Taci fusion protein and use thereof

Abstract

The present invention relates to the field of biomedicine, and in particular to an antibody that specifically recognizes MASP3 and a use thereof. The antibody has high affinity for human MASP3 and inhibitory activity against an alternative pathway. In addition, the present invention further relates to a multi-specific antibody specifically binding to MASP3 and MASP2, and a pharmaceutical composition comprising an antibody specifically binding to MASP3 and an antibody specifically binding to MASP2 and / or the multi-specific antibody. The antibody or the composition is used for preparing a drug for treating autoimmune diseases, transplant-related diseases, inflammatory diseases, hematologic diseases, coagulation diseases, angiogenesis-dependent diseases and / or viral infectious diseases.

Description

TACI fusion protein and its applications

[0001] Reference to electronic sequence listing

[0002] The contents of the electronic sequence list (text name: TACI-MASP3-20251204.xml, record date: 2025.12.04, size: 83KB) are incorporated into this document by reference in their entirety. Technical Field

[0003] This application relates to a TACI fusion protein comprising: (i) an extracellular domain of TACI or a variant thereof capable of binding BlyS and / or APRIL; and (ii) an antigen-binding domain specifically binding to MASP3. It also includes pharmaceutical compositions comprising the TACI fusion protein, methods of preparation thereof, and uses thereof, including methods of using them for the prevention and treatment of, for example, autoimmune diseases. Background Technology

[0004] The complement system plays a crucial role in both innate and adaptive immune responses. Complement clears immune complexes and cellular debris and rapidly generates highly efficient and tightly regulated inflammatory and cytolytic immune responses against infectious organisms, including bacteria, viruses, and protozoan parasites (Dunkelberger, Jason R, and Wen-Chao Song. Cell Research vol. 20, 1(2010): 34-50.). Complement activation leads to a proteolytic cascade, ultimately resulting in the recruitment, phagocytosis, and lysis of inflammatory cells (Kjaer, Troels R et al. Molecular Immunology vol. 56, 4(2013): 413-22.). It also produces anaphylatoxins (C3a, C4a, C5a) that effectively promote inflammatory responses, as well as opsonins (C3b and C4b) that coat the surface of pathogens and mediate phagocytosis. The endpoint of the cascade reaction is the assembly of the membrane attack complex (MAC) on the cell membrane, forming pores that lead to cell lysis (Dunkelberger, Jason R, and Wen-Chao Song. Cell Research vol. 20, 1(2010): 34-50.).

[0005] The complement system can be activated via three pathways: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). The classical pathway begins with C1q. When C1q recognizes target molecules (such as immune complexes or surface-bound pentameric proteins), its conformation changes, allowing C1q to interact with C1r and C1s to form activated C1qr2s2 (Carroll, MC. Annual review of immunology vol. 16 (1998): 545-68.). The enzyme's active site is on C1s. Subsequently, activated C1qr2s2 cleaves C4 and subsequent C2 to generate the C3 convertase C4b2a in the classical pathway. C4b2a can induce proteolytic activity, activating the common terminal pathway (Sim, RB, and SA Tsiftsoglou. Biochemical Society transactions vol. 32, Pt 1 (2004): 21-7.).

[0006] The lectin pathway can be triggered by circulating pattern recognition receptors (PRRs) that recognize carbohydrates on the surface of microorganisms, including mannose-binding lectin (MBL), collagen lectin, and fibrinogen. Fibrinogens are PRRs with fibrin-like domains, such as M-fibrinogen (ficolin-1), L-ficolin (ficolin-2), and H-ficolin (ficolin-3). MBL-associated serine proteases (MASP-1 and MASP-2) are evolutionarily related to C1r and C1s and are functionally similar. MBL forms a complex with MASPs, which subsequently cleaves C4 and C2 to form the C3 convertase C4b2a in the lectin pathway (Garred, Peter et al. Immunological reviews vol.274,1(2016):74-97.).

[0007] When C3b is present on the surface of the activator, the alternative pathway can be initiated (Lachmann, Peter J. Immunobiology vol. 223, 8-9 (2018): 519-523.). After C3b captures FB (Factor B), the C3b-bound FB can be cleaved by FD (Factor D) into Ba and Bb. In the blood, MASP-3 can cleave pro-FD, continuously providing FD for the alternative pathway, even before any activation signal appears (Oroszlán, Gábor et al. Journal of Immunology (Baltimore, Md.: 1950) vol. 196, 2 (2016): 857-65.; Dobó, József et al. Scientific reports vol. 6 31877. 18 Aug. 2016). C3b and Bb form the C3 convertase C3bBb in the alternative pathway, which can cleave more C3, thereby producing more C3 convertase complexes. This positive feedback mechanism amplifies complement activation initiated by either the classical or alternative pathways (Harboe, M et al. Clinical and experimental immunology vol. 138, 3(2004): 439-46.). The alternative pathway can also be initiated spontaneously via a so-called "slow transport" mechanism (Pangburn, MK et al. The Journal of experimental medicine vol. 154, 3(1981): 856-67.). C3 is slowly hydrolyzed in the cycle to produce C3(H2O), which is similar to C3b. C3(H2O) binds to FB and is then cleaved by FD. C3(H2O)Bb is a liquid C3 convertase that can generate C3b near any surface. On the cell's own surface, it can be protected from complement-mediated damage by various complement inhibitors. On unprotected surfaces (such as bacterial surfaces), deposited C3b can initiate the amplified loop in the alternative pathway, leading to complete complement activation.

[0008] When the density of deposited C3b reaches a certain point, C3b combines with C3 convertase (C4b2a or C3bBb) to form C5 convertase (C4b2aC3b or C3bBbC3b) (Mannes, Marco et al. Blood vol. 137, 4(2021): 443-455.; Roumenina, Lubka T. Blood vol. 137, 4(2021): 431-432.). From this point, three activation pathways converge into a common terminal pathway (TP). C5 convertase cleaves C5 into C5a and C5b, where C5a is a highly potent anaphylatoxin, while C5b binds to C6 and C7 to form C5b67, exposing its membrane-binding site. This allows C5b to bind non-specifically to nearby cell membranes and capture C8 and multiple C9 molecules to form the C5b6789n complex, also known as the membrane attack complex (MAC). MACs inserted into the cell membrane can form hydrophilic pores that penetrate the membrane, leading to the lysis and destruction of target cells (Tegla, Cosmin A et al. Immunologic research vol.51,1(2011):45-60.).

[0009] B lymphocyte stimulator (BlyS) can affect the survival and maturation of peripheral B cells. In autoimmune diseases, serum BlyS levels are elevated, and BlyS concentration is associated with autoantibody titers and disease progression.

[0010] BLyS, also known as BAFF, TALL-1, zTNF4, and THANK, is a member of the tumor necrosis factor (TNF) superfamily (TNFSF13B). It is mainly produced and secreted by myeloid cells, but also by non-lymphocyte types and epithelial cells. (Mackay F, Browning JL. BAFF: a fundamental survival factor for B cells. Immunology 2002; 2:465–75; Moisini I, Davidson A. BAFF: a local and systemic target in auto-immune diseases. Clin Exp Immunol 2009; 158:155–63.; Ng LG, et al. The BAFF / APRIL system: life beyond B lymphocytes. Mol Immunol 2005; 42:763–72). It is expressed as a type II transmembrane protein (a molecule with biological activity of 17 kDa), which mainly has three receptors: two high-affinity receptors, the B cell-activating factor receptor (BAFF-R) and the transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI), and a low-affinity receptor, the B cell maturation antigen (BCMA). 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 (MZ) B cells. BAFF binding to it has high specificity and high affinity, and the two regulate B cell survival, development, and differentiation. BCMA and TACI are mainly expressed on the membrane surface of activated B cells, memory B cells, and plasma cells. While recognizing BAFF, they also recognize proliferation-inducing ligand (APRIL), another member of the TNF ligand superfamily, with even higher affinity. Unlike BAFF-R, BCMA and TACI are associated with inflammatory responses and innate immunity.

[0011] The term "APRIL" in this application, also known as a proliferation-inducing ligand, is the 13th member of the tumor necrosis factor ligand superfamily. It plays a crucial role in the regulation of activated B cells, the survival of long-lived plasma cells, and immunoglobulin (Ig) isotype switching. APRIL shares approximately 30% homology with its family member BAFF (Wallweber, HJ et al. The crystal structure of a proliferation-inducing ligand, APRIL. J. Mol. Biol. 2004, 343, 283–290). Both APRIL and BAFF can bind to TACI and BCMA (Medema, JP et al. The uncertain glory of APRIL. Cell Death Differ. 2003, 10, 1121–1125). APRIL promotes and participates in lymphocyte proliferation, differentiation, and survival through binding to its receptors (BCMA and TACI).

[0012] The term "TACI" in this application, meaning Transmembrane activator, calcium modulator, and cyclophilin ligand interactor, is a receptor expressed on B cells, particularly CD27+ marginal zone B cells, memory B cells, and plasma cells. TACI is a regulatory factor that influences multiple events in the immune response. APRIL and BAFF are ligands of TACI, and their activation of TACI promotes the upregulation of activation-induced cytidine deaminase (AICDA) mRNA, isotype switching, and B cell maturation, thereby producing T cell-independent antibodies (Castigli E, et al. TACI is mutant in common variable immunodeficiency and IgA deficiency. Nat Genet. (2005) 37:829–34.10.1038 / ng1601). TACI is a type III transmembrane protein. Human TACI is a polypeptide of 293 amino acids, including a region located at the N-terminus (amino acid residues 1-165), a transmembrane region (amino acid residues 166-186), and an intracellular region (amino acid residues 187-293).

[0013] B cell-mediated humoral immunity is a key mechanism in many autoimmune diseases. When autoreactive B cells are overactivated, the production of autoantibodies and the formation of immune complexes surge, promoting tissue and organ damage and leading to various autoimmune diseases. (X. Lin, et al. B cell-mediated autoimmune diseases, Adv. Exp. Med. Biol. 1254 (2020) 145–160; ZXXiao, et al. An updated advance of autoantibodies in autoimmune diseases, Autoimmun. Rev. 20 (2) (2021) 102743). Meanwhile, due to the broad functional scope of complement, complement dysregulation can lead to a variety of human diseases. In particular, inappropriate, uncontrolled, and / or chronic complement activation can induce inflammation or autoimmune diseases, or exacerbate the pathological effects of non-complement, tissue-destructive triggers (Pouw RB, et al. Tipping the balance: intricate roles of the complement system in disease and therapy. Semin Immunopathol. 2021; 43(6):757–71.). These diseases include systemic lupus erythematosus, lupus nephritis, IgA nephropathy, Sjögren's syndrome, idiopathic membranous nephropathy, antiphospholipid syndrome, rheumatoid arthritis, transplant rejection, multiple sclerosis, etc. For example, systemic lupus erythematosus (SLE) is an autoimmune disease that endangers a variety of human tissues and organs (such as facial skin, kidneys, etc.), characterized by excessive activity of B cells and T cells, destruction of immune self-tolerance, deposition of immune complexes, and organ damage. The pathogenesis of SLE is intertwined with factors such as the production of autoantibodies, the clearance of apoptotic remnants, the deposition of immune complexes in tissues, complement activation, and excessive secretion of cytokines (DCSalazar-Camarena, et al. Association of BAFF, APRIL serum levels, BAFF-R, TACI and BCMA expression on peripheral B-cell subsets with clinical manifestations in systemic lupus erythematosus, Lupus 25(6)(2016)582–592). BlyS levels affect survival signals and selective apoptosis of B cells that produce autoantibodies.High levels of BlyS may relax B cell selection and promote the production of autoantibodies, thereby exacerbating the disease state of SLE (Cancro MP, et al. The role of B lymphocyte stimulator (BLyS) in systemic lupus erythematosus. J Clin Invest. 2009 May; 119(5):1066-73). Inhibition of BlyS or APRIL can reduce the number of abnormal B cells that lead to SLE and reduce disease activity in patients. Meanwhile, the activation of the complement pathway plays a key role in the pathogenesis of SLE, and all three pathways of complement activation may be involved in inflammation and tissue damage in SLE.

