binding activity with human, monkey, murine APRIL.

[0150] In some embodiments, the trimeric protein complex has good drugability, for example, has good stability (for example, the peptide bond between one or more amino acid residues at the terminal (N-terminal and / or C-terminal) has good stability) and / or reduced amino acid modification (for example, glycosylation modification, hydroxylation modification).

[0151] In a third aspect, the present application provides an isolated nucleic acid molecule encoding the polypeptide of the first aspect, or the trimeric protein complex of the second aspect.

[0152] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide of the first aspect.

[0153] In some embodiments, the isolated nucleic acid molecule encodes the trimeric protein complex of the second aspect, which comprises a first polypeptide, a second polypeptide, and a third polypeptide; the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first polypeptide, a second nucleotide sequence encoding the second polypeptide, and a third nucleotide sequence encoding the third polypeptide.

[0154] In some embodiments, the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are the same or different from each other.

[0155] In some embodiments, the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are present on the same or different isolated nucleic acid molecules.

[0156] In a fourth aspect, the present application provides a vector comprising the isolated nucleic acid molecule of the third aspect.

[0157] In some embodiments, the vector comprises a nucleotide sequence encoding the polypeptide of the first aspect.

[0158] In some embodiments, the vector encodes the trimeric protein complex of the second aspect, which comprises a first polypeptide, a second polypeptide, and a third polypeptide; the vector comprises a first nucleotide sequence encoding the first polypeptide, a second nucleotide sequence encoding the second polypeptide, and a third nucleotide sequence encoding the third polypeptide.

[0159] In some embodiments, the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are the same or different from each other.

[0160] In certain embodiments, the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence are present on the same or different vector molecules.

[0161] In a fifth aspect, the present application provides a host cell comprising the isolated nucleic acid molecule of the third aspect or the vector of the fourth aspect.

[0162] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

[0163] The polypeptide or trimeric protein complex of the present application can be prepared in various methods known in the art, for example, by genetic engineering recombination techniques. For example, a DNA molecule encoding the polypeptide or trimeric protein complex of the present application is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, which is then transfected into a host cell. Then, the transfected host cell is cultured under specific conditions, and the polypeptide or trimeric protein complex of the present application is expressed.

[0164] In a sixth aspect, the present application provides a method of preparing the polypeptide of the first aspect, or the trimeric protein complex of the second aspect, comprising culturing the host cell of the third aspect under conditions permitting expression of the protein, and recovering the polypeptide or the trimeric protein complex from the culture of the host cell.

[0165] In a seventh aspect, the present application provides a pharmaceutical composition comprising the polypeptide of the first aspect, the trimeric protein complex of the second aspect, the isolated nucleic acid molecule of the third aspect, the vector of the fourth aspect, or the host cell of the fifth aspect.

[0166] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0167] In certain embodiments, the pharmaceutical composition comprises the trimeric protein complex of the second aspect, the isolated nucleic acid molecule encoding the trimeric protein complex, the vector, or the host cell.

[0168] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an anti-inflammatory drug or an immunosuppressant.

[0169] In an eighth aspect, the present application also provides use of the polypeptide of the first aspect, the trimeric protein complex of the second aspect, the isolated nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, or the pharmaceutical composition of the seventh aspect for the manufacture of a medicament for preventing and / or treating a B-cell related disease in a subject. The present application also provides a method for preventing and / or treating a B-cell related disease in a subject, comprising administering to a subject in need thereof an effective amount of the polypeptide of the first aspect, the trimeric protein complex of the second aspect, the isolated nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, or the pharmaceutical composition of the seventh aspect.

[0170] In certain embodiments, the treatment of the B-cell related disease can benefit from the inhibition of BLys and / or APRIL mediated signal transduction.

[0171] In certain embodiments, the B-cell related disease comprises an autoimmune disease.

[0172] In certain embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, Sjogren's syndrome, IgA nephropathy, membranous nephropathy, lupus nephritis, rheumatoid arthritis, neuromyelitis optica, multiple sclerosis, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, ankylosing spondylitis, graft versus host disease, cold agglutinin disease, autoimmune hemolytic anemia, autoimmune cytopenia, pemphigus, pemphigoid, and any combination thereof.

[0173] In certain embodiments, the subject is a mammal.

[0174] In certain embodiments, the polypeptide, the trimeric protein complex, the isolated nucleic acid molecule, the vector, the host cell, or the pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., an anti-inflammatory drug or an immunosuppressive agent).

[0175] The polypeptide or trimeric protein complex or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The polypeptide or trimeric protein complex or pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the polypeptide or trimeric protein complex or pharmaceutical composition of the present application in the required amount in an appropriate solvent with one or more of the other ingredients, as described below, as desired, followed by filtered sterilization. Furthermore, sterile injection solutions can be prepared as sterile lyophilized powders for reconstitution with a suitable vehicle, e.g., water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), dextrose solution (e.g., 5% dextrose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered saline), Ringer's solution, and any combination thereof.

[0176] The polypeptide or trimeric protein complex or pharmaceutical composition of the present application can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracerebroventricular, inguinal, intravesical, local (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration / means of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route of administration and / or means of administration will vary depending on the intended purpose. In certain embodiments, the polypeptide or trimeric protein complex or pharmaceutical composition of the present application is administered by intravenous injection or bolus.

[0177] Single-domain antibody or antigen-binding fragment thereof

[0178] In a ninth aspect, the present application provides a single-domain antibody or antigen-binding fragment thereof that specifically binds to serum albumin, comprising:

[0179] a CDR1 or variant thereof, a CDR2 or variant thereof, a CDR3 or variant thereof comprised by a VHH as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21, 220-224;

[0180] said variant has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions or additions in comparison to the sequence from which it is derived.

[0181] In certain embodiments, the CDRs are defined by the Kabat, IMGT, Chothia, or AbM numbering system.

[0182] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises:

[0183] (1) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 7; and, a CDR3 as set forth in SEQ ID NO: 8;

[0184] (2) a CDR1 as set forth in SEQ ID NO: 10; a CDR2 as set forth in SEQ ID NO: 11; and, a CDR3 as set forth in SEQ ID NO: 12;

[0185] (3) a CDR1 as set forth in SEQ ID NO: 14; a CDR2 as set forth in SEQ ID NO: 15; and, a CDR3 as set forth in SEQ ID NO: 16;

[0186] (4) a CDR1 as set forth in SEQ ID NO: 14; a CDR2 as set forth in SEQ ID NO: 233; and, a CDR3 as set forth in SEQ ID NO: 16;

[0187] (5) a CDR1 as set forth in SEQ ID NO: 18; a CDR2 as set forth in SEQ ID NO: 19; and, a CDR3 as set forth in SEQ ID NO: 20;

[0188] (6) a CDR1 as set forth in SEQ ID NO: 18; a CDR2 as set forth in SEQ ID NO: 234; and, a CDR3 as set forth in SEQ ID NO: 20; or,

[0189] (7) a CDR1 as set forth in SEQ ID NO: 22; a CDR2 as set forth in SEQ ID NO: 23; and, a CDR3 as set forth in SEQ ID NO: 24.

[0190] In certain embodiments, the CDRs are defined by the Kabat numbering system.

[0191] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region derived from an immunoglobulin.

[0192] As will be readily understood by those skilled in the art, the heavy chain framework region derived from an immunoglobulin includes both a heavy chain framework region directly derived from an immunoglobulin, as well as a heavy chain framework region that has been engineered or modified from a heavy chain framework region derived from an immunoglobulin. Without being limited by theory, the immunoglobulin can be derived from any species, e.g., a mammal (e.g., a human or a camelid).

[0193] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises:

[0194] (i) a sequence as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21;

[0195] (ii) a sequence that has one or several (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions compared to the sequence as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21; or

[0196] (iii) a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21.

[0197] In certain embodiments, the substitution is a conservative substitution.

[0198] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21.

[0199] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof is humanized.

[0200] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline antibody gene).

[0201] As will be readily understood by those skilled in the art, the heavy chain framework region derived from a human immunoglobulin includes both a heavy chain framework region directly derived from a human immunoglobulin, as well as a heavy chain framework region that has been engineered or modified from a heavy chain framework region derived from a human immunoglobulin.

[0202] In certain embodiments, the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from a human-derived residue to a camelid-derived residue.

[0203] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises:

[0204] (i) a sequence as set forth in any one of SEQ ID NOs: 220-224;

[0205] (ii) a sequence that has one or several (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions compared to the sequence as set forth in any one of SEQ ID NOs: 220-224; or

[0206] (iii) a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 220-224.

[0207] In certain embodiments, the substitution is a conservative substitution.

[0208] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NOs: 220-224.

[0209] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof has cross-binding activity to human, murine (e.g., mouse, rat), monkey serum albumin.

[0210] In a tenth aspect, the present application provides a polypeptide construct that specifically binds to serum albumin, comprising the single-domain antibody or antigen-binding fragment thereof of the ninth aspect, and an immunoglobulin Fc domain.

[0211] In certain embodiments, the immunoglobulin Fc domain is directly linked or optionally linked via a peptide linker to the N-terminus and / or C-terminus (e.g., C-terminus) of the single-domain antibody or antigen-binding fragment thereof.

[0212] In certain embodiments, the immunoglobulin Fc domain is an Fc domain of IgG (e.g., an Fc domain of IgG1).

[0213] In certain embodiments, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 25, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared thereto.

[0214] In certain embodiments, the polypeptide construct comprises a first peptide chain comprising: a first single-domain antibody or antigen-binding fragment thereof, and a first Fc domain, wherein the first single-domain antibody or antigen-binding fragment thereof is as defined in the ninth aspect. In certain embodiments, the first peptide chain comprises, in N-terminal to C-terminal order: the first single-domain antibody or antigen-binding fragment thereof, and the first Fc domain.

[0215] In certain embodiments, the polypeptide construct further comprises a second peptide chain comprising a second Fc domain. In certain embodiments, the second peptide chain further comprises a second single-domain antibody or antigen-binding fragment thereof. In certain embodiments, the second single-domain antibody or antigen-binding fragment thereof is as defined in the ninth aspect.

[0216] In certain embodiments, the second peptide chain comprises, in N-terminal to C-terminal order: the second single-domain antibody or antigen-binding fragment thereof, and the second Fc domain.

[0217] In certain embodiments, the polypeptide construct has one or more of the features selected from:

[0218] (i) the first single-domain antibody or antigen-binding fragment thereof is the same as or different from the second single-domain antibody or antigen-binding fragment thereof;

[0219] (v) the first Fc domain and the second Fc domain are the same or different;

[0220] (vi) the first peptide chain is the same as or different from the second peptide chain;

[0221] (vii) the first peptide chain and the second peptide chain are connected by disulfide bond formation between them.

[0222] In certain embodiments, the polypeptide construct comprises a sequence as set forth in any one of SEQ ID NOs: 26-30.

[0223] In certain embodiments, the first peptide chain and / or the second peptide chain of the polypeptide construct each independently comprises a sequence as set forth in any one of SEQ ID NOs: 26-30.

[0224] In an eleventh aspect, the present application provides a fusion protein comprising the single domain antibody or antigen binding fragment thereof of the ninth aspect or the polypeptide construct of the tenth aspect, and, a further peptide domain.

[0225] In certain embodiments, the further peptide domain is selected from a polypeptide domain having a therapeutic effect.

[0226] In certain embodiments, the further polypeptide domain is directly linked or optionally linked via a peptide linker to the N-terminus and / or C-terminus of the single domain antibody or antigen binding fragment thereof or the polypeptide construct.

[0227] In a twelfth aspect, the present application provides an isolated nucleic acid molecule encoding the single domain antibody or antigen binding fragment thereof of the ninth aspect, the polypeptide construct of the tenth aspect, or the fusion protein of the eleventh aspect.

[0228] In certain embodiments, the isolated nucleic acid molecule encodes the single domain antibody or antigen binding fragment thereof of the ninth aspect.

[0229] In certain embodiments, the isolated nucleic acid molecule encodes the polypeptide construct of the tenth aspect.

[0230] In certain embodiments, the isolated nucleic acid molecule encodes the fusion protein of the eleventh aspect.

[0231] In certain embodiments, the polypeptide construct comprises a first peptide chain and a second peptide chain, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first peptide chain and a second nucleotide sequence encoding the second peptide chain, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecules comprise a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0232] In a thirteenth aspect, the present application provides a vector comprising the isolated nucleic acid molecule of the twelfth aspect.

[0233] In certain embodiments, the vector comprises a nucleotide sequence encoding the single domain antibody or antigen binding fragment thereof of the ninth aspect.

[0234] In certain embodiments, the vector comprises a nucleotide sequence encoding the polypeptide construct of the tenth aspect.

[0235] In certain embodiments, the vector comprises a nucleotide sequence encoding the fusion protein of the eleventh aspect.

[0236] In certain embodiments, the polypeptide construct comprises a first peptide chain and a second peptide chain, the vector comprises a first nucleotide sequence encoding the first peptide chain and a second nucleotide sequence encoding the second peptide chain, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0237] In a fourteenth aspect, the present application provides a host cell comprising the isolated nucleic acid molecule of the twelfth aspect or the vector of the thirteenth aspect.

[0238] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

[0239] The single domain antibody or antigen-binding fragment thereof, polypeptide construct, or fusion protein of the present application can be prepared in various methods known in the art, for example, by genetic engineering recombination techniques. For example, a DNA molecule encoding the single domain antibody or antigen-binding fragment thereof, polypeptide construct, or fusion protein of the present application is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, which is then transfected into a host cell. The transfected host cell is then cultured under specific conditions, and the single domain antibody or antigen-binding fragment thereof, polypeptide construct, or fusion protein of the present application is expressed.

[0240] In a fifteenth aspect, the present application provides a method of preparing the single domain antibody or antigen-binding fragment thereof of the ninth aspect, the polypeptide construct of the tenth aspect, or the fusion protein of the eleventh aspect, comprising culturing the host cell of the fourteenth aspect under conditions permitting expression of the protein, and recovering the single domain antibody or antigen-binding fragment thereof or the polypeptide construct or the fusion protein from the culture of the host cell.

[0241] In a sixteenth aspect, the present application provides a bispecific or multispecific antibody comprising the single domain antibody or antigen-binding fragment thereof of the ninth aspect or the polypeptide construct of the tenth aspect.

