WO2022258045A1 - Bifunctional fusion protein molecule containing anti-human il-17 antibody and taci - Google Patents

Abstract

A bifunctional fusion protein molecule containing an anti-human IL-17 antibody and TACI, comprising (a) an anti-human IL-17 antibody or antigen-binding fragment capable of blocking IL-17A/IL17RA binding, and (b) a TACI fusion protein or fragment capable of binding to BAFF/APRIL, and a preparation method therefor and an application thereof (for example, being used for the treatment of autoimmune diseases such as autoimmune encephalomyelitis).

Description

Bifunctional fusion protein molecule of anti-human IL-17 antibody and TACI

This disclosure requires that the Chinese patent application with the application number 202110655750.2 and the title of the invention "bifunctional fusion protein molecule of anti-human IL-17 antibody and TACI" be submitted to the China Patent Office on June 11, 2021, and on May 12, 2022. The priority of the Chinese patent application with the application number 202210512398.1 and the invention title "Bifunctional Fusion Protein Molecule of Anti-human IL-17 Antibody and TACI" filed with the China Patent Office on 11th, the entire content of which is incorporated by reference in this disclosure.

technical field

The present invention relates to a bifunctional fusion protein molecule comprising (a) an antibody or an antigen-binding fragment thereof that binds to IL-17, and (b) a cell membrane receptor TACI protein fragment that binds to BAFF/APRIL, the application of said molecule (e.g. , for the treatment of autoimmune diseases such as systemic lupus erythematosus), and the method for preparing the molecule.

Background technique

IL-17 (Interleukin-17) is an inflammatory cytokine produced by Th17 cells, which can promote the activation of T cells and stimulate epithelial cells, endothelial cells, and fibroblasts to produce various cytokines such as IL-6, IL- 8. GM-CSF, which leads to inflammation. IL-17 plays an important role in various autoimmune diseases (such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, etc.). Studies have shown that the level of IL-17 in the serum of patients with systemic lupus erythematosus is significantly increased. (N Engl J Med.2009; 361:888-98.Nat Rev Immunol.2014;14:585-600.Nat Immunol.2009;10(7):778-85.)

BAFF (B cell activating factor) and APRIL (A proliferation-inducing ligand) are B cell activation and regulatory factors belonging to the TNF family, which can promote the development and proliferation of B cells, increase the expression of various immunoglobulins in serum, and have The body's immune response plays an important regulatory role. They bind to cell membrane receptors TACI (Transmembrane activator and CAML-interactor) and BCMA (B cell maturation antigen). In addition, BAFF can also bind to another receptor, BAFFR (B cell-activating factor receptor). BAFF and APRIL regulate the activation, development and proliferation of lymphocytes through the signal transmission of these receptors. The overexpression of BAFF and APRIL is one of the causes of many autoimmune diseases. Studies have shown that the concentrations of BAFF and APRIL in the serum of patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis are significantly increased. Therefore, targeting BAFF/APRIL has become an effective way to treat autoimmune diseases. (J Immunol Res.2015; 2015:247426. Exp Cell Res.2011, 317(9)1270-1277.)

Systematic Lupus Erythematosus (SLE) is a systemic autoimmune disease that affects multiple systems and organs throughout the body. Extensive organ damage can be caused in the early stage of the disease, seriously affecting the quality of life of the patient; if not treated in time, it will cause irreversible damage to the affected organs and eventually lead to the death of the patient. The global prevalence of the disease is about (12-39) people/100,000 people, while the prevalence in China is as high as (30.13-70.41) people/100,000 people. With the continuous improvement of diagnosis and treatment, the survival rate of SLE patients has been significantly improved. However, the survival rate over 10 years has an obvious inflection point, and the survival rate between 25 and 30 years has dropped from 89% to 30%. At present, Anti-BAFF monoclonal antibody Belimumab and TACI fusion protein RC-18 targeting BAFF/APRIL have been approved for the treatment of SLE, both of which are drugs targeting B cells. The pathogenesis of SLE is complicated, and a variety of immune cells are involved. In addition to B cells, abnormal T cells are also key pathogenic factors.

The present invention aims to construct a bifunctional molecule targeting IL-17 and BAFF/APRIL at the same time, inhibit the excessive activation of B cells and T cells, and may show a better therapeutic effect on SLE.

Contents of the invention

The present invention provides TACI domain fragments or variants, bifunctional fusion protein molecules targeting IL-17 and TACI, fusion proteins and pharmaceutical compositions comprising TACI domain fragments, and their use in the treatment of systemic lupus erythematosus, autoimmune Use in autoimmune diseases such as encephalomyelitis.

In a first aspect, the present disclosure provides a bifunctional fusion protein molecule comprising a first domain and a second domain; wherein the first domain comprises a TACI extracellular domain Fragment or variant; said second domain comprises an antibody or an antigen-binding fragment specifically binding to human IL-17.

In a preferred embodiment, the first domain of the bifunctional fusion protein molecule comprises a TACI extracellular domain fragment or variant, and the TACI extracellular domain fragment or variant has SEQ ID NO: 2 (TACI amino acids 1-159 of the full-length fragment), SEQ ID NO: 3 (amino acids 68-109 of the full-length TACI fragment), SEQ ID NO: 4 (amino acids 21-127 of the full-length TACI fragment) or The sequence shown in SEQ ID NO: 5 (amino acids 1-116 of the full-length fragment of TACI).

In one embodiment, the antibody or antigen-binding fragment is selected from: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or, (3) a fully human antibody or fragment thereof.

In a specific embodiment, the antibody or antigen-binding fragment is selected from one or more of F(ab) ₂ , Fab', Fab, Fv, scFv, bispecific antibody, nanobody and antibody minimum recognition unit.

In a specific embodiment, said antibody or antigen-binding fragment that specifically binds human IL-17 comprises Vunakizumab, Ixekizumab or Secukinumab.

In a specific embodiment, the heavy chain and light chain of the antibody or antigen-binding fragment specifically binding to human IL-17 have the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

In another embodiment, the TACI extracellular domain fragment or variant is linked to the N-terminal or C-terminal of the heavy chain or light chain of an antibody or antigen-binding fragment of human IL-17.

In a preferred embodiment, the TACI extracellular domain fragment or variant is fused with an antibody or an antigen-binding fragment of human IL-17 through a linking peptide; preferably using SEQ ID NO.6, SEQ ID NO.7, SEQ ID The connecting peptide shown in NO.8 or SEQ ID NO.9.

In another embodiment, the bifunctional fusion protein molecule comprises:

(1) The heavy chain has the sequence shown in SEQ ID NO: 13; the light chain has the sequence shown in SEQ ID NO: 12;

(2) The heavy chain has the sequence shown in SEQ ID NO: 14; the light chain has the sequence shown in SEQ ID NO: 12;

(3) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 15;

(4) The heavy chain has the sequence shown in SEQ ID NO: 16; the light chain has the sequence shown in SEQ ID NO: 12;

(5) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 17;

(6) The heavy chain has the sequence shown in SEQ ID NO: 18; the light chain has the sequence shown in SEQ ID NO: 12;

(7) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 19;

(8) The heavy chain has the sequence shown in SEQ ID NO: 20; the light chain has the sequence shown in SEQ ID NO: 12;

(9) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 21;

(10) The heavy chain has the sequence shown in SEQ ID NO: 22; the light chain has the sequence shown in SEQ ID NO: 12;

(11) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 23;

(12) The heavy chain has the sequence shown in SEQ ID NO: 24; the light chain has the sequence shown in SEQ ID NO: 12;

(13) The heavy chain has the sequence shown in SEQ ID NO: 25; the light chain has the sequence shown in SEQ ID NO: 12;

(14) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 26;

