Fc fusion protein which blocks action of BAFF and april on receptors thereof

Abstract

Provided in the present invention are an Fc fusion protein capable of inhibiting BAFF and APRIL signaling, a pharmaceutical composition containing the Fc fusion protein, and a related use.

Description

Fc fusion proteins that block the action of BAFF, APRIL on their receptors

[0001] This application claims the benefit of Chinese application No. 202411319512.4, filed on September 20, 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 specifically, the present application relates to Fc fusion proteins capable of inhibiting BAFF and APRIL signaling, pharmaceutical compositions containing the Fc fusion proteins, and related uses. BACKGROUND

[0003] B cells are associated with autoimmune diseases, such as systemic lupus erythematous (SLE), and the like. This is due to their ability to 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 the production of pathogenic autoantibodies. Thus, 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, and the blockade 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 achieve 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 an Fc fusion protein molecule capable of efficiently blocking the binding of Blys and / or APRIL to its receptor, efficiently inhibiting the activity of Blys and APRIL, thereby being able to inhibit the signal transduction mediated by the binding of Blys and / or APRIL to its receptor, and further being able to reduce the amount of autoantibodies, which is expected to be used for the treatment of B cell-related diseases (e.g., autoimmune diseases). Further, the Fc 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), high expression, easy purification, low production cost, and can be better applied to large-scale industrial production.

[0009] Therefore, in a first aspect, the present application provides a polypeptide comprising a first peptide segment, a second peptide segment and a third peptide segment; wherein the first peptide segment comprises or consists of the extracellular domain of BCMA or a fragment thereof; the second peptide segment comprises or consists of the extracellular domain of TACI or a fragment thereof; and the third peptide segment comprises or consists of an Fc peptide derived from an immunoglobulin and an optional hinge peptide.

[0010] In certain embodiments, the first peptide segment, the second peptide segment, and the third peptide segment are arranged in any order.

[0011] In certain embodiments, the polypeptide comprises, in order from N-terminus to C-terminus:

[0012] (i) the first peptide segment, the second peptide segment, and the third peptide segment;

[0013] (ii) the first peptide segment, the third peptide segment, and the second peptide segment;

[0014] (iii) the second peptide segment, the first peptide segment, and the third peptide segment;

[0015] (iv) the second peptide segment, the third peptide segment, and the first peptide segment;

[0016] (v) the third peptide segment, the first peptide segment, and the second peptide segment; or

[0017] (vi) the third peptide segment, the second peptide segment, and the first peptide segment.

[0018] In particular, the present inventors have found that when the BCMA ectodomain or a fragment thereof is located at the N-terminus of the Fc peptide and the TACI ectodomain or a fragment thereof is located at the C-terminus of the Fc peptide, the Fc fusion protein has superior target binding activity and / or Blys and / or APRIL blocking activity to other Fc fusion proteins.

[0019] In certain embodiments, the polypeptide comprises, in order from N-terminus to C-terminus: the first peptide segment, the third peptide segment, and the second peptide segment.

[0020] In certain embodiments, the polypeptide comprises, in order from N-terminus to C-terminus: the second peptide segment, the third peptide segment, and the first peptide segment.

[0021] In certain embodiments, the polypeptide comprises, in order from N-terminus to C-terminus: the first peptide segment, the second peptide segment, and the third peptide segment.

[0022] In certain embodiments, the polypeptide comprises, in order from N-terminus to C-terminus: the second peptide segment, the first peptide segment, and the third peptide segment.

[0023] In certain embodiments, each adjacent peptide segment of the polypeptide is optionally connected by or without a linker (e.g., connected by a linker or directly connected).

[0024] In certain embodiments, each of the linkers is independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker).

[0025] In certain embodiments, the peptide linker is independently selected from the group consisting of a peptide linker comprising one or more glycine (G) and / or serine (S).

[0026] In certain embodiments, the peptide linker is independently selected from the group consisting of a peptide linker having a structure as set forth in (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. n

[0027] In certain embodiments, the peptide linker is independently selected from the group consisting of a peptide linker having a structure as set forth in (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. n

[0028] In certain embodiments, the peptide linker independently comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 47-52.

[0029] In certain embodiments, the polypeptide comprises, in order from N-terminus to C-terminus:

[0030] (1) the first peptide segment, the third peptide segment, and the second peptide segment;

[0031] (2) the second peptide segment, the third peptide segment, and the first peptide segment;

[0032] (3) the first peptide segment, the second peptide segment, and the third peptide segment; or,

[0033] (4) the second peptide segment, the first peptide segment, and the third peptide segment;

[0034] and the third peptide segment is C-terminally linked to a peptide linker as set forth in SEQ ID NO: 50.

[0035] In certain embodiments, the first peptide segment comprises or consists of a ligand binding domain of the BCMA.

[0036] In certain embodiments, the BCMA ectodomain is selected from the group consisting of a wild-type BCMA ectodomain and a variant thereof; wherein the BCMA ectodomain variant has a ligand binding activity of the wild-type BCMA ectodomain from which it is derived.

[0037] In certain embodiments, the BCMA ectodomain variant has one or more of the following features:

[0038] ​​(i) the BCMA ectodomain variant has reduced glycosylation sites (e.g., N-glycosylation sites) compared to the wild-type BCMA ectodomain, or the BCMA ectodomain variant does not contain glycosylation sites (e.g., N-glycosylation sites); in certain embodiments, the BCMA ectodomain variant does not comprise any signature sequence N-X-(S or T), 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: 28 replaced by an amino acid residue other than an asparagine residue 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: 28 replaced by a glutamine residue compared to the wild-type BCMA ectodomain;

[0039] (ii) the BCMA ectodomain variant has the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 28 replaced by an amino acid residue other than a methionine residue 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 4 of SEQ ID NO: 28 replaced by a glycine residue or a proline residue compared to the wild-type BCMA ectodomain;

[0040] (iii) the BCMA ectodomain variant has the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 28 replaced by an amino acid residue other than a lysine residue 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 50 of SEQ ID NO: 28 replaced by a glutamic acid residue or a glycine residue compared to the wild-type BCMA ectodomain.

[0041] It is readily understood by one skilled in the art that 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:

[0042] (1) the N residue is deleted or replaced by one or more other amino acid residues (e.g., one non-N amino acid residue);

[0043] (2) the (S or T) residue is deleted or replaced by one or more other amino acid residues (e.g., one non-S and non-T amino acid residue);

[0044] (3) the X residue is deleted or replaced by a proline residue;

[0045] (4) one or more amino acid residues (e.g., one amino acid residue other than N) are added between the N residue and the X residue; and

[0046] (5) one or more amino acid residues (e.g., one amino acid residue other than S and other than T) are added between the X residue and the (S or T) residue;

[0047] Thus, the obtained BCMA ectodomain variant does not comprise any of the signature sequences N-X-(S or T).

[0048] In certain embodiments, the BCMA ectodomain variant does not comprise any of the signature sequences N-X-(S or T) as compared to the wild-type BCMA ectodomain, and in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 28 (e.g., a methionine residue) is replaced by an amino acid residue other than a methionine residue.

[0049] In certain embodiments, the BCMA ectodomain variant does not comprise any of the signature sequences N-X-(S or T) as compared to the wild-type BCMA ectodomain, and in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 28 (e.g., a methionine residue) is replaced by a glycine residue or a proline residue.

[0050] In certain embodiments, the BCMA ectodomain variant comprises a substitution of the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 28 by an amino acid residue other than lysine as compared to the wild-type BCMA ectodomain. In certain embodiments, the BCMA ectodomain variant comprises a substitution of the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 28 by a glutamic acid or a glycine residue as compared to the wild-type BCMA ectodomain.

[0051] In certain embodiments, the BCMA ectodomain variant does not comprise any of the signature sequences N-X-(S or T) as compared to the wild-type BCMA ectodomain, and in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 28 (e.g., a lysine residue) is replaced by an amino acid residue other than a lysine residue.

[0052] In certain embodiments, the BCMA ectodomain variant does not comprise any of the signature sequences N-X-(S or T) as compared to the wild-type BCMA ectodomain, and in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 28 (e.g., a lysine residue) is replaced by a glutamic acid residue or a glycine residue.

[0053] In certain embodiments, in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 28 (e.g., a methionine residue) is replaced with an amino acid residue other than a methionine residue, and the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 28 (e.g., a lysine residue) is replaced with an amino acid residue other than a lysine residue, as compared to the wild-type BCMA ectodomain.

[0054] In certain embodiments, in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 28 (e.g., a methionine residue) is replaced with a glycine residue or a proline residue, and the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 28 (e.g., a lysine residue) is replaced with a glutamic acid residue or a glycine residue, as compared to the wild-type BCMA ectodomain.

[0055] In certain embodiments, the BCMA ectodomain variant does not comprise any of the signature sequences N-X-(S or T), and, in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 28 (e.g., a methionine residue) is replaced with an amino acid residue other than a methionine residue, and the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 28 (e.g., a lysine residue) is replaced with an amino acid residue other than a lysine residue, as compared to the wild-type BCMA ectodomain.