[0242] In certain embodiments, the bispecific or multispecific antibody specifically binds to serum albumin and additionally specifically binds to one or more other targets.

[0243] In certain embodiments, the bispecific or multispecific antibody further comprises at least one second antibody or antigen binding fragment thereof having a second binding specificity for a second target.

[0244] In a seventeenth aspect, the present application provides a conjugate comprising the single domain antibody or antigen binding fragment thereof of the ninth aspect or the polypeptide construct of the tenth aspect, and a therapeutic agent linked to the single domain antibody or antigen binding fragment thereof or the polypeptide construct.

[0245] In an eighteenth aspect, the present application provides a pharmaceutical composition comprising the single domain antibody or antigen binding fragment thereof of the ninth aspect, the polypeptide construct of the tenth aspect, the fusion protein of the eleventh aspect, the isolated nucleic acid molecule of the twelfth aspect, the vector of the thirteenth aspect, the host cell of the fourteenth aspect, the bispecific or multispecific antibody of the sixteenth aspect, or the conjugate of the seventeenth aspect.

[0246] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0247] In certain embodiments, the pharmaceutical composition comprises the single domain antibody or antigen binding fragment thereof of the ninth aspect, the polypeptide construct of the tenth aspect, or a nucleic acid molecule, vector or host cell encoding the single domain antibody or antigen binding fragment thereof or the polypeptide construct.

[0248] In certain embodiments, the pharmaceutical composition comprises the fusion protein of the eleventh aspect, or a nucleic acid molecule, vector or host cell encoding the fusion protein.

[0249] In certain embodiments, the pharmaceutical composition comprises the bispecific or multispecific antibody of the sixteenth aspect, or a nucleic acid molecule, vector or host cell encoding the bispecific or multispecific antibody.

[0250] In certain embodiments, the pharmaceutical composition comprises the conjugate of the seventeenth aspect.

[0251] In a nineteenth aspect, the present application provides use of the single domain antibody or antigen binding fragment thereof of the ninth aspect, the polypeptide construct of the tenth aspect, the fusion protein of the eleventh aspect, the isolated nucleic acid molecule of the twelfth aspect, the vector of the thirteenth aspect, the host cell of the fourteenth aspect, the bispecific or multispecific antibody of the sixteenth aspect, the conjugate of the seventeenth aspect, or the pharmaceutical composition of the eighteenth aspect for the manufacture of a medicament. The present application also provides a method for preventing and / or treating a disease in a subject, comprising administering to a subject in need thereof an effective amount of the single domain antibody or antigen binding fragment thereof of the ninth aspect, the polypeptide construct of the tenth aspect, the fusion protein of the eleventh aspect, the isolated nucleic acid molecule of the twelfth aspect, the vector of the thirteenth aspect, the host cell of the fourteenth aspect, the bispecific or multispecific antibody of the sixteenth aspect, the conjugate of the seventeenth aspect, or the pharmaceutical composition of the eighteenth aspect.

[0252] In certain embodiments, the medicament is for preventing and / or treating a serum albumin related disease in a subject.

[0253] In certain embodiments, the serum albumin related disease is a disease related to abnormal serum albumin content (e.g., hypoalbuminemia or hyperalbuminemia).

[0254] In certain embodiments, the medicament is capable of directly or indirectly participating in FcRn-mediated serum albumin circulation in a subject.

[0255] In certain embodiments, the medicament exhibits an extended half-life in vivo relative to a corresponding medicament lacking the single domain antibody or antigen binding fragment thereof.

[0256] In certain embodiments, the medicament is a macromolecular medicament (e.g., a polypeptide medicament).

[0257] In certain embodiments, the subject is a mammal.

[0258] The single-domain antibody or its antigen-binding fragment, polypeptide construct, fusion protein, conjugate, or pharmaceutical composition of this application can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The single-domain antibody or its antigen-binding fragment, polypeptide construct, fusion protein, conjugate, or pharmaceutical composition of this invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating an appropriate dose of the single-domain antibody or its antigen-binding fragment or polypeptide construct or fusion protein or conjugate or pharmaceutical composition of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives, stabilizers, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0259] The single-domain antibody or its antigen-binding fragment or polypeptide construct or fusion protein or conjugate or pharmaceutical composition of this application may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, topical (e.g., powder, ointment or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the single-domain antibody or its antigen-binding fragment or polypeptide construct or fusion protein or conjugate or pharmaceutical composition of the present invention is administered by intravenous injection or bolus.

[0260] In a twentieth aspect, this application provides a method for prolonging the in vivo half-life of a drug, comprising: linking a single-domain antibody or its antigen-binding fragment thereof (as described in the ninth aspect) or a polypeptide construct (as described in the tenth aspect) to the drug.

[0261] In certain embodiments, the prolongation of the in vivo half-life is relative to the in vivo half-life of the drug in the absence of the single-domain antibody or antigen-binding fragment thereof.

[0262] In certain embodiments, the drug is selected from a macromolecular drug (e.g., a polypeptide drug).

[0263] In certain embodiments, the subject is a mammal.

[0264] In a twenty-first aspect, the present application provides a conjugate comprising the single-domain antibody or antigen-binding fragment thereof of the ninth aspect or the polypeptide construct of the tenth aspect, and a detectable label linked to the single-domain antibody or antigen-binding fragment thereof or the polypeptide construct.

[0265] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0266] In a twenty-second aspect, the present application provides a kit comprising the single-domain antibody or antigen-binding fragment thereof of the ninth aspect, the polypeptide construct of the tenth aspect, or the conjugate of the twenty-first aspect.

[0267] In certain embodiments, the kit comprises the conjugate of the twenty-first aspect.

[0268] In certain embodiments, the kit comprises the single-domain antibody or antigen-binding fragment thereof of the ninth aspect or the polypeptide construct of the tenth aspect, and a second antibody or antigen-binding fragment thereof that specifically recognizes the single-domain antibody or antigen-binding fragment thereof or the polypeptide construct; optionally, the second antibody or antigen-binding fragment thereof further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0269] In a twenty-third aspect, the present application also provides a method for detecting the presence or level of serum albumin in a sample, comprising using the single-domain antibody or antigen-binding fragment thereof of the ninth aspect, the polypeptide construct of the tenth aspect, or the conjugate of the twenty-first aspect.

[0270] In certain embodiments, the method is an immunological detection, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

[0271] In certain embodiments, the method comprises using the conjugate of the twenty-first aspect.

[0272] In certain embodiments, the method comprises using the single-domain antibody or antigen-binding fragment thereof of the ninth aspect or the polypeptide construct of the tenth aspect, and the method further comprises using a secondary antibody or antigen-binding fragment thereof carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin) to detect the single-domain antibody or antigen-binding fragment thereof or the polypeptide construct.

[0273] In certain embodiments, the method comprises: (1) contacting the sample with the single-domain antibody or antigen-binding fragment thereof or the polypeptide construct or the conjugate; (2) detecting the formation of antigen-antibody immune complex or detecting the amount of the immune complex.

[0274] The present application also provides a method of diagnosing a disease related to serum albumin (e.g., a disease associated with abnormal serum albumin level) comprising detecting the presence of serum albumin or the level thereof in a sample from a subject using the method of the twenty-third aspect.

[0275] In certain embodiments, the disease related to serum albumin is a disease associated with abnormal serum albumin content (e.g., hypoalbuminemia or hyperalbuminemia).

[0276] In a twenty-fourth aspect, the present application also provides use of the single-domain antibody or antigen-binding fragment thereof of the ninth aspect, the polypeptide construct of the tenth aspect, or the conjugate of the twenty-first aspect in the manufacture of a detection reagent for detecting the presence of serum albumin or the level thereof in a sample and / or diagnosing a disease related to serum albumin.

[0277] In certain embodiments, the disease related to serum albumin is a disease associated with abnormal serum albumin content (e.g., hypoalbuminemia or hyperalbuminemia).

[0278] Definitions of terms

[0279] In the present application, unless otherwise indicated, the scientific and technical terms used herein have the meanings that would be generally understood by one of ordinary skill in the art. Also, the viral, biochemical, immunological laboratory procedures used herein are in accordance with conventional techniques in the respective fields. In addition, for better understanding of the present application, the definitions and explanations of relevant terms are provided as follows.

[0280] When the terms "for example", "for instance", "such as" or "like", "including" or "containing" or variations thereof are used in this document, such terms are not to be interpreted in an exclusive sense unless otherwise indicated.

[0281] The terms "a" and "an" and "the" and similar referents in the context of describing the application (especially in the context of the following claims) are to be interpreted in an inclusive rather than an exclusive sense unless otherwise indicated.

[0282] As used herein, the term "Cartilage Matrix Protein", also referred to herein as "CMP", has the meaning commonly understood by those skilled in the art, and is also known in the art as matrilin-1 or CRTM.

[0283] As used herein, the term "CMP trimerization domain" or "trimerization domain derived from Cartilage Matrix Protein" are used interchangeably herein, and refer to a polypeptide structure capable of associating with two similar or identical polypeptides (e.g., a polypeptide containing the CMP trimerization domain) to form a stable trimer. In certain embodiments, the CMP trimerization domain comprises the amino acid residues in cartilage matrix protein corresponding to positions 454-496 of SEQ ID NO: 229.

[0284] As used herein, the term "Procollagen" has the meaning commonly understood by those skilled in the art, and is the precursor or immature form of collagen. In contrast to mature collagen, procollagen generally contains additional N- and C-propeptide fragments at its N- and C-termini, respectively, which are removed during collagen maturation by a series of enzymatic reactions. After the N- and C-propeptides are cleaved, the collagen molecule spontaneously forms a stable triple helical structure, which is the predominant form of collagen and has high mechanical strength and stability. Mature collagen chains form collagen fibrils through intermolecular interactions, which are further strengthened by specific enzyme-catalyzed chemical cross-linking.

[0285] Generally, the C-propeptide of procollagen is a globular non-triple helical domain that is capable of self-assembly into a trimer. In contrast to the triple helical region of collagen, the C-propeptide does not contain any glycine repeats.

[0286] As used herein, the term "single-domain antibody" (sdAb) has the meaning generally understood by those skilled in the art, and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region, VHH), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or shark antibody). Single-domain antibodies are also known as nanobodies, and the two terms are used interchangeably. Typically, a nanobody consists of 4 framework regions and 3 complementarity determining regions, with the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or to lack one or both of those framework regions, so long as antigen binding and specificity are substantially maintained.

[0287] As used herein, the term "antigen binding fragment" of a single-domain antibody refers to a polypeptide comprising a fragment of a single-domain antibody that retains the ability to specifically bind the same antigen to which the single-domain antibody binds, and / or competes with the single-domain antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen binding fragments of the nanobodies of the application can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the nanobodies of the application. In some embodiments, the "antigen binding fragment" of the single-domain antibody can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or to lack one or both of those framework regions, compared to the full-length single-domain antibody, so long as antigen binding and specificity are substantially maintained.

[0288] Antigen binding fragments of single-domain antibodies can be obtained from a given single-domain antibody (e.g., a nanobody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and screened for specificity in the same manner as for the intact nanobody.

[0289] Herein, unless the context clearly indicates otherwise, when referring to the term "single-domain antibody", this includes not only intact single-domain antibodies, but also antigen binding fragments of single-domain antibodies.

[0290] As used herein, the term "complementarity determining region" or "CDR" refers to amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in a Nanobody, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, e.g. according to the definition in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003) or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given Nanobody, a skilled person will readily identify the CDRs as defined by the various numbering systems. Moreover, the correspondence between the different numbering systems is well known to the skilled person (see e.g. Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In the present application, the CDRs contained by the single-domain antibody or antigen-binding fragment thereof of the application can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the single-domain antibody or antigen-binding fragment thereof of the application are preferably determined by the Kabat, Chothia, IMGT or AbM numbering system.

[0291] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0292] As used herein, the term "bispecific antibody" or "bispecific molecule" refers to an antibody having binding specificity for two different antigens (or epitopes). The term "multispecific antibody" or "multispecific molecule" refers to an antibody having binding specificity for at least two more (e.g., three or four) different antigens (or epitopes). Bispecific antibodies or multispecific antibodies comprise multiple antigen binding domains having binding specificity for different antigens (or epitopes), enabling binding to at least two different binding sites and / or target molecules.

[0293] The term "antibody", as used herein, refers to a molecule derived from an immunoglobulin that is capable of specifically binding to a target antigen through at least one antigen-combining site located in its variable region. An "intact antibody" typically comprises two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a different class of antibody, as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable region and the constant region are joined by a "J" region of about 12 or more amino acids, and the heavy chain further includes a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains are not directly involved in binding of an antibody to an antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antigen binding site. Assignment of amino acids to each region or domain can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0294] The term "polypeptide construct" is used herein in its broadest sense. Generally, the polypeptide construct is intended to mean a construct comprising one or more polypeptide or protein components, wherein the one or more polypeptide or protein components can each independently have different origins or different biological activities or functions, and are linked by covalent and / or non-covalent means (e.g., covalent linkage by covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds, and / or non-covalent linkage by hydrogen bonds). The polypeptide construct of the present application is not limited in the number of molecular chains (e.g., peptide chains) it contains, for example, the polypeptide construct of the present application can contain only one molecular chain (e.g., peptide chain), or contain two or more molecular chains (e.g., peptide chains) covalently and / or non-covalently linked (e.g., covalent linkage by covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds, and / or non-covalent linkage by hydrogen bonds) between the two or more molecular chains (e.g., peptide chains). Likewise, it is readily understood by one skilled in the art that in embodiments comprising multiple polypeptide or protein components, the multiple polypeptide or protein components contained in the polypeptide construct of the present application can be located in the same molecular chain (e.g., peptide chain) in whole or in part, or can each be located in a different molecular chain (e.g., peptide chain).

[0295] As used herein, the term "identity" is used in reference to the matching of sequences between two polypeptides or between two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions x 100%). In certain embodiments, the two sequences are the same length.

[0296] The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0297] As used herein, the term "variant", in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by the introduction of an amino acid residue substitution, deletion, or addition. In certain instances, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not by way of limitation, a polypeptide can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivatized polypeptide or peptide can be produced by chemical modification using techniques known to those of ordinary skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Furthermore, a variant has similar, the same, or improved function as the polypeptide or peptide from which it is derived.