(15) The heavy chain has the sequence shown in SEQ ID NO: 27; the light chain has the sequence shown in SEQ ID NO: 12;

(16) The heavy chain has the sequence shown in SEQ ID NO: 28; the light chain has the sequence shown in SEQ ID NO: 12;

(17) The heavy chain has the sequence shown in SEQ ID NO: 29; the light chain has the sequence shown in SEQ ID NO: 12;

(18) The heavy chain has the sequence shown in SEQ ID NO: 30; the light chain has the sequence shown in SEQ ID NO: 12;

(19) The heavy chain has the sequence shown in SEQ ID NO: 31; the light chain has the sequence shown in SEQ ID NO: 12;

(20) The heavy chain has a sequence shown in SEQ ID NO: 32; the light chain has a sequence shown in SEQ ID NO: 12;

(21) The heavy chain has the sequence shown in SEQ ID NO: 33; the light chain has the sequence shown in SEQ ID NO: 12;

(22) The heavy chain has the sequence shown in SEQ ID NO: 34; the light chain has the sequence shown in SEQ ID NO: 12;

(23) The heavy chain has the sequence shown in SEQ ID NO: 35; the light chain has the sequence shown in SEQ ID NO: 12;

(24) The heavy chain has the sequence shown in SEQ ID NO: 36; the light chain has the sequence shown in SEQ ID NO: 12;

(25) The heavy chain has the sequence shown in SEQ ID NO: 37; the light chain has the sequence shown in SEQ ID NO: 12;

(26) The heavy chain has a sequence shown in SEQ ID NO: 38; the light chain has a sequence shown in SEQ ID NO: 12; or

(27) An amino acid sequence having at least 90% identity to the sequence shown in (1)-(26) above, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98%, 99% identity.

In another specific embodiment, the bifunctional fusion protein molecule has the TACI extracellular domain fragment or variant described in the first aspect above, and competitively binds to BAFF/APRIL protein, and has the following characteristics:

(1) specifically binding to IL-17A protein;

(2) Blocking the binding of IL-17A to IL-17A receptor protein IL-17RA;

(3) Blocking the binding of BAFF to the BAFF receptor BAFFR;

(4) Blocking the binding of BAFF to BAFF receptor BCMA;

(5) Blocking the binding of APRIL to the APRIL receptor BCMA;

(6) neutralize the secretion of CXCL1 induced by human IL-17;

(7) mediates proliferation inhibition of human B cells; and/or,

(8) Treatment of autoimmune diseases.

In a second aspect, the disclosure of the present invention provides the TACI extracellular domain fragment or variant described in the first aspect, which is a transmembrane activator and CAML Interactor (Transmembrane Activator and CAML Interactor, TACI) Extracellular domain fragment or variant, the TACI extracellular domain fragment or variant has the sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

In a third aspect, the present disclosure provides an isolated nucleic acid molecule encoding the bifunctional fusion protein molecule described in the first aspect or the TACI extracellular domain fragment or variant described in the second aspect .

In a fourth aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule isolated in the aforementioned third aspect.

In a fifth aspect, the present disclosure provides a host cell, the host cell comprising the isolated nucleic acid molecule in the aforementioned third aspect, or the expression vector described in the aforementioned fourth aspect; preferably, the The host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis; more preferably, the host cell is selected from Expi293 cells .

In the sixth aspect, the present invention provides a method for preparing a bifunctional fusion protein molecule, culturing or culturing the host cell described in the fifth aspect under appropriate conditions, and isolating the bifunctional fusion protein molecule.

In the seventh aspect, the present disclosure provides a pharmaceutical composition comprising the bifunctional fusion protein molecule described in the aforementioned first aspect, the TACI extracellular domain fragment described in the aforementioned second aspect, or variant, the isolated nucleic acid molecule in the aforementioned third aspect, the expression vector described in the aforementioned fourth aspect, the cell described in the fifth aspect, or the product prepared by the method described in the sixth aspect; and a pharmaceutically acceptable Acceptable carrier, diluent or adjuvant; preferably, the pharmaceutical composition further comprises an additional autoimmune disease therapeutic agent.

In the eighth aspect, the present disclosure provides the bifunctional fusion protein molecule described in the aforementioned first aspect, the TACI extracellular domain fragment or variant described in the aforementioned second aspect, and the isolated TACI in the aforementioned third aspect. The nucleic acid molecule, the expression vector described in the aforementioned fourth aspect, the cell described in the fifth aspect, or the product prepared by the method described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect is used in the preparation of prevention and/or treatment Use in autoimmune diseases;

Preferably, the disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenia Purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, Reynauld's syndrome, Sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, autoimmune encephalomyelitis, and autoimmune thyroiditis.

In the ninth aspect, the present invention provides a method for preventing and/or treating autoimmune diseases, comprising administering the bifunctional fusion protein molecule described in the aforementioned first aspect, the aforementioned bifunctional fusion protein molecule described in the second aspect, to a patient in need thereof. The TACI extracellular domain fragment or variant, the isolated nucleic acid molecule in the third aspect, the expression vector in the fourth aspect, the cell in the fifth aspect, or the method in the sixth aspect The prepared product, or the pharmaceutical composition described in the seventh aspect;

Preferably, the disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenia Purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, Reynauld's syndrome, Sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, autoimmune encephalomyelitis, and autoimmune thyroiditis.

In the tenth aspect, the present invention provides the bifunctional fusion protein molecule described in the aforementioned first aspect, the TACI extracellular domain fragment or variant described in the aforementioned second aspect, and the isolated nucleic acid molecule in the aforementioned third aspect , the expression vector described in the aforementioned fourth aspect, the cell described in the fifth aspect, or the product prepared by the method described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect for preventing and/or treating autoimmunity disease;

Preferably, the disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenia Purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, Reynauld's syndrome, Sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, autoimmune encephalomyelitis, and autoimmune thyroiditis.

In the eleventh aspect, the present invention provides a kit comprising the bifunctional fusion protein molecule described in the aforementioned first aspect, the TACI extracellular domain fragment or variant described in the aforementioned second aspect, the aforementioned The isolated nucleic acid molecule in the third aspect, the expression vector described in the aforementioned fourth aspect, the cell described in the fifth aspect, or the product prepared by the method described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect , and instructions for use.

Definitions and Explanations of Terms

As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with an antigen of interest, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including but not Limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (e.g. bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies, antibody conjugates) and Antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments). Furthermore, unless otherwise stated, the term "monoclonal antibody" (mAb) is intended to include antibodies capable of specific Intact antibody molecules, as well as incomplete antibody fragments (e.g., Fab and F(ab')2 fragments, which lack the Fc fragment of intact antibodies and thus lack Fc-mediated effector functions) that selectively bind target proteins (see Wahl et al., J .Nucl.Med.24:316, 1983; the contents of which are incorporated herein by reference).

An "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas , proto-ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fishes (eg sharks).

The term "antigen-binding fragment" herein refers to one or more fragments of an antibody that retain the ability to specifically bind a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibody fragment may be a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, Nanobody, aptamer or domain antibody. Examples of binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab)2 Fragment, a bivalent fragment comprising two Fab fragments connected at the hinge region by disulfide bonds; (iii) Fd fragment consisting of VH and CH1 domains; (iv) VL and VH domains consisting of a single arm of the antibody (V) dAb comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 341:544-546, 1989); (vii) consisting of VH or VL (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, which CDRs may optionally be joined by a synthetic linker. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be joined using recombinant methods through a linker that enables them to be made in which the VL and VH regions pair to form A single protein chain of a monovalent molecule (termed a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 , 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as whole antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.