[0014] IgA nephropathy (IgAN) is characterized by the deposition of IgA1 (especially galactose-deficient IgA1, Gd-IgA1) in the glomerular mesangium. The high molecular weight IgA deposited in the mesangial region activates the complement system via the lectin pathway or alternative pathway (Daha, Mohamed R, and Cees van Kooten. Journal of Nephrology vol. 29, 1(2016): 1-4.). Simultaneously, BlyS and APRIL bind to receptors on the surface of B cells, activating B cells and initiating their proliferation and differentiation, a process that leads to the production of Gd-IgA1. Inhibiting BlyS and APRIL can reduce Ig levels and proteinuria in patients with IgA nephropathy, providing a new avenue for exploring the treatment of IgA nephropathy (Meng Wang, et al. Efficacy and safety of telitacicept, a BLyS / APRIL dual inhibitor, in the treatment of IgA nephropathy: a retrospective case-control study, Clinical Kidney Journal, Volume 17, Issue 10, October 2024, sfae285).

[0015] Regarding autoimmune diseases, it is generally believed that multiple signaling pathways may be involved in the development and progression of the disease. Therefore, treatments targeting only a single target or signaling pathway may be insufficient and have limitations in clinical application. Thus, there is an urgent need to develop drugs that can simultaneously target multiple targets or signaling pathways, thereby achieving a more comprehensive therapeutic effect.

[0016] All publications, patents, patent applications and disclosed information in published patent applications mentioned in this application are incorporated herein by reference in their entirety.

[0017] Application Overview

[0018] On the one hand, this application relates to a TACI fusion protein comprising: (i) an extracellular domain of TACI or a variant thereof capable of binding BAFF and / or APRIL; and (ii) an antigen-binding domain specifically binding MASP3.

[0019] In some embodiments, the TACI fusion protein described in this application, wherein the antigen-binding domain that specifically binds to MASP3 comprises: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:2; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:5; LC-CDR2, which contains the amino acid sequence SEQ ID NO:6; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:8.

[0020] In some embodiments, the TACI fusion protein described in this application, wherein the antigen-binding domain that specifically binds to MASP3 comprises: (i) V H It comprises the amino acid sequence SEQ ID NO:14 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:14; and V L It contains the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:20; (ii) V H It comprises the amino acid sequence SEQ ID NO:13 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:13; and V L It comprises the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:21; or (iii) V H It comprises the amino acid sequence SEQ ID NO:14 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:14; and V LIt contains the amino acid sequence SEQ ID NO:22 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:22.

[0021] In some embodiments, the TACI fusion protein described in this application further includes an antigen-binding domain that specifically binds to MASP2.

[0022] In some embodiments, the TACI fusion protein of this application includes an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP2 includes: (i) V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:23; HC-CDR2, which contains the amino acid sequence SEQ ID NO:24; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:25; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:26; LC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:28; or (ii) V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:23; HC-CDR2, which contains the amino acid sequence SEQ ID NO:24; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:25; and V. L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:26; LC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:29.

[0023] In some embodiments, the TACI fusion protein of this application includes an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP2 includes: (i) V H It comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V L It comprises the amino acid sequence shown in SEQ ID NO:36 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:36; or (ii) V HIt comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V L It contains the amino acid sequence shown in SEQ ID NO:37 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:37.

[0024] In some embodiments, the TACI fusion protein of this application comprises an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP3 comprises: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:2; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:5; LC-CDR2, which contains the amino acid sequence SEQ ID NO:6; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:8; and an antigen-binding domain therein that specifically binds to MASP2, comprising: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:23; HC-CDR2, which contains the amino acid sequence SEQ ID NO:24; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:25; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:26; LC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:29.

[0025] In some embodiments, the TACI fusion protein of this application comprises an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, wherein (i) the antigen-binding domain that specifically binds to MASP3 comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:14 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:14; and V LIt comprises the amino acid sequence shown in SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:20; and wherein the antigen-binding domain specifically binding to MASP2 comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V L It comprises the amino acid sequence shown in SEQ ID NO:37 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:37; or (ii) the antigen-binding domain that specifically binds to MASP3 comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:13 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:13; and V L It comprises the amino acid sequence shown in SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:21; and an antigen-binding domain specifically binding to MASP2 comprising: V H It comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V L It contains the amino acid sequence shown in SEQ ID NO:37 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:37.

[0026] In some embodiments, the TACI fusion protein of this application, wherein the antigen-binding domain specifically binding to MASP3 or MAPS2 comprises an Fc fragment. In some embodiments, the antigen-binding domain specifically binding to MASP3 or MAPS2 is a full-length IgG antibody. In some embodiments, the antigen-binding domain specifically binding to MASP3 or MAPS2 is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antigen-binding domain specifically binding to MASP3 or MAPS2 is a chimeric, fully human, or humanized antibody. In some embodiments, the antigen-binding domain specifically binding to MASP3 or MAPS2 is selected from Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, nanobody, diabody, or linear antibody.

[0027] In some embodiments, the TACI fusion protein of this application, wherein the extracellular domain of TACI or a variant thereof capable of binding BAFF and / or APRIL comprises the amino acid sequence SEQ ID NO:64 or a fragment or variant thereof.

[0028] In some embodiments, the TACI fusion protein described in this application comprises one, two, three, or four extracellular domains of TACI or variants thereof capable of binding BAFF and / or APRIL.

[0029] In some embodiments, the TACI fusion protein of this application, wherein the extracellular domain of TACI or a variant thereof capable of binding BAFF and / or APRIL is linked to the heavy chain variable region (V) of the antigen-binding domain specifically binding MASP3 or MASP2. H ) or light chain variable region (V L The N-terminus or C-terminus of ).

[0030] In some embodiments, the TACI fusion protein of this application, wherein the extracellular domain of TACI or a variant thereof capable of binding BAFF and / or APRIL is attached to the N-terminus or C-terminus of the Fc fragment.

[0031] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, two of which contain V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure, with the other two polypeptide chains containing V from the N-terminus to the C-terminus. L -C L Structure, where V H 1 and V L These are the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domains.

[0032] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H 1-C H The 1-Fc structure, with the other two polypeptide chains containing V from the N-terminus to the C-terminus. L -C L Structure, where V H 1 and V L These are the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and CH 3. Structural domain, preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0033] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H 2-C L -Fc-TACI structure, a polypeptide chain containing V from N-terminus to C-terminus L 1-C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively; V H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domain, preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0034] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H The 2-CL-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. L 1-C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively; V H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domain. Preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0035] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H 2-C L -Fc structure, a polypeptide chain containing V from N-terminus to C-terminus. L 1-C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively; V H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domain. Preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0036] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H The 2-CL-Fc structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. L 1-C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively; V H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domain. Preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0037] In some embodiments, the TACI fusion protein described in this application comprises three polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc structure is a polypeptide chain containing V from the N-terminus to the C-terminus.L 1-C L The structure, and another polypeptide chain containing a TACI-Fc structure from the N-terminus to the C-terminus, wherein V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domain, preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0038] In some embodiments, the TACI fusion protein of this application comprises an amino acid sequence SEQ ID NO:70 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:70; and an amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:54.

[0039] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:71 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:71; and the amino acid sequence SEQ ID NO:67 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:67; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:54.

[0040] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:66 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:66; and the amino acid sequence SEQ ID NO:52 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:52; and the amino acid sequence SEQ ID NO:67 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:67; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:54.

[0041] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:74 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:74; and the amino acid sequence SEQ ID NO:52 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:52; and the amino acid sequence SEQ ID NO:75 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:75; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:54.

[0042] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:66 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:66; and the amino acid sequence SEQ ID NO:52 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:52; and the amino acid sequence SEQ ID NO:60 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:60; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:54.

[0043] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:71 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:71; and the amino acid sequence SEQ ID NO:72 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:72; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:54.

[0044] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:76 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:76; and the amino acid sequence SEQ ID NO:77 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:77; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:54.

[0045] In some embodiments, a method of treating a desired individual disease or condition is provided, comprising administering to the individual an effective amount of any of the TACI fusion proteins described above or a pharmaceutical composition comprising thereas. In some embodiments, the use of any of the TACI fusion proteins described above or a pharmaceutical composition comprising thereas is provided in the preparation of a pharmaceutical composition for treating a desired individual disease or condition. In some embodiments, the disease or condition includes autoimmune diseases, transplant-related diseases, inflammatory diseases, blood disorders, coagulation disorders, angiogenesis-dependent diseases, and / or viral infections or conditions. In some embodiments, the diseases or conditions include ischemia-reperfusion injury, atherosclerosis, nephrotic syndrome, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, acute post-infectious glomerulonephritis, cryoglobulinemia glomerulonephritis, lupus nephritis, systemic lupus erythematosus (SLE), Henoch-Schönlein purpura nephritis, IgA nephropathy, ischemic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), transplant-associated TMA, Upshaw-Schulman syndrome, arthritis, traumatic brain injury, aspiration pneumonia, optic nerve disease. The following conditions may be associated with acute respiratory distress syndrome (ARDS): myelitis, multiple sclerosis, amyotrophic lateral sclerosis (ALS), chronic obstructive pulmonary disease (COPD), C3 glomerulonephropathy, transplant rejection, graft-versus-host disease (GVHD), sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, myasthenia gravis, Degos disease, disseminated intravascular coagulation (DIC), angiogenesis-dependent cancer, hematopoietic stem cell transplantation, cold agglutinin disease, Sjögren's syndrome, age-related macular degeneration, retinopathy, proliferative diabetes mellitus, retinopathy secondary to vitreous hemorrhage, neovascular glaucoma, corneal neovascularization, retinopathy of prematurity, and respiratory distress syndrome or pneumonia caused by coronavirus infection.

[0046] In some embodiments, this application provides an isolated nucleic acid molecule encoding any of the TACI fusion proteins described above. In some embodiments, a vector is provided comprising any of the nucleic acid molecules described above. In some embodiments, a host cell is provided comprising any of the TACI fusion proteins, any nucleic acid molecules, or any vectors described above. In some embodiments, a method for preparing a TACI fusion protein is provided, comprising: a) culturing any of the host cells described above under conditions for effective expression of the TACI fusion protein; and b) obtaining the expressed TACI fusion protein in the host cell.