[0056] In certain embodiments, the BCMA ectodomain variant does not comprise any of the signature sequences N-X-(S or T), and, in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 28 (e.g., a methionine residue) is replaced with a glycine residue or a proline residue, and the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 28 (e.g., a lysine residue) is replaced with a glutamic acid residue or a glycine residue, as compared to the wild-type BCMA ectodomain.

[0057] In certain embodiments, the wild-type BCMA ectodomain has: (a) an amino acid sequence as set forth in SEQ ID NO: 28; (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: 28; 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: 28.

[0058] In certain embodiments, the BCMA ectodomain variant has an amino acid sequence as set forth in any one of SEQ ID NOs: 29-33, 39, 41.

[0059] In certain embodiments, the BCMA is human BCMA.

[0060] In certain embodiments, the first peptide segment comprises the amino acid residues at positions corresponding to positions 8-41 of SEQ ID NO: 28 in the BCMA (e.g., the wild-type BCMA ectodomain or the BCMA ectodomain variant).

[0061] As used herein, the expression “the amino acid residues at positions corresponding to positions 8-41 of SEQ ID NO: 28 in the BCMA (e.g., wild-type BCMA ectodomain or the BCMA ectodomain variant)” means the amino acid positions / residues in the sequence of the BCMA (e.g., wild-type BCMA ectodomain or the BCMA ectodomain variant) under comparison that are located at equivalent positions to the amino acid residues at positions 8-41 of SEQ ID NO: 28 when the sequence of the BCMA (e.g., wild-type BCMA ectodomain or the BCMA ectodomain variant) is optimally aligned with SEQ ID NO: 28, i.e., when the sequence of the BCMA (e.g., wild-type BCMA ectodomain or the BCMA ectodomain variant) is aligned with SEQ ID NO: 28 to obtain the highest percentage identity.

[0062] Unless specifically indicated otherwise or apparently contradicted by context, the meaning of the remaining similar expressions herein is defined by analogy with the above.

[0063] In certain embodiments, the first peptide segment comprises or consists of 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: 28 in the BCMA (e.g., the wild type BCMA ectodomain or the BCMA ectodomain variant).

[0064] In certain embodiments, the first peptide segment comprises or consists of amino acid residues at positions corresponding to positions 1-51, 2-51, 1-49, 1-45, or 1-54 of SEQ ID NO: 28 in the BCMA (e.g., the wild type BCMA ectodomain or the BCMA ectodomain variant).

[0065] In certain embodiments, the first peptide segment has an amino acid sequence as set forth in any one of SEQ ID NOs: 28-46.

[0066] In certain embodiments, the second peptide segment comprises or consists of the ligand binding domain of the TACI.

[0067] In certain embodiments, the TACI extracellular domain has: (a) an amino acid sequence as set forth in SEQ ID NO: 25; (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: 25; 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: 25.

[0068] In certain embodiments, the TACI is a human TACI.

[0069] In certain embodiments, the second peptide segment comprises the amino acid residues in the TACI (e.g., the TACI extracellular domain) at positions corresponding to positions 71-104 of SEQ ID NO: 25.

[0070] In certain embodiments, the second peptide segment comprises or consists of the amino acid residues in the TACI (e.g., the TACI extracellular domain) 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: 25.

[0071] In certain embodiments, the second peptide segment comprises or consists of the amino acid residues in the TACI (e.g., the TACI extracellular domain) at positions corresponding to positions 68-110 of SEQ ID NO: 25.

[0072] In certain embodiments, the second peptide segment comprises the amino acid residues in the TACI (e.g., the TACI extracellular domain) at positions corresponding to positions 35-60 of SEQ ID NO: 25.

[0073] In certain embodiments, the second peptide segment comprises or consists of the amino acid residues in the TACI corresponding to positions 35-60, or 35-67, or 35-110, or 35-165, or 30-60, or 30-67, or 30-110, or 30-165, or 13-60, or 13-67, or 13-110, or 13-165, or 1-60, or 1-67, or 1-110, or 1-165 of SEQ ID NO: 25.

[0074] In certain embodiments, the second peptide segment comprises or consists of the amino acid residues in the TACI corresponding to positions 30-67 of SEQ ID NO: 25.

[0075] In certain embodiments, the second peptide segment has an amino acid sequence as set forth in any one of SEQ ID NOs: 25-27.

[0076] In certain embodiments, the Fc peptide is an Fc peptide derived from an immunoglobulin (e.g., an IgG, such as IgG1, IgG2, IgG3, IgG4).

[0077] In certain embodiments, the Fc peptide is an Fc peptide derived from a human immunoglobulin (e.g., a human IgG, such as human IgG1, human IgG2, human IgG3, human IgG4).

[0078] As will be readily understood by those skilled in the art, the Fc peptide derived from an immunoglobulin (e.g., a human immunoglobulin) includes both an Fc peptide directly derived from an immunoglobulin (e.g., a human immunoglobulin) and an Fc peptide (e.g., an Fc peptide variant) obtained by engineering or modification of an Fc peptide derived from an immunoglobulin (e.g., a human immunoglobulin).

[0079] In certain embodiments, the Fc peptide is selected from the group consisting of an Fc peptide of a wild-type human immunoglobulin and a variant thereof; wherein the variant has an altered property as compared to the Fc peptide of the wild-type human immunoglobulin.

[0080] In certain embodiments, the Fc peptide variant has an altered (e.g., enhanced or reduced or ablated) Fc effector function (e.g., ADCC, CDC and / or ADCP activity), an extended half-life, and / or, an enhanced stability (e.g., reduced aggregation, fragmentation and / or degradation) as compared to the Fc peptide of the wild-type human immunoglobulin.

[0081] In certain embodiments, the Fc peptide variant has reduced or ablated Fc effector functions (e.g., ADCC, CDC, and / or ADCP activity) compared to the Fc peptide of a wild-type human immunoglobulin.

[0082] In certain embodiments, the Fc peptide is selected from the Fc peptide of wild-type human immunoglobulin IgGl and variants thereof; wherein the Fc peptide variant has altered properties compared to the Fc peptide of wild-type human immunoglobulin IgGl.

[0083] In certain embodiments, the Fc peptide variant has altered (e.g., enhanced or reduced or ablated) Fc effector functions (e.g., ADCC, CDC, and / or ADCP activity), prolonged half-life, and / or enhanced stability (e.g., reduced aggregation, fragmentation, and / or degradation) compared to the Fc peptide of wild-type human immunoglobulin IgGl.

[0084] In certain embodiments, the Fc peptide variant has reduced or ablated Fc effector functions (e.g., ADCC, CDC, and / or ADCP activity) compared to the Fc peptide of wild-type human immunoglobulin IgGl.

[0085] In certain embodiments, the Fc peptide variant has reduced FcyR binding activity, reduced serum complement molecule (Clq) binding activity, enhanced FcRn binding activity, and / or enhanced stability (e.g., reduced aggregation, fragmentation, and / or degradation) compared to the Fc peptide of wild-type human immunoglobulin IgGl.

[0086] In certain embodiments, the Fc peptide variant comprises mutations selected from the group consisting of L234A, L235E, G237A mutations, C-terminal lysine deletion (e.g., K447 deletion), and any combination thereof compared to the Fc peptide of wild-type human immunoglobulin IgGl. In certain embodiments, the positions of the mutations are defined by the Eu numbering system.

[0087] In certain embodiments, the Fc peptide variant comprises mutations selected from the group consisting of (i) L234A, L235E, and G237A, (ii) C-terminal lysine deletion (e.g., K447 deletion), and (iii) a combination of (i) and (ii) compared to the Fc peptide of wild-type human immunoglobulin IgGl. In certain embodiments, the positions of the mutations are defined by the Eu numbering system.

[0088] In certain embodiments, the Fc peptide variant comprises mutations L234A, L235E, and G237A, as compared to the Fc peptide of wild-type human immunoglobulin IgGl. In certain embodiments, the Fc peptide variant further comprises a C-terminal lysine deletion (e.g., K447 deletion). In certain embodiments, the positions of the mutations are defined by the Eu numbering system.

[0089] In certain embodiments, the Fc peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 54, and 55.

[0090] In certain embodiments, the third peptide segment comprises or consists of the Fc peptide and a hinge peptide derived from an immunoglobulin.

[0091] In certain embodiments, the hinge peptide is optionally connected to the N-terminus of the Fc peptide with or without a linker (e.g., connected with a linker or directly connected).

[0092] In certain embodiments, the hinge peptide is a hinge peptide derived from an immunoglobulin (e.g., IgG, such as IgGl, IgG2, IgG3, IgG4).

[0093] In certain embodiments, the hinge peptide is a hinge peptide derived from a human immunoglobulin (e.g., human IgG, such as human IgGl, human IgG2, human IgG3, human IgG4).

[0094] As readily understood by one skilled in the art, the hinge peptide derived from an immunoglobulin (e.g., human immunoglobulin) includes both a hinge peptide directly derived from an immunoglobulin (e.g., human immunoglobulin) and a hinge peptide obtained by engineering or modification of a hinge peptide derived from an immunoglobulin (e.g., human immunoglobulin) (e.g., an artificially designed hinge peptide).