As used herein, the term "chimeric" antibody refers to an antibody that has variable sequences derived from immunoglobulins of one source organism (such as a rat or mouse) and immunoglobulins derived from a different organism (such as a human). The constant region of an immunoglobulin. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202; incorporated by reference above and into this article.

As used herein, the term "bispecific antibody" refers to an antibody, typically a human or humanized antibody, that has monoclonal binding specificities for at least two different antigens.

The term "humanized antibody" herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody). Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.

The term "fully human antibody" herein refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibody" herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.

As used herein, the term "nanobody" refers to the natural heavy chain antibody that lacks the light chain in camels, and its variable region can be cloned to obtain a single domain antibody consisting of only the variable region of the heavy chain, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

The term "antibody minimum recognition unit" herein refers to the smallest unit that an antibody can recognize an antigen in an antigen-antibody binding reaction.

The term "IL-17 binding molecule" herein refers to any molecule capable of binding to human IL-17 antigen alone or in combination with other molecules, specifically an anti-human IL-17 antibody. In some embodiments of the disclosed methods, protocols, kits, processes, uses and compositions, an IL-17 binding molecule is employed, such as an anti-human IL-17 antibody.

The term "TACI" herein refers to Transmembrane Activator and CAML Interactor (TACI) (von Bülow and Bram, Science 228:138 (1997); Bram and von Bülow, U.S. Patent 5,969,102 (1999)) . TACI is a membrane-bound receptor that has an extracellular region containing two cysteine-rich pseudo-repeats, a transmembrane region, and CAML (calcium regulator and affinity The cytoplasmic region where CAML interacts, CAML is an integral membrane protein localized in intracellular vesicles and is a co-inducer of NFAT activation when overexpressed in Jurkat cells.

The term "BAFF" in this article is also called B cell activating factor, which is a member of the tumor necrosis factor ligand superfamily and is mainly expressed in myeloid family cells including monocytes, macrophages, and dendritic cells. , neutrophils, malignant B cells, etc. B-lymphocyte-stimulating factors are expressed in the form of trimers on the surface of these cell membranes, and then released into the microcirculation after being cleaved by Furin protease, and then bind to receptors on the surface of B-cell membranes. BAFF mainly has three receptors, transmembrane activator and CAML Interactor (Transmembrane Activator and CAML Interactor, TACI), B cell maturation antigen (B cell maturation antigen, BCMA) and B cell activating factor receptor (B cell-activating factor receptor, BAFF-R), these three receptors are expressed on the surface of B cells at different developmental stages, and BAFF binds to different receptors to mediate different biological functions. BAFF-R is mainly expressed on transitional B cells, including follicular (FO) B cells and marginal zone (marginal zone, MZ) B cells, and the binding of BAFF to it has high specificity and high affinity. Regulates the survival, development and differentiation of B cells. BCMA and TACI are mainly expressed on the membrane surface of activated B cells, memory B cells, and plasma cells, and can recognize each other with BAFF and another member of the TNF ligand superfamily, proliferation-inducing ligand (APRIL). And the binding affinity is higher. Unlike BAFF-R, BCMA and TACI are associated with inflammatory responses and innate immunity.

The term "APRIL" in this paper is also called proliferation-inducing ligand (A proliferation-inducing ligand), which is a member of the tumor necrosis factor ligand superfamily and is mainly expressed on a variety of immune cells, such as dendritic cells, macrophages, Monocytes, T lymphocytes, etc. APRIL has about 30% homology with BAFF, a member of the same family, and can also be released into the microcirculation after being cleaved by Furin protease, usually in the form of a trimer. APRIL promotes and participates in the proliferation, differentiation and survival of lymphocytes by binding to its receptors (BCMA and TACI).

The term "fusion protein" herein refers to the protein product obtained by linking the coding regions of two or more genes by gene recombination methods, chemical methods or other appropriate methods, and expressing gene recombination under the control of the same regulatory sequence. In the fusion protein of the present invention, the coding regions of two or more genes may be fused at one or several positions by sequences encoding peptide linkers or connecting peptides. Peptide linkers or connecting peptides can also be used to construct fusion proteins of the invention. The term "fusion protein" of the present invention further includes (a) the extracellular domain of TACI or a variant or fragment thereof capable of binding BAFF and/or APRIL; and (b) an anti-human IL-17 antibody.

As used herein, the term "percent (%) sequence identity" or "sequences that are percent (%) identical" refers to sequences that have been aligned for maximum percent sequence identity and introduced gaps, if necessary (e.g., for optimal alignment). Yes, gaps can be introduced in one or both of the candidate and reference sequences, and non-homologous sequences can be ignored for comparison purposes), after which the amino acid (or nucleotide) residues of the candidate sequence are identical to the reference The percentage of identical amino acid (or nucleotide) residues in a sequence. For purposes of determining percent sequence identity, alignment can be achieved in a number of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50% to 100% sequence identity. The length of a candidate sequence aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence . When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

Herein the term "nucleic acid" includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually expressed 5' to 3'. In this context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of antibodies of the invention in vitro and/or in vivo, for example in a host or patient. Such DNA (eg cDNA) or RNA (eg mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see e.g. Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1).

As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (eg, plasmids), RNA vectors, viruses or other suitable replicons (eg, viral vectors). A variety of vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. Expression vectors of the invention contain polynucleotide sequences together with additional sequence elements, eg, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the invention include plasmids that contain regulatory sequences, such as promoter and enhancer regions, that direct transcription of the gene. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA resulting from transcription of the genes. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosomal entry sites (IRES), and polyadenylation signal sites to direct the efficient transcription of genes carried on the expression vector. The expression vector of the present invention may also contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourthricin.

The term "host cell" herein refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

As used herein, the term "pharmaceutical composition" refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. additional ingredients.

The terms "subject", "subject" and "patient" herein refer to an organism receiving treatment for a particular disease or condition as described herein, such as cancer or an infectious disease. Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, members of the bovid family (such as domestic cattle, bison, buffalo, elk, and yaks), sheep, and horses.

As used herein, the term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) an undesired physiological change or pathology in the subject being treated, such as a cell proliferative disorder (such as cancer or infectious disease). progression of the disease). Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable. Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented. When referring to the terms slow down, lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.

The term "appropriate conditions" herein refers to conditions suitable for culturing various host cells, including eukaryotic cells and prokaryotic cells.

The term "effective amount" herein refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject. "Effective amount" also refers to an amount of a compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition. When the active ingredient is administered to a subject alone, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.

The term "autoimmune disease" is defined herein as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive responses to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes (type 1), dystrophic bullous epidermis Lysis, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, malignant anemia, ulcerative colitis, etc.

The term "EC50" herein refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.

Description of drawings

Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

Figure 1-Figure 4, ELISA binding experiments of bifunctional molecules and IL-17A, in which the positive control is Secukinumab.

Figure 5-Figure 8, bifunctional molecule blocking IL-17A/IL-17RA binding experiment, wherein the positive control is Secukinumab.

Figure 9-Figure 11, the ELISA binding experiment of bifunctional molecules and BAFF, in which the positive control is RC-18.

Fig. 12-Fig. 14, ELISA binding experiments of bifunctional molecules and APRIL, wherein the positive control is RC-18.

Figure 15-Figure 17, bifunctional molecule blocking BAFF/BAFFR binding experiment, wherein the positive control is RC-18.

Figure 18-Figure 20, bifunctional molecule blocking BAFF/BCMA binding experiment, wherein the positive control is RC-18.

Figure 21-Figure 23, bifunctional molecule blocking APRIL/BCMA binding experiment, wherein the positive control is RC-18.

Figure 24. The experiment of bifunctional molecule neutralizing IL-17, in which the positive control is Secukinumab.

Fig. 25 . The experiment of inhibiting the proliferation of B cells by bifunctional molecules, wherein the positive control is RC-18.