[0298] As used herein, the term "specifically binds" refers to a nonrandom binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (K D ) of the interaction. In the present application, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0299] The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation. Both the "association rate constant" (kaor kon) and the "dissociation rate constant" (kdisor koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 : 186-187). The ratio of kdis / kon is equal to the dissociation constant K D(see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). The K D , konand kdisvalues can be measured using any effective method. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition to Biacore, the dissociation constant can also be measured using bioluminescence interferometry or Kinexa.

[0300] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the host cell assumes the genetic material carried by the vector and expresses the polynucleotide carried by the vector. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages, such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0301] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0302] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue for another with similar side chains, e.g., substitutions that take place within a family of amino acid residues that are physicochemically or functionally similar, e.g., substitutions having similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a preferred substitution is one in which the replaced amino acid residue is replaced with another amino acid residue from the same side chain family. Methods of identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0303] The writing of the twenty conventional amino acids referred to herein follows the conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by the one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0304] Unless otherwise indicated herein, or otherwise clearly contradicted by context, "A, B, and / or C" or like references should be understood as "A, B, C, or any combination thereof," e.g., as being understood to be selected from any one of A, B, C, A and B, A and C, B and C, A and B and C.

[0305] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, in pharmacology and / or physiology, with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives, stabilizers. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Adjuvants include, but are not limited to, inorganic adjuvants (such as aluminum hydroxide, vanadate), biological adjuvants (such as Mycobacterium tuberculosis, BCG, Corynebacterium parvum, Bordetella pertussis, Gram-negative bacterial endotoxin, B subunit of cholera toxin, muramyl dipeptide, cytokines), synthetic adjuvants (such as double-stranded polyribonucleotides, uridine acid), oil agents (such as Freund's complete adjuvant, arachis oil emulsion), and nano-adjuvants, etc. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial agents and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carriers or excipients include sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0306] As used herein, the term "prevent" refers to a method taking place in order to stop or delay the occurrence of a disease or disorder or a symptom in a subject. As used herein, the term "treat" refers to a method taking place in order to obtain a beneficial or desired clinical result. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0307] As used herein, the term "subject" refers to a mammal, for example, a human. In certain embodiments, the subject (e.g., human) has an autoimmune disease, or is at risk of having the disease.

[0308] As used herein, the term "effective amount" refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., an autoimmune disease) refers to an amount that is sufficient to prevent, stop, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount that is sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease. Determining such effective amounts is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, body weight, and gender, the mode of administration of a drug, and other therapies being administered concurrently, and the like.

[0309] Advantages of the Invention

[0310] The present inventors have innovatively designed and researched trimeric fusion protein targeting TACI and BCMA, and found that the trimeric fusion protein molecules obtained by the design and construction have higher activity than Fc fusion protein molecules, can block the binding of ligands and receptors (e.g., block the binding of Blys and / or APRIL to their receptors) with high activity, thereby reducing the amount of autoantibodies, and can be used for treating B cell-related diseases (e.g., autoimmune diseases). Further, the trimeric fusion protein of the present application also has improved drugability, for example, has better stability and / or reduced amino acid modification (e.g., glycosylation modification, hydroxylation modification), low production cost, and can be better applied to large-scale industrial production.

[0311] Embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will appreciate that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the following detailed description of the preferred embodiments, when taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0312] Figure 1A: Detection results of SZ0384 binding activity to serum albumin by ELISA.

[0313] Figure IB: Detection results of SZ0387 binding activity to serum albumin by ELISA.

[0314] Figure 1C: Detection results of SZ0388 binding activity to serum albumin by ELISA.

[0315] Figure ID: Detection results of SZ0390 binding activity to serum albumin by ELISA.

[0316] Figure IE: Detection results of SZ0396 binding activity to serum albumin by ELISA.

[0317] Figure 2A: Schematic diagram of exemplary trimeric fusion protein structure of the present application.

[0318] Figure 2B: Schematic diagram of exemplary Fc fusion protein structure of the present application.

[0319] Figure 3A: Detection results of blocking activity of C040 and control molecules to hAPRIL.

[0320] Figure 3B: Detection results of blocking activity of C040 and control molecules to hBLys.

[0321] Figure 4A: Detection results of blocking activity of C043 and control molecules to hAPRIL.

[0322] Figure 4B: Detection results of blocking activity of C043 and control molecules to hBLys.

[0323] Figure 5A: Detection results of blocking activity of C035 and control molecules to hAPRIL.

[0324] Figure 5B: Detection results of blocking activity of C035 and control molecules to hBLys.

[0325] Figure 6A: Detection results of blocking activity of C097 and control molecules to hAPRIL.

[0326] Figure 6B: Detection results of blocking activity of C097 and control molecules to hBLys.

[0327] Figure 7A: Results of detection of blocking activity of C035, C398, N018, N019, N007 against hAPRIL.

[0328] Figure 7B: Results of detection of blocking activity of C035, C398, N018, N019, N007 against hBLys.

[0329] Figure 8A: Results of detection of blocking activity of C097, C399 against hAPRIL.

[0330] Figure 8B: Results of detection of blocking activity of C097, C399 against hBLys.

[0331] Figure 9A: Results of detection of blocking activity of C035, C297, C298, C299, C300, C301, C302, C303, C304, C305, C306 against hAPRIL.

[0332] Figure 9B: Results of detection of blocking activity of C035, C297, C298, C299, C300, C301, C302, C303, C304, C305, C306 against hBLys.

[0333] Figure 10A: Results of detection of blocking activity of C035, C093, C095 against hAPRIL.

[0334] Figure 10B: Results of detection of blocking activity of C035, C093, C095 against hBLys.

[0335] Figure 10C: Results of detection of blocking activity of C035, C102, C103, C104, C105, C106, C107, C108 against hAPRIL.

[0336] Figure 10D: Results of detection of blocking activity of C035, C102, C103, C104, C105, C106, C107, C108 against hBLys.

[0337] Figure 11A: Results of detection of blocking activity of C095, C096 against hAPRIL.

[0338] Figure 11B: Results of detection of blocking activity of C095, C096 against hBLys.

[0339] Figure 12A: Results of detection of blocking activity of C035, C158, C159, C160, C162, C163, C164, C165, C166 against hAPRIL.

[0340] Figure 12B: Results of detecting blocking activity of C035, C158, C159, C160, C162, C163, C164, C165, C166 against hBLys.

[0341] Figure 12C: Results of detecting blocking activity of C035, C217 against hAPRIL.

[0342] Figure 12D: Results of detecting blocking activity of C035, C217 against hBLys.

[0343] Figure 13A: Results of detecting blocking activity of C203, C327, C328, C329, C331, C332, C333, C334, C335, C336 against hAPRIL.

[0344] Figure 13B: Results of detecting blocking activity of C203, C327, C328, C329, C331, C332, C333, C334, C335, C336 against hBLys.

[0345] Figure 13C: Results of mass spectrometry detection of C203.

[0346] Figure 13D: Results of mass spectrometry detection of C327.

[0347] Figure 13E: Results of mass spectrometry detection of C334.

[0348] Figure 13F: Results of mass spectrometry detection of C335.

[0349] Figure 13G: Results of mass spectrometry detection of C336.

[0350] Figure 13H: Results of detecting blocking activity of C374, C439, C440, C441, C442, C443, C444, C449, C450, C451, C452, C453, C454, C455, C456 against hAPRIL.

[0351] Figure 131: Results of detecting blocking activity of C374, C439, C440, C441, C442, C443, C444, C449, C450, C451, C452, C453, C454, C455, C456 against hBLys.

[0352] Figure 13J: Results of mass spectrometry detection of C374.

[0353] Figure 13K: Results of mass spectrometry detection of C456.

[0354] Figure 14A: Results of detection of blocking activity of C035, C203, C307, C311, C372, C373, C374, C375, C376, C377 against hAPRIL.

[0355] Figure 14B: Results of detection of blocking activity of C035, C203, C307, C311, C372, C373, C374, C375, C376, C377 against hBLys.

[0356] Figure 14C: Results of detection of blocking activity of C035, C275, C317, C321, C378, C379, C380, C381, C382, C383, C384, C385, C386 against hAPRIL.

[0357] Figure 14D: Results of detection of blocking activity of C035, C275, C317, C321, C378, C379, C380, C381, C382, C383, C384, C385, C386 against hBLys.

[0358] Figure 14E: Results of detection of blocking activity of C035, C296, C387, C388, C389, C390, C391, C392, C393, C394, C395, C396, C397 against hAPRIL.

[0359] Figure 14F: Results of detection of blocking activity of C035, C296, C387, C388, C389, C390, C391, C392, C393, C394, C395, C396, C397 against hBLys.

[0360] Figure 14G: Results of detection of blocking activity of C374, C554, C555, C556, C557, C558, C559, C560, C561, C562, C563, C564, C565 against hAPRIL.

[0361] Figure 14H: Results of detection of blocking activity of C374, C554, C555, C556, C557, C558, C559, C560, C561, C562, C563, C564, C565 against hBLys.

[0362] Figure 14I: Results of detection of blocking activity of C382, C566, C567, C568, C569, C570, C571, C572, C573, C574, C575, C576, C577 against hAPRIL.

[0363] Figure 14J: Results of detection of blocking activity of C382, C566, C567, C568, C569, C570, C571, C572, C573, C574, C575, C576, C577 against hBlys.

[0364] Figure 14K: Results of detection of blocking activity of C395, C531, C532, C533, C534, C536, C539, C542 against hAPRIL.

[0365] Figure 14L: Results of detection of blocking activity of C395, C531, C532, C533, C534, C536, C539, C542 against hBlys.

[0366] Figure 14M: Results of detection of blocking activity of C395, C578, C579, C580, C581, C582, C583, C584 against hAPRIL.

[0367] Figure 14N: Results of detection of blocking activity of C395, C578, C579, C580, C581, C582, C583, C584 against hBlys.

[0368] Figure 14O: Results of mass spectrometry detection of C533.

[0369] Figure 14P: Results of mass spectrometry detection of C534.

[0370] Figure 14Q: Results of mass spectrometry detection of C567.

[0371] Figure 15A: Results of detection of blocking activity of C035, C164, C203 against mAPRIL.

[0372] Figure 15B: Results of detection of blocking activity of C035, C164, C203 against mBLys.

[0373] Figure 15C: Results of detection of blocking activity of C533, C567 against mAPRIL.

[0374] Figure 15D: Results of detection of blocking activity of C533, C567 against mBLys.

[0375] Figure 16A: Results of detection of anti-KLH IgG antibody on day 20.

[0376] Figure 16B: Results of detection of anti-KLH IgM antibody on day 20.

[0377] Figure 16C: Results of detection of anti-KLH IgM antibody on day 20.

[0378] Figure 16D: Day 20 anti-KLH IgG antibody detection results.

[0379] Figure 17A: NZBWF1 / J model mouse survival results.

[0380] Figure 17B: NZBWF1 / J model mouse 40 week urine biochemistry UACR (mALB / UCREA) results.

[0381] Figure 17C: NZBWF1 / J model mouse blood biochemistry SCREA results.

[0382] Figure 17D: NZBWF1 / J model mouse blood biochemistry BUN data.

[0383] Figure 17E: NZBWF1 / J model mouse endpoint spleen weight results.

[0384] Figure 17F: NZBWF1 / J model mouse endpoint kidney pathology score results.

[0385] Figure 18A: MRL / lpr model mouse 83 day endpoint mALB results.

[0386] Figure 18B: MRL / lpr model mouse 83 day endpoint anti-dsDNA IgG antibody results.

[0387] Figure 18C: MRL / lpr model mouse 83 day endpoint kidney pathology score results.

[0388] Figure 18D: MRL / lpr model mouse 83 day endpoint spleen flow cytometry results.

[0389] Figure 18E: MRL / lpr model mouse day 71 anti-dsDNA IgG antibody results.

[0390] Figure 18F: MRL / lpr model mouse 98 day endpoint kidney pathology score results.

[0391] Figure 19A: Cynomolgus monkey PK with cIgM detection results.

[0392] Figure 19B: Cynomolgus monkey PK with cIgA detection results.

[0393] Figure 19C: Cynomolgus monkey PK with cIgG detection results. DETAILED DESCRIPTION

[0394] The application will now be described with reference to the following examples, which are intended to be illustrative only and not limiting of the application.

[0395] Unless otherwise indicated, molecular biology and immunological techniques used in the present application are performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples describe the present application by way of example only and are not intended to limit the scope of the application as claimed.

[0396] Preparation Example:

[0397] 1. Preparation of antigen for detection

[0398] 1.1 Transient expression of antigen for detection and control protein

[0399] A transient expression vector for expressing hBlys ECD-N-His, hBlys ECD-N-mIgG1 Fc, mBlys ECD-N-mIgG1 Fc, hApril-N-mIgG1 Fc was constructed, wherein the amino acid sequences of hBlys ECD-N-His, hBlys ECD-N-mIgG1 Fc, mBlys ECD-N-mIgG1 Fc, hApril-N-mIgG1 Fc are shown in SEQ ID NOs: 1-4, respectively, and hBlys ECD-N-His is a fusion protein formed by fusing a His tag to the N-terminus of the extracellular domain of hBlys, and hBlys ECD-N-mIgG1 Fc, mBlys ECD-N-mIgG1 Fc, hApril-N-mIgG1 Fc are fusion proteins formed by fusing the Fc domain of mouse IgG1 to the N-terminus of the extracellular domain of hBlys, mBlys, and hApril, respectively.

[0400] Transient transfection expression of the antigen was performed using 293E cells in Freestyle medium. 24 hours before transfection, 0.5 x 10 6 HEK293E cells at 300 ml were inoculated in a 1000 ml cell culture flask at 0.5 x 10

[0401] In addition, other exemplary antigens, control molecules used in the present application can be obtained commercially, and their corresponding manufacturers, item numbers are as follows:

[0402] hApril-His (ACRO, APL-H52D1); mBlys (Bio-Techne, 8876-BF-010 / CF); mApril (ImmunoChemistry Technologies, 6490); cApril-His-Flag-Avi (MCE, A0A2K5TJA1); cBlys-His-Flag-Avi (MCE, A0A2K5V2X4); P01 / Taltacip (Rongchang Bio).

[0403] Among them, hApril, mApril and cApril represent human, mouse and monkey April proteins, respectively, and hBlys, mBlys and cBlys represent human, mouse and monkey Blys proteins, respectively.