[0095] In certain embodiments, the hinge peptide is selected from the group consisting of a hinge peptide of a wild-type human immunoglobulin (e.g., human IgG, such as human IgGl, human IgG2, human IgG3, human IgG4) and a variant thereof.

[0096] In certain embodiments, the hinge peptide variant comprises a mutation that reduces protein aggregation and improves drugability, such as a C220S mutation, as compared to the wild-type hinge peptide from which it is derived. In certain embodiments, the position of the mutation is defined by the Eu numbering system.

[0097] In certain embodiments, the hinge peptide comprises an amino acid sequence as set forth in SEQ ID NO: 23 or 53.

[0098] In certain preferred embodiments, the hinge peptide is at the N-terminus of the Fc peptide.

[0099] In some embodiments, the first peptide segment, the second peptide segment, and the third peptide segment are each independently derived from the same or different species.

[0100] In some embodiments, the polypeptide further comprises a signal peptide and / or a detectable label (e.g., a tag protein).

[0101] In some 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.

[0102] In some embodiments, the polypeptide has a sequence as set forth in any one of SEQ ID NOs: 5-7, 9, 12-18, 56-60.

[0103] In some embodiments, the polypeptide is capable of blocking the binding of human BLys and / or APRIL to its receptor.

[0104] In some embodiments, the polypeptide is capable of blocking the binding of murine BLys and / or APRIL to its receptor.

[0105] In some embodiments, the polypeptide is capable of blocking the binding of monkey BLys and / or APRIL to its receptor.

[0106] In some embodiments, the polypeptide has cross-binding activity to human, monkey, and murine BLys.

[0107] In some embodiments, the polypeptide has cross-binding activity to human, monkey, and murine APRIL.

[0108] In some embodiments, the polypeptide has good drugability, e.g., has good stability (e.g., has reduced aggregation, fragmentation, and / or degradation; e.g., the peptide bond between one or more amino acid residues at the termini (N-terminus and / or C-terminus) has good stability) and / or reduced amino acid modification (e.g., glycosylation modification, hydroxylation modification).

[0109] In a second aspect, the present application provides a polypeptide dimer comprising at least one polypeptide of the first aspect.

[0110] In some embodiments, the polypeptide dimer comprises a first polypeptide and a second polypeptide; the first polypeptide is selected from the polypeptides of the first aspect.

[0111] In some embodiments, the first polypeptide and the second polypeptide are each independently selected from the polypeptides of the first aspect.

[0112] In certain embodiments, said first polypeptide, said second polypeptide comprise the same or different said first peptide segment, the same or different said second peptide segment, and / or, the same or different said third peptide segment.

[0113] In certain embodiments, said first polypeptide is the same as said second polypeptide.

[0114] In certain embodiments, said polypeptide dimer is a homomeric polypeptide dimer.

[0115] In certain embodiments, said second polypeptide comprises or consists of an Fc peptide derived from an immunoglobulin and optionally a hinge peptide.

[0116] In certain embodiments, said second polypeptide comprises an Fc peptide as defined in the first aspect, and / or, said second polypeptide comprises a hinge peptide as defined in the first aspect.

[0117] In certain embodiments, said second polypeptide comprises the same Fc peptide as said first polypeptide, and / or, said second polypeptide comprises the same hinge peptide as said first polypeptide.

[0118] In certain embodiments, said second polypeptide further comprises an extracellular domain or a fragment thereof selected from a member of the TNF receptor family; preferably, said member of the TNF receptor family is selected from one or more of the following group: TNFR1, TNFR2, BAFFR, BCMA, TACI, OX40, GITR, CD27, 4-1 BB, CD40, DcR1, DcR2, DR3, HVEM, LTβR, RANK, Fn14, Fas, TRAILR1, TRAILR2.

[0119] In certain embodiments, said second polypeptide further comprises: (i) an extracellular domain or a fragment thereof of BCMA, and / or, (ii) an extracellular domain or a fragment thereof of TACI.

[0120] In certain embodiments, said second polypeptide comprises an extracellular domain or a fragment thereof of BCMA selected from the first peptide segment as defined in the first aspect, and / or, said second polypeptide comprises an extracellular domain or a fragment thereof of TACI selected from the second peptide segment as defined in the first aspect.

[0121] In certain embodiments, said second polypeptide comprises the same extracellular domain or fragment thereof of BCMA as said first polypeptide comprises of the first peptide segment, and / or, said second polypeptide comprises the same extracellular domain or fragment thereof of TACI as said first polypeptide comprises of the second peptide segment.

[0122] In certain embodiments, each of the adjacent peptide segments of the second polypeptide is optionally connected by or without a linker (e.g., connected by a linker or directly connected). In certain embodiments, each of the linkers is independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, each of the peptide linkers is independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, each of the peptide linkers is independently selected from a peptide linker having a structure as set forth in (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 1, 2, 3, or 4. In certain embodiments, each of the peptide linkers independently comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 47-52. n In certain embodiments, each of the adjacent peptide segments of the second polypeptide is connected by or without a linker (e.g., connected by a linker or directly connected). In certain embodiments, each of the linkers is independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, each of the peptide linkers is independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, each of the peptide linkers is independently selected from a peptide linker having a structure as set forth in (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 1, 2, 3, or 4. In certain embodiments, each of the peptide linkers independently comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 47-52.

[0123] In certain embodiments, the polypeptide dimer comprises a covalent and / or non-covalent linkage between the polypeptides.

[0124] In certain embodiments, the polypeptide dimer comprises a covalent linkage between the polypeptides.

[0125] In certain embodiments, the polypeptide dimer comprises the first polypeptide and the second polypeptide; wherein the first polypeptide comprises a sequence as set forth in any one of SEQ ID NOs: 5-7, 9, 12-18, 56-60, and / or the second polypeptide comprises a sequence as set forth in any one of SEQ ID NOs: 5-7, 9, 12-18, 56-60.

[0126] In certain embodiments, the polypeptide dimer comprises the first polypeptide and the second polypeptide; wherein:

[0127] (1) each of the first polypeptide and the second polypeptide independently comprises a sequence as set forth in SEQ ID NO: 5;

[0128] (2) each of the first polypeptide and the second polypeptide independently comprises a sequence as set forth in SEQ ID NO: 6;

[0129] (3) each of the first polypeptide and the second polypeptide independently comprises a sequence as set forth in SEQ ID NO: 7;

[0130] (4) each of the first polypeptide and the second polypeptide independently comprises a sequence as set forth in SEQ ID NO: 9;

[0131] (5) each of the first polypeptide and the second polypeptide independently comprises a sequence as set forth in SEQ ID NO: 12;

[0132] (6) the first polypeptide and the second polypeptide each independently comprises a sequence as set forth in SEQ ID NO: 13;

[0133] (7) the first polypeptide and the second polypeptide each independently comprises a sequence as set forth in SEQ ID NO: 14;

[0134] (8) the first polypeptide and the second polypeptide each independently comprises a sequence as set forth in SEQ ID NO: 15

[0135] (9) the first polypeptide and the second polypeptide each independently comprises a sequence as set forth in SEQ ID NO: 16;

[0136] (10) the first polypeptide and the second polypeptide each independently comprises a sequence as set forth in SEQ ID NO: 17;

[0137] (11) the first polypeptide and the second polypeptide each independently comprises a sequence as set forth in SEQ ID NO: 18;

[0138] (12) the first polypeptide and the second polypeptide each independently comprises a sequence as set forth in SEQ ID NO: 56;

[0139] (13) the first polypeptide and the second polypeptide each independently comprises a sequence as set forth in SEQ ID NO: 57;

[0140] (14) the first polypeptide and the second polypeptide each independently comprises a sequence as set forth in SEQ ID NO: 58;

[0141] (15) the first polypeptide and the second polypeptide each independently comprises a sequence as set forth in SEQ ID NO: 59; or,

[0142] (16) the first polypeptide and the second polypeptide each independently comprises a sequence as set forth in SEQ ID NO: 60.

[0143] In certain embodiments, the polypeptide dimer is capable of blocking the binding of human BLys and / or APRIL to its receptor.

[0144] In certain embodiments, the polypeptide dimer is capable of blocking the binding of murine BLys and / or APRIL to its receptor.

[0145] In certain embodiments, the polypeptide dimer is capable of blocking the binding of monkey BLys and / or APRIL to its receptor.

[0146] In certain embodiments, the polypeptide dimer possesses cross-binding activity to human, monkey, murine BLys.

[0147] In some embodiments, the polypeptide dimer has cross-binding activity with human, monkey, murine APRIL.

[0148] In some embodiments, the polypeptide dimer has good drugability, for example, has good stability (e.g., has reduced aggregation, fragmentation and / or degradation; for example, the peptide bond between one or more amino acid residues of the terminus (N-terminus and / or C-terminus) has good stability) and / or reduced amino acid modification (e.g., glycosylation modification, hydroxylation modification).

[0149] In a third aspect, the present application provides an isolated nucleic acid molecule encoding the polypeptide of the first aspect, or the polypeptide dimer of the second aspect.

[0150] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide of the first aspect.