Fig. 26-Fig. 29. ELISA binding experiment of linker-optimized bifunctional molecules and IL-17A, in which the positive control is Secukinumab.

Fig. 30-Fig. 33. ELISA binding experiment of linker-optimized bifunctional molecules and BAFF, in which the positive control is RC-18.

Figure 34-Figure 37, the ELISA binding experiment of the bifunctional molecule after linker optimization and APRIL, in which the positive control is RC-18.

Figure 38. The bifunctional molecule neutralizes IL-17 experiment after linker optimization, in which the positive control is Secukinumab and the negative control is hIgG1.

Fig. 39 . B cell proliferation inhibition experiment of bifunctional molecules after linker optimization, wherein the positive control is RC-18, and the negative control is hIgG1.

Fig. 40. Effects of bifunctional molecules on serum Cxcl-1 levels in mice injected with IL-17. The positive control is Secukinumab, the negative control is blank vehicle (PBS), and the normal control is mice not injected with IL-17.

Figure 41. Effects of bifunctional molecules on serum IgA levels in mice injected with BAFF, wherein the positive control is RC-18, the negative control is blank vehicle (PBS), and the normal control is mice not injected with BAFF.

Figure 42. The drug effect of bifunctional molecules in the mouse DTH model, wherein the positive control is Secukinumab and RC-18, the negative control is blank vehicle (PBS), and the normal control is unmodeled mice.

Figure 43. The drug effect of bifunctional molecules in the mouse EAE model, wherein the positive control is Secukinumab and RC-18, the negative control is blank vehicle (PBS), and the normal control is unmodeled mice.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The embodiments of the present invention are merely exemplary, and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Expression and Purification of Bifunctional Molecules

According to the full-length sequence of human TACI protein (SEQ ID NO: 1, the sequence is cited in Uniprot#O14836) and structure, different TACI fragments are designed and connected to the heavy chain of IL-17 antibody Secukinumab (SEQ ID NO: 11, the sequence is cited in WHO drug information) or the N-terminal or C-terminal of the light chain (SEQ ID NO: 12, the sequence is cited in WHO drug information) to construct a plasmid for producing IL-17-TACI bifunctional molecules. The specific information on the construction of bifunctional molecules is shown in Table 1 , the amino acid sequence information is shown in Table 2. Anti-IL-17 monoclonal antibody Secukinumab and TACI fusion protein RC-18 (SEQ ID NO: 10, sequence cited in WHO drug information) were used as positive controls.

Table 1. Combination information of heavy and light chains for bifunctional molecules

name	heavy chain	light chain
1-1-(G4S)3	TACI(1-159)-(G4S)3-Secukinumab HC	Secukinumab LC
1-3-(G4S)3	Secukinumab HC-(G4S)3-TACI(1-159)	Secukinumab LC
1-4-(G4S)3	Secukinumab HC	Secukinumab LC-(G4S)3-TACI(1-159)
3-1-(G4S)3	TACI(68-109)-(G4S)3-Secukinumab HC	Secukinumab LC
3-2-(G4S)3	Secukinumab HC	TACI(68-109)-(G4S)3-Secukinumab LC
3-3-(G4S)3	Secukinumab HC-(G4S)3-TACI(68-109)	Secukinumab LC
3-4-(G4S)3	Secukinumab HC	Secukinumab LC-(G4S)3-TACI(68-109)
4-1-(G4S)3	TACI(21-127)-(G4S)3-Secukinumab HC	Secukinumab LC
4-2-(G4S)3	Secukinumab HC	TACI(21-127)-(G4S)3-Secukinumab LC
4-3-(G4S)3	Secukinumab HC-(G4S)3-TACI(21-127)	Secukinumab LC
4-4-(G4S)3	Secukinumab HC	Secukinumab LC-(G4S)3-TACI(21-127)
5-1-(G4S)3	TACI(1-116)-(G4S)3-Secukinumab HC	Secukinumab LC
5-3-(G4S)3	Secukinumab HC-(G4S)3-TACI(1-116)	Secukinumab LC
5-4-(G4S)3	Secukinumab HC	Secukinumab LC-(G4S)3-TACI(1-116)

Table 2. Specific amino acid sequence information of bifunctional molecules and control antibodies

Add the plasmid and transfection reagent PEI (Polysciences, Cat. No.: 24765-1) into OPTI-MEM (Gibco, Cat. %CO ₂ , 120 rpm, 37°C shaker culture. On the second day after transfection, OPM-293ProFeed (Shanghai Opma, Cat. No.: F081918-001) and 6 g/L glucose (Sigma, Cat. No.: G7528) were added. On the sixth day of transfection, the cell supernatant was collected and purified with Protein A (GE, catalog number: 28985254), and the eluted sample was dialyzed to PBS, pH 7.4. The purity of the samples was determined by SEC-HPLC. 1-1-(G4S)3, 3-1-(G4S)3, 3-2-(G4S)3, 4-2-(G4S)3, 5-1-(G4S)3, 5-4-( G4S)3 purity is better, SEC monomer > 85%. The yield and SEC purity of bifunctional molecules are shown in Table 3.

Table 3. Yield and Purity of Bifunctional Molecules

Example 2 Detection of Binding Activity and Blocking Activity of Bifunctional Molecules

After the anti-IL-17-TACI bifunctional molecule was obtained, the binding activity and blocking activity of the IL-17 end and the TACI end were measured respectively.

2.1 Human IL-17A ELISA binding experiment

Human IL-17A protein (Acro Biosystems, product number: ILA-H5118) was coated overnight at 4°C, rinsed three times with 0.05% Tween 20-PBS solution, added 3% BSA blocking solution, and blocked at 37°C for 1.5h. Rinse 3 times with 0.05% Tween 20-PBS solution, add the doubly diluted samples, and incubate at 37°C for 1h. Rinse 3 times with 0.05% Tween 20-PBS solution, add secondary antibody HRP goat anti-human IgG Fc (Merck, catalog number: AP113), and incubate at 37°C for 1h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, add TMB solution (Seracare, product number: 5120-0077), react at room temperature for 10 minutes, then add 1M hydrochloric acid to terminate the reaction, and read the plate with a microplate reader at a wavelength of 450nM. The experimental results are shown in Figures 1-4, the bifunctional molecule has strong binding to human IL-17A. Except for 3-2-(G4S)3, the EC50 of other molecules is close to IL-17 monoclonal antibody Secukinumab. The combined EC50 values are shown in Table 4.

Table 4. Binding activity of bifunctional molecules to IL-17A

sample name	EC50(nM)
1-1-(G4S)3	0.076
1-3-(G4S)3	0.102
1-4-(G4S)3	0.064
3-1-(G4S)3	0.045
3-2-(G4S)3	0.190
3-3-(G4S)3	0.077
3-4-(G4S)3	0.083
4-1-(G4S)3	0.033
4-2-(G4S)3	0.027
4-3-(G4S)3	0.036
4-4-(G4S)3	0.045
5-1-(G4S)3	0.050
5-3-(G4S)3	0.029
5-4-(G4S)3	0.038
Secukinumab	0.055

2.2 IL-17A and IL-17RA binding blocking experiment

Human IL-17A protein (Acro Biosystems, product number: ILA-H5118) was coated overnight at 4°C, rinsed three times with 0.05% Tween 20-PBS solution, added 2% BSA blocking solution, and blocked at 37°C for 1.5h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, Biotin-IL-17RA (Acro Biosystems, product number: ILA-H5257) and doubly diluted samples were added, and incubated at 37°C for 1.5h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, the secondary antibody SA-HRP (Sigma, catalog number: S2438) was added and incubated at 37°C for 1h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, add TMB solution (Seracare, product number: 5120-0077), react at room temperature for 10 minutes, then add 1M hydrochloric acid to terminate the reaction, and read the plate with a microplate reader at a wavelength of 450nM. The experimental results are shown in Figures 5-8, and the bifunctional molecules can effectively block the binding of IL-17A and IL-17RA. Except for 3-2-(G4S)3, the ICSO of other molecules was close to or slightly better than that of IL-17 monoclonal antibody Secukinumab. See Table 5 for the IC50 values of blocking experiments.