[0047] Pharmaceutical compositions, kits, and products comprising any of the TACI fusion proteins, nucleic acid molecules, vectors, or host cells as described above are also provided. Attached Figure Description

[0048] Figures 1A-1B show the inhibitory activity of humanized M3-K2 antibodies in the complement alternative pathway-mediated hemolysis assay. Humanized antibodies M3-K2-2, M3-K2-3, M3-K2-5 (Figure 1A) and M3-K2-6 to M3-K2-11 (Figure 1B) all inhibited hemolytic activity mediated by the complement alternative pathway.

[0049] Figure 2 shows a schematic diagram of the structure of the Hetero H,CrossMab bispecific antibody.

[0050] Figures 3A-3F are schematic diagrams of the structures of different types of TACI fusion proteins.

[0051] Figure 4 shows the results of the urinary microalbumin to creatinine ratio (UACR) in the lupus nephritis mouse model group and various treatment groups.

[0052] Figure 5 shows the kidney-to-body weight ratio in the lupus nephritis mouse model group and each treatment group.

[0053] Figure 6 shows the results of serum urea (URE) levels in the blank control group, model group, and various treatment groups of lupus nephritis mice.

[0054] Figure 7 shows the results of the urinary microalbumin to creatinine ratio (UACR) in the blank group, model group, and various treatment groups of lupus nephritis mice.

[0055] Figure 8A shows the kidney weight ratio results of the blank group, model group, and various treatment groups in mice with lupus nephritis.

[0056] Figure 8B shows the spleen weight ratio in the blank group, model group, and various treatment groups of lupus nephritis mice.

[0057] Detailed description of this application

[0058] definition

[0059] As described herein, “treatment” or “treating” is a method of obtaining a beneficial or desired outcome, including clinical outcomes. For the purposes of this application, the beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms caused by a disease, reduction of disease severity, stabilization of the disease (e.g., prevention or delay of disease exacerbation), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of disease status, relief of the disease (partial or complete), reduction of the dosage of one or more other medications required to treat the disease, delay of disease progression, improvement or enhancement of quality of life, weight gain, and / or prolongation of survival. Simultaneously, “treatment” also includes reductions in disease pathological outcomes (e.g., reduction of immune complex deposition, inhibition of thrombus formation). The methods of this application consider any one or more aspects of these treatments.

[0060] The term "prevent," and similar words such as "prevented," "preventing," "prevention," or "prophylactic," refers to a method of preventing, suppressing, or reducing the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition before it occurs or recurs. As used herein, "prevention" and similar words also include reducing the risk and susceptibility to the occurrence or recurrence of a disease or condition.

[0061] As described herein, the term "antibody" is used broadly to encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies, and their antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Full-length antibodies consist of two heavy chains and two light chains. Variable regions in both the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically include three hypervariable loops, referred to as complementarity-determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibody or antigen-binding fragments disclosed herein can be defined or identified according to the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). Three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDR regions and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by having α, δ, ε, γ, and μ-type heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0062] As described herein, the term "antigen-binding fragment" includes an antibody fragment, including, for example, a double-chain antibody (diabody), Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized double-chain antibody (ds double-chain antibody), single-chain antibody (scFv), scFv dimer (bivalent double-chain antibody), multispecific antibody, single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment capable of binding to an antigen but not containing a complete antibody structure. Wherein, Fab (fragment antigen-binding) as described in this application is a V containing an antibody. L Structural domain, V H Domain, C L Domain, and CH A monovalent fragment of a 1-domain structure. The antigen-binding fragment also includes a fusion protein comprising the antibody fragment described above. The antigen-binding fragment is capable of binding the same antigen to a parent antibody or a parent antibody fragment (such as a parental scFv). In some embodiments, the antigen-binding fragment may include one or more CDRs from a specific human antibody that are grafted into the frame region from one or more different human antibodies.

[0063] As described herein, the term "multispecific antibody" refers to an antibody molecule (e.g., a bispecific antibody) that has binding specificity to at least two different antigens or epitopes in a single molecule. Preferably, a multispecific antibody is a bispecific antibody. As described in this application, the term "bispecific antibody" refers to an antibody molecule that has binding specificity to two different antigens or epitopes in a single molecule. The production process of a multispecific antibody (e.g., a bispecific antibody) includes the design of the complete molecule, the synthesis and cloning of the nucleotide sequence of each domain, expression in mammalian cells, and purification of the final product. Exemplary structures of multispecific antibodies (e.g., bispecific antibodies) include structures known in the art, such as DVD-Ig structures, Bs4Ab structures, Hetero H, CrossMab structures, CrossMab2+1 structures, IgG-(scFv)2 structures, or scFv-Fab IgG structures, etc. (e.g., see review article Labrijn AF, et al. Nat Rev Drug Discov. 2019 Aug; 18(8):585-608).

[0064] As described in this application, the term "antigen-binding domain" refers to the portion of an antigen-binding molecule that specifically binds to an antigen. More specifically, the term "antigen-binding domain" refers to a portion of an antibody that includes a region that specifically binds to and is complementary to some or all of the antigen. In the case of a large antigen, the antigen-binding molecule may bind only a specific portion of the antigen, referred to as an antigenic epitope. For example, the antigen-binding domain may be provided by one or more variable regions (also called variable domains). Preferably, the antigen-binding domain includes an antibody light chain variable region (V... L ) and antibody heavy chain variable region (V H On the one hand, an antigen-binding domain can bind to its antigen and block or partially block the function of said antigen. Antigen-binding domains that specifically bind to MASP3 or MASP2 include antibodies and antigen-binding fragments as further defined in this application.

[0065] As described in this application, the term "epitope" refers to a specific atom or amino acid group on an antigen that an antibody or antibody portion binds to. If two antibodies or antibody portions exhibit competitive binding to an antigen, they may bind to the same epitope on the antigen.

[0066] As described in this application, when the first antibody inhibits the binding of the second antibody to the MASP3 target by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) at an equimolar concentration, the first antibody "competitively" binds to the MASP3 target with the second antibody, and vice versa. PCT Publication WO 03 / 48731 describes a high-throughput antibody "epitope binning" method based on cross-competition.

[0067] As described in this application, the terms "specifically binding," "specifically recognizing," or "specific to..." refer to a measurable and reproducible interaction, such as the binding of a target to an antibody that confirms the presence of the target in a heterogeneous group of molecules, including biomolecules. For example, an antibody's ability to specifically recognize a target (which may be an epitope) means that the antibody binds to that target with higher affinity, stronger binding, and / or more persistent binding compared to binding to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with a binding affinity at least 10 times greater than its binding affinity to other targets.

[0068] As described in this application, an “isolated” antibody means an antibody that (1) is not related to a naturally occurring protein, (2) does not contain other proteins of the same origin, (3) is expressed by cells of a different species, or (4) does not exist in nature.

[0069] As described in this application, the term "isolated nucleic acid" refers to a nucleic acid or combination thereof of genomic, cDNA, or synthetic origin. Depending on its origin, the "isolated nucleic acid" (1) is not related to all or part of the polynucleotides in "isolated nucleic acids" found in nature, (2) can be operatively linked to polynucleotides not naturally associated with it, or (3) does not exist in nature as part of a longer sequence.

[0070] As described in this application, the term "CDR" or "complementarity-determining region" refers to a discontinuous antigen-binding site found within the variable region of a heavy-chain or light-chain polypeptide. In the literature Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al. al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc MPet These specific regions have been described in al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where these definitions include overlaps or subsets of amino acid residues when compared with each other. However, any definition used to indicate a CDR of an antibody or transplanted antibody or its variants is included within the scope of the terminology defined and used in this application. Table 1 lists the positions of the amino acid residues included in the CDRs defined by the above-cited references for comparison. Algorithms and interfaces for CDR prediction are known in the art, including, for example, those described in Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The entire contents of the references cited in this paragraph are incorporated herein by reference for use in this application and in one or more claims that may be included herein.

[0071] Table 1: CDR Definition 1 The amino acid residue numbering follows the nomenclature method described in Kabat et al. above.2 The amino acid residue numbering follows the nomenclature method described in Chothia et al. above. 3 The amino acid residue numbering follows the nomenclature method described in MacCallum et al. above. 4 The amino acid residue numbering follows the nomenclature method described in Lefranc et al. above. 5 The amino acid residue numbering follows the nomenclature method described in Honegger and Plückthun.

[0072] The term "chimeric antibody" refers to an antibody whose heavy chain and / or light chain portion is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody species or subclass, while the remaining portion of this chain(s) is identical or homologous to the corresponding sequence in an antibody from another genus or belonging to another antibody species or subclass, as well as fragments of such antibodies, provided they possess the biological activity described in this application (see US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0073] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment is a dimer formed by a heavy chain variable region and a light chain variable region tightly and non-covalently linked. The folding of these two domains derives six hypervariable rings (three in the light chain and three in the heavy chain), which provide the antibody with amino acid residues for binding to the antigen and confer specificity for antigen binding. However, even a single variable region (or half of the Fv fragment, containing only three antigen-specific CDRs) can recognize and bind to antigens, although its affinity is lower than that of the complete binding site.

[0074] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a type of Fv containing V molecules linked together to form a single polypeptide chain. H and V L Antibody fragments of antibody domains. In some embodiments, the scFv polypeptide further includes V H and V L The linker polypeptide between the domains allows scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0075] The term "diabodies" refers to antibodies that are formed in V... H and V L A bivalent antibody is a small antibody fragment prepared by constructing scFv fragments (see above) using short linkers (e.g., 5-10 residues). This allows the variable regions to pair between chains rather than within chains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Multispecific bichain antibodies are heterodimers of two "crossover" scFv fragments, where the V0 of the two antibodies... H and V L The domains are located on different polypeptide chains. Double-chain antibodies are fully described in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0076] The “humanized” form of non-human (e.g., rodent) antibodies is a chimeric antibody, which includes a minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), where the hypervariable region (HVR) residues of the receptor antibody are replaced by hypervariable region residues from non-human species such as mice, rats, rabbits, or non-human primates (donor antibodies) that possess desirable antibody specificity, affinity, and performance. In some cases, residues in the human immunoglobulin framework region (FR) are replaced by corresponding non-human residues. Additionally, humanized antibodies may include residues not present in either the receptor or donor antibody. These modifications can further improve antibody performance. Typically, humanized antibodies contain substantially at least one, usually two, variable regions, where all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. Humanized antibodies may also optionally include at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. For details, please refer to Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0077] The "percentage of amino acid sequence homology (%)" or "homology" of the peptide and antibody sequences identified in this application is defined as the percentage of identical amino acid residues in a candidate sequence compared to a peptide sequence, assuming that conserved substitutions are part of sequence homology. The percentage of amino acid sequence homology can be determined using various alignment methods within the scope of the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required to maximize alignment across the full length of the compared sequences. However, for the purposes of this application, the percentage of amino acid sequence homology values ​​are generated using the sequence alignment computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0078] The term "Fc (fragment crystallizable)" or "Fc region" refers to a polypeptide containing the complete antibody constant region, excluding the CH1 domain, and in some cases containing a partial hinge, whether in monomeric or multimeric form. The original immunoglobulin source of natural Fc is preferably human, and can be any immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4. Natural Fc consists of monomeric polypeptides, which can be covalently (i.e., disulfide bonds) and non-covalently linked into dimers or multimers. The Fc region of an immunoglobulin generally contains the C1 domain of the heavy chain constant region. H 2. Structural Domains and C H 3 structural domains, and optionally include C H 4. Structural domains.