[0151] In some embodiments, the isolated nucleic acid molecule encodes the polypeptide dimer of the second aspect, the polypeptide dimer comprising a first polypeptide and a second polypeptide; the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first polypeptide, and a second nucleotide sequence encoding the second polypeptide.

[0152] In some embodiments, the first nucleotide sequence, the second nucleotide sequence are the same or different from each other.

[0153] In some embodiments, the first nucleotide sequence, the second nucleotide sequence are present on the same or different isolated nucleic acid molecules.

[0154] In a fourth aspect, the present application provides a vector comprising the isolated nucleic acid molecule of the third aspect.

[0155] In some embodiments, the vector comprises a nucleotide sequence encoding the polypeptide of the first aspect.

[0156] In some embodiments, the vector encodes the polypeptide dimer of the second aspect, the polypeptide dimer comprising a first polypeptide and a second polypeptide; the vector comprises a first nucleotide sequence encoding the first polypeptide, and a second nucleotide sequence encoding the second polypeptide.

[0157] In some embodiments, the first nucleotide sequence, the second nucleotide sequence are the same or different from each other.

[0158] In some embodiments, the first nucleotide sequence, the second nucleotide sequence are present on the same or different vector molecules.

[0159] 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.

[0160] 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.

[0161] The polypeptide or polypeptide dimer of the present application can be produced in various methods known in the art, for example, by genetic engineering recombination techniques. For example, a DNA molecule encoding the polypeptide or polypeptide dimer 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 polypeptide or polypeptide dimer of the present application is expressed.

[0162] In a sixth aspect, the present application provides a method of producing the polypeptide of the first aspect, or the polypeptide dimer of the second aspect, comprising culturing the host cell of the fifth aspect under conditions permitting expression of the protein, and recovering the polypeptide or the polypeptide dimer from the culture of the host cell.

[0163] In a seventh aspect, the present application provides a pharmaceutical composition comprising the polypeptide of the first aspect, the polypeptide dimer 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.

[0164] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0165] In certain embodiments, the pharmaceutical composition comprises the polypeptide dimer of the second aspect, an isolated nucleic acid molecule encoding the polypeptide dimer, a vector, or a host cell.

[0166] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an anti-inflammatory drug or an immunosuppressant.

[0167] In an eighth aspect, the present application provides use of the polypeptide of the first aspect, the polypeptide dimer 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.

[0168] In certain embodiments, the prevention and / or treatment of the B cell-related disease can benefit from the inhibition of BLys and / or APRIL-mediated signal transduction.

[0169] In certain embodiments, the B-cell related disease comprises an autoimmune disease.

[0170] 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.

[0171] In certain preferred embodiments, the autoimmune disease is systemic lupus erythematosus or IgA nephropathy.

[0172] In certain embodiments, the subject is a mammal.

[0173] In certain embodiments, the polypeptide, polypeptide dimer, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition is used alone or in combination (e.g., concurrently or sequentially) with another pharmaceutically active agent (e.g., an anti-inflammatory drug or an immunosuppressive agent).

[0174] In a ninth aspect, the present application provides a method of 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 polypeptide dimer 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.

[0175] In certain embodiments, the prevention and / or treatment of the B-cell related disease can benefit from the inhibition of BLys and / or APRIL-mediated signal transduction.

[0176] In certain embodiments, the B-cell related disease comprises an autoimmune disease.

[0177] 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.

[0178] In certain preferred embodiments, the autoimmune disease is systemic lupus erythematosus or IgA nephropathy.

[0179] In certain embodiments, the subject is a mammal.

[0180] In certain embodiments, the polypeptide, polypeptide dimer, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition is used alone or in combination with (e.g., concurrently or sequentially administered with) another pharmaceutically active agent (e.g., an anti-inflammatory drug or an immunosuppressant).

[0181] The polypeptide or polypeptide dimer 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 polypeptide dimer 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 polypeptide dimer 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 (e.g., by vacuum drying or freeze-drying), for ease of storage and use. Such lyophilized powders can be reconstituted into sterile injection solutions just prior to use, e.g., by the addition of 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, or any combination thereof.

[0182] The polypeptide or polypeptide dimer 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, intracerebrospinal, inguinal, intravesical, local (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration / mode 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 and / or mode of administration will vary depending upon the desired goal. In certain embodiments, the polypeptide or polypeptide dimer or pharmaceutical composition of the present application is administered by intravenous injection or bolus.

[0183] Terminology Definitions

[0184] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. Also, the virology, biochemistry, immunology laboratory procedures used herein are conventional procedures well established in the respective fields. Also, for better understanding of the present application, the definitions and explanations of the related terms are provided below.

[0185] When the terms "for example", "for instance", "such as", "including", "containing" or variations thereof are used in this document, these terms are not to be interpreted in an exclusive sense, but are to be interpreted as specifying the presence of the stated features, steps or components, but not precluding the presence of one or more additional features, steps or components.

[0186] Unless otherwise indicated herein, or in the context of a contradiction with the context, the terms "a" and "an" and the like are to be construed to cover both the singular and the plural. Thus, for example, reference to "a" or "an" entity also means "one or more" of that entity.

[0187] As used herein, "BCMA", B-cell maturation antigen, has the meaning commonly understood by one of ordinary 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. The specific amino acid sequence of the wild-type BCMA protein can be obtained from public databases (e.g., GenBank database). One of ordinary 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: 28, but also includes various species of origin having BCMA with an extracellular region different from that shown in SEQ ID NO: 28.

[0188] As used herein, the term "ligand binding domain of BCMA" has the meaning understood by one of ordinary skill in the art, which generally means a part or all of the extracellular region of the extracellular region of BCMA responsible for binding to APRIL or BLys or other ligands.

[0189] As used herein, "TACI", i.e., transmembrane activator and calcium-modulator and cyclophilin ligand interactor, has the meaning generally understood by those skilled in the art, and is important in the activation, proliferation and immunoregulation of B cells, etc. The specific amino acid sequence of wild-type TACI protein can be obtained from public databases (e.g., GenBank database). Those skilled in the art understand 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 includes not only TACI having an extracellular region as shown in SEQ ID NO: 25, but also various species of origin having TACI with an extracellular region different from that shown in SEQ ID NO: 25.

[0190] As used herein, the term "ligand binding domain of TACI" has the meaning understood by those skilled in the art, which generally means a portion or all of the extracellular region of TACI in the extracellular region of TACI responsible for binding to APRIL or BLys or other ligands.

[0191] As used herein, the term "Fc peptide", "Fc", "Fc fragment" or "Fc region" means an antibody fragment formed by the second constant region (CH2) and the third constant region (CH3) of the immunoglobulin heavy chain. The Fc fragment of an antibody has various functions, but is not involved in the binding of antigens. For example, the Fc peptide can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system.

[0192] As used herein, the term "hinge peptide" or "hinge region" means the hinge peptide of the immunoglobulin heavy chain, which is located at the C-terminus of the CH1 region of the immunoglobulin heavy chain, for connecting the CH2 region of the immunoglobulin heavy chain. The hinge peptide usually contains cysteine, which can be used to form a disulfide bond between two heavy chains containing the hinge peptide of the immunoglobulin, to mediate dimerization between two Fc peptides contained in the two heavy chains.

[0193] As used herein, the term "identity" is used in reference to the match 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 sequence 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.

[0194] 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, modified as 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.

[0195] As used herein, the term "variant", in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide that comprises 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 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.

[0196] 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 elements 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 (YACs), bacterial artificial chromosomes (BACs) or P1 -derived artificial chromosomes (PACs); 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 virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0197] 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.

[0198] 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).

[0199] 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.

[0200] 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.

[0201] 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, vanadium), 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 polyadenylic acid, uridylic acid), oil agents (such as Freund's complete adjuvant, peanut 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.

[0202] 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.

[0203] 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.

[0204] 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 already having the disease. Determining such effective amounts is well within the capability of those skilled in the art. For example, an amount effective for therapeutic purposes 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 sex, the mode of administration of a drug, and other therapies being administered concurrently, and the like.

[0205] Advantages of the Invention

[0206] The Fc fusion protein molecules provided herein are capable of inhibiting the signal transduction mediated by the binding of Blys and / or APRIL to its receptors, and thus are capable of reducing the production of autoantibodies, and are expected to be useful for treating B cell related diseases (e.g., autoimmune diseases). Further, the Fc fusion proteins of the present application also have improved drugability, for example, have better stability and / or reduced amino acid modifications (e.g., glycosylation modification, hydroxylation modification), low production cost, and are capable of better application in large-scale industrial production.

[0207] Embodiments of the present application will be described in detail with reference to the attached drawings and examples, but it will be understood that the 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, taken in conjunction with the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0208] Figure 1A: Results of detection of blocking activity of C241, C242, C243 and control molecules against hAPRIL.

[0209] Figure 1B: Results of detection of blocking activity of C241, C242, C243 and control molecules against hBLys.

[0210] Figure 2A: Results of detection of blocking activity of C271, C272, C266 and control molecules against hAPRIL.

[0211] Figure 2B: Results of detection of blocking activity of C271, C272, C266 and control molecules against hBLys.

