CN117529506A

CN117529506A - Bifunctional fusion protein molecules of anti-human IL-17 antibodies and TACI

Info

Publication number: CN117529506A
Application number: CN202280038470.XA
Authority: CN
Inventors: 付雅媛; 柴晓鹃; 曹卓晓; 唐任宏; 任晋生
Original assignee: Hainan Simcere Pharmaceutical Co ltd
Current assignee: Hainan Simcere Pharmaceutical Co ltd
Priority date: 2021-06-11
Filing date: 2022-06-10
Publication date: 2024-02-06
Also published as: WO2022258045A1

Abstract

The present invention relates to bifunctional fusion protein molecules of anti-human IL-17 antibodies and TACI. Specifically, the invention discloses (a) an antibody or antigen binding fragment of anti-human IL-17 capable of blocking IL-17A/IL17RA binding and (b) a TACI fusion protein or fragment capable of binding BAFF/APRIL, as well as methods of making and using the same (e.g., for the treatment of autoimmune diseases such as autoimmune encephalomyelitis).

Description

Bifunctional fusion protein molecules of anti-human IL-17 antibodies and TACI

The present disclosure claims priority from the chinese patent office filed at day 6 and 11 of 2021, application number 202110655750.2, entitled "bifunctional fusion protein molecule of anti-human IL-17 antibody and TACI", and from the chinese patent office filed at day 5 and 12 of 2022, application number 202210512398.1, entitled "bifunctional fusion protein molecule of anti-human IL-17 antibody and TACI", the entire contents of which are incorporated herein by reference.

Technical Field

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

Background

IL-17 (Interlukin-17) is an inflammatory cytokine produced by Th17 cells, and can promote activation of T cells, stimulate epithelial cells, endothelial cells, and fibroblasts to produce various cytokines such as IL-6, IL-8, and GM-CSF, and thus cause inflammation. IL-17 plays an important role in a variety of autoimmune diseases (e.g., psoriasis, rheumatoid arthritis, systemic lupus erythematosus, etc.). Studies have shown that IL-17 levels are significantly elevated in serum from patients with systemic lupus erythematosus. ( 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 production-reduction ligand) are B cell activating and regulating factors belonging to TNF family, can promote the development and proliferation of B cells, can increase the expression level of various immunoglobulins in serum, and has important regulating effect on immune response of organisms. They bind to the cell membrane receptors TACI (Transmembrane activator and CAML-interface) and BCMA (B cell maturation antigen). In addition, BAFF is also able to bind to another receptor BAFFR (B cell-activating factor receptor). BAFF and APRIL regulate lymphocyte activation, development and proliferation via signaling by these several receptors. Overexpression of BAFF and APRIL is one of the etiologies of a variety of autoimmune diseases. The study shows that the serum BAFF and APRIL concentration of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and the like are obviously increased. Targeting BAFF/APRIL is therefore an effective approach to the treatment of autoimmune diseases. ( J Immunol Res.2015;2015:247426.Exp Cell Res.2011, 317 (9) 1270-1277. )

Systemic lupus erythematosus (Systematic Lupus Erythematosus, SLE) is a systemic autoimmune disease affecting the systemic multisystem and organs. Extensive organ damage can be caused in early stage of disease occurrence, and the life quality of patients is seriously affected; if not treated in time, irreversible damage to the affected viscera can be caused, and finally death of the patient is caused. The global prevalence of the disease is about (12-39) per 10 ten thousand, while the prevalence of china is as high as (30.13-70.41) per 10 ten thousand. With increasing diagnosis and treatment levels, survival rates of SLE patients are significantly improved. However, survival rates above 10 years have seen a clear inflection point, with survival rates from 25 years to 30 years dropping from 89% to 30%. Currently, anti-BAFF monoclonal antibody Belimumab and TACI fusion protein RC-18 targeting BAFF/APRIL are obtained and used for treating SLE, and are all drugs targeting B cells. While SLE has a complex pathogenesis, a variety of immune cells are involved, and besides B cells, T cell abnormalities are also a critical pathogenesis.

The invention aims to construct a bifunctional molecule targeting IL-17 and BAFF/APRIL simultaneously, inhibit the overactivation of B cells and T cells, and possibly show better treatment effect on SLE.

Disclosure of Invention

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

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 ectodomain fragment or variant; the second domain comprises an antibody or antigen binding fragment that specifically binds human IL-17.

In a preferred embodiment, the first domain of the bifunctional fusion protein molecule comprises a TACI ectodomain fragment or variant having the amino acid sequence of SEQ ID NO:2 (amino acids 1-159 of the full-length TACI 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 SEQ ID NO:5 (amino acids 1-116 of the full-length TACI fragment).

In one embodiment, the antibody or antigen binding fragment is selected from the group consisting of: (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 the group consisting of F (ab) ₂ One or more of Fab', fab, fv, scFv, bispecific antibodies, nanobodies, and antibody minimal recognition units.

In a specific embodiment, the antibody or antigen binding fragment that specifically binds human IL-17 comprises Vunakizumab, ixekizumab or securinumab.

In a specific embodiment, the heavy and light chains of the antibody or antigen binding fragment, respectively, that specifically binds human IL-17 have the amino acid sequence of SEQ ID NO:11 and SEQ ID NO: 12.

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

In a preferred embodiment, the TACI ectodomain fragment or variant is fused to an antibody or antigen-binding fragment of human IL-17 by a linker peptide; preferably, the linker peptide shown as SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 is used.

In another embodiment, the bifunctional fusion protein molecule comprises:

(1) The heavy chain has the amino acid sequence of SEQ ID NO: 13; the light chain has the amino acid sequence of SEQ ID NO: 12;

(2) The heavy chain has the amino acid sequence of SEQ ID NO:14, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(3) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO:15, a sequence shown in seq id no;

(4) The heavy chain has the amino acid sequence of SEQ ID NO:16, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(5) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO:17, a sequence shown in seq id no;

(6) The heavy chain has the amino acid sequence of SEQ ID NO:18, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(7) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO: 19;

(8) The heavy chain has the amino acid sequence of SEQ ID NO:20, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(9) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO:21, a sequence shown in seq id no;

(10) The heavy chain has the amino acid sequence of SEQ ID NO: 22; the light chain has the amino acid sequence of SEQ ID NO: 12;

(11) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO: 23;

(12) The heavy chain has the amino acid sequence of SEQ ID NO:24, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(13) The heavy chain has the amino acid sequence of SEQ ID NO:25, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(14) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO: 26;