Table 5. Blocking activity of bifunctional molecules on IL17A-IL17RA binding

sample name	IC50(nM)
1-1-(G4S)3	6.14
1-3-(G4S)3	2.32
1-4-(G4S)3	2.45
3-1-(G4S)3	6.46
3-2-(G4S)3	19.44
3-3-(G4S)3	6.05
3-4-(G4S)3	5.60
4-1-(G4S)3	2.79
4-2-(G4S)3	6.38
4-3-(G4S)3	5.00
4-4-(G4S)3	5.23
5-1-(G4S)3	4.99
5-3-(G4S)3	2.71
5-4-(G4S)3	5.77
Secukinumab	5.54

2.3 ELISA binding experiment of BAFF or APRIL

Human BAFF protein (Acro Biosystems, Cat. No.: BAF-H52D4) or human APRIL protein (Acro Biosystems, Cat. No.: APL-H52D1) was coated overnight at 4°C, rinsed 3 times with 0.05% Tween 20-PBS solution, added 3% BSA to block liquid, 37 ° C for 1.5 h. Rinse 3 times with 0.05% Tween 20-PBS solution, add the doubly diluted samples, and incubate at 37°C for 1h. Rinse 3 times with 0.05% Tween 20-PBS solution, add secondary antibody HRP goat anti-human IgG Fc (Merck, catalog number: AP113), and incubate at 37°C for 1h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, add TMB solution (Seracare, product number: 5120-0077), react at room temperature for 10 minutes, then add 1M hydrochloric acid to terminate the reaction, and read the plate with a microplate reader at a wavelength of 450nM. The experimental results are shown in Figure 9-Figure 14, the bifunctional molecule has strong binding to human BAFF and APRIL, among which 3-1-(G4S)3, 3-2-(G4S)3, 4-1-( The EC50 values of G4S)3, 4-2-(G4S)3, 5-1-(G4S)3 combined with BAFF and APRIL are better than those of TACI fusion protein RC-18, and the combined EC50 values are shown in Table 6.

Table 6. Binding activity of bifunctional molecules to BAFF or APRIL

sample name	BAFF EC50(nM)	APRIL EC50(nM)
1-1-(G4S)3	0.121	0.33
1-3-(G4S)3	0.302	1.15
1-4-(G4S)3	0.106	0.87

3-1-(G4S)3	0.028	0.06
3-2-(G4S)3	0.029	0.09
3-3-(G4S)3	0.102	0.51
3-4-(G4S)3	0.099	0.64
4-1-(G4S)3	0.042	0.03
4-2-(G4S)3	0.027	0.03
4-3-(G4S)3	0.140	0.74
4-4-(G4S)3	0.110	0.76
5-1-(G4S)3	0.047	0.06
5-3-(G4S)3	0.168	0.73
5-4-(G4S)3	0.115	0.62
RC-18	0.074	0.42

2.4 BAFF and BAFFR binding blocking experiment

Human BAFF protein (Acro Biosystems, catalog number: BAF-H52D4) was coated overnight at 4°C, rinsed three times with 0.05% Tween 20-PBS solution, added 2% BSA blocking solution, and blocked at 37°C for 1.5h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, Biotin-BAFFR (Acro Biosystems, product number: BAR-H5257) and doubly diluted samples were added, and incubated at 37°C for 1.5h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, the secondary antibody SA-HRP (Sigma, catalog number: S2438) was added and incubated at 37°C for 1h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, TMB solution (Seracare, product number: 5120-0077) was added, reacted at room temperature for 10 minutes, and then 1M hydrochloric acid was added to stop the reaction, and the plate was read with a microplate reader at a wavelength of 450nM. The experimental results are shown in Figure 15-Figure 17, the bifunctional molecules can effectively block the binding of BAFF and BAFFR, in which 3-1-(G4S)3, 3-2-(G4S)3, 5-1-(G4S The blocking activity of )3 is significantly better than that of TACI fusion protein RC-18 (if the IC50 value differs by more than 2 times, it is considered significantly better). See Table 7 for the IC50 values of blocking experiments.

2.5 BAFF and BCMA binding blocking experiment

Human BAFF protein (Acro Biosystems, catalog number: BAF-H52D4) was coated overnight at 4°C, rinsed three times with 0.05% Tween 20-PBS solution, added 2% BSA blocking solution, and blocked at 37°C for 1.5h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, Biotin-BCMA (Acro Biosystems, catalog number: BC7-H5254) and doubly diluted samples were added, and incubated at 37°C for 1.5h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, the secondary antibody SA-HRP (Sigma, catalog number: S2438) was added and incubated at 37°C for 1h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, TMB solution (Seracare, product number: 5120-0077) was added, reacted at room temperature for 10 minutes, and then 1M hydrochloric acid was added to stop the reaction, and the plate was read with a microplate reader at a wavelength of 450nM. The experimental results are shown in Figures 18-20. The bifunctional molecules can effectively block the binding of BAFF and BCMA, among which 3-1-(G4S)3, 3-2-(G4S)3, 3-3-(G4S) 3. The blocking activity of 4-2-(G4S)3 and 5-1-(G4S)3 is significantly better than that of TACI fusion protein RC-18 (if the IC50 value differs by more than 2 times, it is considered significantly better). See Table 7 for the IC50 values of blocking experiments.

2.6 BCMA and APRIL binding blocking experiment

Human BCMA protein (Acro Biosystems, catalog number: BC7-H5254) was coated overnight at 4°C, rinsed three times with 0.05% Tween 20-PBS solution, added 2% BSA blocking solution, and blocked at 37°C for 1.5h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, add Biotin-APRIL (Acro Biosystems, product number: APL-H82F5) and doubly diluted samples, and incubate at 37°C for 1.5h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, the secondary antibody SA-HRP (Sigma, catalog number: S2438) was added and incubated at 37°C for 1h. After rinsing with 0.05% Tween 20-PBS solution for 3 times, TMB solution (Seracare, product number: 5120-0077) was added, reacted at room temperature for 10 minutes, and then 1M hydrochloric acid was added to stop the reaction, and the plate was read with a microplate reader at a wavelength of 450nM. The experimental results are shown in Figures 21-23. The bifunctional molecules can effectively block the combination of BCMA and APRIL, among which 3-1-(G4S)3, 3-2-(G4S)3, 3-3-(G4S) 3. The blocking activity of 4-1-(G4S)3, 4-2-(G4S)3, and 5-1-(G4S)3 is significantly better than that of TACI fusion protein RC-18 (IC50 values differ by more than 2 times, considered to be significantly better than). See Table 7 for the IC50 values of blocking experiments.

Table 7. Blocking activity of bifunctional molecules on BAFF-BAFFR, BAFF-BCMA, APRIL-BCMA

Example 3 Detection of neutralizing activity of bifunctional molecules to IL-17 and BAFF/APRIL

3.1 IL-17 neutralization assay to detect the activity of bifunctional molecule IL-17

IL-17 can stimulate HT-29 cells to secrete CXCL1. Add HT-29 cells (Cell Bank of Chinese Academy of Sciences, Cat. No.: TCHu103) into the 96-well plate, add human IL-17 (R&D Systems, Cat. No.: 317-ILB-050) and the sample diluted at the same time, mix well and total After incubation for 48 h, the cell supernatant was collected, and the human CXCL1 level in the supernatant was detected with an ELISA kit (R&D Systems, catalog number: SGR00B). The experimental results are shown in Figure 24, the bifunctional molecules can effectively neutralize IL-17, thereby inhibiting the secretion of CXCL1. Except for 3-2-(G4S)3, 4-2-(G4S)3, the neutralizing activity of other molecules is close to that of IL-17 monoclonal antibody Secukinumab. See Table 8 for the IC50 values of neutralization experiments.