[0212] Figure 3A: Results of detection of blocking activity of C248, C249 and control molecules against hAPRIL.

[0213] Figure 3B: Results of detection of blocking activity of C248, C249 and control molecules against hBLys.

[0214] Figure 4A: Results of detection of blocking activity of C271, C272, C264, C269 and control molecules against hAPRIL.

[0215] Figure 4B: Results of detection of blocking activity of C271, C272, C264, C269 and control molecules against hBLys.

[0216] Figure 5A: Results of detection of blocking activity of C269, C338, C339, C340 against hAPRIL.

[0217] Figure 5B: Results of detection of blocking activity of C269, C338, C339, C340 against hBLys.

[0218] Figure 6A: Results of detection of blocking activity of C340, C341, C405, C406, C407 against hAPRIL.

[0219] Figure 6B: Results of detection of blocking activity of C340, C341, C405, C406, C407 and control molecules against hBLys.

[0220] Figure 7A: Results of mass spectrometric detection of C406.

[0221] Figures 7B-7F are results of mass spectrometric detection of C683v, C684v, C687v, C695v, C703v, respectively.

[0222] Figure 8A: Results of detection of blocking activity of C683v, C684v, C687v, C695v, C703v against hAPRIL.

[0223] Figure 8B: Results of detection of blocking activity of C683v, C684v, C687v, C695v, C703v against hBLys.

[0224] Figure 9A: Results of detection of mouse anti-KLH IgM antibodies of each group.

[0225] Figure 9B: Results of detection of mouse anti-KLH IgG antibodies of each group.

[0226] Figure 10: Results of detection of spleen cell number at the end point of experiment of each group of mice. DETAILED DESCRIPTION

[0227] The present application will now be described with reference to the following examples which are intended to be illustrative, but not limiting, of the present application.

[0228] Unless otherwise indicated, the molecular biology and immunological techniques utilized 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 in terms of preferred embodiments, and that the present application is not intended to be limited to the preferred embodiments described in the examples.

[0229] Preparation Example

[0230] 1. Preparation of antigen hBlys ECD-N-His, hBlys ECD-N-mIgG1 Fc, mBlys ECD-N-mIgG1 Fc, hApril-N-mIgG1 Fc molecules for detection

[0231] 1.1 Transient expression of antigen, protein for detection

[0232] The transient expression vector for expressing hBlys ECD-N-His, hBlys ECD-N-mIgG1Fc, mBlys ECD-N-mIgG1Fc, hApril-N-mIgG1Fc was constructed, wherein the amino acid sequences of hBlys ECD-N-His, hBlys ECD-N-mIgG1Fc, mBlys ECD-N-mIgG1Fc, hApril-N-mIgG1Fc are respectively shown as SEQ ID NO: 19-22, hBlys ECD-N-His is a fusion protein formed by fusing His tag at the N terminus of hBlys extracellular domain, and hBlys ECD-N-mIgG1Fc, mBlys ECD-N-mIgG1Fc, hApril-N-mIgG1Fc are respectively fusion proteins formed by fusing Fc domain of mouse IgG1 at the N terminus of hBlys extracellular domain, mBlys extracellular domain, and hApril extracellular domain.

[0233] The transient transfection expression of the antigen was carried out using 293E cells in Freestyle medium. 24 hours before transfection, 0.5×10 6 The cells were inoculated in 1000ml cell culture bottles at 300ml of 0.5×10

[0234] In addition, other exemplary antigens used in the present application can be commercially available, and the corresponding manufacturers and item numbers are as follows:

[0235] hApril-His (purchased from ACRO, item number: APL-H52D1); mBlys (purchased from Bio-Techne, item number: 8876-BF-010 / CF); mApril (purchased from ImmunoChemistry Technologies, item number: 6490); cApril (purchased from MCE, item number: A0A2K5TJA1); cBlys (purchased from MCE, item number: A0A2K5V2X4).

[0236] Wherein, hApril, mApril, cApril represent human, mouse, cynomolgus April protein respectively, hBlys, mBlys, cBlys represent human, mouse, cynomolgus Blys protein respectively.

[0237] 1.2 Purification and detection of antigen protein

[0238] His tag protein purification: collect supernatant, filter the supernatant with 0.22 micron filter membrane, use Ni column affinity chromatography, load into the Ni chromatography column which has been equilibrated with 20mM PB-500mM NaCl, pH7.4 buffer, after equilibration with 20mM PB-500mM NaCl, pH7.4 buffer, elute, elution conditions are as follows: (1) use A liquid: (20mM PB-500mM NaCl, pH7.4) + 4% B liquid: (20mM PB-500mM NaCl-500mM imidazole, pH7.4) for elution, collect samples according to the ultraviolet absorption peak type, after the ultraviolet absorption peak is stable and does not fluctuate, carry out the second step elution; (2) use A liquid: (20mM PB-500mM NaCl, pH7.4) + 20% B liquid: (20mM PB-500mM NaCl-500mM imidazole, pH7.4) for elution, collect samples according to the ultraviolet absorption peak type, after the ultraviolet absorption peak is stable and does not fluctuate, carry out the third step elution; (3) use A liquid: (20mM PB-500mM NaCl, pH7.4) + 60% B liquid: (20mM PB-500mM NaCl-500mM imidazole, pH7.4) for elution, collect samples according to the ultraviolet absorption peak type, after the ultraviolet absorption peak is stable and does not fluctuate, stop collection. The purified sample is detected by SDS-PAGE with 4-20% gradient gel.

[0239] mFc tag protein purification: collect supernatant, filter the supernatant with 0.22 micron filter membrane, pass through Prism A (GE) affinity chromatography, elute with 20mM citric acid-citric acid sodium, pH 3.0, adjust the pH to neutral with 1M Tris base. The purified sample is detected by SDS-PAGE with 4-20% gradient gel to detect the purified protein sample.

[0240] 1.3 Preparation of positive control protein

[0241] Telitacicept (P01, SEQ ID NO: 1) was used as a positive control protein. The original drug of Telitacicept (Rongchang Biotech) was purchased, dissolved according to the instructions, and stored at -80°C after sub-packaging.

[0242] 2. Preparation of CHO-K1 cells expressing hTACI and mTACI proteins on cell surface

[0243] The gene encoding human TACI (hTACI) protein (Gene ID: AF023614.1) and the gene encoding mouse TACI (mTACI) protein (Gene ID: AF257673.1) were cloned into the pLvx-Puro-IRES plasmid, respectively, to construct overexpression lentivirus plasmids of hTACI and mTACI proteins.

[0244] A three-plasmid system (envelope plasmid pCMV-VSVG, packaging plasmid pCMV-dr8.91, and target plasmid pLvx master plasmid) was used for lentivirus packaging and concentration to obtain a lentivirus solution. The human TACI series was infected with the lentivirus solution, and the mouse TACI series was infected with the lentivirus solution. After infection, the infected cells were subjected to pressure screening with puromycin (Puro) to obtain a resistant pool (Pool). The expression of hTACI and mTACI proteins was detected by FACs. The identified positive Pool cells were subjected to single cloning by FACs sorting method, and overexpression hTACI CHO-K1 cell lines and overexpression mTACI Jurkat cell lines were obtained after expansion and identification.

[0245] Example: Fc fusion protein design and expression

[0246] Preparation of TACI and BCMA Fc fusion proteins

[0247] 1. Design of TACI and BCMA Fc fusion proteins

[0248] The Fc fusion protein monomer of the present application comprises a functional domain (e.g., an extracellular ligand binding domain) selected from TACI and / or BCMA, and an immunoglobulin Fc domain. Specifically, the amino acid sequences of the Fc fusion proteins and control molecules of the present application are shown in Table 1.

[0249] Table 1, Fc fusion protein molecule number and sequence information

[0250] 2. Purification and detection of Fc fusion proteins

[0251] 2.1 Transfection, expression and detection in mammalian cell ExpiCHO-S

[0252] Transient transfection expression of candidate molecules was performed using ExpiCHO-S in ExpiCHO Expression Medium. 24 hours before transfection, 3-4 x 10 6 cells / ml of ExpiCHO-S cells were inoculated in 500 mL cell culture flask and incubated at 37°C, 8% CO2 incubator, 120 rpm shaker. Cell density was determined before transfection, when reached 6-8 x 10 6 cells / ml, the cell density was adjusted to 6 x 10 6 cells / ml for transfection. 100 μg of expression plasmid (constructed on pcDNA3.4) for candidate molecules was diluted to 4 ml using OptiPRO SFM; 320 μl of ExpiFectamine CHO Reagent was added to 3.68 ml OptiPRO SFM and mixed well, the diluted transfection reagent and plasmid were mixed immediately and incubated at room temperature for 2 minutes, then the mixture was added to the cells and mixed well, and incubated at 37°C, 8% CO2 incubator, 120 rpm shaker. 600 μl of ExpiFectamine CHO Enhancer was added to the culture system 18-22 hours after transfection. 16 ml of ExpiCHO Feed, and the culture conditions were adjusted to 32°C, 5% CO2, 120 rpm. 16 ml of ExpiCHO Feed was added on the fifth day after transfection. Incubated for 6-7 days.