(15) The heavy chain has the amino acid sequence of SEQ ID NO: 27; the light chain has the amino acid sequence of SEQ ID NO: 12;

(16) The heavy chain has the amino acid sequence of SEQ ID NO:28, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(17) The heavy chain has the amino acid sequence of SEQ ID NO: 29; the light chain has the amino acid sequence of SEQ ID NO: 12;

(18) The heavy chain has the amino acid sequence of SEQ ID NO:30, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(19) The heavy chain has the amino acid sequence of SEQ ID NO: 31; the light chain has the amino acid sequence of SEQ ID NO: 12;

(20) The heavy chain has the amino acid sequence of SEQ ID NO: 32; the light chain has the amino acid sequence of SEQ ID NO: 12;

(21) The heavy chain has the amino acid sequence of SEQ ID NO:33, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(22) The heavy chain has the amino acid sequence of SEQ ID NO: 34; the light chain has the amino acid sequence of SEQ ID NO: 12;

(23) The heavy chain has the amino acid sequence of SEQ ID NO:35, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(24) The heavy chain has the amino acid sequence of SEQ ID NO:36, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(25) The heavy chain has the amino acid sequence of SEQ ID NO:37, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(26) The heavy chain has the amino acid sequence of SEQ ID NO: 38; the light chain has the amino acid sequence of SEQ ID NO: 12; or (b)

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

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

(1) Specifically binds to IL-17A protein;

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

(3) Blocking BAFF binding to BAFF receptor BAFFR;

(4) Blocking BAFF binding to BAFF receptor BCMA;

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

(6) Neutralizing human IL-17-induced CXCL1 secretion;

(7) Mediate inhibition of proliferation of human B cells; and/or the number of the groups of groups,

(8) Treating autoimmune diseases.

In a second aspect, the present disclosure provides a TACI ectodomain fragment or variant of the first aspect that is a transmembrane activator and CAML interacting factor (Transmembrane Activator and CAML Interactor, TACI) ectodomain fragment or variant having the amino acid sequence of 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 a bifunctional fusion protein molecule of the first aspect or a TACI ectodomain fragment or variant of the second aspect.

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

In a fifth aspect, the present disclosure provides a host cell comprising the isolated nucleic acid molecule of the third aspect, or the expression vector of the fourth aspect; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, escherichia coli and/or bacillus subtilis; more preferably, the host cell is selected from the group consisting of an Expi293 cell.

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

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

In an eighth aspect, the present disclosure provides the use of a bifunctional fusion protein molecule of the first aspect, a TACI ectodomain fragment or variant of the second aspect, an isolated nucleic acid molecule of the third aspect, an expression vector of the fourth aspect, a cell of the fifth aspect, or a product prepared by a method of the sixth aspect, or a pharmaceutical composition of the seventh aspect, for the preparation of a medicament for the prevention and/or treatment of an autoimmune disease;

preferably, the disease is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic Lupus Erythematosus (SLE), lupus Nephritis (LN), wegener's disease, inflammatory bowel disease, idiopathic Thrombocytopenic Purpura (ITP), thrombotic Thrombocytopenic Purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, igA nephropathy, igM polyneuropathy, myasthenia gravis, vasculitis, diabetes mellitus, reynaud's syndrome, sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, autoimmune encephalomyelitis and autoimmune thyroiditis.

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

In a tenth aspect, the present invention provides a bifunctional fusion protein molecule of the first aspect, a TACI ectodomain fragment or variant of the second aspect, an isolated nucleic acid molecule of the third aspect, an expression vector of the fourth aspect, a cell of the fifth aspect, or a product prepared by a method of the sixth aspect, or a pharmaceutical composition of the seventh aspect, for use in the prevention and/or treatment of an autoimmune disease;

In an eleventh aspect, the invention provides a kit comprising a bifunctional fusion protein molecule of the first aspect, a fragment or variant of the TACI ectodomain of the second aspect, an isolated nucleic acid molecule of the third aspect, an expression vector of the fourth aspect, a cell of the fifth aspect, or a product of the method of the sixth aspect, or a pharmaceutical composition of the seventh aspect, and instructions for use.

Definition and description of terms

As used herein, the term "antibody" (Ab) refers to immunoglobulin molecules that specifically bind to or are immunoreactive with an antigen of interest, including polyclonal, monoclonal, genetically engineered and other modified forms of the antibody (including, but not limited to, chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (e.g., bispecific, trispecific and tetraspecific antibodies, diabodies, trispecific and tetraspecific antibodies, antibody conjugates) and antigen-binding fragments of the antibody (including, e.g., fab ', F (Ab ') 2, fab, fv, rIgG and scFv fragments) — furthermore, unless otherwise specified, the term "monoclonal antibody" (mAb) is intended to include intact antibody molecules capable of specifically binding to the target protein as well as incomplete antibody fragments (e.g., fab and F (Ab ') 2 fragments, which lack the Fc fragment of the intact antibody and thus lack Fc-mediated effector function) (see Wahl et al, j. Nucl. Med.24:316, 1983; the disclosure of which is incorporated herein by reference).

The "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, primo-ostris, alpacas, sheep, rabbits, mice, rats or chondrilleids (e.g. shark).

The term "antigen binding fragment" herein refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen binding function of an antibody may be performed by a fragment of a full-length antibody. The antibody fragment may be a Fab, F (ab') 2, scFv, SMIP, diabody, triabody, affibody (affibody), nanobody, aptamer, or domain antibody. Examples of binding fragments that encompass the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL and CHl domains; (ii) A F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked at a hinge region by a disulfide bond; (iii) an Fd fragment consisting of VH and CHl domains; (iv) Fv fragments consisting of the VL and VH domains of the antibody single arm; (V) a dAb comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al Nature 341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) an isolated Complementarity Determining Region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be connected by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, these two domains can be joined, using recombinant methods, by a linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as 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 may 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 having a variable sequence derived from an immunoglobulin of one origin organism (e.g., rat or mouse) and constant regions derived from an immunoglobulin of a different organism (e.g., human). 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; gilles et al, 1985J Immunol Methods 125:191-202; the above is incorporated by reference herein.

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

The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or part of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies generally retain or partially retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune responses, and the like.

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

As used herein, the term "nanobody" refers to a heavy chain antibody in which the naturally occurring light chain is deleted in a camelid, the variable region of which is cloned to give a single domain antibody consisting of only the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen binding fragment.

The term "antibody minimal recognition unit" as used herein refers to the smallest unit that an antibody can recognize an antigen in a binding reaction of an antigen to an antibody.