Table 8. IL-17 neutralization experiments

sample name	IC50(nM)
1-1-(G4S)3	0.57
3-1-(G4S)3	0.27
3-2-(G4S)3	7.50
3-3-(G4S)3	0.25
3-4-(G4S)3	0.40
4-1-(G4S)3	0.41
4-2-(G4S)3	2.48
4-3-(G4S)3	0.17
4-4-(G4S)3	0.23
5-1-(G4S)3	0.36
Secukinumab	0.25

3.2 B cell proliferation assay to detect the activity of TACI terminal

BAFF and APRIL can promote the proliferation of B cells. Extract human B cells (human B cell isolation kit, Stemcell, catalog number: 17954) and IL4 (Peprotech, catalog number: 200-04) from human PBMC cells (Allcells, catalog number: PB004F-C), anti-IgM (JaksonImmuno, catalog number : 109-007-043), BAFF (Acro Biosystems, Cat. No.: BAF-H52D4), APRIL (R&D Systems, Cat. No.: 5860-AP-010) were co-incubated, and the samples to be tested were added at the same time. After incubation for 4 days, Ki67- APC (eBioscience, catalog number: 17-5699-42) was used to detect the proliferation of B cells. The experimental results are shown in Figure 25. The inhibitory activity of the bifunctional molecule is generally better than that of the TACI fusion protein RC-18, and both can effectively inhibit the proliferation of B cells induced by BAFF and APRIL.

Comparison of Linkers in Example 4 Bifunctional Molecules

There are many options for linkers connecting Secukinumab and TACI fragments, and (G4S)3 is used in Example 1. In addition, we also tried (G4S)4, (GS)10 and (GSG)5, the information of these bifunctional molecules is shown in Table 9, and the specific amino acid sequence information is shown in Table 10.

Table 9. Details of the optimized structure of the bifunctional molecular linker

name	heavy chain	light chain
3-1-(G4S)4	TACI(68-109)-(G4S)4-Secukinumab HC	Secukinumab LC (SEQ ID NO: 12)
3-1-(GS)10	TACI(68-109)-(GS)10-Secukinumab HC	Secukinumab LC
3-1-(GSG)5	TACI(68-109)-(GSG)5-Secukinumab HC	Secukinumab LC
3-3-(G4S)4	Secukinumab HC-(G4S)4-TACI(68-109)	Secukinumab LC
3-3-(GS)10	Secukinumab HC-(GS)10-TACI(68-109)	Secukinumab LC
3-3-(GSG)5	Secukinumab HC-(GSG)5-TACI(68-109)	Secukinumab LC
4-1-(G4S)4	TACI(21-127)-(G4S)4-Secukinumab HC	Secukinumab LC
4-1-(GS)10	TACI(21-127)-(GS)10-Secukinumab HC	Secukinumab LC
4-1-(GSG)5	TACI(21-127)-(GSG)5-Secukinumab HC	Secukinumab LC
5-1-(G4S)4	TACI(1-116)-(G4S)4-Secukinumab HC	Secukinumab LC
5-1-(GS)10	TACI(1-116)-(GS)10-Secukinumab HC	Secukinumab LC
5-1-(GSG)5	TACI(1-116)-(GSG)5-Secukinumab HC	Secukinumab LC

Table 10. Amino acid sequence information of different linkers and their bifunctional molecules

The purification results are shown in Table 11. These bifunctional molecules were subjected to binding experiments of human IL-17A, BAFF and APRIL according to the method in Example 2. The experimental methods refer to Examples 2.1 and 2.3. The experimental results are shown in Figures 26-37. The results showed that changing the linker had no significant effect on the binding activity of the IL-17 end and the TACI end.

Table 11. Yield and Purity of Bifunctional Molecular Linkers Optimized

In addition, IL-17 neutralization experiments and B cell proliferation experiments were also performed on the bifunctional molecules after linker optimization, and the experimental methods refer to Example 3. The results of IL-17 neutralization experiments are shown in Figure 38, bifunctional molecules 3-1-(GSG)5, 3-3-(G4S)4, 4-1-(GS)10 and 5-1-(GSG) 5 can effectively neutralize IL-17, thereby inhibiting the secretion of CXCL1, and the neutralizing activity of 4-1-(GS)10 and 5-1-(GSG)5 is better than that of IL-17 monoclonal antibody Secukinumab. The results of B cell proliferation experiments are shown in Figure 39. The bifunctional molecules 3-1-(GSG)5, 3-3-(G4S)4, 4-1-(GS)10 and 5-1-(GSG)5 all It can effectively inhibit the proliferation of B cells induced by BAFF and APRIL, and the inhibitory activity is better than that of TACI fusion protein RC-18. See Table 12 for the IC50 values of IL17 neutralization assay and B cell proliferation assay.

Table 12. IL17 neutralization assay and B cell proliferation assay

Example 5 Effect of Bifunctional Molecules on Cxcl-1 Levels in Mice After IL-17 Injection

6-8 weeks old C57BL/6N female mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were taken, and the mice were randomly divided into normal control group, negative control group, positive control group and bifunctional molecule group. The dosage of the bifunctional molecule group is 16 mg/kg; the positive control group is IL-17 monoclonal antibody Secukinumab (15 mg/kg) at an equimolar dose; the negative control group is an equal volume of blank vehicle (PBS), and the normal control group is Mice that were not injected with IL-17 were administered once by intraperitoneal injection at the same time point. 24 hours after administration, mice in each group were intraperitoneally injected with human IL-17 protein (ACROBiosystems) at a dose of 3 μg/mouse; 26 hours after administration, mice in each group were sacrificed and whole blood was collected, left at room temperature and then centrifuged to take The concentration of cytokine Cxcl-1 in serum was detected by ELISA kit (Mouse CXCL1 ELISA Kit; R&D).

The experimental results are shown in Figure 40. After administration of the bifunctional molecules 3-1-(GSG)5 and 3-3-(G4S)4, they can effectively neutralize IL-17 and increase the level of Cxcl-1 in mouse serum. Significantly reduced, compared with the negative control group, there is a statistical difference (* represents the significance of each experimental group compared with the negative control group, the statistical method is one-way ANOVA, **** represents the P value＜0.0001). The bifunctional molecule inhibited Cxcl-1 levels better than the positive control secukinumab.

Example 6 Effect of Bifunctional Molecules on IgA and IgG Levels in Mice After Injection of BAFF

6-8 weeks old Balb/c female mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were taken, and the mice were randomly divided into normal control group, negative control group, positive control group and bifunctional molecule group. Wherein the dosage of bifunctional molecule group is 3-1-(GSG)5 of 32mg/kg; The positive control group is the RC-18 (15mg/kg) of equimolar dose; The negative control group is the blank vehicle (PBS) of equal volume ), the normal control group was mice not injected with BAFF, and they were administered once by intraperitoneal injection at the same time point. At 2h, 26h, 50h and 74h after administration, mice in each group were injected with human BAFF protein (Human BAFF protein; Sino Biological) via tail vein at a dose of 3 mg/kg. 98 hours after administration, the mice in each group were sacrificed and whole blood was collected. After standing at room temperature, the supernatant was collected by centrifugation. The concentration of cytokine IgA in the serum was detected with an ELISA kit (Mouse IgA ELISA Kit; Abcam).