[0404] 1.2 Purification and detection of antigen proteins

[0405] His-tag protein purification: collect the supernatant, filter the supernatant with a 0.22 micron filter, use a Ni column affinity chromatography, load into a Ni chromatography column that has been equilibrated with 20 mM PB-500 mM NaCl, pH 7.4 buffer, and then equilibrated with 20 mM PB-500 mM NaCl, pH 7.4 buffer, and then eluted under the following conditions: (1) elute with A liquid (20 mM PB-500 mM NaCl, pH 7.4) + 4% B liquid (20 mM PB-500 mM NaCl-500 mM imidazole, pH 7.4), collect samples according to the ultraviolet absorption peak type, and when the ultraviolet absorption peak is stable and does not fluctuate, proceed to the second step of elution; (2) elute with A liquid (20 mM PB-500 mM NaCl, pH 7.4) + 20% B liquid (20 mM PB-500 mM NaCl-500 mM imidazole, pH 7.4), collect samples according to the ultraviolet absorption peak type, and when the ultraviolet absorption peak is stable and does not fluctuate, proceed to the third step of elution; (3) elute with A liquid (20 mM PB-500 mM NaCl, pH 7.4) + 60% B liquid (20 mM PB-500 mM NaCl-500 mM imidazole, pH 7.4), collect samples according to the ultraviolet absorption peak type, and when the ultraviolet absorption peak is stable and does not fluctuate, stop collecting. The purified sample is detected by SDS-PAGE using 4-20% gradient gel.

[0406] mFc tag protein purification: collect the supernatant, filter the supernatant with 0.22 micron filter membrane, pass through Prism A (GE) affinity chromatography, elute with 20 mM citric acid-sodium citrate, pH 3.0, adjust the pH to neutral with 1 M Tris base. The purified sample is detected by SDS-PAGE with 4-20% gradient gel to detect the purified protein sample.

[0407] 2. Preparation of CHO-K1 cells overexpressing hTACI and mTACI proteins on cell surface

[0408] The human TACI (hTACI) protein coding gene (NCBI database, gene number: AF023614.1) and the mouse TACI (mTACI) protein coding gene (NCBI database, gene number: AF257673.1) were respectively connected into the pLvx-Puro-IRES plasmid to construct the overexpression lentivirus plasmid of hTACI protein and mTACI protein (i.e. pLvx master plasmid).

[0409] The three-plasmid system (envelope plasmid pCMV-VSVG, packaging plasmid pCMV-dr8.91, and target plasmid pLvx master plasmid) was used for lentivirus packaging, concentration, and lentivirus solution was obtained. The CHO-K1 cells were infected with the lentivirus solution. After infection, the infected cells were subjected to pressure screening by puromycin (Puro) to obtain a resistant pool (Pool), and the expression of hTACI and mTACI proteins was detected by FACs. The identified positive Pool cells were subjected to monoclonalization by FACs sorting method, and the CHO-K1 cell strain overexpressing hTACI and mTACI proteins was obtained after expansion and identification.

[0410] 3. Screening of anti-HSA VHH antibody

[0411] 3.1 Immunization, library construction, and screening

[0412] The alpaca and camel were immunized with a mixture of human serum albumin (HSA), cynomolgus serum albumin (CSA), and rattus serum albumin (RSA) in equal proportions, and the peripheral blood was taken after 4 immunizations for constructing the alpaca and camel immune library.

[0413] Fresh peripheral blood 50ml of the titer reached by extraction of immunity, separation of PBMC, extraction of total RNA, reverse transcription of total cDNA. The reverse transcription product was used as a template for two rounds of PCR: the first round of PCR designed primers in the vector signal peptide and antibody heavy chain constant region, and two bands of about 1000bp and 750bp were amplified, and the 750bp band was recovered as a template for the second round of PCR; the second round of PCR was designed in the antibody heavy chain FR1 and FR4 region, and the restriction enzyme cutting site of the phagemid vector was introduced. The second round of PCR product was digested and recovered by agarose gel, and then loaded into the digested phagemid vector, and the TG1 E. coli was electrotransformed to complete the construction of the immune library.

[0414] The immune library was packaged into a phage library, and biotinylated HSA (HSA-biotin) or RSA (RSA-biotin) was used as an antigen for liquid phase panning. The bacteria solution after the first to second round of panning was diluted and plated, and cultured overnight; single colonies were picked from the plate, packaged with helper phage M13KO7, and the phage supernatant was subjected to ELISA screening, and the positive clones were sequenced.

[0415] The ELISA detection steps are as follows: coating the antigens HSA, CSA, RSA, mouse serum albumin (MSA) respectively, blocking with 5% gelatin, adding library panning phage supernatant, incubating at room temperature for 1 hour. After washing the plate with PBST, adding anti-M13-HRP (sino biological: 11973-MM05T-H) antibody diluted at a ratio of 1:10000, and reading the OD450 value with an enzyme label instrument. The results are shown in Table 1.

[0416] Table 1 ELISA detection results

[0417] The results show that the anti-HSA nanobodies AlpGS002HRM002, AlpGS002HRM039, AlpGS002HRMG002, AlpGS002HRMG005, CA019A4RMH116 obtained by screening have binding activity to HSA, RSA, MSA and CSA.

[0418] The nanobodies obtained by the above ELISA screening were sequenced, and the corresponding VHH and CDR1-3 sequence information is shown in Table 2, wherein the CDRs are defined by the Kabat numbering system.

[0419] Table 2 Nanobody sequence information

[0420] 3.2 anti-HSA VHH-human IgG1 Fc fusion protein construction and affinity detection

[0421] 3.2.1 anti-HSA VHH-human IgGl Fc fusion protein construction

[0422] The above-mentioned AlpGS002HRM002, AlpGS002HRM039, AlpGS002HRMG002, AlpGS002HRMG005, and CA019A4RMH116, a total of 5 VHHs, were selected for VHH-human IgGl Fc fusion protein design, and CHO cells were used for protein expression. The protein names and sequence information are shown in Table 3.

[0423] Table 3 anti-HSA VHH-human IgGl Fc fusion protein design

[0424] 3.2.2 anti-HSA fusion protein in vitro activity detection

[0425] 3.2.2.1 ELISA detection of anti-HSA fusion protein binding activity with HSA, CSA, RSA, and MSA

[0426] The anti-HSA fusion protein was coated, and after blocking, 5-fold gradient dilution of biotinylated HSA-Biotin, CSA-Biotin, RSA-Biotin, and MSA-Biotin was added, and incubated at room temperature for 1 hour. After washing the plate with PBST, anti-Streptavidin-HRP antibody (Acro, STN-NH913) was added at a dilution ratio of 1:10,000. The results are shown in Figures 1A-1E and Table 4. The results showed that the anti-HSA fusion protein molecules SZ0384, SZ0387, SZ0388, and SZ0396 had strong binding activity with HSA, RSA, MSA, and CSA, and SZ0390 had strong binding activity with HSA and CSA, and weak binding activity with RSA and MSA.

[0427] Table 4 ELISA detection results

[0428] 3.2.2.2 ForteBio Octet HTX detection of anti-HSA fusion protein affinity with HSA, CSA, RSA, and MSA

[0429] Anti-HSA fusion protein 200s were captured using AHC (Sartorius; 18-5060) probes, and then the affinity of anti-HSA fusion protein to HSA, CSA, RSA, MSA was analyzed with HSA / CSA / RSA / MSA as analytes, binding reaction 120s, dissociation reaction 180s. The results are shown in Table 5. The results show that anti-HSA fusion protein molecules SZ0384, SZ0387, SZ0388, SZ0396 have good affinity to HSA, RSA, MSA and CSA, and SZ0390 has good affinity to HSA and CSA, and weak affinity to RSA and MSA.

[0430] Table 5 Octet detection results

[0431] 4. Preparation of trimeric fusion protein

[0432] 4.1 Design of trimeric fusion protein

[0433] The monomer of the trimeric fusion protein of the present application comprises a functional domain (e.g., an extracellular ligand binding domain) selected from TACI and BCMA, a trimerization domain selected from a trimerization domain derived from CMP (cartilage matrix protein) and a trimerization domain derived from C-propeptide of procollagen (hereinafter referred to as T2), and an optional serum albumin binding domain (e.g., an anti-serum albumin nanobody). Specifically, the structures of the trimeric fusion protein of the present application and the Fc fusion protein control molecule are shown in FIGS. 2A-2B, respectively, and the corresponding amino acid sequences are shown in Table 6.

[0434] Table 6 Number and sequence information of molecules of the present application

[0435] 4.2 Purification and detection of trimeric fusion protein

[0436] 4.2.1 Transfection, expression and detection in mammalian cells ExpiCHO-S

[0437] ExpiCHO-S was used for transient transfection expression of fusion protein molecules in ExpiCHO Expression Medium culture medium. 24 hours before transfection, 3-4 x 10 6 The cells were cultured in a 500 mL cell culture flask at 37°C, 8% CO2 incubator, 120 rpm shaker. The cell density was determined before transfection, and the cell density reached 6-8 x 106 The cell density was adjusted to 6 x 105cells / ml when the cell density was 1 x 106cells / ml. 6 The cell density was adjusted to 6 x 105cells / ml when the cell density was 1 x 106cells / ml. The fusion protein molecule was constructed into the expression plasmid pcDNA3.4 100 μg, diluted with OptiPRO SFM to 4 ml; 320 μl of ExpiFectamine CHO Reagent was added to 3.68 ml of OptiPRO SFM and mixed, and the above diluted transfection reagent and plasmid were mixed immediately, incubated at room temperature for 2 minutes, and then the mixture was added to the cells, mixed, and cultured at 37°C, 8% CO2incubator, 120 rpm shaker. 600 μl of ExpiFectamine CHO Enhancer was added to the culture system 18-22 hours after cell transfection. The culture conditions were adjusted to 32°C, 5% CO2, 120 rpm. 16 ml of ExpiCHO Feed was added on the fifth day of transfection. Cultured for 6-7 days.

[0438] 4.2.2 Purification and detection of trimer fusion protein

[0439] The fusion protein molecule expression supernatant was collected, the supernatant was filtered with a 0.22 micron filter, and the Prism A (GE) affinity chromatography was performed, eluted with 20 mM citric acid-sodium citrate, pH 3.0, and the pH was adjusted to neutral with 1 M Tris base. The samples with a purity of less than 90% were purified by molecular sieve chromatography, and the purified samples were detected by SDS-PAGE and HPLC-SEC to ensure that the protein purity for detection was greater than 90%.

[0440] Example: Biological activity evaluation

[0441] 1 Reporter gene method for detecting the in vitro activity of trimer fusion protein molecules

[0442] CHO-K1-hTACI-NFκB-Luc cells (constructed by ourselves, the CHO-K1 cells express hTACI and contain the NF-κB luciferase reporter system) were used as experimental culture medium with 10% FBS (Gibco, 10091-148), 3 μg / mL puromycin (Gibco, A1113803), 250 μg / mL hygromycin B (Invitrogen, 10687010) in F-12K medium (Gibco, 21127-022) to adjust the cell density to 3 x 105cells / ml. 5The cells were inoculated in 96-well plates at a density of 100 μl / well, and incubated in a 37 °C, 5% CO2 incubator for 18-20 h. The test medium was diluted with the fusion protein molecules to be tested, 50 μl / well was added to the 96-well plate, and the final concentration was 600 nM, 3-fold dilution, 10 gradients. The test medium was diluted with hApril-His or hBlys ECD-N-His, 80 ng / mL, 50 μl / well was added to the corresponding experimental wells. The total experimental system was 100 μl / well, which was incubated in a 37 °C, 5% CO2 incubator for 4 h. Bright-Lite (Norgen, DD1204-02) 100 μl / well was added, and the light value was detected by SpectraMax iD5 multifunctional enzyme label instrument (Molecular Devices) after 3 min of light protection at room temperature. The reporter gene inhibition rate calculation formula: Reporter gene inhibition (%) = 100% x [(RLU APRIL / BLyS -RLU SAMPLE ) / (RLU APRIL / BLyS -RLU cell )].

[0443] 1) Construction of trimer fusion protein using different functional domains (TACI, BCMA)

[0444] The structure of the fusion protein molecules constructed in this example is shown in Table 7. The trimer fusion protein comprises, from N-terminus to C-terminus, a TACI / BCMA functional domain, a linker1, a CMP trimerization domain, a linker2, an anti-HSA VHH, and the Fc fusion protein molecule comprises, from N-terminus to C-terminus, a TACI / BCMA functional domain, a linker3, and a Fc dimerization domain. The CMP trimerization domain sequence used in the construction of the trimer fusion protein is shown in SEQ ID NO: 230, the anti-serum albumin single-domain antibody ALB8 VHH is derived from patent US8703131B2, the VHH sequence is shown in SEQ ID NO: 225, and the CDR1-3 sequences are shown in SEQ ID NO: 226-228.

[0445] Table 7 Structure of fusion protein molecules

[0446] The linker1 is a peptide linker between the N-terminal functional domain and the trimerization domain in the trimer fusion protein, the linker2 is a peptide linker between the trimerization domain and the C-terminal anti-HSA VHH in the trimer fusion protein, and the linker3 is a peptide linker between the N-terminal functional domain and the Fc domain in the Fc fusion protein.

[0447] Fc fusion protein P01 (i.e., Telitacicept, SEQ ID NO: 31), trimeric fusion protein C040 (SEQ ID NO: 35) were constructed using TACI 13-118 (i.e., TACI aa. 13-118, SEQ ID NO: 171) sequence. The above molecules were compared by in vitro activity detection, and the results are shown in FIGS. 3A-3B. It can be seen from the results that the trimeric fusion protein C040 can simultaneously block the activities of hAPRIL and hBLys, and its blocking activity of hAPRIL is better than that of the Fc fusion protein P01, wherein the highest inhibition rate of C040 to hAPRIL is better than that of P01.

[0448] Fc fusion protein C242 (SEQ ID NO: 61), trimeric fusion protein C043 (SEQ ID NO: 36) were constructed using TACI 68-110 (i.e., TACI aa. 68-110, SEQ ID NO: 172) sequence. The above molecules were compared by in vitro activity detection, and the results are shown in FIGS. 4A-4B. It can be seen from the results that the trimeric fusion protein C043 can simultaneously block the activities of hAPRIL and hBLys, and its blocking activity of hAPRIL is better than that of the Fc fusion protein C242, wherein the highest inhibition rate of C043 to hAPRIL is better than that of C242.