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

The term "TACI" as used herein refers to transmembrane activators and CAML interacting factors (Transmembrane Activator and CAML Interactor, TACI) (von Bulow and Bram, science 228:138 (1997); bram and von Bulow, U.S. Pat. No. 5,969,102 (1999)). TACI is a membrane-bound receptor with an extracellular region containing two cysteine-rich pseudo-repeats, a transmembrane region and a cytoplasmic region that interacts with CAMLs (calcium modulator and cyclophilin ligands), an integral membrane protein located in intracellular vesicles, which when overexpressed in Jurkat cells is a synergistic inducer of NFAT activation.

The term "BAFF" herein also known as B lymphocyte stimulating factor (B cell activating factor), which is one of the members of the tumor necrosis factor ligand superfamily, is expressed primarily in bone marrow family cells including monocytes, macrophages, dendritic cells, neutrophils, malignant B cells, and the like. B lymphocyte stimulating factors are expressed on the surface of the cell membranes in a form of a trimer, and then released into microcirculation after being cut by Furin protease, so that the B lymphocyte stimulating factors are combined with receptors on the surface of the cell membranes. BAFF has mainly three receptors, transmembrane activator and CAML interacting factor (Transmembrane Activator and CAML Interactor, TACI), B cell maturation antigen (B cell maturation antigen, BCMA) and B cell activator receptor (B cell-activating factor receptor, BAFF-R), which are expressed on the surface of B cells at different developmental stages, respectively, and binding of BAFF to different receptors mediates different biological functions. BAFF-R is expressed predominantly on transitional B cells, including Follicular (FO) B cells, marginal Zone (MZ) B cells, with which BAFF binds with high specificity and high affinity, which bind to regulate B cell survival, development and differentiation. BCMA and TACI are expressed mainly on the membrane surface of activated B cells, memory B cells and plasma cells, recognize BAFF and also recognize proliferation-inducing ligands (APRIL) which are another member of the TNF ligand superfamily, and bind with higher affinity. Unlike BAFF-R, BCMA and TACI are associated with inflammatory responses and innate immunity.

The term "APRIL" herein also known as proliferation-inducing ligand (a pro-duction ligand) is one of the members 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 to the co-family member BAFF and can also be cleaved by Furin protease and released into the microcirculation, usually in the form of a trimer. APRIL promotes and participates in lymphocyte proliferation, differentiation and survival through binding to its receptors (BCMA and TACI).

The term "fusion protein" as used herein refers to a protein product obtained by gene recombination in which the coding regions of two or more genes are linked by genetic recombination methods, chemical methods or other suitable methods, and the gene recombination is expressed under the control of the same regulatory sequence. In the fusion proteins of the invention, the coding regions of two or more genes may be fused at one or more positions by sequences encoding peptide linkers or connecting peptides. Peptide linkers or linker peptides may also be used to construct fusion proteins of the invention. The term "fusion protein" according to the invention further comprises (a) an 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.

The term "percent (%) sequence identity" or "sequence of percent (%) identity" herein refers to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to amino acid (or nucleotide) residues of a reference sequence after aligning the sequences and introducing gaps, if desired, for maximum percent sequence identity (e.g., gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). For the purpose of determining percent sequence identity, the alignment may be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. One skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm that requires maximum alignment over the full length of the sequences being compared. For example, a reference sequence for comparison to a candidate sequence may show that the candidate sequence exhibits from 50% to 100% sequence identity over the entire length of the candidate sequence or over selected portions of consecutive amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequences 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.

The term "nucleic acid" herein includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, in particular 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 a phosphate group. In general, a nucleic acid molecule is 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 represented as 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), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. The nucleic acid molecule may 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 having derivatized sugar or phosphate backbone bonded or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and/or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule, so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., stadler et al, nature Medicine 2017,published online 2017, 6 months 12, 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 (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vectors of the invention contain polynucleotide sequences and additional sequence elements, for example, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that may be used to express the antibodies and antibody fragments of the invention include plasmids containing regulatory sequences (e.g., promoter and enhancer regions) that direct transcription of genes. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5 'and 3' untranslated regions, internal Ribosome Entry Sites (IRES) and polyadenylation signal sites, in order to direct efficient transcription of genes carried on expression vectors. The expression vectors of the invention may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding antibiotic (e.g., ampicillin, chloramphenicol, kanamycin, or nociceptin) resistance.

The term "host cell" as used herein refers to a cell into which exogenous nucleic acid has been introduced, and includes the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cells.

The term "pharmaceutical composition" herein refers to a formulation which exists in a form which allows for the biological activity of the active ingredient contained therein to be effective and which does not contain additional ingredients which have unacceptable toxicity to the subject to whom the pharmaceutical composition is administered.

The terms "subject," "subject," and "patient" herein refer to an organism that is receiving treatment for a particular disease or disorder (e.g., cancer or infectious disease) as described herein. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovine family (e.g., cattle, bison, buffalo, elk, and yaks, etc.), sheep, and horses, etc., that are treated for a disease or disorder (e.g., a cell proliferative disorder such as cancer or an infectious disease).

The term "treatment" herein refers to a surgical or pharmaceutical treatment (surgical or therapeutic treatment) aimed at preventing, slowing (reducing) the progression of an undesired physiological change or disorder, such as a cell proliferative disorder (e.g., cancer or infectious disease), in a subject. 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 complete), whether detectable or undetectable. Subjects in need of treatment include subjects already with the condition or disease and subjects prone to the condition or disease or subjects intended to prevent the condition or disease. When referring to terms slow down, alleviate, attenuate, mitigate, alleviate, etc., the meaning also includes eliminating, vanishing, non-occurrence, etc.

The term "suitable conditions" herein refers to conditions suitable for culturing a variety of host cells, including eukaryotic cells and prokaryotic cells.

The term "effective amount" herein refers to an amount of a therapeutic agent that is effective to prevent or ameliorate a disease condition or progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject. An "effective amount" also refers to an amount of a compound that is sufficient to alleviate symptoms, such as treating, curing, preventing or alleviating a related medical condition, or an increase in the rate of treating, curing, preventing or alleviating such conditions. When an active ingredient is administered to an individual alone, a therapeutically effective dose is referred to as the 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 parotitis, crohn's disease, diabetes (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, graves ' disease, gillin-barre 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, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxoedema, pernicious anemia, ulcerative colitis, and the like.

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

Drawings

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meaning as understood by one of ordinary skill in the art.

FIGS. 1-4, ELISA binding experiments of bifunctional molecules to IL-17A, wherein the positive control was Secukinumab.