The experimental results are shown in Figure 41. After administration of the bifunctional molecule 3-1-(GSG)5, it can effectively neutralize BAFF and significantly reduce the level of IgA in the serum of mice, which is statistically significant compared with the negative control group. Differences (* indicates the significance of the statistical difference between each experimental group and the negative control group, the statistical method is one-way ANOVA, *** indicates the P value <0.001, **** indicates the P value <0.0001). The inhibitory effect of 3-1-(GSG)5 on IgA level was significantly better than that of RC-18 in equimolar dose (IgA: P value<0.001).

Example 7 Pharmacodynamics of Bifunctional Molecules in Mouse Delayed Hypersensitivity (DTH) Model

Take 6-8 week-old IL-17A humanized C57BL/6N female mice (Shanghai Southern Model Biotechnology Co., Ltd.), and subcutaneously inject 100 μl of fully emulsified C57BL containing 100 μg key Emulsion of porin hemocyanin (KLH; Sigma), 50 μl per spot injection. The preparation method of the emulsion is: dissolving the KLH protein with PBS into a protein solution with an initial concentration of 3 mg/ml; using a full plastic syringe to draw an equal volume of the protein solution, complete Freund's adjuvant (CFA, Complete Freund's adjuvant; Sigma-Aldrich ) and incomplete Freund's adjuvant (IFA, Incomplete Freund's adjuvant; Sigma-Aldrich), connected with a connecting tube with a 100-mesh screen, pushed back and forth on ice for about 15 minutes, so that the emulsion was evenly emulsified, and the liquid was dropped on the water surface If it does not disperse, it can be used for subcutaneous injection modeling. The final concentration of KLH protein in the emulsion was 1 mg/ml. On the 5th day after the model was immunized, 10 μl of KLH protein solution dissolved in PBS to a concentration of 1 mg/ml was injected intradermally into the right ear of each mouse for stimulation, and a dial thickness gauge (PEACOCK) was used to stimulate before and after stimulation. The mouse right ear thickness was measured at 24h, 48h, 72h, and 96h after stimulation to obtain the mouse right ear thickness change value Δear thickness=(right ear thickness after stimulation)−(right ear thickness before stimulation).

The mice were divided into normal control group, negative control group, positive control group and bifunctional molecule group. Among them, the dosage of the bifunctional molecule group was 64 mg/kg of 3-1-(GSG)5; the positive control group was Secukinumab and RC-18 in equimolar doses, and the corresponding dosages were: 60 mg/kg of Secukinumab, 60 mg/kg of RC-18, -18 is 30mg/kg; the negative control group is blank vehicle (PBS), and the normal control group is mice without model. From the day of modeling and immunization, administration was administered by intraperitoneal injection, administered once every two days, and administered 5 times in total.

The experimental results are shown in Figure 42. After administration of the bifunctional molecule 3-1-(GSG)5, the change in thickness of the right ear of DTH mice was significantly reduced, and there was a statistical difference compared with the negative control group (* indicates that each experiment The significance of the statistical difference between the negative control group and the negative control group, the statistical method is two-way ANOVA, ** means P value <0.01, **** means P value <0.0001). The drug effect of 3-1-(GSG)5 was significantly better than that of the positive control RC-18 at an equimolar dose (P value<0.0001).

Example 8 Pharmacodynamics of Bifunctional Molecules in Mouse Experimental Autoimmune Encephalomyelitis (EAE) Model

Take 6-8 week-old IL-17A humanized C57BL/6N female mice (Shanghai Southern Model Biotechnology Co., Ltd.), and subcutaneously inject 150 μl at three points evenly on both sides of the back of the neck and the dorsal midline of the base of the tail. A well-emulsified emulsion containing 225 μg of MOG (29-156) protein was injected in 50 μl per spot. The preparation method of the emulsion is: dissolving the MOG (29-156) protein with PBS into a protein solution with an initial concentration of 3 mg/ml; using two full plastic syringes to draw equal volumes of the protein solution and complete Freund's adjuvant (CFA, Complete Freund's adjuvant; Sigma-Aldrich), connected with a connecting tube with a 100-mesh screen, pushed back and forth on ice for about 15 minutes to make the emulsion evenly emulsified, subject to the fact that the droplets do not disperse on the water surface, it can be used for subcutaneous injection modeling. The final concentration of MOG protein in the emulsion was 1.5 mg/ml. PTX (Pertussis Toxins; List Biological Laboratories) was prepared into a 1 μg/ml solution with physiological saline, and 0.2 ml was intraperitoneally injected into the mice on the day of model-making immunization and 48 hours after immunization, and each mouse was injected with a total of 400 ng of PTX. After about 9 days after modeling, the mice entered the onset period. The mice were observed continuously for 28 days, and the body weight of the mice was recorded every day, and the clinical symptoms of the disease were scored blindly by an independent second person. The scoring criteria are as follows: 0 points, no obvious disease symptoms; 1 point, loss of tail tension or weakness of hind limbs; 2 points, loss of tail tension and weakness of hind limbs; 3 points, partial paralysis of hind limbs; 4 points, complete paralysis of hind limbs; Dead state or death.

The mice were divided into normal control group, negative control group, positive control group and bifunctional molecule group. Among them, the dosage of the bifunctional molecule group was 10.6 mg/kg of 3-1-(GSG)5; the positive control group was Secukinumab and RC-18 in equimolar doses, and the corresponding dosages were: Secukinumab was 10 mg/kg, RC-18 is 5 mg/kg; the negative control group is blank vehicle (PBS), and the normal control group is unmodeled mice. From the day of modeling and immunization, administration was administered by intraperitoneal injection, administered once every two days, and treated continuously for 28 days.

The experimental results are shown in Figure 43. After administration of the bifunctional molecule 3-1-(GSG)5, the clinical symptoms of the disease in EAE mice were significantly improved, and the clinical score values were statistically different from those of the negative control group (* indicates The significance of the statistical difference between each experimental group and the negative control group, the statistical method is two-way ANOVA, **** means P value <0.0001). The efficacy of 3-1-(GSG)5 was significantly better than that of the positive controls Secukinumab and RC-18 in equimolar doses (P value<0.0001).

Claims (16)

1. A bifunctional fusion protein molecule, characterized in that, the bifunctional fusion protein molecule comprises a first domain and a second domain; wherein the first domain comprises a TACI extracellular domain fragment or variant; the The second domain of comprises an antibody or antigen-binding fragment that specifically binds human IL-17.
2. The bifunctional fusion protein molecule according to claim 1, wherein the first domain of the bifunctional fusion protein molecule comprises a TACI extracellular domain fragment or a variant, and the TACI extracellular domain fragment or variant The body has the sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
3. The bifunctional fusion protein molecule according to any one of claims 1-2, wherein the antibody or antigen-binding fragment is selected from:

   (1) Chimeric antibodies or fragments thereof;

   (2) a humanized antibody or fragment thereof; or,

   (3) Fully human antibodies or fragments thereof;

   Preferably, the antibody or antigen-binding fragment is selected from one or more of F(ab) ₂ , Fab', Fab, Fv, scFv, bispecific antibody, nanobody and antibody minimum recognition unit;

   Preferably, the antibody or antigen-binding fragment specifically binding to human IL-17 comprises Vunakizumab, Ixekizumab or Secukinumab;

   More preferably, the heavy chain and light chain of the antibody or antigen-binding fragment specifically binding to human IL-17 have the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
4. The bifunctional fusion protein molecule according to any one of claims 1-3, wherein the TACI extracellular domain fragment or variant is connected to the heavy chain or light chain of an antibody or antigen-binding fragment of human IL-17 N-terminal or C-terminal of the chain;