[0449] Fc fusion protein P09 (SEQ ID NO: 33), trimeric fusion protein C035 (SEQ ID NO: 34) were constructed using BCMA 1-51 (i.e., BCMA aa. 1-51, SEQ ID NO: 174) sequence. The above molecules were compared by in vitro activity detection, and the results are shown in FIGS. 5A-5B. It can be seen from the results that the trimeric fusion protein C035 can simultaneously block the activities of hAPRIL and hBLys, and its blocking activity of hBlys is significantly better than that of the Fc fusion protein P09.

[0450] Fc fusion protein P08 (SEQ ID NO: 32), trimeric fusion protein C097 (SEQ ID NO: 41) were constructed using BCMA 1-54 (i.e., BCMA aa. 1-54, SEQ ID NO: 173) sequence. The above molecules were compared by in vitro activity detection, and the results are shown in FIGS. 6A-6B. It can be seen from the results that the trimeric fusion protein C097 can simultaneously block the activities of hAPRIL and hBLys, and its blocking activity of hBlys is significantly better than that of the Fc fusion protein P08.

[0451] 2) Construction of trimeric fusion proteins using different trimeric bridge structures

[0452] Based on C035, C097 molecules, the CMP trimerization domain was replaced with trimerization domain T2 (SEQ ID NO: 232), and the linker2 was replaced with the peptide linker shown in SEQ ID NO: 208, to obtain C398, C399 molecules (SEQ ID NO: 109 and 110), respectively. Based on the C035 molecule, the molecule N007 (SEQ ID NO: 111, which comprises a linker2 as shown in SEQ ID NO: 208) was constructed without the functional domain BCMA sequence, based on the C398 molecule, the molecule N018 (SEQ ID NO: 112) was constructed without the anti-HSA VHH sequence, and based on the C398 molecule, the molecule N019 (SEQ ID NO: 113) was constructed without the functional domain BCMA sequence.

[0453] The C035, C398, N007, N018, N019 molecules were compared by in vitro activity detection, and the results are shown in Figures 7A-7B. As can be seen from the results, the trimer fusion protein molecules C035 and C398 constructed using different trimer bridging structures CMP and T2 can simultaneously block the activity of hAPRIL and hBLys, and the blocking activity of hApril and hBlys is better than that of Fc fusion protein P01, wherein the blocking activity of C035 is better than that of C398, and N018 without anti-HSA VHH can also block the activity of hAPRIL and hBLys, while N007 and N019 without functional domain BCMA have no blocking activity.

[0454] The C097, C399 molecules were compared by in vitro activity detection, and the results are shown in Figures 8A-8B. As can be seen from the results, after replacing the functional domain with different BCMA sequences, the trimer bridging structure is replaced from the CMP trimerization domain to the trimerization domain T2, i.e. the C097, C399 molecules can simultaneously block the activity of hAPRIL and hBLys, and the blocking activity of hApril is better than that of Fc fusion protein P01, wherein the blocking activity of C097 is better than that of C399.

[0455] 3) Use different forms of linker1 to construct trimer fusion proteins

[0456] Based on C035 molecule, and using linker2 as shown in SEQ ID NO: 208, different forms were tried for linker1 connecting BCMA functional domain and CMP trimerization domain, respectively using ASTKGPSVFPLAP (SEQ ID NO: 206), ASTKGPSVFPLAPGGGGS (SEQ ID NO: 207), RNTGRGGEEKGGGGS (SEQ ID NO: 208), RNTGRGGEEKKKEKEKEEQEERETKTPE (SEQ ID NO: 209), EPKSSDKTHTSPPSP (SEQ ID NO: 210), EPKSSDKTHTSPPSPGGGGS (SEQ ID NO: 211), AKTTPKLEEGEFSEARV (SEQ ID NO: 212), GGGGS (SEQ ID NO: 213), GGGGSGGGGS (SEQ ID NO: 214), GGGGSGGGGSGGGGS (SEQ ID NO: 215), C297, C298, C299, C300, C301, C302, C303, C304, C305, C306 molecules (SEQ ID NO: 64-73) were constructed.

[0457] The above molecules were detected for in vitro activity comparison, and the results are shown in Figures 9A-9B. The results show that after replacing the linker1 connecting the BCMA and CMP domains, the trimer fusion protein can still maintain high blocking activity against hAPRIL and hBlys.

[0458] 4) Construction of trimer fusion protein using different forms of linker2

[0459] Based on C035 molecule, different forms were tried for linker2 connecting CMP trimerization domain and anti-HSA VHH, respectively using ASDYKDDDKSGGGGS (SEQ ID NO: 217), GGGGSGGGGS (SEQ ID NO: 214), ASTKGPSVFPLAPGGGGS (SEQ ID NO: 207), RNTGRGGEEKGGGGS (SEQ ID NO: 208), RNTGRGGEEKKKEKEKEEQEERETKTPE (SEQ ID NO: 209), EPKSSDKTHTSPPSP (SEQ ID NO: 210), EPKSSDKTHTSPPSPGGGGS (SEQ ID NO: 211), AKTTPKLEEGEFSEARV (SEQ ID NO: 212), ASTDDDKSVFPLAPGGGGS (SEQ ID NO: 218), C093, C095, C102, C103, C104, C105, C106, C107, C108 molecules (SEQ ID NO: 37, 39, 43-49) were constructed.

[0460] The above molecules were detected for in vitro activity comparison, and the results are shown in Figures 10A-10D. The results show that after replacing linker2 connecting CMP domain and anti-HSA VHH, the trimer fusion protein can still maintain high blocking activity against hAPRIL and hBlys.

[0461] 5) Using BCMA deglycosylation design to construct trimer fusion protein

[0462] The BCMA functional domain of C093 and C095 molecules was designed for deglycosylation (the amino acid residue N at position 42 was mutated to Q), and C094 and C096 molecules (SEQ ID NO: 38 and 40) were obtained, and the above molecules were detected for in vitro activity. The detection results of C095 and C096 are shown in Figures 11A-11B. As can be known from the results, the trimer fusion protein constructed based on deglycosylation mutation can still maintain high blocking activity against hAPRIL and hBlys.

[0463] 6) Using different anti-HSA VHH sequences to construct trimer fusion protein

[0464] Based on C103 molecule, anti-HSA VHH ALB8 in the molecule was replaced by anti-HSA VHH camel sequence AlpGS002HRM002 (SEQ ID NO: 5), AlpGS002HRM039 (SEQ ID NO: 9), AlpGS002HRMG002 (SEQ ID NO: 13), AlpGS002HRMG005 (SEQ ID NO: 17), CA019A4RMH116 (SEQ ID NO: 21) screened in the application to construct C158, C159, C160, C161, C162 molecules (SEQ ID NO: 50-54). In addition, based on the corresponding anti-HSA VHH humanized molecules (SEQ ID NO: 220-224), C163, C164, C165, C166, C217 molecules (SEQ ID NO: 55-58, 60) were constructed respectively. The above molecules were detected for in vitro activity, and the results are shown in FIGS. 12A-12D. It can be known from the results that the trimer fusion proteins constructed based on different anti-HSA VHH can maintain high blocking activity against hAPRIL and hBlys.

[0465] 7) Using different truncated and mutated BCMA functional domains to construct trimer fusion proteins

[0466] The BCMA side of C164 molecule was deglycosylated (i.e., the amino acid residue N at position 42 of BCMA was mutated to Q) to obtain C203 molecule (SEQ ID NO: 59). Mass spectrometry detection was performed on C203 molecule, and it was found that the molecule had a break between the 3rd and 4th amino acids at the N-terminal of the BCMA domain. Based on C203 molecule, the N-terminal sequence of BCMA domain was truncated by 1, 2, 3, and 6 amino acids to obtain C327, C328, C329, and C331 molecules (SEQ ID NO: 74-77); based on C203 molecule, the 4th amino acid M of BCMA was mutated to amino acids L, V, G, P, and S to obtain C332, C333, C334, C335, and C336 molecules (SEQ ID NO: 78-82). The above molecules were detected for in vitro activity and mass spectrometry, and the results are shown in FIGS. 13A-13G. It can be known from the results that the trimer fusion proteins after the above truncation and mutation of the BCMA functional domain can maintain high blocking activity against hAPRIL and hBlys, and it is found that when the N-terminal sequence of BCMA domain is truncated by 1 amino acid or the 4th amino acid M of BCMA is mutated to amino acids P, G, and S, respectively, the break can be effectively prevented, and the drugability can be better improved.

[0467] Based on the C335 molecule, the linker1 is replaced with the linker1 shown in SEQ ID NO: 206 to obtain the C374 molecule (SEQ ID NO: 85). Mass spectrometry PTM modification detection is performed on the C374 molecule, and it is found that the 4th amino acid P and the 50th amino acid K of the BCMA domain of the molecule have a "+16D" modification (hydroxylation modification). Based on the C374 molecule, the C-terminal of the BCMA domain is truncated, and the 1st-49th, 1st-48th, 1st-46th, 1st-45th, 1st-44th, 1st-43rd, 1st-42nd, 1st-41st are truncated to obtain C449, C450, C451, C452, C453, C454, C455, C456 molecules (SEQ ID NO: 124-131); the 50th amino acid K of BCMA is mutated to amino acids R, S, Q, E, G, A, respectively, to obtain C439, C440, C441, C442, C443, C444 molecules (SEQ ID NO: 118-123). The above molecules are subjected to in vitro activity detection and mass spectrometry reduced molecular weight detection, and the results are shown in FIGS. 13H-13K. It can be known from the results that the trimer fusion proteins obtained by the above truncation and mutation of the BCMA functional domain can maintain the high blocking activity of hAPRIL and hBlys, and it is found that the molecules can reduce the proportion of "+16D" modification (hydroxylation modification) when the C-terminal sequence of the BCMA domain is truncated by 2-10 amino acids or the 50th amino acid K of the BCMA is mutated, and can better improve the drugability.

[0468] 8) Use of different optimized sequences of BCMA, different linkers1 and linkers2, and different anti-HSA VHHs to construct trimer fusion proteins

[0469] The combined molecular structure is shown in Table 8, which comprises, from N-terminal to C-terminal, BCMA functional domain, linker1, CMP trimerization domain, linker2, anti-HSA VHH, and the sequence is shown in SEQ ID NO: 114-115, 83, 84, 86-88, 62, 116-117, 89-97, 63, 98-108, 132-169. The above molecules are subjected to in vitro activity detection and mass spectrometry reduced molecular weight detection, and the results are shown in FIGS. 14A-14Q. It can be known from the results that all the trimer fusion proteins obtained by the combination can maintain high blocking activity against hAPRIL and hBlys, and have good drugability.

[0470] Table 8: Combined optimized molecular structure

[0471] 9) Detection of species cross-reactivity of trimer fusion proteins

[0472] The C035, C164, C203, C533, C567 molecules were tested by reporter gene assay using CHO-K1-mTACI-NFκB-Luc (the CHO-K1 cells express mTACI and contain an NF-κB luciferase reporter system), and the results are shown in Figures 15A-15D. The results show that the trimer fusion proteins C035, C164, C203, C533, C567 molecules have high activity in blocking the action of hApril and hBlys on the receptor, and also have high activity in blocking the action of mApril and mBlys on the mTACI reporter gene cell line, and have good human-mouse cross-reactivity.

[0473] 2 Affinity determination of trimer fusion protein molecules binding to hBlys, hApril, cApril, and cBlys

[0474] The affinities of C035, C096, C164, C203, C533, C567 molecules binding to hBlys, hApril, cApril, and cBlys were detected using a Biacore 8K instrument. The specific method is as follows: an anti-His antibody (Cytiva, 28995056) was coupled to a CM5 biosensor chip (Cytiva, BR-1005-30), and hBlys, hApril, cBlys, and cApril containing His tags were used as ligands, with an antigen capture amount of 0.1-0.5 μg / ml. The trimer fusion protein molecules were diluted by a 2-fold concentration gradient, and 7 concentration gradients were diluted using 1x HBS-EP+ Buffer (10x, Cytiva, BR-1006-69) at a ratio of 1:1. The starting concentration of etanercept was 100 nM, and the starting concentration of the trimer fusion protein molecules was 10-40 nM. The chip was flowed at a rate of 30 μL / min, the binding time was 120 s, the dissociation time was 120-600 s, the regeneration condition was Gly 1.5, and the time was 25-30 s. The results are shown in Table 9.

[0475] Table 9. Affinity determination results

[0476] The results show that the C035, C096, C164, C203, C533, C567 molecules have good affinities with hBlys, hApril, cApril, and cBlys, and have good human-mouse cross-reactivity.

[0477] 3 Affinity determination of trimer fusion protein molecules with HSA, MSA, and CSA

[0478] The Biacore 8K device was used to detect the affinity of C103, C158, C159, C160, C162, C163, C164, C165, C217, C203, C275, C533, C567 molecules binding to HSA, MSA, CSA.

[0479] The specific method is as follows: using CM5-22 (anti-mouse antibody) chip to capture hBlys ECD-N-mIgG1Fc, further capturing trimer fusion protein molecules by hBlys ECD-N-mIgG1Fc, trimer fusion protein molecules capturing HSA / MSA / CSA for analysis, hBlys ECD-N-mIgG1Fc antigen capture amount is 0.5-1.5 μg / ml, trimer fusion protein molecule capture amount is 5 μg / ml, HSA / MSA / CSA as analyte, 2-fold concentration gradient dilution method, using 1x HBS-EP+Buffer (10x, Cytiva, BR-1006-69) dilution 7-8 concentration gradients, starting concentration is 500 nM or 1000 nM, binding time 120 s, dissociation time 300 s, regeneration conditions are Gly 1.5, 35-90 s. The results are shown in Tables 10-12.

[0480] From the data, it can be seen that the affinities of C103, C158, C159, C160, C162, C163, C164, C165, C217, C203, C275, C533, C567 molecules to different species of albumin including HSA, MSA, CSA are good, and have good species cross-reactivity.