FIGS. 5-8, bifunctional blocking IL-17A/IL-17RA binding experiments, wherein the positive control was Secukinumab.

FIGS. 9-11, ELISA binding experiments of bifunctional molecules to BAFF, wherein the positive control was RC-18.

FIGS. 12-14, ELISA binding experiments of bifunctional molecules to APRIL, wherein the positive control was RC-18.

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

FIGS. 18-20, bifunctional blocking BAFF/BCMA binding experiments, wherein the positive control was RC-18.

FIGS. 21-23, bifunctional molecule blocking APRIL/BCMA binding experiments, wherein the positive control was RC-18.

FIG. 24, bifunctional molecule neutralization IL-17 assay, wherein the positive control is Secukinumab.

FIG. 25, proliferation inhibition assay of B cells by bifunctional molecule, wherein positive control is RC-18.

FIGS. 26-29, linker-optimized ELISA binding experiments for bifunctional molecules to IL-17A, wherein the positive control was Secukinumab.

FIG. 30-FIG. 33, ELISA binding experiments of linker optimized bifunctional molecules to BAFF, wherein the positive control was RC-18.

FIGS. 34-37, ELISA binding experiments of linker optimized bifunctional molecules to APRIL, wherein the positive control was RC-18.

FIG. 38, linker optimized bifunctional neutralizing IL-17 assay, wherein positive control was Secukinumab and negative control was hIgG1.

FIG. 39 shows an experiment of proliferation inhibition of B cells by a bifunctional molecule after linker optimization, wherein the positive control is RC-18 and the negative control is hIgG1.

FIG. 40, effect of bifunctional molecules on serum Cxcl-1 levels after IL-17 injection in mice, where positive control was Secukinumab, negative control was blank vehicle (PBS), and normal control was non-IL-17 injected mice.

FIG. 41, effect of bifunctional molecules on serum IgA levels after BAFF injection in mice, wherein positive control was RC-18, negative control was blank vehicle (PBS), and normal control was non-BAFF injected mice.

FIG. 42, efficacy of bifunctional molecules in a mouse DTH model, wherein positive controls are Secukinumab and RC-18, negative controls are blank vehicle (PBS), and normal controls are non-model mice.

FIG. 43, efficacy of bifunctional molecules in a mouse EAE model, wherein positive controls are Secukinumab and RC-18, negative controls are blank vehicle (PBS), and normal controls are non-model mice.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The present embodiments are merely examples and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

EXAMPLE 1 expression and purification of bifunctional molecules

Based on the full length sequence (SEQ ID NO:1, sequence is referenced to Uniprot#O14836) and structure of human TACI protein, different TACI fragments were designed and ligated to the N-or C-terminus of the heavy chain (SEQ ID NO:11, sequence is referenced to WHO drug information) or light chain (SEQ ID NO:12, sequence is referenced to WHO drug information) of the IL-17 antibody Secukinumab, plasmids were constructed to produce IL-17-TACI bifunctional molecules, specific information for the construction of bifunctional molecules is shown in Table 1, and amino acid sequence information is shown in Table 2.Anti-IL-17 mab Secukinumab and TACI fusion proteins RC-18 (SEQ ID NO:10, sequence referenced WHO drug information) served as positive controls.

TABLE 1 heavy and light chain combination information for bifunctional molecules

Name of the 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 for bifunctional molecules and control antibodies

Adding plasmid and transfection reagent PEI (Polysciences, cat# 24765-1) into OPTI-MEM (Gibco, cat# 11058021), mixing, standing for 15min, adding into Expi293 cells (thermosipher, cat# A14527), adding 5% CO ₂ Shaking culture at 120rpm and 37 ℃. The next day of transfection, OPM-293Profeed (Shanghai Ao Pu Mai, cat# F081918-001) and 6G/L glucose (Sigma, cat# G7528) were added. On day six of transfection, cell supernatants were collected, purified by ProteinA (GE, cat# 28985254) and eluted samples dialyzed against PBS, pH7.4. The purity of the samples was determined by SEC-HPLC. 1-1- (G4S) 3,3-2- (G4S) 3,4-2- (G4S) 3,5-1- (G4S) 3,5-4- (G4S) 3 have better purity, and the SEC monomer is more than 85 percent. The yields and SEC purities of the 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 obtaining the anti-IL-17-TACI bifunctional molecule, the binding activity and blocking activity of the IL-17 end and the TACI end are respectively determined.

2.1 human IL-17A ELISA binding experiments

Human IL-17A protein (Acro Biosystems, cat# ILA-H5118) was coated overnight at 4℃and rinsed 3 times with 0.05% Tween 20-PBS, 3% BSA blocking solution was added and blocked at 37℃for 1.5H. After rinsing 3 times with 0.05% Tween 20-PBS, the diluted sample was added and incubated at 37℃for 1h. After rinsing 3 times with 0.05% Tween 20-PBS, secondary antibody HRP coat anti-human IgG Fc (Merck, cat# AP 113) was added and incubated at 37℃for 1h. After 3 times rinsing with 0.05% Tween 20-PBS, TMB solution (Seracare, cat# 5120-0077) was added, the reaction was stopped by adding 1M hydrochloric acid after 10min at room temperature, and plates were read at 450nM wavelength by an ELISA reader. The experimental results are shown in figures 1-4, and the bifunctional molecules are strongly combined with human IL-17A. Except 3-2- (G4S) 3, the EC50 of the remaining molecules was similar to that of IL-17 mab Secukinumab. The binding 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 assay

Human IL-17A protein (Acro Biosystems, cat# ILA-H5118) was coated overnight at 4℃and rinsed 3 times with 0.05% Tween 20-PBS, 2% BSA blocking solution was added and blocked at 37℃for 1.5H. The samples were rinsed 3 times with 0.05% Tween 20-PBS and then added with Biotin-IL-17RA (Acro Biosystems, cat# ILA-H5257) and a double dilution, and incubated at 37℃for 1.5H. After rinsing 3 times with 0.05% Tween 20-PBS, secondary antibody SA-HRP (Sigma, cat# S2438) was added and incubated at 37℃for 1h. After 3 times rinsing with 0.05% Tween 20-PBS, TMB solution (Seracare, cat# 5120-0077) was added, the reaction was stopped by adding 1M hydrochloric acid after 10min at room temperature, and plates were read at 450nM wavelength by an ELISA reader. The results of the experiment are shown in FIGS. 5-8, and the bifunctional molecules can effectively block the binding of IL-17A and IL-17 RA. ICS0 of the remaining molecules, except 3-2- (G4S) 3, was close to or slightly superior to that of IL-17 mab Secukinumab. The IC50 values of the blocking experiments are shown in table 5.