[0253] 2.2 Purification and detection of Fc fusion protein

[0254] The supernatant was collected and filtered using a 0.22 micron filter, and then subjected to PrismA (GE) affinity chromatography, eluted using 20 mM citric acid-sodium citrate, pH 3.0, and the pH was adjusted to neutral using 1 M Tris base. The purified sample was subjected to SDS-PAGE detection using 4-20% gradient gel. The results are shown in Table 2.

[0255] Table 2, Fc fusion protein molecule expression and purification detection results

[0256] Example: Evaluation of biological activity of Fc fusion protein

[0257] 1 Reporter gene method for detecting Fc fusion protein molecule in vitro activity detection

[0258] CHO-K1-hTACI-NFκB-Luc cells (constructed by ourselves, the CHO-K1 cells express hTACI and contain NF-κB luciferase reporter system) were adjusted to 3x10 5 cells / ml, 100 μl / well were inoculated in 96-well full white plates (CORNING, 3917), i.e. 3x10 4 cells / well, and incubated in a 37°C, 5% CO2 incubator for 18-20 h. The experimental medium was diluted with the sample 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 experimental medium was diluted with hApril-His or BLyS protein (hBlys ECD-N-His, self-made), 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 (Nanjing Zannan Biotechnology Co., Ltd., DD1204-02) 100 μl / well was added, and the light value was detected by SpectraMax iD5 multifunctional enzyme labeler (Molecular Devices) after 3 min of dark room temperature standing.

[0259] Reporter gene inhibition (%) = 100% x [(RLU APRIL / BLyS -RLU SAMPLE ) / (RLU APRIL / BLyS -RLU cell )].

[0260] 1) In vitro activity detection of TACI and BCMA individual domain Fc fusion proteins

[0261] Based on TACI aa.30-67, TACI aa.68-110, and BCMA aa.1-51, TACI aa.30-67, TACI aa.68-110, and BCMA aa.1-51 fusion proteins were constructed at the N-terminal or C-terminal of Fc domain (which contains the hinge region as shown in SEQ ID NO: 53 and the Fc region as shown in SEQ ID NO: 55). The corresponding molecular names are C241, C242, C243, C271, C272, and C266, respectively.

[0262] The in vitro activity of P01, C241, C242, C243, C271, C272, and C266 was detected, and the results are shown in FIGS. 1A-1B and 2A-2B. According to the results, C241 is worse than P01 in inhibiting April activity and BLys activity. C242 is worse than P01 in inhibiting April activity, and is equivalent to P01 in inhibiting BLys activity. C243 is worse than P01 in inhibiting BLys activity, and is superior to the P01 molecule in inhibiting April activity. C266 is equivalent to P01 in inhibiting BLys activity, and is significantly superior to the P01 molecule in inhibiting April activity. C271 is worse than P01 in inhibiting April activity and BLys activity. C272 is worse than P01 in inhibiting April activity, and is significantly superior to the P01 molecule in inhibiting BLys activity.

[0263] According to the above results, the molecules constructed in the present application, in the case of only containing TACI or BCMA connected with Fc, cannot simultaneously enhance the inhibitory activity against April and BLys relative to P01.

[0264] 2) In vitro activity detection of TACI, BCMA domain combination Fc fusion protein

[0265] TACI domain selection TACI aa.68-110, BCMA selection BCMA aa.1-51, combination with Fc domain (containing hinge region as shown in SEQ ID NO: 53 and Fc region as shown in SEQ ID NO: 55) to construct Fc fusion protein molecules, TACI(aa.68-110)-BCMA(aa.1-51)-Fc, BCMA(aa.1-51)-TACI(aa.68-110)-Fc, TACI(aa.68-110)-Fc-BCMA(aa.1-51), BCMA(aa.1-51)-Fc-TACI(aa.68-110) molecules were constructed, and the corresponding molecular names were C248, C249, C264, and C269, respectively.

[0266] Result analysis

[0267] 1) In vitro activity detection of P01, C248, C249, C264, and C269 molecules, and the results are shown in FIGS. 3A-3B and 4A-4B. According to the results, C248, C249, C264, and C269 are significantly superior to the P01 molecule in inhibiting BLys and April activity.

[0268] 2) Comparison of C248 and C249 results

[0269] Table 3, C248, C249 structure and inhibitory activity

[0270] C248 and C249 only differ in the connection order of the domains. As shown in Table 3 and Figure 3A-3B, C248 has better inhibitory activity against April than C249, and has similar inhibitory activity against Blys. Meanwhile, C248 and C249 have higher inhibitory activity against BLys and April than P01. This indicates that when BCMA (aa. 1-51) and TACI (aa. 68-110) are both located at the N-terminus of Fc, it is more advantageous to locate TACI (aa. 68-110) at the N-terminus of BCMA (aa. 1-51) to improve the inhibitory activity of the constructed molecule against April.

[0271] 3) Comparison of C264 and C269 results

[0272] Table 4, C264, C269 structure and inhibitory activity

[0273] C264 and C269 differ in the connection order of the domains. As shown in Table 4 and Figure 4A-4B, C269 has better inhibitory activity against April and Blys than C264. Meanwhile, C264 and C269 have higher inhibitory activity against BLys and April than P01. This indicates that when BCMA (aa. 1-51) and TACI (aa. 68-110) are located at both ends of Fc, especially when BCMA (aa. 1-51) is connected to the N-terminus of Fc and TACI (aa. 68-110) is connected to the C-terminus of Fc (i.e., BCMA-Fc-TACI), it can greatly improve the inhibitory activity against BLys and April.

[0274] 4) Comparison of inhibitory activity of C264, C269, C271 and C272

[0275] Meanwhile, the inhibitory activity of C264, C269, C271, C272 and P01 was also compared, as shown in Figure 4A and Figure 4B and Table 5.

[0276] Table 5, comparison of inhibitory activity of C264, C269, C271, C272

[0277] Note: "-" represents that it cannot be fitted.

[0278] As can be seen from Figure 4A, C269 is significantly better than C264 in inhibiting April activity, and both C269 and C264 are significantly better than P01, while C271 and C272 are worse than P01. That is, among C264, C269, C271 and C272, C269 has the strongest inhibitory activity against April, and both C269 and C264 are significantly better than P01.

[0279] Similarly, for the inhibitory activity against BLys (Figure 4B), C269 is significantly better than C264 and C272, and C269, C264 and C272 are stronger than P01, while C271 is worse than P01. That is, among C269, C264, C271 and C272, C269 has the strongest inhibitory activity against BLys, and both C269 and C264 are significantly better than P01.

[0280] In summary, when the construct contains both BCMA (aa. 1-51) and TACI (aa. 68-110) connected to Fc (e.g., BCMA (aa. 1-51) and TACI (aa. 68-110) are located at both ends of Fc, in particular, BCMA (aa. 1-51) is connected to the N-terminus of Fc, and TACI (aa. 68-110) is connected to the C-terminus of Fc (i.e., BCMA-Fc-TACI)), it can greatly improve the inhibitory activity against BLys and April relative to the construct containing only BCMA (aa. 1-51) or TACI (aa. 68-110) connected to Fc.

[0281] At the same time, the construct containing only TACI (aa. 68-110) or BCMA (aa. 1-51) connected to Fc cannot simultaneously enhance the inhibitory activity against April and BLys relative to P01. In contrast, when the constructed molecule contains BCMA (aa. 1-51), TACI (aa. 68-110) and Fc, the constructed molecule can significantly enhance the inhibitory activity against April and BLys at the same time.

[0282] 3) In vitro activity detection of BCMA domain deglycosylation optimized molecules

[0283] Select molecules C248, C249, C269 for optimization of the 42nd glycosylation site of BCMA, and mutate the 42nd site N to Q for deglycosylation design, respectively obtain molecules C338, C339, C340.

[0284] The in vitro activity of P01, C269, C338, C339, C340 molecules was detected, and the results are shown in Figures 5A-5B and Table 6. As can be seen from the results, C338, C339, C340 all maintain high activity to block the action of April and Blys.

[0285] Table 6, C338, C339, C340 structure and inhibition activity

[0286] C338, C339, C340, these three molecules only differ in the connection mode of BCMA, TACI and Fc. As can be seen from Table 6 and Figures 5A-5B, the three molecules have equivalent inhibition activity against April. In terms of inhibition activity against Blys, C340 and C339 are superior to C338, indicating that the molecule produced by using the connection mode of BCMA-Fc-TACI has the strongest inhibition effect against April and Blys, especially the inhibition activity against Blys is improved.

[0287] Table 7, C269, C340 structure and inhibition activity comparison

[0288] The difference between C269 and C340 is only the mutation of N42Q on BCMA, i.e. the N at the 42nd site is mutated to Q, and the deglycosylation design is performed. As can be seen from Table 7 and Figures 5A-5B, C269 and C340 are equivalent in terms of inhibition activity against April and Blys, indicating that the removal of glycosylation does not affect the blocking activity of the molecule against April and Blys.