[0481] Table 10 Affinity determination results of trimer fusion protein molecules and HSA

[0482] Table 11 Affinity determination results of trimer fusion protein molecules and CSA

[0483] Table 12 Affinity determination results of trimer fusion protein molecules and MSA

[0484] 4 KLH mouse immunization model for evaluating the in vivo efficacy of trimer fusion protein

[0485] A mouse KLH immunization model was used to evaluate the effect of immunomodulatory molecules on antigen-specific responses against the cell-dependent antigen KLH after one or two injections of KLH. To initiate the KLH study, 10-week-old female C57BL / 6N mice were randomized into 5 groups of 6 mice each. Mice were administered 0.25 mg KLH (Keyhole limpet hemocyanin (KLH): Biosyn; Cat: KLH-NP) via intraperitoneal (IP) injection on days 0 and 12; KLH solution was diluted to 1.25 pg / pL with PBS prior to injection. Test articles were dosed to mice via IP injection on days 4 and 11 as described in Table 13, at the dosing amounts listed in Table 13. On days 5 (24 h after the 1stdose), 12 (24 h after the 2nddose / KLH boost), and 20, mice were anesthetized with 1-4% isoflurane and blood was collected via retro-orbital plexus, and blood samples were collected to evaluate anti-KLH antibody levels. Results are shown in Figures 16A-16D.

[0486] Table 13-1 KLH mouse immunization model dose schedule and groupings

[0487] Table 13-2 KLH mouse immunization model dose schedule and groupings

[0488] Results show that C096, C203, C533, and C567 molecules were able to suppress the concentration of KLH-induced anti-KLH IgG antibodies and anti-KLH IgM antibodies, and the level of suppression of anti-KLH IgG antibodies was superior to P01.

[0489] 5 NZBWF1 / J SLE spontaneous model evaluation of trimer fusion proteins in vivo efficacy

[0490] NZBWF1 / J background mice are a spontaneous SLE model mouse purchased from Jakson Laboratories (Strain No: 100008), which have high circulating concentrations of anti-dsDNA antibodies starting at about 20 weeks of age, and the first clinical signs of disease are detectable at about 23 weeks of age. The mice develop hemolytic anemia, proteinuria, and progressive glomerulonephritis mediated by immune complex deposition in the glomerular basement membrane.

[0491] The model mice were randomly divided into 4 groups according to body weight at 21 weeks of age. The mice will be subcutaneously administered twice a week from the 22nd week, and the P01 and C096 molecules will be administered in an equimolar manner, for 20 weeks. The dose regimen and grouping are shown in Table 14. The body weight of the mice was recorded every week during the experiment, and blood was collected at 21 / 25 / 30 / 34 / 40 weeks to detect ds-DNA, urea nitrogen (BUN), and serum creatinine (SCREA), and metabolic cages were used to collect urine to detect urinary protein (UTP), microalbumin (mALB), and urinary creatinine (UCREA). At the end of the experiment, the spleen, kidney, and lymph node were collected and weighed to calculate the organ index. Kidney tissue was collected for histological embedding, and HE staining was used to detect pathological changes. The results are shown in Figures 17A-17E.

[0492] Table 14 Dose regimen and grouping of NZBWF1 / J SLE spontaneous model mice

[0493] The results show that from the survival rate, urine biochemical indicators, blood biochemical indicators, spleen weight, and kidney pathology, it can be found that the trimer molecule C096 has a significant effect on inhibiting the development of the disease and is superior to P01 in terms of efficacy. One death occurred in the P01 group during the experiment, and no deaths occurred in the C096 group; the 40-week urine biochemical indicator UACR results show that the C096 group is superior to the P01 group, and there is a significant difference compared with the Model; the blood biochemical indicators SCREA and BUN show that the C096 group is superior to the P01 group, and the end-point data are similar to the Normal group; the spleen weight shows that the C096 group is superior to the P01 group, and there is a significant difference compared with the Model group; the kidney pathology evaluation also shows that the C096 group is superior to the P01 group, and there is a significant difference compared with the Model.

[0494] 6 MRL / lpr pharmacodynamic model validation of trimer fusion protein in vivo

[0495] Experiment one:

[0496] MRL / lpr background mice are spontaneous SLE model mice, purchased from Jakson Laboratory (strain number: 000485). And at 8-9 weeks of age, they are randomly divided into 4 groups according to body weight. The mice will be subcutaneously administered from the 10th-11th week, and the administration frequency of C203 molecules adopts the administration mode of twice a week and once a week, which lasts for 12 weeks, and the endpoint is the 83rd day (D83). The dose scheme and grouping are shown in Table 15-1. During the experiment, the body weight of the mice is recorded every week, and the blood is collected at the 2nd / 4th / 6th / 8th / 12th / 14th week to detect ds-DNA, mouse IgG (mIgG) and serum creatinine (SCREA), and the urine is collected in a metabolic cage to detect urine protein (UTP), microalbumin (mALB) and urine creatinine (UCREA). At the end of the experiment, the spleen is collected to weigh and calculate the organ index, and the spleen cells are detected by flow cytometry to calculate the number of plasma cells. The kidney tissue is collected for tissue embedding, and HE staining is used to detect pathological changes.

[0497] Table 15-1 Dose scheme and grouping of MRL / lpr pharmacodynamic model mice

[0498] The results are shown in Figures 18A-18D, and the results show that the C203 molecule twice a week group and the once a week group both exhibit pharmacodynamics relative to the Model group. At the endpoint of the 83rd day, the mALB, anti-dsDNA IgG antibody index of the C203 group is significantly decreased relative to the Model group, the kidney tissue pathological score at the endpoint of the C203 group is significantly different from the Model group, and the FACS detection of CD138+ plasma cells also shows that the C203 group is significantly different from the Model group.

[0499] Experiment two:

[0500] MRL / lpr background mice are spontaneous SLE model mice, purchased from Jakson Laboratory (strain number: 000485). And at 8-9 weeks of age, they are randomly divided into 4 groups according to body weight. The mice will be subcutaneously administered from the 10th-11th week, and the administration frequency of C203 molecules adopts the administration mode of twice a week and once a week, which lasts for 12 weeks, and the endpoint is the 83rd day (D83). The dose scheme and grouping are shown in Table 15-1. During the experiment, the body weight of the mice is recorded every week, and the blood is collected at the 2nd / 4th / 6th / 8th / 12th / 14th week to detect ds-DNA, mouse IgG (mIgG) and serum creatinine (SCREA), and the urine is collected in a metabolic cage to detect urine protein (UTP), microalbumin (mALB) and urine creatinine (UCREA). At the end of the experiment, the spleen is collected to weigh and calculate the organ index, and the spleen cells are detected by flow cytometry to calculate the number of plasma cells. The kidney tissue is collected for tissue embedding, and HE staining is used to detect pathological changes.

[0501] Table 15-2 Dose scheme and grouping of MRL / lpr pharmacodynamic model mice

[0502] Results are shown in Figures 18E-18F, which show that both C533 and C567 molecules inhibited the production of anti-dsDNA relative to the Model group, and were effective in reducing the end point kidney histopathology score at the end point of day 98.

[0503] 7PK detection of trimeric fusion proteins in cynomolgus monkeys

[0504] To test the half-life of C035 and C203 in cynomolgus monkeys, a pharmacokinetic test was performed in cynomolgus monkeys using a 2-dose method. Two cynomolgus monkeys per group, female, were subcutaneously administered C035 and C203 at a dose of 3 mg / kg and 1.5 mg / kg, respectively, by weight. Blood was collected at 30 min (0 h) before administration, and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 96 h, 120 h, 168 h, 240 h, 336 h, 408 h, 504 h, and 672 h after administration (i.v.). At each time point, 1.2 mL of whole blood was collected, serum was prepared, divided into 3 portions of 0.2 mL each, and stored at -80°C until analysis. One sample was used for cynomolgus monkey PK detection. The second dose was administered to the monkeys on day 30 after the first dose, and the dose of C035 and C203 was 3 mg / kg. Blood was collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 96 h, 120 h, 168 h, 240 h, 336 h, 408 h, 504 h, and 672 h after administration (s.c.). At each time point, 1.2 mL of whole blood was collected, serum was prepared, divided into 3 portions of 0.2 mL each, and stored at -80°C until analysis.

[0505] The PK parameters were analyzed based on the blood concentrations, as shown in Table 16. The PK data included HL_Lambda_z(h) elimination phase half-life, which represents the time required for the blood concentration to decrease by half in the elimination phase; AUCINF-obs(hr*ug / ml), which represents the AUC from the start of administration to the theoretically extrapolated infinite time; Cl_F_obs(mL / h / kg) clearance, which represents the apparent distribution volume of the drug cleared from the body per unit time.

[0506] Table 16 PK parameters

[0507] From the detection data, the first administration C035, C203 clearance rate was 2.22 mL / h / kg, 0.55 mL / h / kg, although the first administration C203 administration was lower than C035, but the clearance rate was still better than C035 molecule; second administration C035, C203 clearance rate was 1.87 mL / h / kg, 0.27 mL / h / kg, still can see C203 molecule is better than C035 molecule.

[0508] 8Trimer fusion protein cynomolgus PK with IgM, IgA, IgG detection

[0509] Experiment one:

[0510] In order to test the C035, C203 in cynomolgus monkey in vivo efficacy, in the development of cynomolgus monkey PK at the same time accompanied by cIgM, cIgG, cIgA detection. First administration of 30 min (0h) before administration, 24h, 48h, 96h, 120h, 168h, 240h, 336h, 408h, 504h, 672h time point of serum cIgM, cIgG, cIgA detection, second administration of 24h, 48h, 96h, 120h, 168h, 240h, 336h, 408h, 504h, 672h time point of serum cIgM, cIgG, cIgA detection, detection results as shown in figure 19A-19C.

[0511] The results show that the trimer fusion protein molecule C035, C203 administration of cynomolgus monkeys can produce reduce cIgM, cIgA, cIgG efficacy, in which C203 molecule is better. First administration, C203 administration dose was 1.5 mg / kg, 17 days after administration, cIgM, cIgA, cIgG was at the lowest, the percentage of reduction compared with baseline was 54.55%, 50.85%, 22.8%; C035 administration dose was 3 mg / kg, 17 days after administration, cIgM, cIgA, cIgG was at the lowest, the percentage of reduction compared with baseline was 39.8%, 33.0%, 18.4%. Second administration C203 administration dose was 3 mg / kg, 17 days after administration, cIgM, cIgA, cIgG was at the lowest, the percentage of reduction compared with baseline was 71.85%, 68.3%, 33.45%; second administration C035 administration dose was 3 mg / kg, 10 days after administration, cIgM, cIgA, cIgG was at the lowest, the percentage of reduction compared with baseline was 53.45%, 46.9%, 30.45%.

[0512] Experiment two:

[0513] In order to test the C533 and C567 in vivo efficacy in cynomolgus monkeys, the trimer fusion protein inhibits the detection of cynomolgus IgM (cIgM), IgG (cIgG), IgA (cIgA). Two cynomolgus monkeys in each group, female, P01 and C533 are respectively subcutaneously administered according to the body weight, and the administration dose is 3 mg / kg. The serum collected at the time points of before administration (0 h), 24 h, 48 h, 96 h, 120 h, 168 h, 240 h, 336 h, 408 h, 504 h, 672 h, 840 h, 1008 h, 1176 h, 1344 h after administration (s.c.) is detected for cIgM, cIgG, and cIgA, and the detection results are shown in Table 17.

[0514] The results show that the trimer fusion protein molecules C533 and C567 produce the efficacy of reducing cIgM, cIgA, and cIgG after subcutaneous injection in cynomolgus monkeys. Among them, the cIgM, cIgA, and cIgG of C533 are at the lowest on the 35th day after administration, and the highest percentage of reduction from the baseline is 82.17%, 67.18%, and 39.96%, respectively; the cIgM, cIgA, and cIgG of C567 are at the lowest on the 21st-28th day after administration, and the highest percentage of reduction from the baseline is 73.59%, 58.66%, and 30.98%, respectively; the cIgM, cIgA, and cIgG of P01 are at the lowest on the 5th-10th day after administration, and the highest percentage of reduction from the baseline is 46.74%, 25.92%, and 14.29%, respectively. According to the data, C533 and C567 are superior to P01 in reducing the percentage of monkey IgM, IgA, and IgG and in prolonging the time of reducing monkey IgM, IgA, and IgG.

[0515] Table 17-1 Percentage of cIgM reduction over time after administration

[0516] Table 17-2 Percentage of cIgA reduction over time after administration

[0517] Table 17-3 Percentage of cIgG reduction over time after administration

[0518] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details without departing from the spirit and scope of the present application according to all the teachings disclosed herein. The entire scope of the present application is given by the appended claims and any equivalents thereof.

[0519] Sequence information:

Claims

1. A polypeptide comprising a first peptide segment and a second peptide segment; wherein, the first peptide segment comprises or consists of an extracellular domain of a member of the tumor necrosis factor (TNF) receptor family, or a fragment thereof; and the second peptide segment comprises or consists of a trimerization domain derived from cartilage matrix protein or a trimerization domain derived from procollagen.

2. The polypeptide of claim 1, wherein, the first peptide segment comprises a ligand binding domain of the TNF receptor family member.

3. The polypeptide of claim 1 or 2, wherein, the TNF receptor family member is selected from one or more of the following group: TNFR1, TNFR2, BAFFR, BCMA, TACI, OX40, GITR, CD27, 4-1BB, CD40, DcR1, DcR2, DR3, HVEM, LTβR, RANK, Fn14, Fas, TRAILR1, TRAILR2.

4. The polypeptide of any one of claims 1-3, wherein, the first peptide segment comprises or consists of an extracellular domain of BCMA, or a fragment thereof; preferably, the first peptide segment comprises or consists of a ligand binding domain of the BCMA.