TABLE 5 blocking Activity of bifunctional molecules against 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 experiments with BAFF or APRIL

Human BAFF protein (Acro Biosystems, cat# BAF-H52D 4) or human APRIL protein (Acro Biosystems, cat# APL-H52D 1) was coated overnight at 4℃and rinsed 3 times with 0.05% Tween 20-PBS, and blocked with 3% BSA blocking solution at 37℃for 1.5H. After rinsing 3 times with 0.05% Tween 20-PBS, the diluted sample was added and incubated at 37℃for 1h. After rinsing 3 times with 0.05% Tween 20-PBS, secondary antibody HRP coat anti-human IgG Fc (Merck, cat# AP 113) was added and incubated at 37℃for 1h. After 3 times rinsing with 0.05% Tween 20-PBS, TMB solution (Seracare, cat# 5120-0077) was added, the reaction was stopped by adding 1M hydrochloric acid after 10min at room temperature, and plates were read at 450nM wavelength by an ELISA reader. As shown in FIGS. 9-14, the bifunctional molecules bound to human BAFF and APRIL strongly, wherein the EC50 values of 3-1- (G4S) 3, 3-2- (G4S) 3, 4-1- (G4S) 3, 4-2- (G4S) 3, 5-1- (G4S) 3 bound to BAFF and APRIL were superior to TACI fusion proteins RC-18, and the binding 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 experiments

Human BAFF protein (Acro Biosystems, cat# BAF-H52D 4) was coated overnight at 4℃and rinsed 3 times with 0.05% Tween 20-PBS, 2% BSA blocking solution was added and blocked at 37℃for 1.5H. The samples were rinsed 3 times with 0.05% Tween 20-PBS and then added with Biotin-BAFFR (Acro Biosystems, cat# BAR-H5257) and a double dilution, and incubated at 37℃for 1.5H. After rinsing 3 times with 0.05% Tween 20-PBS, secondary antibody SA-HRP (Sigma, cat# S2438) was added and incubated at 37℃for 1h. After 3 times rinsing with 0.05% Tween 20-PBS, TMB solution (Seracare, cat# 5120-0077) was added, the reaction was stopped by adding 1M hydrochloric acid after 10min at room temperature, and plates were read at 450nM wavelength by an ELISA reader. As shown in FIGS. 15-17, the bifunctional molecules were each able to effectively block the binding of BAFF to BAFFR, wherein the blocking activity of 3-1- (G4S) 3, 3-2- (G4S) 3, 5-1- (G4S) 3 was significantly better than that of TACI fusion protein RC-18 (IC 50 values differ by more than 2-fold, considered to be significantly better). The blocking IC50 values are shown in table 7.

2.5 BAFF and BCMA binding blocking experiments

Human BAFF protein (Acro Biosystems, cat# BAF-H52D 4) was coated overnight at 4℃and rinsed 3 times with 0.05% Tween 20-PBS, 2% BSA blocking solution was added and blocked at 37℃for 1.5H. The samples were rinsed 3 times with 0.05% Tween 20-PBS and then added to Biotin-BCMA (Acro Biosystems, cat# BC 7-H5254) and diluted in a double ratio, and incubated at 37℃for 1.5H. After rinsing 3 times with 0.05% Tween 20-PBS, secondary antibody SA-HRP (Sigma, cat# S2438) was added and incubated at 37℃for 1h. After 3 times rinsing with 0.05% Tween 20-PBS, TMB solution (Seracare, cat# 5120-0077) was added, the reaction was stopped by adding 1M hydrochloric acid after 10min at room temperature, and plates were read at 450nM wavelength by an ELISA reader. As shown in FIGS. 18-20, the blocking activity of 3-1- (G4S) 3, 3-2- (G4S) 3, 3-3- (G4S) 3, 4-2- (G4S) 3, 5-1- (G4S) 3 was significantly better than that of TACI fusion protein RC-18 (IC 50 values differ by more than 2-fold, considered to be better) as the results of the experiments. The blocking IC50 values are shown in table 7.

2.6 BCMA and APRIL binding blocking assay

Human BCMA protein (Acro Biosystems, cat# BC 7-H5254) was coated overnight at 4℃and rinsed 3 times with 0.05% Tween 20-PBS, 2% BSA blocking solution was added and blocked at 37℃for 1.5H. The samples were rinsed 3 times with 0.05% Tween 20-PBS and then added with Biotin-APRIL (Acro Biosystems, cat# APL-H82F 5) and a double dilution, and incubated at 37℃for 1.5H. After rinsing 3 times with 0.05% Tween 20-PBS, secondary antibody SA-HRP (Sigma, cat# S2438) was added and incubated at 37℃for 1h. After 3 times rinsing with 0.05% Tween 20-PBS, TMB solution (Seracare, cat# 5120-0077) was added, the reaction was stopped by adding 1M hydrochloric acid after 10min at room temperature, and plates were read at 450nM wavelength by an ELISA reader. As shown in FIGS. 21-23, the blocking activity of 3-1- (G4S) 3, 3-2- (G4S) 3, 3-3- (G4S) 3, 4-1- (G4S) 3, 4-2- (G4S) 3, 5-1- (G4S) 3 was significantly better than that of TACI fusion protein RC-18 (IC 50 values differing by more than 2-fold, considered to be better). The blocking IC50 values are shown in table 7.

TABLE 7 blocking Activity of bifunctional molecules against BAFF-BAFFR, BAFF-BCMA, APRIL-BCMA

EXAMPLE 3 detection of neutralization Activity of bifunctional molecules on IL-17 and BAFF/APRIL

3.1 IL-17 neutralization assay to detect the Activity of the IL-17 terminus of bifunctional molecules

IL-17 stimulates secretion of CXCL1 by HT-29 cells. HT-29 cells (stock of China academy of sciences: TCHu 103) were added to a 96-well plate, together with human IL-17 (R & D Systems, stock: 317-ILB-050) and a double diluted sample, mixed well and incubated for 48 hours, cell supernatants were collected, and human CXCL1 levels in the supernatants were detected using ELISA kits (R & D Systems, stock: SGR 00B). As shown in FIG. 24, the bifunctional molecules were both effective in neutralizing IL-17, thereby inhibiting CXCL1 secretion. The neutralizing activity of the remaining molecules was similar to that of IL-17 mab Secukinumab except 3-2- (G4S) 3,4-2- (G4S) 3. The neutralization assay IC50 values are shown in Table 8.