[0079] In some embodiments, each of the two Fc monomers in the Fc dimer contains an amino acid substitution that promotes heterodimerization of the two monomers. In some embodiments, heterodimerization of the Fc monomers can be promoted by introducing different but compatible substitutions, such as “knob-into-hole” residue pairs, into the two Fc monomers. The “knob-into-hole” technique is also disclosed in U.S. Patent Publication No. 8,216,805. In some embodiments, one Fc monomer contains the knockb mutation T366W, and the other Fc monomer contains the hole mutations T366S, L368A, and Y407V. In some embodiments, two Cys residues (S354C on the “knob” side and Y349C on the “hole” side) are introduced to form a stabilizing disulfide bridge.

[0080] The term "Fc receptor" or "FcR" is used to describe receptors that bind to the Fc region of antibodies. In some embodiments, the FcR described in this application is an FcR that binds to IgG antibodies (a type of γ receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA (activating receptor) and FcγRIIB (inhibiting receptor), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The cytoplasmic domain of the activating receptor FcγRIIA contains the immunoreceptor tyrosine activation motif (ITAM). The cytoplasmic domain of the inhibitory receptor FcγRIIB contains the immunoreceptor tyrosine inhibition motif (ITIM) (see M.in.). Annu.Rev.Immunol.15:203-234 (1997)). The term also includes allotypes, such as FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131 and / or FcγRIIA-H131. FcRs are described in Ravetch and Kinet, Annu.Rev.Immunol 9:457-92 (1991) and Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J.Lab.Clin.Med.126:330-41 (1995). In this application, the term FcR covers other types of FcRs, including those to be identified in the future. The term FcR also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgGs to the newborn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0081] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC), consisting of an α-chain non-covalently bound to β2-microglobulin. The various functions of the neonatal Fc receptor FcRn are described in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays an important role in the passive transport of immunoglobulin IgGs from the mother to the newborn and in regulating serum IgG levels. As a rescue receptor, FcRn can bind and transport endocytotic IgG in its intact form both intracellularly and intercellularly, protecting it from the default degradation pathway.

[0082] The "C" in the constant region of the human IgG heavy chain H The "1 domain" typically extends from amino acid 118 to amino acid 215 (EU numbering system).

[0083] The "hinge region" is typically defined as extending from Glu at position 216 to Pro at position 230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). By placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide bond at the same positions as IgG1, it is possible to align the hinge regions of other IgG isotypes with the IgG1 sequence.

[0084] The "C" in the Fc region of human IgG H The "2-domain" typically extends from amino acid position 231 to amino acid position 340. C H The unique feature of the 2-domain is that it does not pair tightly with another region. Instead, it is located between the two C-domains of the intact natural IgG molecule. H Two N-terminal branched sugar chains were inserted between the two structural domains. It is speculated that the sugars may serve as a substitute for domain-to-domain pairing, helping to maintain C... H 2. The structural domain is stable. Burton, Molec Immunol. 22:161-206 (1985).

[0085] “C H The 3” domain includes a region extending from the C-terminal residue to the C-terminal region within the Fc region. H 2. Structural domain (from amino acid 341 to the C-terminus of the antibody sequence, usually amino acid residue 446 or 447 of IgG).

[0086] "Functional Fc fragments" possess the "effective functions" inherent in the natural Fc region sequence. Exemplary "effective functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (such as B cell receptors; BCR). These effector functions typically require the Fc region to bind to a binding domain (such as the antibody variable region) and can be evaluated using a variety of experimental methods known in the art.

[0087] Antibodies with "altered" FcR binding affinity or ADCC activity exhibit enhanced or weakened FcR binding and / or ADCC activity compared to the parent peptide or a peptide containing the native IgG Fc sequence. Fc variants exhibiting "enhanced binding" to FcRs have a higher binding affinity (e.g., lower apparent Kd or IC50 value) to at least one FcR compared to the parent peptide or a peptide containing the native IgG Fc sequence. In some embodiments, the binding ability is enhanced by 3-fold, such as 5, 10, 25, 50, 60, 100, 150, 200, or even up to 500-fold, or by 25% to 1000% compared to the parent peptide. Fc variants exhibiting "decreased binding" to FcRs have a lower affinity (e.g., higher apparent Kd or IC50 value) to at least one FcR compared to the parent peptide. Their binding ability is decreased by 40% or more compared to the parent peptide. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells, such as natural killer (NK) cells, neutrophils, and macrophages. This allows these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are essential for this killing. Among the main cell types mediating ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). The ADCC activity of target molecules can be assessed by in vitro ADCC experiments, described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the target molecule can also be assessed in vivo, as described, for example, in animal models disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0088] Peptides containing Fc variants exhibit "enhanced ADCC activity" or are more effective at mediating ADCC effects in the presence of human effector cells compared to peptides containing wild-type IgG Fc or parental peptides. These Fc variants mediate ADCC more effectively, both in vitro and in vivo, when the experimental amounts are substantially the same as those containing wild-type IgG Fc (or parental peptides). Such variants are typically identified using any in vitro ADCC assay known in the art, such as experiments or methods for identifying ADCC activity, for example, in animal models. In some embodiments, such variants mediate ADCC with a 5 to 100-fold increase in efficiency, for example, 25 to 50-fold, compared to wild-type Fc (or parental peptide).

[0089] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (a subclass with a suitable structure) that binds to a homologous antigen. To assess complement activation, CDC experiments can be performed, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Peptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described in U.S. Patent Nos. 6,194,551B1 and WO99 / 51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0090] The "fusion protein" described in this application refers to a protein product obtained by linking the coding regions of two or more genes through gene recombination, chemical methods, or other suitable methods, and expressing the gene recombination under the control of the same regulatory sequence. In the fusion protein of this application, the coding regions of two or more genes may be fused at one or more positions by sequences encoding peptide linkers or linking peptides. Peptide linkers or linking peptides can also be used to construct the fusion protein of this application. The fusion protein of this application further includes a TACI fusion protein, which comprises: (i) an extracellular domain of TACI or a variant thereof capable of binding BlyS and / or APRIL; and (ii) an antigen-binding domain that specifically binds to MASP3.

[0091] Unless otherwise stated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins or RNA may also include introns; for example, nucleotide sequences encoding proteins may contain introns in certain forms.

[0092] The term "operationally ligated" refers to a functional link between a regulatory sequence and a heterologous nucleotide sequence, thereby enabling the latter to be expressed. For example, the first and second nucleotide sequences are operably ligated when they are in a functional relationship. Similarly, if a promoter affects the transcription or expression of a coding sequence, the promoter and coding sequence are operably ligated. Typically, operably ligated DNA sequences are contiguous, and, if necessary, two protein-coding regions can be ligated within the same reading frame.

[0093] "Homology" refers to the sequence similarity or sequence homology between two polypeptides or two nucleic acid molecules. If the same position in two compared sequences is the same base or amino acid monomer subunit, for example, if both DNA molecules have adenine at the same position, then the two DNA molecules are homologous at that position. The percentage of homology between two sequences is a function calculated by multiplying the number of matching or homologous positions in the two sequences by the total number of positions, and then multiplying by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. For instance, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, when comparing two sequences, the goal is to obtain the maximum homology.

[0094] The “effective amount” of the TACI fusion protein or compositions comprising it disclosed in this application refers to an amount sufficient to achieve a particular purpose. The “effective amount” can be determined empirically and by known methods associated with said purpose.

[0095] The term "therapeutic effective amount" refers to the amount of the TACI fusion protein described in this application or a composition containing it that is effective in treating an individual's disease or symptoms. This includes an amount sufficient to reduce or improve the severity and / or duration of the disease or one or more of its symptoms; prevent disease progression; induce symptom resolution; prevent recurrence, development, onset, or progression of one or more disease-related symptoms; detect the disease; or enhance / improve the preventive or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent). In some embodiments, a therapeutic effective amount refers to an amount capable of prolonging patient survival. In some embodiments, a therapeutic effective amount refers to an amount capable of improving patient progression-free survival.

[0096] As used in this application, "pharmaceutical acceptable" or "pharmacologically compatible" means a material that has no biological activity or other undesirable properties, such as the ability to be incorporated into a pharmaceutical composition given to a patient without causing significant adverse biological reactions, or that does not interact harmfully with any other components contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicological or manufacturing testing and / or are included in the inactive ingredient guidelines prepared by the U.S. Food and Drug Administration.

[0097] The embodiments of this application described herein should be understood to include embodiments that are “composed of” and / or “substantially composed of”.

[0098] In this application, the reference to “about” as a numerical value or parameter includes (and describes) variations for that value or parameter itself. For example, a description relating to “about X” includes a description of “X”.

[0099] As used in this application, the reference to "not" a numerical value or parameter generally indicates and describes something other than a certain numerical value or parameter. For example, the statement that this method cannot be used to treat type X infection means that this method is generally used to treat other types of infection besides type X.

[0100] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used in this application and the claims include plural objects.

[0101] TACI fusion protein

[0102] On the one hand, this application relates to a TACI fusion protein comprising: (i) an extracellular domain of TACI or a variant thereof capable of binding BlyS and / or APRIL; and (ii) an antigen-binding domain that specifically binds to MASP3.

[0103] (i) The extracellular domain of TACI or its variants that can bind BlyS and / or APRIL

[0104] On the one hand, the term "extracellular domain of TACI or a variant thereof capable of binding BlyS and / or APRIL" as used in this application also refers to any variant having at least 80% or 85%, preferably at least 90% or 95% or 99% identity with the extracellular domain of TACI (e.g., SEQ ID NO: 64). In some embodiments, it also includes variants containing no more than 50 or 40 or 30 or 20 or 10 or 5 or 3 or 2 or 1 conserved amino acid substitutions. Any such variant can bind BlyS and / or APRIL, and / or any BlyS-APRIL heterotrimer. Preferably, such variants can also inhibit the biological activity of BlyS and / or APRIL and / or any BlyS / APRIL heterotrimer. The biological activity of BlyS or APRIL is, for example, B cell proliferation.

[0105] On the other hand, fragments (active fragments) and variants of the TACI extracellular domain can also be used in this application, as long as these fragments can bind to BlyS and / or APRIL, and / or BlyS-APRIL heterotrimers. Preferably, such fragments can also inhibit or weaken the biological activity of BlyS and / or APRIL and / or BlyS / APRIL heterotrimers.

[0106] In some embodiments, the TACI fusion protein described in this application comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 extracellular domains of TACI or variants thereof capable of binding BAFF and / or APRIL.

[0107] In some embodiments, the extracellular domain of TACI described in this application or a variant thereof capable of binding BlyS and / or APRIL comprises the amino acid sequence SEQ ID NO:64.

[0108] (ii) The antigen-binding domain that specifically binds to MASP3.