[0289] Table 8, Fc fusion protein molecule expression and purification detection results

[0290] Table 8 is extracted from Table 2, and the difference between C269 and C340 is only the N42Q mutation of the BCMA domain in C340. As can be seen from Table 8, from the C269 molecule, it can be seen that the deglycosylation mutation of N42Q greatly improves the purity of the fusion protein molecule after purification (from 75% to more than 90%), indicating that the deglycosylation mutation at the N42 site helps the purification of the Fc fusion protein molecule.

[0291] 4) Mutational optimization for BCMA

[0292] Based on C340 molecule, the Fc domain C-terminal linker was optimized to "RNTGRGGEEKGGGGS" (SEQ ID NO: 50), and the Fc domain N-terminal sequence was designed as BCMA (aa. 1-51, N42Q), BCMA (aa. 2-51, N42Q), BCMA (aa. 1-51, M4P, N42Q), BCMA (aa. 1-51, M4G, N42Q) respectively to obtain molecules C341, C405, C406, C407.

[0293] The in vitro activity of P01, C340, C341, C405, C406, C407 molecules was detected, and the results are shown in FIGS. 6A-6B. As can be seen from the results, C341, C405, C406, C407 all maintain high activity to block the action of April and Blys.

[0294] Table 9 below is extracted from Table 2 and shows the effect of the linker from another aspect.

[0295] Table 9, replacement of the linker leads to an increase in expression

[0296] C340 and C341 differ in that the Fc C-terminal linker of C341 is replaced by RNTGRGGEEKGGGGS (SEQ ID NO: 50). Table 9 shows that the expression of the fusion protein of C341 with this linker is 1.8 times that of C340 (without this linker). C405, C406 and C407 all have this linker, and the expression is also greatly improved.

[0297] This experiment shows that the Fc C-terminal linker "RNTGRGGEEKGGGGS" (SEQ ID NO: 50) can lead to a significant increase in the protein expression of the constructed fusion protein molecules.

[0298] Table 10, comparison of C341, C406, C407 structure and effect

[0299] Compared with C341, C406 contains M4P mutation in the BCMA domain, and C407 contains M4G mutation in the BCMA domain. As can be seen from Table 10 and FIGS. 6A-6B, the inhibition effect of C406 and C407 on April and Blys is significantly stronger than that of C341, especially the inhibition activity on Blys, which shows that the mutation of the 4th position of BCMA significantly enhances the inhibition effect of the fusion protein on April and Blys.

[0300] 5) Drug optimization

[0301] Mass spectrometry PTM modification detection was performed on C406 molecules, and it was found that the molecule had modification leading to heterogeneity, and the 50th amino acid K might have a "+16D" modification (hydroxylation modification). The C-terminal of the BCMA domain was truncated to the 1st-49th position, or the 50th amino acid K of BCMA was mutated to amino acid E, G, respectively, and the linker was replaced, and BCMA (aa.1-51) was replaced with BCMA (aa.1-54), to obtain C683v, C684v, C687v, C695v, C703v molecules, and the structure is shown in Table 11.

[0302] Table 11, C683v, C684v, C687v, C695v, C703v structure

[0303] In vitro activity detection and mass spectrometry reduced molecular weight detection were performed on the above molecules, and the results are shown in Figures 7B-7F and 8A-8B. It can be seen from the results that C683v, C684v, C687v, C695v, C703v all maintain high activity to block April and Blys, and can reduce the proportion of "+16D" modification (hydroxylation modification), and can better improve the drugability.

[0304] 6) KLH pharmacodynamic model verifies the in vivo pharmacodynamics of Fc fusion protein

[0305] A mouse KLH immune model was used to evaluate the effect of immunomodulatory molecules on antigen-specific responses to B cells, T cell-dependent antigen KLH after one or two injections of KLH. To start the KLH study, 10-week-old female C57BL / 6N mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were randomly divided into 10 groups, 6 mice in each group. On day 0 and day 12, mice were administered 0.25 mg KLH (Keyhole limpet hemocyanin; Biosyn; Cat: KLH-NP) via intraperitoneal (IP) injection; the KLH solution was diluted to 1.25 μg / μL with PBS before injection. The test article was administered to the mice via IP injection on days 4 and 11 as outlined in Table 12, and the test article was administered at an equivalent molar molecular weight. On day 20, after the mice were anesthetized with 1-4% isoflurane, blood was collected via the orbital venous plexus, and the blood sample was collected to evaluate the anti-KLH antibody level. At the end of the experiment, the animals were euthanized by carbon dioxide and decapitation, and the spleens of all experimental animals were collected and weighed, and the spleen cell count was performed.

[0306] Table 12, mouse dose regimen and grouping

[0307] The results are shown in Figures 9A-9B and Figure 10. The results show that the C341 and C406 molecules are superior to P01 in terms of IgM, IgG inhibition and the magnitude of the decrease in splenocytes.

[0308] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alterations to the details can be made within the scope of the application as disclosed in the above teachings without departing from the spirit and scope of the application. The application is defined by the following claims and any equivalents thereof.

[0309] Sequence information:

Claims

1. A polypeptide comprising a first peptide segment, a second peptide segment, and a third peptide segment; wherein, the first peptide segment comprises or consists of an extracellular domain of BCMA or a fragment thereof; the second peptide segment comprises or consists of an extracellular domain of TACI or a fragment thereof; and the third peptide segment comprises or consists of an Fc peptide derived from an immunoglobulin and optionally a hinge peptide.

2. The polypeptide of claim 1 or 2, wherein, the first peptide segment, the second peptide segment and the third peptide segment are arranged in any order; preferably, the polypeptide comprises, in order from N-terminus to C-terminus: the first peptide segment, the third peptide segment and the second peptide segment; preferably, the polypeptide comprises, in order from N-terminus to C-terminus: the second peptide segment, the third peptide segment and the first peptide segment; preferably, the polypeptide comprises, in order from N-terminus to C-terminus: the first peptide segment, the second peptide segment and the third peptide segment; preferably, the polypeptide comprises, in order from N-terminus to C-terminus: the second peptide segment, the first peptide segment and the third peptide segment.

3. The polypeptide of claim 1 or 2, wherein, each adjacent peptide segment of the polypeptide is optionally connected by or without a linker (e.g., connected by a linker or directly connected); preferably, the linkers are each independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker); preferably, the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S); For example, the peptide linker is each independently selected from a peptide linker having the structure of (GGGGS)n n (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 1, 2, 3, or 4. for example, the peptide linkers are each independently comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 47-52.

4. The polypeptide of any one of claims 1-3, 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 the 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) or does not contain glycosylation sites (e.g., N-glycosylation sites) as compared to the wild-type BCMA extracellular domain; 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: 28 in the BCMA extracellular domain variant is replaced by an amino acid residue other than asparagine residue as compared to the wild-type BCMA extracellular domain; preferably, the amino acid residue at the position corresponding to position 42 of SEQ ID NO: 28 in the BCMA extracellular domain variant is replaced by a glutamine residue as compared to the wild-type BCMA extracellular domain; (ii) the BCMA extracellular domain variant has reduced immunogenicity as compared to the wild-type BCMA extracellular domain; (ii) in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 28 is replaced by an amino acid residue other than a methionine residue, compared to the wild-type BCMA ectodomain; preferably, in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 4 of SEQ ID NO: 28 is replaced by a glycine residue or a proline residue, compared to the wild-type BCMA ectodomain; (iii) in the BCMA ectodomain variant, the amino acid residue at the position corresponding to position 50 of SEQ ID NO: 28 is replaced by an amino acid residue other than a lysine, 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: 28 is replaced by a glutamic acid or a glycine 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: 28; (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: 28; 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: 28; Preferably, the BCMA ectodomain variant has an amino acid sequence as set forth in any one of SEQ ID NOs: 29-33, 39, 41.

5. The polypeptide of any one of claims 1-4, wherein, the first peptide segment comprises the amino acid residues at the positions corresponding to positions 8-41 of SEQ ID NO: 28 in the BCMA (e.g., 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: 28 in the BCMA (e.g., the wild-type BCMA ectodomain or the BCMA ectodomain variant). More preferably, the first peptide segment comprises the amino acid residues at positions corresponding to positions 1-51, 2-51, 1-49, 1-45, or 1-54 of SEQ ID NO: 28 in the BCMA (e.g., the wild-type BCMA ectodomain or the BCMA ectodomain variant).

6. The polypeptide of any one of claims 1-5, wherein, The first peptide segment has an amino acid sequence as set forth in any one of SEQ ID NOs: 28-46.

7. The polypeptide of any one of claims 1-6, wherein, The TACI ectodomain has: (a) an amino acid sequence as set forth in SEQ ID NO: 25; (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: 25; 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:

25.

8. The polypeptide of any one of claims 1-7, wherein, the second peptide segment comprises amino acid residues in the TACI (e.g., the TACI extracellular domain) at positions corresponding to positions 71-104 of SEQ ID NO: 25; preferably, the second peptide segment comprises amino acid residues in the TACI (e.g., the TACI extracellular domain) 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: 25, more preferably, the second peptide segment comprises amino acid residues in the TACI (e.g., the TACI extracellular domain) at positions corresponding to positions 68-110 of SEQ ID NO:

25.