IL-17 neutralization assay

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 TACI terminal Activity

BAFF and APRIL promote proliferation of B cells. Human B cells (human B cell isolation kit, stemcell, cat# 17954) were extracted from human PBMC cells (Allcells, cat# 109-007-043), anti-IgM (Jakson immunoo, cat# 109-007-043), BAFF (Acro Biosystems, cat# BAF-H52D 4), APRIL (R & D Systems, cat# 5860-AP-010) were incubated with IL4 (Peprotech, cat# 200-04), and after 4 days incubation, the proliferation of B cells was detected with Ki67-APC (eBioscience, cat# 17-5699-42) with the addition of the test sample. As shown in the experimental result in FIG. 25, the inhibition activity of the bifunctional molecule is overall better than that of TACI fusion protein RC-18, and can effectively inhibit B cell proliferation induced by BAFF and APRIL,

Example 4 comparison of the linker in bifunctional molecules

There are various options for the linker linking the Secukinumab and TACI fragments, and (G4S) 3 was used in example 1. Furthermore, we have tried (G4S) 4, (GS) 10 and (GSG) 5, information on these bifunctional molecules is shown in Table 9, and information on specific amino acid sequences is shown in Table 10.

TABLE 9 details of optimized Structure of bifunctional molecular linkers

Name of the 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 bifunctional molecules thereof

The purification results are shown in Table 11. These bifunctional molecules were subjected to binding experiments for human IL-17A, BAFF and APRIL, respectively, as in example 2, with reference to examples 2.1 and 2.3, and the experimental results are shown in FIGS. 26 to 37. The results indicate that changing the linker has no significant effect on the binding activity of the IL-17 and TACI ends.

TABLE 11 yield and purity of optimized bifunctional molecule linkers

In addition, the linker-optimized bifunctional molecules were also tested for IL-17 neutralization and B cell proliferation assays, as described in example 3.IL-17 neutralization assay results As shown in FIG. 38, bifunctional molecules 3-1- (GSG) 5, 3-3- (G4S) 4, 4-1- (GS) 10 and 5-1- (GSG) 5 were each effective in neutralizing IL-17, thereby inhibiting CXCL1 secretion, wherein the neutralizing activity of 4-1- (GS) 10, 5-1- (GSG) 5 was superior to that of IL-17 monoclonal antibody Secukinumab. The B cell proliferation test results are shown in FIG. 39, and the bifunctional molecules 3-1- (GSG) 5, 3-3- (G4S) 4, 4-1- (GS) 10 and 5-1- (GSG) 5 can effectively inhibit B cell proliferation induced by BAFF and APRIL, and the inhibition activity is superior to that of TACI fusion protein RC-18. The IC50 values for IL17 neutralization experiments and B cell proliferation experiments are shown in table 12.

TABLE 12 neutralization assay and B cell proliferation assay of IL17

EXAMPLE 5 Effect of bifunctional molecules on Cxcl-1 levels after mice injected with IL-17

Female mice (Beijing Vitre Lihua laboratory animal technologies Co., ltd.) of 6-8 weeks old C57BL/6N were randomly grouped into a normal control group, a negative control group, a positive control group and a bifunctional molecule group. Wherein the administration dosage of the bifunctional molecule group is 16mg/kg; the positive control group is IL-17 monoclonal antibody Secukinumab (15 mg/kg) with equimolar dosage; the negative control group was an equal volume of blank vehicle (PBS), and the normal control group was mice not injected with IL-17, all administered at the same time point by intraperitoneal injection. 24 hours after administration, each group of mice was intraperitoneally injected with human IL-17 protein (ACRObiosystems) at a dose of 3. Mu.g/mouse; 26 hours after administration, each group of mice was sacrificed and whole blood was collected, and after standing at room temperature, the supernatant was centrifuged and the concentration of cytokine Cxcl-1 in serum was detected with ELISA Kit (Mouse CXCL1 ELISA Kit; R & D).

As shown in fig. 40, the administration of bifunctional molecules 3-1- (GSG) 5 and 3-3- (G4S) 4 was effective to neutralize IL-17, resulting in a significant decrease in the level of Cxcl-1 in the serum of mice, and a statistical difference compared to the negative control group (the statistical difference between each experimental group and the negative control group was significant, and the statistical method was one-wayANOVA, which indicates P-value < 0.0001). The inhibition of Cxcl-1 level by the bifunctional molecule is superior to that of the positive control Secukinumab.

Example 6 Effect of bifunctional molecules on IgA and IgG levels after mice were injected with BAFF

Female mice (Beijing Veitz laboratory animal technologies Co., ltd.) of 6-8 weeks old were randomly grouped into a normal control group, a negative control group, a positive control group, and a bifunctional molecule group. Wherein the administration dose of the bifunctional molecule group is 32mg/kg of 3-1- (GSG) 5; the positive control group was RC-18 (15 mg/kg) at an equimolar dose; the negative control group was an equal volume of blank vehicle (PBS), and the normal control group was BAFF-naive mice, all given once at the same time point by intraperitoneal injection. At 2h, 26h, 50h and 74h post-dose, each group of mice was injected with human BAFF protein (Human BAFF protein; sino Biological) by tail vein at a dose of 3 mg/kg. 98h after dosing, each group of mice was sacrificed and whole blood was collected, and the supernatant was centrifuged after standing at room temperature and the concentration of cytokine IgA in serum was detected using ELISA kit (Mouse IgA ELISA Kit; abcam).

As shown in fig. 41, the administration of bifunctional molecule 3-1- (GSG) 5 effectively neutralized BAFF, resulting in a significant reduction in serum IgA levels in mice compared to the negative control group (statistical significance of statistical differences between each experimental group and the negative control group, statistical method was one-way ANOVA, P value < 0.001, P value < 0.0001). The inhibition effect of 3-1- (GSG) 5 on IgA level is significantly better than that of RC-18 (IgA: P value < 0.001) with equimolar dosage.