[0109] On one hand, the antigen-binding domain that specifically binds to MASP3 as described in this application includes, but is not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy chain and / or light chain CDRs or heavy chain variable regions and / or light chain variable regions as described in this application. On the other hand, the antigen-binding domain is a separate antibody that binds to MASP3. Antigen-binding domains that specifically bind to MASP3 include all or fragments of full-length antibodies (such as full-length IgG1, IgG2, or IgG4) that specifically bind to MASP3, single-chain antibodies that specifically bind to MASP3, multispecific (e.g., bispecific) antibodies that specifically bind to MASP3, immunoconjugates that specifically bind to MASP3, and the like. In some embodiments, the antigen-binding domain that specifically binds to MASP3 is Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, nanobody, or diabody or linear antibody.

[0110] In some embodiments, the antigen-binding domain that specifically binds to MASP3 in the TACI fusion protein includes: (i) V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:2; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:4; LC-CDR2, which contains the amino acid sequence SEQ ID NO:6; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:7, or the V L Variants whose LC-CDRs contain substitutions of up to about 5 amino acids; or (ii) V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:2; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V LContains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:5; LC-CDR2, which contains the amino acid sequence SEQ ID NO:6; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:8, or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0111] In some embodiments, the antigen-binding domain that specifically binds to MASP3 in the TACI fusion protein includes: (i) V H It comprises the amino acid sequence SEQ ID NO:11 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:11; and V L It comprises the amino acid sequence SEQ ID NO:16 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:16; (ii) V H It comprises the amino acid sequence SEQ ID NO:12 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:12; and V L It comprises the amino acid sequence SEQ ID NO:17 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:17; (iii) V H It comprises the amino acid sequence SEQ ID NO:12 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:12; and V L It comprises the amino acid sequence SEQ ID NO:18 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:18; (iv) V HIt comprises the amino acid sequence SEQ ID NO:13 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:13; and V L It comprises the amino acid sequence SEQ ID NO:17 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:17; (v)V H It comprises the amino acid sequence SEQ ID NO:13 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:13; and V L It comprises the amino acid sequence SEQ ID NO:18 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:18; (vi)V H It comprises the amino acid sequence SEQ ID NO:14 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:14; and V L It comprises the amino acid sequence SEQ ID NO:17 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:17; (vii)V H It comprises the amino acid sequence SEQ ID NO:14 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:14; and V L It comprises the amino acid sequence SEQ ID NO:18 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:18; (viii)V HIt comprises the amino acid sequence SEQ ID NO:11 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:11; and V L It comprises the amino acid sequence SEQ ID NO:19 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:19; (ix)V H It comprises the amino acid sequence SEQ ID NO:13 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:13; and V L It comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence SEQ ID NO:20; (x)V H It comprises the amino acid sequence SEQ ID NO:15 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:15; and V L It comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence SEQ ID NO:20; (xi)V H It comprises the amino acid sequence SEQ ID NO:14 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:14; and V L It comprises the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence SEQ ID NO:20; (xii)V HIt comprises the amino acid sequence SEQ ID NO:12 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:12; and V L It comprises the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence SEQ ID NO:21; (xiii)V H It comprises the amino acid sequence SEQ ID NO:12 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:12; and V L It contains the amino acid sequence SEQ ID NO:22 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence SEQ ID NO:22; (xiv)V H It comprises the amino acid sequence SEQ ID NO:13 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:13; and V L It comprises the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence SEQ ID NO:21; (xv)V H It comprises the amino acid sequence SEQ ID NO:13 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:13; and V L It comprises the amino acid sequence SEQ ID NO:22 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence SEQ ID NO:22; (xvi)V HIt comprises the amino acid sequence SEQ ID NO:14 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:14; and V L It comprises the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:21; or (xvii)V H It comprises the amino acid sequence SEQ ID NO:14 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:14; and V L It contains the amino acid sequence SEQ ID NO:22 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:22.

[0112] In some embodiments, the amino acid substitutions described above are limited to the “Exemplary Substitutions” shown in Table A of this application. In some embodiments, the amino acid substitutions are limited to the “Preferred Substitutions” shown in Table A of this application.

[0113] Exemplary antigen-binding domain sequences that specifically bind to MASP3 are shown in Tables 2 and 3, where CDRs are numbered according to the Kabat definition. Those skilled in the art will recognize that various known algorithms exist for predicting the location of CDRs and defining the variable regions of the antibody light and heavy chains. CDRs comprising antibodies as described in this application, CDRs comprising antibodies as described in this application, V H and / or V L Sequences, but antibodies based on prediction algorithms rather than those exemplified in the table below are also within the scope of this application.

[0114] In some embodiments, the TACI fusion protein further comprises a heavy chain constant region of an immunoglobulin, such as an Fc region. In some embodiments, the Fc contains amino acid substitutions compared to wild-type Fc, thereby imparting enhanced or diminished effector functions of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In some embodiments, the Fc is derived from wild-type human IgG1 Fc. In some embodiments, the C of the Fc... HThe 2-domain contains, but is not limited to, the following amino acid substitutions relative to the wild-type Fc: L234A, L235A, wherein the numbering follows an EU index such as Kabat. In some embodiments, the Fc comprises two associative subunits. In some embodiments, the two subunits comprised of the Fc are the same or different first and second subunits. In some embodiments, the first and second subunits comprise one or more amino acid substitutions that reduce homodimerization. In some embodiments, the first subunit has a knot structure according to the knob-into-Hole technique, and the second subunit has a hole structure according to the knob-into-Hole technique, or the first subunit has a hole structure according to the knob-into-Hole technique, and the second subunit has a knot structure according to the knob-into-Hole technique.

[0115] In some embodiments, the antigen-binding domain that specifically binds to MASP3 is a full-length antibody. In some embodiments, the full-length antibody that specifically binds to MASP3 is IgA, IgD, IgE, IgG, or IgM. In some embodiments, in the TACI fusion protein, the full-length antibody that specifically binds to MASP3 includes an IgG constant region, such as a constant region of IgG1, IgG2, IgG3, IgG4, or a variant thereof. In some embodiments, the IgG refers to human IgG. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:43 or SEQ ID NO:44. In some embodiments, the antigen-binding domain that specifically binds to MASP3 includes a κ light chain constant region. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:45. In some embodiments, the antigen-binding domain that specifically binds to MASP3 includes a λ light chain constant region. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:46.

[0116] In some embodiments, the TACI fusion protein described in this application further includes an antigen-binding domain that specifically binds to MASP2.

[0117] In some embodiments, the TACI fusion protein comprises an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP3 comprises: (i) V H The V HContains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:2; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:4; LC-CDR2, which contains the amino acid sequence SEQ ID NO:6; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:7; or (ii) V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:2; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:5; LC-CDR2, which contains the amino acid sequence SEQ ID NO:6; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:8.

[0118] In some embodiments, the TACI fusion protein comprises an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP2 comprises: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:23; HC-CDR2, which contains the amino acid sequence SEQ ID NO:24; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:25; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:26; LC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and LC-CDR3, which contains any of the amino acid sequences shown in SEQ ID NOs:28-32.

[0119] In some embodiments, the TACI fusion protein comprises an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP3 comprises: (a) V HIt comprises the amino acid sequence shown in SEQ ID NO:13 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:13; and V L (b) V H It comprises the amino acid sequence shown in SEQ ID NO:14 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:14; and V L It comprises the amino acid sequence shown in SEQ ID NO:17 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:17; (c)V H It comprises the amino acid sequence shown in SEQ ID NO:14 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:14; and V L It comprises the amino acid sequence shown in SEQ ID NO:20 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:20; (d)V H It comprises the amino acid sequence shown in SEQ ID NO:12 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:12; and V L It comprises the amino acid sequence shown in SEQ ID NO:21 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:21; (e)V HIt comprises the amino acid sequence shown in SEQ ID NO:13 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:13; and V L It comprises the amino acid sequence shown in SEQ ID NO:21 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:21; or (f)V H It comprises the amino acid sequence shown in SEQ ID NO:14 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:14; and V L It comprises the amino acid sequence shown in SEQ ID NO:22 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence shown in SEQ ID NO:22.

[0120] In some embodiments, the TACI fusion protein comprises an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP2 comprises: V H The V H Contains the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO:34; and V L The V L Contains any of the amino acid sequences shown in SEQ ID NOs:36-41 or variants thereof, wherein the variants have at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology to any of the amino acid sequences shown in SEQ ID NOs:36-41. In some embodiments, the antigen-binding domain that specifically binds to MASP2 comprises: V H The V H Contains the amino acid sequence shown in SEQ ID NO:34; and V L The V L It contains any of the amino acid sequences shown in SEQ ID NOs:36-41.

[0121] In some embodiments, the TACI fusion protein includes an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP3 includes: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:2; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3; and V. L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:5; LC-CDR2, which contains the amino acid sequence SEQ ID NO:6; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:8; and wherein the antigen-binding domain specifically binding to MASP2 contains: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:23; HC-CDR2, which contains the amino acid sequence SEQ ID NO:24; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:25; and V. L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:26; LC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:29.

[0122] In some embodiments, the TACI fusion protein includes an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP3 includes: V H It comprises the amino acid sequence shown in SEQ ID NO:14 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:14; and V L It comprises the amino acid sequence shown in SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:20; and wherein the antigen-binding domain specifically binding to MASP2 comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V LIt contains the amino acid sequence shown in SEQ ID NO:37 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:37.

[0123] In some embodiments, the TACI fusion protein includes an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP3 includes: V H It comprises the amino acid sequence shown in SEQ ID NO:12 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:12; and V L It comprises the amino acid sequence shown in SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:21; and wherein the antigen-binding domain specifically binding to MASP2 comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V L It contains the amino acid sequence shown in SEQ ID NO:37 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:37.

[0124] In some embodiments, the TACI fusion protein includes an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP3 includes: V H It comprises the amino acid sequence shown in SEQ ID NO:13 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:13; and V L It comprises the amino acid sequence shown in SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:21; and wherein the antigen-binding domain specifically binding to MASP2 comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V L It contains the amino acid sequence shown in SEQ ID NO:37 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:37.

[0125] In some embodiments, the TACI fusion protein includes an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, wherein the antigen-binding domain that specifically binds to MASP3 includes: V H It comprises the amino acid sequence shown in SEQ ID NO:14 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:14; and V L It comprises the amino acid sequence shown in SEQ ID NO:22 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:22; and wherein the antigen-binding domain specifically binding to MASP2 comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V L It contains the amino acid sequence shown in SEQ ID NO:37 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:37.