9. The polypeptide of any one of claims 1-7, wherein, the second peptide segment comprises amino acid residues in the TACI (e.g., the TACI extracellular domain) at positions corresponding to positions 35-60 of SEQ ID NO: 25; preferably, the second peptide segment comprises amino acid residues in the TACI (e.g., the TACI extracellular domain) at positions corresponding to positions 30-67 of SEQ ID NO:

25. the second peptide segment has an amino acid sequence as set forth in any one of SEQ ID NOs: 25-27.

10. The polypeptide of any one of claims 1-9, wherein, the Fc peptide is an Fc peptide derived from an immunoglobulin (e.g., IgG, such as IgG1, IgG2, IgG3, IgG4); 11. The polypeptide of any one of claims 1-10, wherein, preferably, the Fc peptide is an Fc peptide derived from a human immunoglobulin (e.g., human IgG, such as human IgG1, human IgG2, human IgG3, human IgG4); preferably, the Fc peptide is an Fc peptide derived from a human immunoglobulin (e.g., human IgG, such as human IgG1, human IgG2, human IgG3, human IgG4); Preferably, the Fc peptide is selected from the Fc peptide of wild-type human immunoglobulin IgG1 and variants thereof; wherein the Fc peptide variant has altered properties compared to the Fc peptide of wild-type human immunoglobulin IgG1 ; Preferably, the Fc peptide variant has altered (e.g., enhanced or reduced or ablated) Fc effector functions (e.g., ADCC, CDC and / or ADCP activities), prolonged half-life, and / or, enhanced stability (e.g., reduced aggregation, fragmentation and / or degradation) compared to the Fc peptide of wild-type human immunoglobulin IgG1 ; Preferably, the Fc peptide variant has reduced or ablated Fc effector functions (e.g., ADCC, CDC and / or ADCP activities) compared to the Fc peptide of wild-type human immunoglobulin IgG1 ; Preferably, the Fc peptide variant has reduced FcyR binding activity, reduced serum complement molecule (C1q) binding activity, enhanced FcRn binding activity, and / or, enhanced stability (e.g., reduced aggregation, fragmentation and / or degradation) compared to the Fc peptide of wild-type human immunoglobulin IgG1 ; Preferably, the Fc peptide variant comprises mutations selected from the group consisting of L234A, L235E, G237A mutations, C-terminal lysine deletion (e.g., K447 deletion), and any combination thereof compared to the Fc peptide of wild-type human immunoglobulin IgG1 ; Preferably, the Fc peptide variant comprises mutations selected from the group consisting of (i) L234A, L235E and G237A, (ii) C-terminal lysine deletion (e.g., K447 deletion), and (iii) a combination of (i) and (ii) compared to the Fc peptide of wild-type human immunoglobulin IgG1 ; Preferably, the Fc peptide variant comprises mutations L234A, L235E and G237A; preferably, the Fc peptide variant further comprises C-terminal lysine deletion (e.g., K447 deletion) compared to the Fc peptide of wild-type human immunoglobulin IgG1 ; Preferably, the Fc peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 54 and 55.

12. The polypeptide of any one of claims 1-11, wherein, The third peptide segment comprises or consists of the Fc peptide and a hinge peptide derived from an immunoglobulin; Preferably, the hinge peptide is optionally linked (e.g., linked by a linker or directly linked) to the N-terminus of the Fc peptide by or without a linker; Preferably, the hinge peptide is a hinge peptide derived from an immunoglobulin (e.g., IgG, such as IgG1, IgG2, IgG3, IgG4); Preferably, the hinge peptide is a hinge peptide derived from a human immunoglobulin (e.g., human IgG, such as human IgG1, human IgG2, human IgG3, human IgG4); Preferably, the hinge peptide is selected from the group consisting of a hinge peptide of wild-type human immunoglobulin (e.g., human IgG, such as human IgG1, human IgG2, human IgG3, human IgG4) and variants thereof; Preferably, the hinge peptide variant comprises a mutation that reduces protein aggregation, such as a C220S mutation compared to the wild-type hinge peptide from which it is derived; Preferably, the hinge peptide comprises an amino acid sequence as set forth in SEQ ID NO: 23 or 53.

13. The polypeptide of any one of claims 1-12, wherein, The polypeptide has a sequence as set forth in any one of SEQ ID NOs: 5-7, 9, 12-18, 56-60.

14. A polypeptide dimer comprising at least one polypeptide according to any one of claims 1-13.

15. The polypeptide dimer according to claim 14, comprising a first polypeptide and a second polypeptide; the first polypeptide being selected from a polypeptide according to any one of claims 1-13.

16. The polypeptide dimer of claim 15, wherein, each of the first polypeptide and the second polypeptide is independently selected from a polypeptide according to any one of claims 1-13; Preferably, the first polypeptide, the second polypeptide have the same or different first peptide segment, the same or different second peptide segment, and / or, the same or different third peptide segment, between each other; Preferably, the polypeptide dimer is a homomultimeric polypeptide dimer.

17. The polypeptide dimer of claim 15, wherein, The second polypeptide comprises or consists of an Fc peptide and optionally a hinge peptide derived from an immunoglobulin; Preferably, the second polypeptide comprises an Fc peptide as defined in claim 11, and / or, the second polypeptide comprises a hinge peptide as defined in claim 12; Preferably, the second polypeptide comprises an Fc peptide identical to the Fc peptide comprised by the first polypeptide, and / or, the second polypeptide comprises a hinge peptide identical to the hinge peptide comprised by the first polypeptide; Preferably, the second polypeptide further comprises an extracellular domain or a fragment thereof selected from a member of the TNF receptor family; preferably, the member of the TNF receptor family 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; Preferably, the second polypeptide further comprises: (i) an extracellular domain or a fragment thereof of BCMA, and / or, (ii) an extracellular domain or a fragment thereof of TACI; Preferably, the extracellular domain or a fragment thereof of BCMA comprised by the second polypeptide is selected from the first peptide segment as defined in any one of claims 1-6, and / or, the extracellular domain or a fragment thereof of TACI comprised by the second polypeptide is selected from the second peptide segment as defined in any one of claims 1-3, 7-10; Preferably, the extracellular domain or a fragment thereof of BCMA comprised by the second polypeptide is identical to the first peptide segment comprised by the first polypeptide, and / or, the extracellular domain or a fragment thereof of TACI comprised by the second polypeptide is identical to the second peptide segment comprised by the first polypeptide.

18. The polypeptide dimer of any one of claims 14-17, wherein, The polypeptides comprised by the polypeptide dimer form covalent and / or non-covalent linkages between each other; Preferably, the polypeptides comprised by the polypeptide dimer form covalent linkages between each other.

19. An isolated nucleic acid molecule encoding a polypeptide according to any one of claims 1-13, or a polypeptide dimer according to any one of claims 14-18.

20. A vector comprising the isolated nucleic acid molecule of claim 19.

21. A host cell comprising the isolated nucleic acid molecule of claim 19 or the vector of claim 20.

22. A method of producing the polypeptide of any one of claims 1 to 13, or the polypeptide dimer of any one of claims 14 to 18, comprising culturing the host cell of claim 21 under conditions permitting expression of the protein, and recovering the polypeptide or the polypeptide dimer from the cultured host cell culture.

23. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 13, the polypeptide dimer of any one of claims 14 to 18, the isolated nucleic acid molecule of claim 19, the vector of claim 20, or the host cell of claim 21; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition comprises the polypeptide dimer of any one of claims 14 to 18, an isolated nucleic acid molecule encoding the polypeptide dimer, 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.

24. Use of the polypeptide of any one of claims 1 to 13, the polypeptide dimer of any one of claims 14 to 18, the isolated nucleic acid molecule of claim 19, the vector of claim 20, the host cell of claim 21, or the pharmaceutical composition of claim 23, for the manufacture of a medicament for the prevention and / or treatment of a B-cell related disease in a subject; Preferably, the prevention and / or treatment of the B-cell related disease can benefit from the inhibition of BLys and / or APRIL mediated signal transduction; 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; More preferably, the autoimmune disease is systemic lupus erythematosus or IgA nephropathy; Preferably, the subject is a mammal; Preferably, the polypeptide, polypeptide dimer, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition is used alone or in combination (e.g., concurrently or sequentially) with an additional pharmaceutically active agent, such as an anti-inflammatory drug or an immunosuppressant.

25. A method of 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 any one of claims 1 to 13, the polypeptide dimer of any one of claims 14 to 18, the isolated nucleic acid molecule of claim 19, the vector of claim 20, the host cell of claim 21, or the pharmaceutical composition of claim 23. Preferably, the prevention and / or treatment of a B-cell related disease can benefit from the inhibition of BLys and / or APRIL-mediated signaling; 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; More preferably, the autoimmune disease is systemic lupus erythematosus or IgA nephropathy; Preferably, the subject is a mammal; Preferably, the polypeptide, polypeptide dimer, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition is used alone or in combination (e.g., concurrently or sequentially) with another pharmaceutically active agent (e.g., an anti-inflammatory drug or an immunosuppressive agent).

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