EXAMPLE 7 efficacy of bifunctional molecules in a mouse delayed hypersensitivity (DTH) model

6-8 week old IL-17A humanized C57BL/6N female mice (Shanghai's model Biotech Co., ltd.) were injected subcutaneously at two sites on the ventral side of each mouse with 100. Mu.l of fully emulsified emulsion containing 100. Mu.g keyhole limpet hemocyanin (KLH; sigma) at 50. Mu.l per site. The preparation method of the emulsion comprises the following steps: dissolving KLH protein into a protein solution with an initial concentration of 3mg/ml by using PBS; the protein solution, complete Freund's adjuvant (CFA; sigma-Aldrich) and incomplete Freund's adjuvant (IFA, incomplete Freund's adjuvant; sigma-Aldrich) are respectively sucked by a plastic syringe in equal volumes, and are connected by a communicating tube with a 100-mesh screen, and pushed back and forth on ice for about 15min to make emulsion uniform, so that the liquid drops are not scattered on the water surface, and the plastic syringe can be used for subcutaneous injection molding. The final concentration of KLH protein in the emulsion was 1mg/ml. On day 5 after the model immunization, 10 μl of KLH protein solution dissolved in PBS to a concentration of 1mg/ml was injected into the skin of each mouse, and the thickness of the right ear of the mouse was measured by a dial thickness gauge (PEACOCK) at 24 hours, 48 hours, 72 hours and 96 hours before and after the stimulation to obtain a change value delta ear thickness= (thickness of right ear after the stimulation) - (thickness of right ear before the stimulation).

Mice were grouped into normal control, negative control, positive control, and bifunctional molecule groups. Wherein the administration dose of the bifunctional molecule group is 64mg/kg of 3-1- (GSG) 5; the positive control group is the equivalent molar doses of Secukinumab and RC-18, and the corresponding administration doses are respectively as follows: secukinumab is 60mg/kg, RC-18 is 30mg/kg; the negative control group was blank vehicle (PBS), and the normal control group was non-model mice. The administration was by intraperitoneal injection starting on the day of the model immunization, once every two days, 5 times in total.

As shown in fig. 42, the thickness change of the right ear of DTH mice was significantly reduced after administration of bifunctional molecule 3-1- (GSG) 5, and there was a statistical difference compared to the negative control group (the statistical method is two-way ANOVA, which represents P value < 0.01, and P value < 0.0001). The efficacy of 3-1- (GSG) 5 was significantly better than that of equimolar doses of positive control RC-18 (P value < 0.0001).

Example 8 efficacy of bifunctional molecules in a mouse Experimental Autoimmune Encephalomyelitis (EAE) model

6-8 week old IL-17A humanized C57BL/6N female mice (Shanghai Nannon model Biotech Co., ltd.) were injected with 150. Mu.l of fully emulsified emulsion containing 225. Mu.g MOG (29-156) protein in three spots uniformly on the posterior cervical and lateral midline of the tail root of each mouse, and 50. Mu.l of each spot. The preparation method of the emulsion comprises the following steps: the MOG (29-156) protein was dissolved in PBS to a protein solution with an initial concentration of 3 mg/ml; two plastic syringes are used to absorb the same volume of protein solution and Complete Freund's adjuvant (CFA; sigma-Aldrich), and a communicating tube with 100 mesh screen is used to connect, and the mixture is pushed back and forth on ice for 15min to make emulsion even, so that the droplets are not scattered on the water surface, and the mixture can be used for subcutaneous injection molding. The final concentration of MOG protein in the emulsion was 1.5mg/ml. PTX (Pertussis Toxins; list Biological Laboratories) was prepared as a 1. Mu.g/ml solution in physiological saline and was intraperitoneally injected into mice on the day of the model immunization and 48 hours after the immunization, respectively, with 400ng of PTX per mouse. Mice after molding entered the morbidity over about 9 days. Mice body weights were recorded daily for 28 days of continuous observation, and disease clinical symptoms were scored blindly by independent second persons. The scoring criteria are as follows: 0 minutes, no obvious disease symptoms; 1 minute, tail tension disappears or the hind limb is weak; 2 minutes, tail tension is relieved and the hind limb is weak; 3 minutes, paralysis of hind limb part; 4 minutes, the hind limb is completely paralyzed; 5 minutes, dying state or death.

Mice were grouped into normal control, negative control, positive control, and bifunctional molecule groups. Wherein the bifunctional molecule group is administered at a dose of 10.6mg/kg of 3-1- (GSG) 5; the positive control group is the equivalent molar doses of Secukinumab and RC-18, and the corresponding administration doses are respectively as follows: secukinumab is 10mg/kg, RC-18 is 5mg/kg; the negative control group was blank vehicle (PBS), and the normal control group was non-model mice. The administration was by intraperitoneal injection, once every two days, starting on the day of the model immunization, for 28 consecutive days.

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

Claims

A bifunctional fusion protein molecule comprising a first domain and a second domain; wherein the first domain comprises a TACI ectodomain fragment or variant; the second domain comprises an antibody or antigen binding fragment that specifically binds human IL-17.
The bifunctional fusion protein molecule of claim 1, wherein the first domain of the bifunctional fusion protein molecule comprises a TACI ectodomain fragment or variant having the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO:4 or SEQ ID NO: 5.
The bifunctional fusion protein molecule of any one of claims 1-2, wherein the antibody or antigen-binding fragment is selected from the group consisting of:

(1) A chimeric antibody or fragment thereof;

(2) A humanized antibody or fragment thereof; or alternatively, the first and second heat exchangers may be,

(3) Fully human antibodies or fragments thereof;

preferably, the antibody or antigen binding fragment is selected from the group consisting of F (ab) ₂ One or more of Fab', fab, fv, scFv, bispecific antibodies, nanobodies, and antibody minimal recognition units;

preferably, the antibody or antigen binding fragment that specifically binds human IL-17 comprises Vunakizumab, ixekizumab or securinumab;

more preferably, the heavy and light chains of the antibody or antigen binding fragment, respectively, that specifically binds human IL-17 have the amino acid sequence of SEQ ID NO:11 and SEQ ID NO: 12.
A bifunctional fusion protein molecule as claimed in any one of claims 1 to 3 wherein the TACI ectodomain fragment or variant is linked to the N-or C-terminus of the heavy or light chain of an antibody or antigen-binding fragment of human IL-17;

Preferably, the TACI ectodomain fragment or variant is fused to an antibody or antigen-binding fragment of human IL-17 by a linker peptide; preferably, the linker peptide shown as SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 is used.
The bifunctional fusion protein molecule of any one of claims 1-4, wherein: the bifunctional fusion protein molecule comprises:

(1) The heavy chain has the amino acid sequence of SEQ ID NO: 13; the light chain has the amino acid sequence of SEQ ID NO: 12;

(2) The heavy chain has the amino acid sequence of SEQ ID NO:14, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(3) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO:15, a sequence shown in seq id no;

(4) The heavy chain has the amino acid sequence of SEQ ID NO:16, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(5) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO:17, a sequence shown in seq id no;