[0126] In some embodiments, the TACI fusion protein comprises an antigen-binding domain specifically binding to MASP3 and an antigen-binding domain specifically binding to MASP2, wherein the antigen-binding domain specifically binding to MASP3 and the antigen-binding domain specifically binding to MASP2 comprise a bispecific antibody specifically binding to MASP3 and MASP2. In some embodiments, the bispecific antibody in the TACI fusion protein of this application may adopt any structure in the prior art, such as the DVD-Ig structure, the Bs4Ab structure, the IgG-(scFv)2 structure, or the scFv-Fab IgG structure, etc. (for example, see the review articles Labrijn AF, et al. Nat Rev Drug Discov. 2019 Aug; 18(8):585-608 and Klein C, et al. The use of CrossMAb technology for the generation of bi-and multispecific antibodies. MAbs. 2016 Aug-Sep; 8(6):1010-20). In some embodiments, the bispecific antibody in the TACI fusion protein of this application adopts the Hetero H, CrossMab structure. Its schematic diagram is shown in Figure 2. It is a bivalent multispecific antibody composed of heterodimers, containing two antigen-binding domains (Fab) and further containing two Fc domains, which include C H2 and C H 3. Structural Domains. In some embodiments, C in one of the Fc H In the 3 domain, amino acid residues are replaced with amino acid residues of larger side chain volume, forming a "knob", and another C in the Fc domain. H In the 3-domain structure, amino acid residues are replaced with smaller side-chain amino acid residues, forming "holes" that promote the binding of heterodimers. In Fab, the light chain constant region (C... L ) and heavy chain constant region C H 1. Structural Domain (C) H 1) The positions can be interchanged; or the variable region of the heavy chain (V) H ) and light chain variable region (V L The positions of ) can be interchanged; or the constant region of the light chain (C) L ) and heavy chain constant region C H 1. Structural Domain (C) H 1) and heavy chain variable region (V H ) and light chain variable region (V L They can be replaced at the same time.

[0127] In some embodiments, the Fc is derived from wild-type human IgG1 Fc. In other embodiments, the C of the Fc... H The 2-domain contains, but is not limited to, the following amino acid substitutions: L234A, L235A, wherein the numbering follows an EU index such as Kabat. In other embodiments, the Fc contains a KIH (Knob-into-hole) mutation, wherein the C of the Fc... H The 3-domain includes, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering follows an EU index as described in Kabat. In some embodiments, the Fc hole includes substitutions of Y349C, T366S, L368A, and Y407V, and the Fc knob includes substitutions of S354C and T366W, wherein the numbering follows an EU index as described in Kabat.

[0128] In some embodiments, the TACI fusion protein described in this application is wherein the antigen-binding domain that specifically binds to MASP3 is a monoclonal antibody that specifically binds to MASP3.

[0129] In some embodiments, the TACI fusion protein described in this application comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) extracellular domains of TACI as described above, or variants thereof capable of binding BlyS and / or APRIL.

[0130] In some embodiments, the TACI fusion protein of this application, wherein the extracellular domain of TACI or a variant thereof capable of binding BlyS and / or APRIL is linked via a linker peptide or directly to an antigen-binding domain that specifically binds MASP3 or MASP2. In some embodiments, the linker peptide comprises a (G4S)n sequence, wherein n is an integer independently selected from 1-6 (e.g., 1, 2, 3, 4, 5, 6). In some embodiments, the linker peptide comprises the amino acid sequence SEQ ID NO:63.

[0131] In some embodiments, the TACI fusion protein described in this application, wherein the extracellular domain of TACI or a variant thereof capable of binding BlyS and / or APRIL is linked to the heavy chain variable region (V) of the antigen-binding domain specifically binding MASP3 or MASP2. H ) or light chain variable region (V L The N-terminus or C-terminus of ).

[0132] In some embodiments, the TACI fusion protein of this application, wherein the extracellular domain of TACI or a variant thereof capable of binding BlyS and / or APRIL is linked to the N-terminus or C-terminus of the Fc fragment.

[0133] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, two of which contain V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure, with the other two polypeptide chains containing V from the N-terminus to the C-terminus. L -C L Structure, where V H 1 and V L These are the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domains.

[0134] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H 1-C H The 1-Fc structure, with the other two polypeptide chains containing V from the N-terminus to the C-terminus. L -C L Structure, where V H 1 and V L These are the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H3. Structural domain, preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0135] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H 2-C L -Fc-TACI structure, a polypeptide chain containing V from N-terminus to C-terminus L 1-C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively; V H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domain, preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0136] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H The 2-CL-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. L 1-C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively; V H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domain. Preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0137] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H 2-C L -Fc structure, a polypeptide chain containing V from N-terminus to C-terminus. L 1-C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively; V H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domain. Preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0138] In some embodiments, the TACI fusion protein described in this application comprises four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H The 2-CL-Fc structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. L 1-C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively; V H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domain. Preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0139] In some embodiments, the TACI fusion protein described in this application comprises three polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc structure is a polypeptide chain containing V from the N-terminus to the C-terminus.L 1-C L The structure, and another polypeptide chain containing a TACI-Fc structure from the N-terminus to the C-terminus, wherein V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domain, preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

[0140] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:70 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:70; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:54.

[0141] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:71 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:71; and the amino acid sequence SEQ ID NO:67 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:67; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:54.

[0142] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:66 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:66; and the amino acid sequence SEQ ID NO:52 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:52; and the amino acid sequence SEQ ID NO:67 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:67; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:67; NO:54 has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology.

[0143] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:74 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:74; and the amino acid sequence SEQ ID NO:52 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:52; and the amino acid sequence SEQ ID NO:75 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:75; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:74; NO:54 has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology.

[0144] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:66 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:66; and the amino acid sequence SEQ ID NO:52 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:52; and the amino acid sequence SEQ ID NO:60 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:60; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:66; NO:54 has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology.

[0145] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:71 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:71; and the amino acid sequence SEQ ID NO:72 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:72; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:54.

[0146] In some embodiments, the TACI fusion protein of this application comprises the amino acid sequence SEQ ID NO:76 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:76; and the amino acid sequence SEQ ID NO:77 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:77; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence SEQ ID NO:54.

[0147] Exemplary antigen-binding domain sequences are shown in Tables 2-8, where CDR numbers are based on the EU index in Kabat. Exemplary TACI fusion protein sequences are shown in Table 10. Those skilled in the art will recognize that various known algorithms exist for predicting CDR locations and defining the variable regions of the antibody light and heavy chains. CDRs and V... (The text abruptly ends here, so the translation stops as well.) H and / or V L Sequences, but antibodies based on prediction algorithms rather than those exemplified in the table below are also within the scope of this application.

[0148] Table 2: Exemplary antibody CDR sequences that specifically bind to MASP3

[0149] Table 3: Exemplary antibodies that specifically bind to MASP3 (V) H / V L sequence

[0150] Table 4: Exemplary antibody CDR sequences that specifically bind to MASP2

[0151] Table 5: Exemplary antibodies that specifically bind to MASP2 H / V L sequence

[0152] Table 6: Exemplary Antibody Constant Region Sequences

[0153] Table 7: Partial heavy and light chain sequences of exemplary multispecific antibodies that structurally bind to MASP3 and MASP2 using Hetero H, CrossMab.

[0154] Table 8: Full-length heavy and light chain sequences of exemplary multispecific antibodies that structurally bind to MASP3 and MASP2 using Hetero H, CrossMab.

[0155] Table 9: Exemplary linker peptide (or adapter) sequences

[0156] Table 10: Exemplary TACI fusion protein sequences

[0157] Combining affinity

[0158] Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA). Kd values ​​can be determined by methods known in the art, such as surface plasmon resonance (SPR) technology or biolayer interference (BLI) technology.

[0159] Binding affinity can be expressed as Kd, Koff, Kon, or Ka. As used herein, the term "Koff" refers to the rate constant for the dissociation of the antigen-binding domain from the antigen-binding domain / antigen complex, determined by a kinetic selection device. The term "Kon" refers to the binding rate constant for the antibody to bind to the antigen to form an antigen-binding domain / antigen complex. The dissociation constant "Kd" used herein refers to the dissociation constant for a specific antibody-antigen interaction, which is the antigen concentration required in the antibody molecule solution for the antigen to occupy half of all antibody-binding domains and reach equilibrium, equal to Koff / Kon. The determination of Kd assumes all binding molecules are in solution. For cases where the antigen-binding domain is attached to the cell wall, such as in a yeast expression system, the corresponding dissociation rate constant is expressed using EC. 50 It is expressed as a good approximation of Kd. The affinity binding constant Ka is the reciprocal of the dissociation constant Kd.

[0160] The equilibrium dissociation constant (Kd) can serve as an indicator of the affinity between the antigen-binding domain and the antigen. For example, it can be easily analyzed using the Scatchard method with antibodies labeled with various markers and a Biacore instrument (manufactured by Amersham Biosciences), following the user manual or accompanying kit, by analyzing the interactions between biomolecules through surface plasmon resonance. The Kd values ​​obtained using these methods are expressed in units of megohms (M). Antibodies that bind specifically to a target may have a Kd value, for example, ≤10. -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 -13 The Kd value of M.

[0161] The binding specificity of antibodies can be experimentally determined using methods known in the art. These methods include, but are not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, BIAcore assays, and peptide scanning.

[0162] In some embodiments, the antigen-binding domain of the TACI fusion protein specifically binds to the MASP3 target, with a Kd value of 10. -7 M to 10 -13 M (e.g., 10) -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10 -12 M).

[0163] Nucleic acid and vector

[0164] Nucleic acid molecules encoding any of the TACI fusion proteins described in this application are also taken into consideration.

[0165] In some embodiments, this application provides (or a group of) nucleic acids encoding any of the TACI fusion proteins described in this application. In some embodiments, the nucleic acid (or group of nucleic acids) described in this application may further include a nucleic acid sequence encoding a polypeptide tag (e.g., a protein purification tag, His-tag, HA tag).

[0166] This application also provides isolated host cells containing a vector carrying the nucleic acid molecules. This application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize under at least moderately stringent hybridization conditions with a nucleic acid sequence encoding an antigen-binding domain or bispecific antibody of this application.

[0167] This application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize with nucleic acid sequences encoding antigen-binding domains or bispecific antibodies of this application at least under moderately stringent hybridization conditions.

[0168] This application also provides a vector into which the nucleic acid sequence of this application can be inserted.

[0169] In short, by inserting a natural or synthetic nucleic acid encoding an antigen-binding domain or bispecific antibody into a suitable expression vector, allowing the nucleic acid to be operatively linked to 5' and 3' regulatory elements, such as promoters (e.g., lymphocyte-specific promoters) and a 3' untranslated region (UTR), the antigen-binding domain or bispecific antibody can be expressed. The vector is suitable for replication and integration in eukaryotic host cells. A typical cloning and expression vector contains transcription and translation terminators, a start sequence, and a promoter that regulate the expression of the target nucleic acid sequence.

[0170] The nucleic acids described in this application can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Nucleic acid delivery methods are known in the art. See, for example, US Pat. Nos. 5,399,346, 5,580,859, 5,589,466, the entire contents of which are incorporated herein by reference. In some embodiments, this application also provides gene therapy vectors.

[0171] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors, including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and Cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0172] In addition, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology or molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector includes an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0173] Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus is then isolated and delivered in vivo or in vitro to the cells of a recipient. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they enable long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over tumor-derived retroviruses, such as murine leukemia virus, because they can transduce non-dividing cells, such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0174] Other promoter elements, such as enhancers, regulate transcription initiation frequency. They are typically located 30-110 bp upstream of the start site, although recent studies have found that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so the promoter retains its function even when elements interchange or move. In the thymidine kinase (TK) promoter, activity begins to decline only when the spacing between promoter elements increases to 50 bp.