   Preferably, the TACI extracellular domain fragment or variant is fused with an antibody or an antigen-binding fragment of human IL-17 through a linker peptide; preferably using SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or Connecting peptide shown in SEQ ID NO.9.
5. The bifunctional fusion protein molecule according to any one of claims 1-4, wherein the bifunctional fusion protein molecule comprises:

   (1) The heavy chain has the sequence shown in SEQ ID NO: 13; the light chain has the sequence shown in SEQ ID NO: 12;

   (2) The heavy chain has the sequence shown in SEQ ID NO: 14; the light chain has the sequence shown in SEQ ID NO: 12;

   (3) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 15;

   (4) The heavy chain has the sequence shown in SEQ ID NO: 16; the light chain has the sequence shown in SEQ ID NO: 12;

   (5) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 17;

   (6) The heavy chain has the sequence shown in SEQ ID NO: 18; the light chain has the sequence shown in SEQ ID NO: 12;

   (7) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 19;

   (8) The heavy chain has the sequence shown in SEQ ID NO: 20; the light chain has the sequence shown in SEQ ID NO: 12;

   (9) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 21;

   (10) The heavy chain has the sequence shown in SEQ ID NO: 22; the light chain has the sequence shown in SEQ ID NO: 12;

   (11) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 23;

   (12) The heavy chain has the sequence shown in SEQ ID NO: 24; the light chain has the sequence shown in SEQ ID NO: 12;

   (13) The heavy chain has the sequence shown in SEQ ID NO: 25; the light chain has the sequence shown in SEQ ID NO: 12;

   (14) The heavy chain has the sequence shown in SEQ ID NO: 11; the light chain has the sequence shown in SEQ ID NO: 26;

   (15) The heavy chain has the sequence shown in SEQ ID NO: 27; the light chain has the sequence shown in SEQ ID NO: 12;

   (16) The heavy chain has the sequence shown in SEQ ID NO: 28; the light chain has the sequence shown in SEQ ID NO: 12;

   (17) The heavy chain has the sequence shown in SEQ ID NO: 29; the light chain has the sequence shown in SEQ ID NO: 12;

   (18) The heavy chain has the sequence shown in SEQ ID NO: 30; the light chain has the sequence shown in SEQ ID NO: 12;

   (19) The heavy chain has the sequence shown in SEQ ID NO: 31; the light chain has the sequence shown in SEQ ID NO: 12;

   (20) The heavy chain has a sequence shown in SEQ ID NO: 32; the light chain has a sequence shown in SEQ ID NO: 12;

   (21) The heavy chain has the sequence shown in SEQ ID NO: 33; the light chain has the sequence shown in SEQ ID NO: 12;

   (22) The heavy chain has the sequence shown in SEQ ID NO: 34; the light chain has the sequence shown in SEQ ID NO: 12;

   (23) The heavy chain has the sequence shown in SEQ ID NO: 35; the light chain has the sequence shown in SEQ ID NO: 12;

   (24) The heavy chain has the sequence shown in SEQ ID NO: 36; the light chain has the sequence shown in SEQ ID NO: 12;

   (25) The heavy chain has the sequence shown in SEQ ID NO: 37; the light chain has the sequence shown in SEQ ID NO: 12;

   (26) The heavy chain has a sequence shown in SEQ ID NO: 38; the light chain has a sequence shown in SEQ ID NO: 12; or

   (27) An amino acid sequence having at least 90% identity to the sequence shown in (1)-(26) above, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98%, 99% identity.
6. The bifunctional fusion protein molecule according to any one of claims 1-5, wherein the bifunctional fusion protein molecule has the TACI extracellular domain fragment or variant described in claim 1, and competes with binding to BAFF/APRIL protein, and has the following properties:

   (1) specifically binding to IL-17A protein;

   (2) Blocking the binding of IL-17A to IL-17A receptor protein IL-17RA;

   (3) Blocking the binding of BAFF to the BAFF receptor BAFFR;

   (4) Blocking the binding of BAFF to BAFF receptor BCMA;

   (5) Blocking the binding of APRIL to the APRIL receptor BCMA;

   (6) neutralize the secretion of CXCL1 induced by human IL-17;

   (7) mediates proliferation inhibition of human B cells; and/or,

   (8) Treatment of autoimmune diseases.
7. TACI extracellular domain fragment or variant in the bifunctional fusion protein molecule described in claim 1, it is a kind of transmembrane activator and CAML interactor (Transmembrane Activator and CAML Interactor, TACI) extracellular domain Fragment or variant, characterized in that the TACI extracellular domain fragment or variant has a sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
8. An isolated nucleic acid molecule, characterized in that it encodes the bifunctional fusion protein molecule of any one of claims 1-6 or the TACI extracellular domain fragment or variant of claim 7.
9. An expression vector comprising the isolated nucleic acid molecule of claim 8.
10. A host cell, characterized in that, the host cell comprises the isolated nucleic acid molecule according to claim 8, or the expression vector according to claim 9; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; More preferably, the host cells are derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis; more preferably, the host cells are selected from Expi293 cells.
11. A method for preparing a bifunctional fusion protein molecule, characterized in that the host cell according to claim 10 is cultivated or cultured under appropriate conditions, and the bifunctional fusion protein molecule is isolated.
12. A pharmaceutical composition, characterized in that, the composition comprises the bifunctional fusion protein molecule described in any one of claims 1-6, the TACI extracellular domain fragment or variant described in claim 7, the claim The isolated nucleic acid molecule of 8, the expression vector of claim 9, the cell of claim 10 or the product prepared according to the method of claim 11; preferably, the composition further comprises a pharmaceutically acceptable carrier (carrier), diluent or adjuvant; preferably, the pharmaceutical composition further comprises an additional autoimmune disease therapeutic agent.
13. The bifunctional fusion protein molecule described in any one of claims 1-6, the TACI extracellular domain fragment or variant described in claim 7, the isolated nucleic acid molecule described in claim 8, the TACI described in claim 9 Use of the expression vector, the cell according to claim 10, the product prepared according to the method according to claim 11, or the pharmaceutical composition according to claim 12 in the preparation of prevention and/or treatment of autoimmune diseases;

    Preferably, the disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenia Purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, Reynauld's syndrome, Sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, autoimmune encephalomyelitis, and autoimmune thyroiditis.
14. A method for preventing and/or treating autoimmune diseases, comprising administering the bifunctional fusion protein molecule of any one of claims 1-6 and the TACI extracellular domain fragment of claim 7 to patients in need thereof or variant, the isolated nucleic acid molecule of claim 8, the expression vector of claim 9, the cell of claim 10, the product prepared by the method of claim 11, or the expression vector of claim 12 pharmaceutical composition;

    Preferably, the disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenia Purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, Reynauld's syndrome, Sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, autoimmune encephalomyelitis, and autoimmune thyroiditis.
15. The bifunctional fusion protein molecule described in any one of claims 1-6, the TACI extracellular domain fragment or variant described in claim 7, the isolated nucleic acid molecule described in claim 8, the TACI described in claim 9 The expression vector, the cell according to claim 10, the product prepared according to the method according to claim 11, or the pharmaceutical composition according to claim 12, is characterized in that it is used for preventing and/or treating autoimmune diseases;

    Preferably, the disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenia Purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, Reynauld's syndrome, Sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, autoimmune encephalomyelitis, and autoimmune thyroiditis.
16. A kit comprising the bifunctional fusion protein molecule described in any one of claims 1-6, the TACI extracellular domain fragment or variant described in claim 7, the isolated nucleic acid molecule described in claim 8 , the expression vector of claim 9, the cell of claim 10, the product prepared according to the method of claim 11, or the pharmaceutical composition of claim 12; optionally, instructions for use are also included.

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