(6) The heavy chain has the amino acid sequence of SEQ ID NO:18, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(7) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO: 19;

(8) The heavy chain has the amino acid sequence of SEQ ID NO:20, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(9) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO:21, a sequence shown in seq id no;

(10) The heavy chain has the amino acid sequence of SEQ ID NO: 22; the light chain has the amino acid sequence of SEQ ID NO: 12;

(11) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO: 23;

(12) The heavy chain has the amino acid sequence of SEQ ID NO:24, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(13) The heavy chain has the amino acid sequence of SEQ ID NO:25, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(14) The heavy chain has the amino acid sequence of SEQ ID NO: 11; the light chain has the amino acid sequence of SEQ ID NO: 26;

(15) The heavy chain has the amino acid sequence of SEQ ID NO: 27; the light chain has the amino acid sequence of SEQ ID NO: 12;

(16) The heavy chain has the amino acid sequence of SEQ ID NO:28, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(17) The heavy chain has the amino acid sequence of SEQ ID NO: 29; the light chain has the amino acid sequence of SEQ ID NO: 12;

(18) The heavy chain has the amino acid sequence of SEQ ID NO:30, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(19) The heavy chain has the amino acid sequence of SEQ ID NO: 31; the light chain has the amino acid sequence of SEQ ID NO: 12;

(20) The heavy chain has the amino acid sequence of SEQ ID NO: 32; the light chain has the amino acid sequence of SEQ ID NO: 12;

(21) The heavy chain has the amino acid sequence of SEQ ID NO:33, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(22) The heavy chain has the amino acid sequence of SEQ ID NO: 34; the light chain has the amino acid sequence of SEQ ID NO: 12;

(23) The heavy chain has the amino acid sequence of SEQ ID NO:35, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(24) The heavy chain has the amino acid sequence of SEQ ID NO:36, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(25) The heavy chain has the amino acid sequence of SEQ ID NO:37, a sequence shown in seq id no; the light chain has the amino acid sequence of SEQ ID NO: 12;

(26) The heavy chain has the amino acid sequence of SEQ ID NO: 38; the light chain has the amino acid sequence of SEQ ID NO: 12; or (b)

(27) Amino acid sequences having at least 90% identity to the sequences set forth in (1) - (26) above, preferably amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
The bifunctional fusion protein molecule of any one of claims 1-5, wherein the bifunctional fusion protein molecule has the TACI ectodomain fragment or variant of claim 1 and competitively binds to the BAFF/APRIL protein and has the following properties:

(1) Specifically binds to IL-17A protein;

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

(3) Blocking BAFF binding to BAFF receptor BAFFR;

(4) Blocking BAFF binding to BAFF receptor BCMA;

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

(6) Neutralizing human IL-17-induced CXCL1 secretion;

(7) Mediate inhibition of proliferation of human B cells; and/or the number of the groups of groups,

(8) Treating autoimmune diseases.
A TACI ectodomain fragment or variant in a bifunctional fusion protein molecule as claimed in claim 1 which is a transmembrane activator and CAML interacting factor (Transmembrane Activator and CAML Interactor, TACI) ectodomain fragment or variant, characterized in that said TACI ectodomain fragment or variant has the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO:4 or SEQ ID NO: 5.
An isolated nucleic acid molecule encoding the bifunctional fusion protein molecule of any one of claims 1-6 or the TACI ectodomain fragment or variant of claim 7.
An expression vector comprising the isolated nucleic acid molecule of claim 8.
A host cell comprising the isolated nucleic acid molecule of claim 8, or the expression vector of claim 9; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, escherichia coli and/or bacillus subtilis; more preferably, the host cell is selected from the group consisting of an Expi293 cell.
A method for the preparation of a bifunctional fusion protein molecule, wherein the host cell of claim 10 is cultured or cultured under appropriate conditions, and the bifunctional fusion protein molecule is isolated.
A pharmaceutical composition comprising the bifunctional fusion protein molecule of any one of claims 1-6, the TACI ectodomain fragment or variant of claim 7, the isolated nucleic acid molecule of claim 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, diluent or adjuvant; preferably, the pharmaceutical composition further comprises an additional autoimmune disease therapeutic agent.
Use of the bifunctional fusion protein molecule of any one of claims 1-6, the TACI ectodomain fragment or variant of claim 7, the isolated nucleic acid molecule of 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 for the preparation of a medicament for the prevention and/or treatment of an autoimmune disease;

Preferably, the disease is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic Lupus Erythematosus (SLE), lupus Nephritis (LN), wegener's disease, inflammatory bowel disease, idiopathic Thrombocytopenic Purpura (ITP), thrombotic Thrombocytopenic Purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, igA nephropathy, igM polyneuropathy, myasthenia gravis, vasculitis, diabetes mellitus, reynaud's syndrome, sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, autoimmune encephalomyelitis and autoimmune thyroiditis.
A method of preventing and/or treating an autoimmune disease comprising administering to a patient in need thereof the bifunctional fusion protein molecule of any one of claims 1-6, the TACI ectodomain fragment or variant of claim 7, the isolated nucleic acid molecule of claim 8, the expression vector of claim 9, the cell of claim 10, the product made according to the method of claim 11, or the pharmaceutical composition of claim 12;

preferably, the disease is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic Lupus Erythematosus (SLE), lupus Nephritis (LN), wegener's disease, inflammatory bowel disease, idiopathic Thrombocytopenic Purpura (ITP), thrombotic Thrombocytopenic Purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, igA nephropathy, igM polyneuropathy, myasthenia gravis, vasculitis, diabetes mellitus, reynaud's syndrome, sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, autoimmune encephalomyelitis and autoimmune thyroiditis.
The bifunctional fusion protein molecule of any one of claims 1-6, the TACI ectodomain fragment or variant of claim 7, the isolated nucleic acid molecule of 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, for use in the prevention and/or treatment of an autoimmune disease;

preferably, the disease is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic Lupus Erythematosus (SLE), lupus Nephritis (LN), wegener's disease, inflammatory bowel disease, idiopathic Thrombocytopenic Purpura (ITP), thrombotic Thrombocytopenic Purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, igA nephropathy, igM polyneuropathy, myasthenia gravis, vasculitis, diabetes mellitus, reynaud's syndrome, sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, autoimmune encephalomyelitis and autoimmune thyroiditis.
A kit comprising the bifunctional fusion protein molecule of any one of claims 1-6, the TACI ectodomain fragment or variant of claim 7, the isolated nucleic acid molecule of 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.