[0175] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a very strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is the elongation factor 1α (EF-1α) promoter. However, other constitutive promoters may also be used, including but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rouss sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. Furthermore, this application should not be limited to the use of only constitutive promoters. Inducible promoters are also considered in this application. The use of inducible promoters provides a molecular switch that initiates the expression of the polynucleotide sequence to which it is operable when such expression is needed, and shuts it off when not needed. Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0176] In some embodiments, the expression of the antigen-binding domain or bispecific antibody is inducible. In some embodiments, the nucleic acid sequence encoding the antigen-binding domain or bispecific antibody is operatively linked to an inducible promoter, including any of the inducible promoters described in this application.

[0177] Inducible promoters

[0178] The use of inducible promoters provides a molecular switch that initiates the expression of a polynucleotide sequence operatively linked to it when expression is desired, and shuts it off when expression is not desired. Exemplary inducible promoters applicable in eukaryotic cells include, but are not limited to, hormone regulatory elements (e.g., see Mader, S. and White, JH (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand regulatory elements (see Spencer, D. M et al (1993) Science 262:1019-1024), and ionizing radiation regulatory elements (see Manome, Y. et al. (1993) Biochemistry 32:10607-10613; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Other exemplary inducible promoters applicable to in vivo or in vitro mammalian systems are described in Gingrich et al. (1998) Annual Rev. Neurosci 21:377-405. In some embodiments, the inducible promoter system for expressing antibodies or antigen-binding fragments or multispecific antibodies is a Tet system, such as the Tet system described by Gossen et al. (1993). In some embodiments, the inducible promoter system for expressing antibodies or antigen-binding fragments or multispecific antibodies is an E. coli lac inhibitory system (see Brown et al., Cell 49:603-612 (1987)).

[0179] To assess the expression of a peptide or its fraction, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells transfected or infected by a viral vector. In other respects, the selectable marker may be carried on a separate DNA fragment and used in co-transfection experiments. Both the selectable marker gene and the reporter gene may be side-mounted to a suitable regulatory sequence to enable its expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.

[0180] Reporter genes can be used to identify potential transfected cells and evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue; it encodes a polypeptide, and its expression exhibits easily detectable properties, such as enzymatic activity. Reporter gene expression is detected at an appropriate time after DNA is introduced into recipient cells. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tel et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or obtained commercially. Typically, a construct with a minimal 5' flanking region capable of displaying the highest expression level of the reporter gene is identified as a promoter. Such promoter regions can be linked to reporter genes and used to assess the ability of certain substances to regulate promoter-driven transcription.

[0181] In some embodiments, a nucleic acid encoding any of the antibodies or antigen-binding fragments or multispecific antibodies described in this application is provided. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of the antibody or antigen-binding fragment or multispecific antibody. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some nucleic acid sequences are contained in the same vector. In some embodiments, all nucleic acid sequences are contained in the same vector. The vector may be selected from, for example, mammalian expression vectors and viral vectors (such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses).

[0182] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian cells, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological methods.

[0183] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, gene gun methods, microinjection, electroporation, and the like. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, polynucleotides are introduced into host cells via calcium phosphate transfection.

[0184] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

[0185] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymeric complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, including oil-in-water emulsions, micelles, hybrid micelles, and liposomes. An exemplary colloidal system used as a delivery carrier in vivo and in vitro is a liposome (e.g., an artificial membrane capsule).

[0186] In the use of non-viral delivery systems, an exemplary delivery vector is the liposome. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid can bind to lipids. Lipid-bound nucleic acids can be encapsulated within the aqueous interior of a liposome, dispersed within a lipid bilayer of the liposome, linked to the liposome by linking molecules that bind to the liposome and oligonucleotides, embedded within the liposome, forming a complex with the liposome, dispersed in a solution containing lipids, mixed with lipids, bound to lipids, suspended in lipids, contained in or mixed with micelles, or otherwise bound to lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions in solution are not limited to any particular structure. For example, they may exist in a bilayer structure, as micelles, or as a “collapsed” structure. They may also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic. For example, lipids include fat droplets that are naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0187] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors of this application, various experiments can be performed to confirm the presence of the recombinant DNA sequence in the host cells. These experiments include, for example, "molecular biology" experiments well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and "biochemical" experiments, such as detecting the presence or absence of a specific polypeptide, for example, identification by immunological methods (ELISAs and Western blots) or by the experiments described in this application, all of which fall within the scope of this application.

[0188] Preparation of antigen-binding domain

[0189] In some embodiments, the TACI fusion protein includes an antigen-binding domain that specifically binds to MASP3, the antigen-binding domain being derived from a monoclonal antibody that specifically binds to MASP3. In some embodiments, the TACI fusion protein includes an antigen-binding domain that specifically binds to MASP3 and an antigen-binding domain that specifically binds to MASP2, the antigen-binding domain being derived from a bispecific antibody that specifically binds to MASP3 and MASP2. In some embodiments, the antigen-binding domain includes a V-type antigen derived from a monoclonal antibody. H and V L Or its variants. In some embodiments, the antigen-binding domain further comprises a C-type antigen from a monoclonal antibody. H 1 and C L Regions, or variations thereof. Monoclonal antibodies can be prepared using methods known in the art, including hybridoma cell methods, yeast display, phage display methods, or recombinant DNA methods. Furthermore, exemplary yeast display and phage display methods are described in this application and the following embodiments. Multispecific antibodies can be prepared using methods known in the art, including chemical conjugation, hybridoma methods, and genetic engineering methods.

[0190] In hybridoma cell methods, hamsters, mice, or other suitable host animals are typically immunized with an immunomodulator to induce the production or ability to produce antibodies that specifically bind to the immunomodulator. Alternatively, lymphocytes can be immunized in vitro. The immunomodulator may include a peptide or fusion protein of the target protein. Peripheral blood lymphocytes (PBLs) are typically used if human cells are desired, while spleen cells or lymph node cells are used if non-human mammalian cells are desired. The lymphocytes are fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells. Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are commonly used. Hybridoma cells can be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma cell culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibits the growth of HGPRT-deficient cells.

[0191] In some embodiments, the immortalized cell lines are efficiently fused, ensuring high-level and stable antibody expression through selected antibody-producing cells, and are sensitive to certain culture media, such as HAT medium. In some embodiments, the immortalized cell lines are mouse myeloma cell lines, obtainable from, for example, the Salk Cell Collection in San Diego, California, and the American Collection of Type Cultures in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines for the preparation of human monoclonal antibodies are also described.

[0192] The presence of monoclonal antibodies against the peptide can then be determined in the culture medium for hybridoma cells. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis as described in Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0193] After identifying the desired hybridoma cells, the target clone can be subcloned using limiting dilution and cultured using standard methods. Suitable media for this purpose include, for example, modified Eagle medium (DMEM) and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in mammals as ascites fluid.

[0194] Subclonal secreted monoclonal antibodies can be isolated or purified from culture medium or ascites using conventional immunoglobulin purification methods, such as protein A-agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0195] In some embodiments, any antibody, antigen-binding fragment, or multispecific antibody according to this application comprises a sequence selected from clones of an antibody library (e.g., a phage library displaying scFv or Fab fragments). The clone can be identified by screening antibody fragment combination libraries with the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries to obtain antibodies with the desired binding properties. These methods have been reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. Further descriptions can be found in al., J.Mol.Biol.340(5):1073-1093(2004); Fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004); and Lee et al., J.Immunol.Methods 284(1-2):119-132(2004).

[0196] In some phage display methods, V is cloned separately using polymerase chain reaction (PCR). H and V LAll components of the gene are randomly recombined in a phage library, and then phages capable of binding antigens are screened, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as scFv fragments or Fab fragments. Phages from immune-derived libraries provide high-affinity antibodies against immunogens without the need to construct hybridoma cells. Alternatively, natural libraries (e.g., from humans) can be cloned to provide a single source of antibodies against multiple non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be prepared by cloning a non-rearranged V-gene fragment from stem cells, encoding the CDR3 hypervariable region using PCR primers containing random sequences, and performing rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, US Patent No. 5,750,373, and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0197] The antibody, antigen-binding fragment, or multispecific antibody is prepared by a method that uses phage display to screen an antigen-binding portion of a library capable of specifically binding to a target (e.g., MASP3 or MASP2). The library may be a human scFv phage display library with at least 1 × 10⁻⁶ cells / mL. 9 (e.g., at least 1×10) 9 2.5×10 9 5×10 9 7.5×10 9 1×10 10 2.5×10 10 5×10 10 7.5×10 10 Or 1×10 11The library contains a variety of unique human antibody fragments. In some embodiments, the library is a natural human library constructed from DNA extracted from PMBCs and spleen of healthy subjects, containing all heavy and light chain subfamilies. In some embodiments, the library is a natural human library constructed from DNA extracted from PMBCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semi-synthetic human library in which the heavy chain CDR3 is completely randomized, and all amino acids (except cysteine) are present at any given position with equal probability. (See, for example, Hoet, R.M et al., Nat. Biotechnol. 23(3):344-348, 2005). In some embodiments, the heavy chain CDR3 length of the semi-synthetic human library is between 5 and 24 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0198] Phage clones with high affinity for a target antigen (e.g., MASP3 or MASP2) can be screened by iterative binding of the phage to the target antigen, which is bound to a solid support (e.g., beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phages and elution of specifically bound phages. The eluted phage clones are then used to infect suitable host cells, such as E. coli XL1-Blue, for expression and purification. Multiple rounds of panning (e.g., 2, 3, 4, 5, 6, or more rounds), such as solution panning, cell panning, or a combination of both, can be used to enrich phage clones that specifically bind to the target antigen. The specific binding of the enriched phage clones to the target antigen can be detected by any method known in the art, including, for example, ELISA and FACS.

[0199] Monoclonal antibodies can also be prepared using recombinant DNA methods, such as those described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibody described in this application can be easily isolated and sequenced using conventional methods, such as using oligonucleotide probes that specifically bind to genes encoding the light and heavy chains of murine antibodies. Hybridoma cells as described above or antigen-specific phage clones of this application can serve as sources of this DNA. After isolation, the DNA can be placed in an expression vector, which is then transfected into host cells, such as simian COS cells, Chinese hamster ovary cancer (CHO) cells, or non-immunoglobulin-producing myeloma cells, to obtain monoclonal antibodies synthesized in recombinant host cells. The DNA can also be modified, for example, by replacing homologous non-human sequences with coding sequences of constant structures and / or framework regions of human heavy and light chains (US Patent No. 4,816,567; Morrison et al., ibid.), or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. This non-immunoglobulin polypeptide can replace the constant region of the antibody in this application, or it can replace an antigen-binding site in the variable region of the antibody in this application, to form a chimeric bivalent antibody. In some embodiments, additional variable regions targeting different epitopes or antigens can be included to generate chimeric multispecific antibodies.

[0200] The antigen-binding domain can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, a recombinant expression method involving immunoglobulin light chains and modified heavy chains. Typically, the heavy chain is truncated at any position in the Fc region to prevent cross-linking between the heavy chains. Alternatively, relevant cysteine ​​residues are substituted with other amino acid residues or deleted to prevent cross-linking.

[0201] In vitro methods are also suitable for preparing monovalent antibodies. Digesting antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.