5. The polypeptide of claim 4, wherein, the BCMA extracellular domain is selected from a wild-type BCMA extracellular domain and a variant thereof; wherein the BCMA extracellular domain variant has a ligand binding activity of the wild-type BCMA extracellular domain from which it is derived; preferably, the BCMA extracellular domain variant has one or more of the following features: (i) the BCMA extracellular domain variant has reduced glycosylation sites (e.g., N-glycosylation sites) compared to the wild-type BCMA extracellular domain, or the BCMA extracellular domain variant does not contain glycosylation sites (e.g., N-glycosylation sites); preferably, the BCMA extracellular domain variant does not comprise any signature sequence N-X-(S or T), wherein N represents asparagine, X represents any one of amino acids except proline, S represents serine, and T represents threonine; preferably, the amino acid residue at the position corresponding to position 42 of SEQ ID NO: 173 in the BCMA extracellular domain variant is replaced by an amino acid residue other than asparagine residue compared to the wild-type BCMA extracellular domain; preferably, the amino acid residue at the position corresponding to position 42 of SEQ ID NO: 173 in the BCMA extracellular domain variant is replaced by a glutamine residue compared to the wild-type BCMA extracellular domain; (ii) the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 173 in the BCMA extracellular domain variant is replaced by an amino acid residue other than methionine residue compared to the wild-type BCMA extracellular domain; preferably, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 173 in the BCMA extracellular domain variant is replaced by a leucine residue, a valine residue, a glycine residue, a proline residue, or a serine residue compared to the wild-type BCMA extracellular domain; (iii) in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 173 is replaced by an amino acid residue other than a lysine residue, compared to the wild-type BCMA ectodomain; preferably, in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 173 is replaced by an arginine residue, a serine residue, a glutamine residue, a glutamic acid residue, a glycine residue, or an alanine residue, compared to the wild-type BCMA ectodomain; Preferably, the wild-type BCMA ectodomain has: (a) an amino acid sequence as set forth in SEQ ID NO: 173; (b) an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 173; or, (c) a sequence that has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) substitutions (preferably conservative substitutions), additions, or deletions of amino acids, compared to the amino acid sequence set forth in SEQ ID NO: 173; Preferably, the BCMA ectodomain variant has a sequence as set forth in any one of SEQ ID NOs: 175, 181, 183, 185, 187, 189, 235-240, 255-260, 275-280.

6. The polypeptide of claim 4 or 5, wherein, the first peptide segment comprises the amino acid residues at the positions corresponding to positions 8-41 of SEQ ID NO: 173 in the wild-type BCMA ectodomain or the BCMA ectodomain variant; Preferably, the first peptide segment comprises the amino acid residues at positions corresponding to positions 8-41, or 8-42, or 8-43, or 8-44, or 8-45, or 8-46, or 8-47, or 8-48, or 8-49, or 8-51, or 8-54, or 7-41, or 7-42, or 7-43, or 7-44, or 7-45, or 7-46, or 7-47, or 7-48, or 7-49, or 7-51, or 7-54, or 4-41, or 4-42, or 4-43, or 4-44, or 4-45, or 4-46, or 4-47, or 4-48, or 4-49, or 4-51, or 4-54, or 3-41, or 3-42, or 3-43, or 3-44, or 3-45, or 3-46, or 3-47, or 3-48, or 3-49, or 3-51, or 3-54, or 2-41, or 2-42, or 2-43, or 2-44, or 2-45, or 2-46, or 2-47, or 2-48, or 2-49, or 2-51, or 2-54, or 1-41, or 1-42, or 1-43, or 1-44, or 1-45, or 1-46, or 1-47, or 1-48, or 1-49, or 1-51, or 1-54 of SEQ ID NO: 173 of the wild-type BCMA extracellular domain or the BCMA extracellular domain variant.

7. The polypeptide of any one of claims 4-6, wherein, The first peptide segment has a sequence as set forth in any one of SEQ ID NOs: 173-204, 235-280.

8. The polypeptide of any one of claims 1-3, wherein, The first peptide segment comprises or consists of an extracellular domain of TACI or a fragment thereof; Preferably, the first peptide segment comprises or consists of a ligand binding domain of the TACI.

9. The polypeptide of claim 8, wherein, The extracellular domain of TACI has: (a) an amino acid sequence as set forth in SEQ ID NO: 170; (b) an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 170; or (c) a sequence that has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence set forth in SEQ ID NO:

170.

10. The polypeptide of claim 8 or 9, wherein, The first peptide segment comprises the amino acid residues at positions corresponding to positions 71-104 of SEQ ID NO: 170 of the TACI. Preferably, the first peptide segment comprises the amino acid residues in the TACI at positions corresponding to positions 71-104, or 71-110, or 71-118, or 71-130, or 71-165, or 68-104, or 68-110, or 68-118, or 68-130, or 68-165, or 50-104, or 50-110, or 50-118, or 50-130, or 50-165, or 30-104, or 30-110, or 30-118, or 30-130, or 30-165, or 13-104, or 13-110, or 13-118, or 13-130, or 13-165, or 1-104, or 1-110, or 1-118, or 1-130, or 1-165, of SEQ ID NO:

170.

11. The polypeptide of any one of claims 8-10, wherein, The first peptide segment has a sequence as set forth in any one of SEQ ID NOs: 170-172.

12. The polypeptide of any one of claims 1-11, wherein, The second peptide segment comprises or consists of a trimerization domain derived from a cartilage matrix protein; Preferably, the second peptide segment comprises the amino acid residues in the cartilage matrix protein at positions corresponding to positions 458-466 (or 458-480, or 458-496, or 454-466, or 454-480, or 454-496, or 445-466, or 445-480, or 445-496) of SEQ ID NO:

229. Preferably, the second peptide segment comprises the amino acid residues in the cartilage matrix protein at positions corresponding to positions 458-466 (or 458-480, or 458-496, or 454-466, or 454-480, or 454-496, or 445-466, or 445-480, or 445-496) of SEQ ID NO:

229.

13. The polypeptide of claim 12, wherein, The cartilage matrix protein has: (a) an amino acid sequence as set forth in SEQ ID NO: 229; (b) an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 229; or (c) a sequence that has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence set forth in SEQ ID NO: 229; Preferably, the second peptide segment has a sequence as set forth in SEQ ID NO:

230.

14. The polypeptide of any one of claims 1-11, wherein, The second peptide segment comprises a trimerization domain derived from a procollagen protein; Preferably, the second peptide segment comprises a trimerization domain derived from a C-propeptide of a procollagen protein; Preferably, the second peptide segment comprises a trimerization domain derived from the C-propeptide of pro.a.1 (II), pro.a.1 (I), pro.a.2 (I), pro.a.1 (III), pro.a.1 (V), pro.a.2 (V), pro.a.1 (XI), pro.a.2 (XI), or pro.a.3 (XI).

15. The polypeptide of claim 14, wherein, the second peptide segment comprises a trimerization domain derived from the C-propeptide of pro.a.1 (I); Preferably, the second peptide segment comprises the amino acid residues at positions corresponding to positions 1220-1282 (or positions 1220-1300, or positions 1220-1350, or positions 1220-1400, or positions 1220-1464) of SEQ ID NO: 231 in the pro.a.1 (I); Preferably, the second peptide segment comprises the amino acid residues at positions corresponding to positions 1220-1464 of SEQ ID NO: 231 in the pro.a.1 (I).

16. The polypeptide of claim 15, wherein, the pro.a.1 (I) has: (a) the amino acid sequence of SEQ ID NO: 231; (b) an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence of SEQ ID NO: 231; or (c) a sequence that differs from the amino acid sequence of SEQ ID NO: 231 by one or a few (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions; Preferably, the second peptide segment further comprises additional amino acid residues R and S; preferably, the second peptide segment comprises amino acid residues R and S at the N-terminus of the trimerization domain derived from the C-propeptide of pro.a.1 (I); preferably, the second peptide segment comprises, in order from N-terminus to C-terminus, one amino acid residue R and one amino acid residue S at the N-terminus of the trimerization domain derived from the C-propeptide of pro.a.1 (I); Preferably, the second peptide segment has the sequence of SEQ ID NO:

232.

17. The polypeptide of any one of claims 1-16, wherein, the second peptide segment is directly linked or optionally linked via a first linker (e.g., a first peptide linker) to the N-terminus or C-terminus (e.g., C-terminus) of the first peptide segment; For example, the first linker is a peptide linker consisting of 3-35 (e.g., 5-30, 10-30, 15-30) amino acid residues; For example, the first linker comprises the sequence of any one of SEQ ID NOs: 205-219.

18. The polypeptide of any one of claims 1-17, further comprising a single-domain antibody or antigen-binding fragment thereof that specifically binds serum albumin.

19. The polypeptide of claim 18, wherein, the single-domain antibody or antigen-binding fragment thereof comprises: the single-domain antibody or antigen-binding fragment thereof comprises: a CDR1, or a variant thereof, a CDR2, or a variant thereof, a CDR3, or a variant thereof, comprised by a VHH as depicted in any one of SEQ ID NOs: 5, 9, 13, 17, 21, 220-225; said variant has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions or additions in comparison to the sequence from which it is derived; Preferably, said single domain antibody or antigen binding fragment thereof comprises: (1) a CDR1 as depicted in SEQ ID NO: 6; a CDR2 as depicted in SEQ ID NO: 7; and, a CDR3 as depicted in SEQ ID NO: 8; (2) a CDR1 as depicted in SEQ ID NO: 10; a CDR2 as depicted in SEQ ID NO: 11; and, a CDR3 as depicted in SEQ ID NO: 12; (3) a CDR1 as depicted in SEQ ID NO: 14; a CDR2 as depicted in SEQ ID NO: 15; and, a CDR3 as depicted in SEQ ID NO: 16; (4) a CDR1 as depicted in SEQ ID NO: 14; a CDR2 as depicted in SEQ ID NO: 233; and, a CDR3 as depicted in SEQ ID NO: 16; (5) a CDR1 as depicted in SEQ ID NO: 18; a CDR2 as depicted in SEQ ID NO: 19; and, a CDR3 as depicted in SEQ ID NO: 20; (6) a CDR1 as depicted in SEQ ID NO: 18; a CDR2 as depicted in SEQ ID NO: 234; and, a CDR3 as depicted in SEQ ID NO: 20; (7) a CDR1 as depicted in SEQ ID NO: 22; a CDR2 as depicted in SEQ ID NO: 23; and, a CDR3 as depicted in SEQ ID NO: 24; or, (8) a CDR1 as depicted in SEQ ID NO: 226; a CDR2 as depicted in SEQ ID NO: 227; and, a CDR3 as depicted in SEQ ID NO:

228.

20. The polypeptide of claim 19, wherein, said single domain antibody or antigen binding fragment thereof comprises: (i) a sequence as depicted in any one of SEQ ID NOs: 5, 9, 13, 17, 21; (ii) a sequence that differs from the sequence as depicted in any one of SEQ ID NOs: 5, 9, 13, 17, 21 by one or several (e.g. 1, 2, 3, 4 or 5) amino acid substitutions, deletions or additions; or (iii) a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence as depicted in any one of SEQ ID NOs: 5, 9, 13, 17, 21; Preferably, said substitution is a conservative substitution; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21.

21. The polypeptide of claim 19 or 20, wherein, The single-domain antibody or antigen-binding fragment thereof is humanized; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline antibody gene); preferably, the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back-mutations from a human-derived residue to a camelid-derived residue; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises: (i) a sequence as set forth in any one of SEQ ID NOs: 220-225; (ii) a sequence with one or several (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions compared to a sequence as set forth in any one of SEQ ID NOs: 220-225; or (iii) a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 220-225; Preferably, the substitution is a conservative substitution; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NOs: 220-225.

22. The polypeptide of any one of claims 18-21, wherein, The single-domain antibody or antigen-binding fragment thereof is directly linked or optionally linked via a second linker (e.g., a second peptide linker) to the N- or C-terminus of the polypeptide; Preferably, the second linker is a peptide linker consisting of 3-35 (e.g., 5-30, 10-25, 10-30) amino acid residues. Preferably, the second linker comprises a sequence as set forth in any one of SEQ ID NOs: 205-219.

23. The polypeptide of any one of claims 18-22, wherein, The second peptide segment is directly linked or optionally linked via a first linker to the C-terminus of the first peptide segment, and the single-domain antibody or antigen-binding fragment thereof is directly linked or optionally linked via a second linker to the C-terminus of the second peptide segment.

24. The polypeptide of any one of claims 1-23, wherein, The polypeptide has a sequence as set forth in any one of SEQ ID NOs: 34-60, 62-110, 112, 114-169.

25. A trimeric protein complex comprising at least one polypeptide according to any one of claims 1-24.

26. The trimeric protein complex of claim 25, comprising a first polypeptide, a second polypeptide, and a third polypeptide each independently selected from any one of claims 1-24; Preferably, the first polypeptide, the second polypeptide, and the third polypeptide have the same or different first peptide segment, the same or different second peptide segment, the same or different first linker, and / or the same or different second linker from each other; Preferably, the first polypeptide, the second polypeptide, and the third polypeptide have the same or different first peptide segment, the same or different second peptide segment, the same or different first linker, and / or the same or different second linker from each other; Preferably, none of said first polypeptide, said second polypeptide, said third polypeptide comprises a single-domain antibody or an antigen-binding fragment thereof that specifically binds to serum albumin; or, at least one of said first polypeptide, said second polypeptide, said third polypeptide comprises a single-domain antibody or an antigen-binding fragment thereof that specifically binds to serum albumin; Preferably, the trimeric protein complex comprises at least two single-domain antibodies or antigen-binding fragments thereof that specifically bind to serum albumin, wherein, said at least two single-domain antibodies or antigen-binding fragments thereof that specifically bind to serum albumin are identical to or different from each other; Preferably, said single-domain antibody or antigen-binding fragment thereof that specifically binds to serum albumin is as defined in any one of claims 19-21. Preferably, said trimeric protein complex is a homotrimeric protein complex.

27. The trimeric protein complex of claim 25, comprising at least one polypeptide selected from any one of claims 1-24, and an additional polypeptide. For example, said trimeric protein complex comprises a first polypeptide selected from any one of claims 1-24, and additional second and third polypeptides. For example, said trimeric protein complex comprises a first polypeptide and a second polypeptide each independently selected from any one of claims 1-24, and an additional third polypeptide. Preferably, said additional polypeptide comprises a trimerization domain, and optionally (i) an extracellular domain of a member of the TNF receptor family or a fragment thereof, and / or (ii) a single-domain antibody or an antigen-binding fragment thereof that specifically binds to serum albumin. Preferably, said additional polypeptide comprises a trimerization domain identical to or different from the second peptide segment comprised by the polypeptide of any one of claims 1-24, said additional polypeptide optionally comprises an extracellular domain of a member of the TNF receptor family or a fragment thereof identical to or different from the first peptide segment comprised by the polypeptide of any one of claims 1-24, and / or, said additional polypeptide optionally comprises a single-domain antibody or an antigen-binding fragment thereof that specifically binds to serum albumin identical to or different from the single-domain antibody or antigen-binding fragment thereof that specifically binds to serum albumin comprised by the polypeptide of any one of claims 1-24. Preferably, said additional polypeptide comprises a trimerization domain identical to the second peptide segment comprised by the polypeptide of any one of claims 1-24.

28. The trimeric protein complex of any one of claims 25-27, wherein, The polypeptides comprised by said trimeric protein complex form covalent or non-covalent linkages between them.

29. An isolated nucleic acid molecule encoding a polypeptide of any one of claims 1-24, or a trimeric protein complex of any one of claims 25-28.

30. A vector comprising the isolated nucleic acid molecule of claim 29.

31. A host cell comprising the isolated nucleic acid molecule of claim 29 or the vector of claim 30.

32. A method of producing a polypeptide of any one of claims 1-24, or a trimeric protein complex of any one of claims 25-28, comprising culturing the host cell of claim 31 under conditions permitting protein expression, and recovering said polypeptide or said trimeric protein complex from the cultured host cell culture.

33. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 24, a trimeric protein complex according to any one of claims 25 to 28, an isolated nucleic acid molecule according to claim 29, a vector according to claim 30, or a host cell according to claim 31 ; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition comprises a trimeric protein complex according to any one of claims 25 to 28, an isolated nucleic acid molecule encoding said trimeric protein complex, a vector, or a host cell; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an anti-inflammatory drug or an immunosuppressant.

34. Use of a polypeptide according to any one of claims 1 to 24, a trimeric protein complex according to any one of claims 25 to 28, an isolated nucleic acid molecule according to claim 29, a vector according to claim 30, a host cell according to claim 31, or a pharmaceutical composition according to claim 33 for the manufacture of a medicament for the prevention and / or treatment of a B-cell related disease in a subject; Preferably, the treatment of the B-cell related disease can benefit from the inhibition of BLys and / or APRIL mediated signalling; Preferably, the B-cell related disease comprises an autoimmune disease; Preferably, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, Sjogren's syndrome, IgA nephropathy, membranous nephropathy, lupus nephritis, rheumatoid arthritis, neuromyelitis optica, multiple sclerosis, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, ankylosing spondylitis, graft versus host disease, cold agglutinin disease, autoimmune hemolytic anemia, autoimmune cytopenia, pemphigus, pemphigoid, and any combination thereof; Preferably, the subject is a mammal; Preferably, the polypeptide, trimeric protein complex, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition is used alone or in combination with an additional pharmaceutically active agent, such as an anti-inflammatory drug or an immunosuppressant.

35. A method for the prevention and / or treatment of a B-cell related disease in a subject, comprising administering to a subject in need thereof an effective amount of a polypeptide according to any one of claims 1 to 24, a trimeric protein complex according to any one of claims 25 to 28, an isolated nucleic acid molecule according to claim 29, a vector according to claim 30, a host cell according to claim 31, or a pharmaceutical composition according to claim 33; Preferably, the treatment of the B-cell related disease can benefit from the inhibition of BLys and / or APRIL mediated signalling; Preferably, the B-cell related disease comprises an autoimmune disease; Preferably, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, Sjogren's syndrome, IgA nephropathy, membranous nephropathy, lupus nephritis, rheumatoid arthritis, neuromyelitis optica, multiple sclerosis, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, ankylosing spondylitis, graft versus host disease, cold agglutinin disease, autoimmune hemolytic anemia, autoimmune cytopenia, pemphigus, pemphigoid, and any combination thereof; Preferably, the subject is a mammal; Preferably, the polypeptide, trimeric protein complex, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition is used alone or in combination with an additional pharmaceutically active agent, such as an anti-inflammatory drug or an immunosuppressant. Preferably, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, Sjogren's syndrome, IgA nephropathy, membranous nephropathy, lupus nephritis, rheumatoid arthritis, neuromyelitis optica, multiple sclerosis, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, ankylosing spondylitis, graft-versus-host disease, cold agglutinin disease, autoimmune hemolytic anemia, autoimmune cytopenia, pemphigus, pemphigoid, and any combination thereof. Preferably, the subject is a mammal. Preferably, the polypeptide, trimeric protein complex, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., an anti-inflammatory drug or an immunosuppressant).

36. A single-domain antibody or antigen-binding fragment thereof that specifically binds to serum albumin, comprising: a CDR1 or variant thereof, a CDR2 or variant thereof, a CDR3 or variant thereof comprised by a VHH as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21, 220-224; said variant having one or several (e.g., 1, 2, or 3) amino acid substitutions, deletions, or additions as compared to the sequence from which it is derived.

37. The single domain antibody or antigen binding fragment thereof of claim 36, wherein, said single-domain antibody or antigen-binding fragment thereof comprises: (1) a CDR1 as set forth in SEQ ID NO: 6; a CDR2 as set forth in SEQ ID NO: 7; and, a CDR3 as set forth in SEQ ID NO: 8; (2) a CDR1 as set forth in SEQ ID NO: 10; a CDR2 as set forth in SEQ ID NO: 11; and, a CDR3 as set forth in SEQ ID NO: 12; (3) a CDR1 as set forth in SEQ ID NO: 14; a CDR2 as set forth in SEQ ID NO: 15; and, a CDR3 as set forth in SEQ ID NO: 16; (4) a CDR1 as set forth in SEQ ID NO: 14; a CDR2 as set forth in SEQ ID NO: 233; and, a CDR3 as set forth in SEQ ID NO: 16; (5) a CDR1 as set forth in SEQ ID NO: 18; a CDR2 as set forth in SEQ ID NO: 19; and, a CDR3 as set forth in SEQ ID NO: 20; (6) a CDR1 as set forth in SEQ ID NO: 18; a CDR2 as set forth in SEQ ID NO: 234; and, a CDR3 as set forth in SEQ ID NO: 20; or, (7) a CDR1 as set forth in SEQ ID NO: 22; a CDR2 as set forth in SEQ ID NO: 23; and, a CDR3 as set forth in SEQ ID NO:

24.

38. The single domain antibody or antigen binding fragment thereof of claim 36 or 37, wherein, said single-domain antibody or antigen-binding fragment thereof comprises: (i) a sequence as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21; (ii) a sequence which differs from the sequence of any one of SEQ ID NO: 5, 9, 13, 17, 21 by one or several substitutions, deletions, or additions of amino acids (e.g., 1, 2, 3, 4, or 5 substitutions, deletions, or additions of amino acids); or (iii) a sequence which has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence of any one of SEQ ID NO: 5, 9, 13, 17, 21; Preferably, the substitutions are conservative substitutions; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NO: 5, 9, 13, 17, 21.

39. The single domain antigen or antigen-binding fragment thereof of any one of claims 36-38, wherein, The single-domain antibody or antigen-binding fragment thereof is humanized; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline antibody gene); preferably, the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from a human-derived residue to a camelid-derived residue; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises: (i) a sequence as set forth in any one of SEQ ID NO: 220-224; (ii) a sequence which differs from the sequence of any one of SEQ ID NO: 220-224 by one or several substitutions, deletions, or additions of amino acids (e.g., 1, 2, 3, 4, or 5 substitutions, deletions, or additions of amino acids); or (iii) a sequence which has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence of any one of SEQ ID NO: 220-224; Preferably, the substitutions are conservative substitutions; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NO: 220-224.

40. A polypeptide construct that specifically binds to serum albumin, comprising a single-domain antibody or antigen-binding fragment thereof of any one of claims 36-39, and an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is directly linked or optionally linked via a peptide linker to the N- and / or C-terminus (e.g., C-terminus) of the single-domain antibody or antigen-binding fragment thereof; Preferably, the immunoglobulin Fc domain is an Fc domain of IgG (e.g., an Fc domain of IgG1); Preferably, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 25, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared thereto; Preferably, the polypeptide construct comprises a sequence as set forth in any one of SEQ ID NOs: 26-30.

41. A fusion protein comprising the single-domain antibody or antigen-binding fragment thereof of any one of claims 36-39 or the polypeptide construct of claim 40, and, an additional peptide domain; Preferably, the additional peptide domain is selected from a polypeptide domain having a therapeutic effect; Preferably, the additional polypeptide domain is directly linked or, optionally, linked via a peptide linker, to the N- and / or C-terminus of the single-domain antibody or antigen-binding fragment thereof or the polypeptide construct.

42. An isolated nucleic acid molecule encoding the single-domain antibody or antigen-binding fragment thereof of any one of claims 36-39, the polypeptide construct of claim 40, or the fusion protein of claim 41.

43. A vector comprising the isolated nucleic acid molecule of claim 42.

44. A host cell comprising the isolated nucleic acid molecule of claim 42 or the vector of claim 43.

45. A method of making the single-domain antibody or antigen-binding fragment thereof of any one of claims 36-39, the polypeptide construct of claim 40, or the fusion protein of claim 41, comprising culturing the host cell of claim 44 under conditions permitting protein expression, and recovering the single-domain antibody or antigen-binding fragment thereof or the polypeptide construct or the fusion protein from the cultured host cell culture.

46. A bispecific or multispecific antibody comprising the single-domain antibody or antigen-binding fragment thereof of any one of claims 36-39 or the polypeptide construct of claim 40; Preferably, the bispecific or multispecific antibody specifically binds serum albumin and additionally specifically binds one or more other targets; Preferably, the bispecific or multispecific antibody further comprises at least one second antibody or antigen-binding fragment thereof having a second binding specificity for a second target.

47. A conjugate comprising the single-domain antibody or antigen-binding fragment thereof of any one of claims 36-39 or the polypeptide construct of claim 40, and a therapeutic agent linked to the single-domain antibody or antigen-binding fragment thereof or the polypeptide construct.

48. A pharmaceutical composition comprising the single domain antibody or antigen binding fragment thereof of any one of claims 36-39, the polypeptide construct of claim 40, the fusion protein of claim 41, the isolated nucleic acid molecule of claim 42, the vector of claim 43, the host cell of claim 44, the bispecific or multispecific antibody of claim 46, or the conjugate of claim 47; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition comprises the single domain antibody or antigen binding fragment thereof of any one of claims 36-39, the polypeptide construct of claim 40, or a nucleic acid molecule, vector or host cell encoding the single domain antibody or antigen binding fragment thereof or the polypeptide construct; Preferably, the pharmaceutical composition comprises the fusion protein of claim 41, or a nucleic acid molecule, vector or host cell encoding the fusion protein; Preferably, the pharmaceutical composition comprises the bispecific or multispecific antibody of claim 46, or a nucleic acid molecule, vector or host cell encoding the bispecific or multispecific antibody; Preferably, the pharmaceutical composition comprises the conjugate of claim 47.

49. Use of the single domain antibody or antigen binding fragment thereof of any one of claims 36-39, the polypeptide construct of claim 40, the fusion protein of claim 41, the isolated nucleic acid molecule of claim 42, the vector of claim 43, the host cell of claim 44, the bispecific or multispecific antibody of claim 46, the conjugate of claim 47, or the pharmaceutical composition of claim 48 for the manufacture of a medicament; Preferably, the medicament is for preventing and / or treating a serum albumin related disease in a subject; Preferably, the serum albumin related disease is a disease associated with abnormal serum albumin content (e.g., hypoalbuminemia or hyperalbuminemia); Preferably, the medicament is capable of directly or indirectly participating in FcRn-mediated serum albumin circulation in a subject; Preferably, the medicament exhibits an extended in vivo half-life relative to a corresponding medicament lacking the single domain antibody or antigen binding fragment thereof; Preferably, the medicament is a macromolecular drug (e.g., a polypeptide drug); Preferably, the subject is a mammal.

50. A method of extending the half-life of a drug in vivo comprising: the single domain antibody or antigen binding fragment thereof of any one of claims 36-39 or the polypeptide construct of claim 40 is linked to the medicament; Preferably, the extension of the in vivo half-life is relative to the in vivo half-life of the medicament lacking the single domain antibody or antigen binding fragment thereof; Preferably, the medicament is selected from the group consisting of macromolecular drugs (e.g., polypeptide drugs); Preferably, the subject is a mammal.

51. A conjugate comprising the single domain antibody or antigen binding fragment thereof of any one of claims 36-39 or the polypeptide construct of claim 40, and a detectable label linked to the single domain antibody or antigen binding fragment thereof or the polypeptide construct. Preferably, the detectable label is selected from an enzyme (e.g. horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g. acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g. fluorescein or a fluorescent protein), a radionuclide or biotin.

52. A kit comprising the single domain antibody or antigen binding fragment thereof of any one of claims 36-39, the polypeptide construct of claim 40, or the conjugate of claim 51 ; Preferably, the kit comprises the conjugate of claim 51 ; Preferably, the kit comprises the single domain antibody or antigen binding fragment thereof of any one of claims 36-39 or the polypeptide construct of claim 40, and a second antibody or antigen binding fragment thereof that specifically recognizes the single domain antibody or antigen binding fragment thereof or the polypeptide construct; optionally, the second antibody or antigen binding fragment thereof further comprises a detectable label, such as an enzyme (e.g. horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g. acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g. fluorescein or a fluorescent protein), a radionuclide or biotin.

53. A method for detecting the presence or level of serum albumin in a sample, comprising using the single domain antibody or antigen binding fragment thereof of any one of claims 36-39, the polypeptide construct of claim 40, or the conjugate of claim 51 ; Preferably, the method is an immunological detection, such as an immunoblotting method, an enzyme immunoassay (e.g. ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay or a radioimmunoassay; Preferably, the method comprises using the conjugate of claim 51 ; Preferably, the method comprises using the single domain antibody or antigen binding fragment thereof of any one of claims 36-39 or the polypeptide construct of claim 40, and the method further comprises using a second antibody or antigen binding fragment thereof carrying a detectable label, such as an enzyme (e.g. horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g. acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g. fluorescein or a fluorescent protein), a radionuclide or biotin, to detect the single domain antibody or antigen binding fragment thereof or the polypeptide construct; Preferably, the method comprises: (1) contacting the sample with the single domain antibody or antigen binding fragment thereof or the polypeptide construct or the conjugate; (2) detecting the formation of antigen-antibody immune complexes or detecting the amount of the immune complexes.

54. Use of the single domain antibody or antigen binding fragment thereof of any one of claims 36-39, the polypeptide construct of claim 40, or the conjugate of claim 51 in the manufacture of a detection reagent for detecting the presence or level of serum albumin in a sample and / or diagnosing a disease associated with serum albumin.

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