[0202] Chemical conjugation was the earliest technique used to prepare multispecific antibodies. In 1985, Brennan first used the chemical conjugation of two monoclonal antibody G1 fragments to prepare multispecific antibodies (Brennan M, et al. Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments[J]. Science, 1985, 229(4708):81-83). There are two main methods of chemical conjugation: one is to directly conjugate two monoclonal antibodies or their derivatives to form multispecific antibodies; the other is to first dissociate the two monoclonal antibodies into free light and heavy chains through various physicochemical methods, and then recombine these light and heavy chains. The advantages of chemical conjugation are its speed, ease of operation, and high recovery rate. However, it can easily damage the antigen-binding domain of the antibody, affecting antibody activity, and it is also prone to forming polymers.

[0203] The preparation of multispecific antibodies using hybridoma cell lines refers to the process of fusing two different hybridoma cell lines using cell fusion technology, followed by the identification and separation of cells that can produce specific therapeutic antibodies (Kohler, G, et al. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. J Immunol., 2005, 174(5):2453-2455). Because two hybridoma cell lines can produce two different light-heavy chains, and these light-heavy chains can combine randomly, the multispecific antibodies prepared by this method have a high degree of randomness and low preparation efficiency.

[0204] Genetic engineering technology is also currently used to prepare a variety of multispecific antibodies (Roland E K. Antibody-cytokine fusion proteins[J]. Arch Biochem Biophys., 2012, 526(2):194-205). Using genetic engineering to edit recombinant antibodies can solve the problem of random combination by restricting the selectivity of light-heavy chain binding in various ways. KiH (Knob into hole) and CrossMab are two commonly used techniques to improve the light-heavy chain pairing problem. KiH technology involves... H 3. The structural domain introduces an asymmetric mutation structure ("knob" mutation refers to a mutation in C...). HIn the 3-domain, a larger amino acid residue replaces a smaller residue, while a "hole" mutation refers to replacing a larger residue with a smaller amino acid residue. The Fc region of modified multispecific antibodies is more prone to heterodimerization than homodimerization due to steric hindrance (Ridgway JB, et al. "Knobs-into-holes" engineering of antibody C). H 3 domains for heavy chain heterodimerization[J]. Protein Eng. 1996, 9(7):617-621). And in glycosylated C... H Introducing a Y349C mutation into the 3 domain can enable the formation of disulfide bonds between glycosylated heavy chains, thereby enhancing the stability of KiH (Kuglstatter A, et al. Structural differences between glycosylated, disulfide-linked heterodimeric knob-into-hole Fc fragment and its homodimeric knob-knob and hole-hole side products[J]. Protein Eng Des Sel., 2017, 30(9): 649-656).

[0205] Besides steric hindrance, the charge effect of amino acid residues has also been used to enhance heterodimerization between the two heavy chains of multispecific antibodies. Through structural simulation and molecular design, a "positive charge" can be generated in one chain and a "negative charge" in the paired chain. This promotes the formation of heterodimers through a repulsion of like charges and attraction of dissimilar charges. Mutations in the two chains, including K409D and D399K, K409D / K392D and D399K / E356K, or E356K / E357K / D399K and K370E / K409D / K439E, can all enhance heterodimer formation to some extent (IGAWA T, et al. Methods for producing polypeptides by regulating polypeptide; association: US, 20100015133A1[P]. 2006). Combining the KiH steric hindrance effect and charge effect is also a strategy to enhance heterodimerization.

[0206] CrossMab technology is a novel antibody pairing technology developed by Roche based on KiH technology. It involves exchanging the domains of one Fab light chain with the heavy chain in a multispecific antibody, while leaving the other chain unexchanged. The exchanged light chain contains fragments of the homologous heavy chain, preventing it from pairing with the unexchanged heavy chain, thus ensuring the correct combination of the light and heavy chains (Schaefer W, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies[J]. Proc Natl Acad Sci USA, 2011, 108(27):11187-11192). This structure includes "CrossMab Fab" and "CrossMab V". H -V L "or "CrossMab C H 1-C L "etc."

[0207] Antibody variable regions with the desired binding specificity (antibody-antigen binding site) can be fused to immunoglobulin constant regions. Preferably, fusion is made with the immunoglobulin heavy chain constant region, which includes at least a partial hinge, C H 2 and C H 3. Structural domains. In some embodiments, the heavy chain constant region C includes the necessary sites for light chain binding. H The 1 domain is present in at least one fusion variant. DNA encoding the immunoglobulin heavy chain fusion variant, and optionally DNA encoding the immunoglobulin light chain, is inserted into a separate expression vector and co-transfected into a suitable host organism. In some embodiments, antibody variable regions targeting different antigenic epitopes or different antigens may be fused with immunoglobulin constant region sequences to generate chimeric multispecific antibodies.

[0208] Humanized and fully humanized antibodies

[0209] In some embodiments, the TACI fusion protein described in this application, wherein the antigen-binding domain that specifically binds to MASP3 or MASP2, may be a humanized antibody or a fully human antibody. The humanized form of the non-human (e.g., mouse) antibody portion is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding sequence of the antibody), which typically comprises a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies comprise human immunoglobulins, immunoglobulin chains, or fragments thereof (receptor antibodies), wherein residues of the receptor CDR are replaced by non-human (donor antibody) CDR residues having the desired specificity, affinity, and performance, such as mouse, rat, or rabbit CDRs. In some embodiments, human immunoglobulin Fv frame region residues are replaced by corresponding non-human residues. Humanized antibodies may also contain amino acid residues that are neither part of the receptor antibody nor present in the introduced CDR or frame region sequence. Typically, humanized antibodies contain at least one, usually two, variable regions, where all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the framework regions are human immunoglobulin common sequences.

[0210] Typically, humanized antibodies contain one or more amino acid residues introduced from non-human sources. These non-human amino acid residues are generally referred to as “shift-in” residues and usually originate from the “shift-in” variable region. According to some embodiments, humanization can be performed essentially as described by Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)) by replacing the corresponding sequence of the human antibody with rodent CDRs or CDR sequences. Thus, this “humanized” antibody portion (US Patent No. 4, 816, 567), which is essentially less than a complete human antibody, has its variable region replaced by the corresponding sequence from a non-human source. In practice, the humanized antibody portion is a typical human antibody portion, in which some CDR residues and possibly some framework region residues are replaced by residues from similar sites in rodent antibodies.

[0211] Producing human antibodies is an alternative to humanization. For example, it is currently possible to produce transgenic animals (e.g., mice) that generate a complete human antibody library after immunization without producing endogenous immunoglobulins. For instance, it has been reported that homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice completely suppresses the production of endogenous antibodies. Transferring human germline immunoglobulin gene arrays into germline mutant mice can produce fully human antibodies upon antigen stimulation, see, for example, akobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); US Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852. Alternatively, fully human antibodies can be prepared by introducing human immunoglobulin gene loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely silenced). Upon antigen stimulation, the production of fully human antibodies is found to be very similar to that in humans in all aspects, including gene rearrangement, assembly, and antibody library formation. This method has been used in, for example, US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and It is described in Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0212] Human antibodies or human antibody fractions can also be generated by activating B cells in vitro (see US Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Techniques by Cole et al. and Boerner et al. can also be used to prepare fully human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991).

[0213] Variants of TACI fusion protein

[0214] In some embodiments, this application provides TACI fusion proteins, wherein the amino acid sequences of variants of antigen-binding domains (e.g., antibodies that specifically bind MASP3 or MASP2, or multispecific antibodies that specifically bind MASP3 and MASP2) or variants of TACI peptides are also considered. For example, it may be necessary to improve the binding affinity and / or other biological activities of the antibody or antigen-binding fragment. The amino acid sequences of the antibody or antigen-binding fragment variants or variants of TACI peptides can be prepared by introducing appropriate modifications into the encoding nucleotide sequence or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antigen-binding entity. The final construction can be accomplished by any combination of amino acid residue deletions, insertions, and substitutions to give it the desired characteristics, such as antigen binding.

[0215] In some embodiments, variants of antibodies or antigen-binding fragments having one or more amino acid substitutions are provided. Target sites for substitution mutations include hypervariable regions (HVRs) and frame regions (FRs). Amino acid substitutions can be introduced into the target antibody to screen for products with desired activities, such as improved biological activity or antibody affinity. In some embodiments, the amino acid substitutions described herein are limited to the “Exemplary Substitutions” in Table A of this application. In some embodiments, the amino acid substitutions are limited to the “Preferred Substitutions” in Table A of this application.

[0216] Conservative substitutions are shown in Table A below.

[0217] Table A: Conservative Substitution

[0218] Amino acids are classified into different categories based on the properties of their side chains:

[0219] a. Hydrophobic amino acids: Norleucine, Methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile);

[0220] b. Neutral hydrophilic amino acids: Cysteine ​​(Cys), Serine (Ser), Threonine (Thr), Asparagine (Asn), Glutamine (Gln);

[0221] c. Acidic amino acids: Aspartic acid (Asp), Glutamic acid (Glu);

[0222] d. Basic amino acids: Histidine (His), Lysine (Lys), Arginine (Arg);

[0223] e. Amino acids that affect chain orientation: glycine (Gly), proline (Pro);

[0224] f. Aromatic amino acids: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[0225] Substitution of non-conservative amino acids involves replacing one class with another.

[0226] An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques. In short, one or more CDR residues are mutated, the variant antibody is partially displayed on a phage, and variants with specific biological activities (e.g., based on RBC lysis inhibition assays or binding affinity) are screened. Alterations (e.g., substitutions) can be made in HVR regions to obtain improved RBC lysis inhibition assays or antibody affinity. Alterations can be made in “hotspot regions” of the HVR, i.e., residues encoded by codons that are frequently mutated during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or at specific determinant residues (SDRs), to detect the resulting variant V. H and V L Binding affinity. Methods for constructing and reselecting affinity maturities from secondary libraries have been described in some literature, for example, Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0227] In some affinity maturation embodiments, diversity is introduced into selected variable genes for affinity maturation using any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. This library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity includes an HVR-mediated approach, where several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, for example, using alanine scan mutagenesis or modeling. Typically, the CDR-H3 and CDR-L3 regions are particularly key targets.

[0228] In some embodiments, substitution, insertion, or deletion may occur within one or more HVRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes that do not substantially reduce binding affinity (e.g., the conserved substitutions provided in this application) may be produced in HVRs. These changes may occur outside the HVR "hotspot" or SDR regions. In some embodiments, variants V provided above... H and V L Each HVR sequence is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0229] One method for identifying target amino acids or regions in antibodies through mutation is called "alanine scanning mutation," as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, one or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are substituted with neutral or negatively charged amino acids (e.g., alanine or glutamic acid) to determine whether the antibody-antigen interaction is affected. Substitutions can be further introduced at the amino acid position to demonstrate that the position is functionally sensitive to the initial substitution. Alternatively / additionally, contact sites between the antibody and antigen can be identified by the crystal structure of the antigen-antibody complex. These contact site residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants are screened to determine if they possess the desired properties.

[0230] Amino acid sequence insertions, including fusions at the amino and / or carboxyl ends, can range in length from one residue to polypeptides containing 100 or more residues, and also include the insertion of one or more amino acid residues within the sequence. Examples of terminal insertions include antigen-binding moieties with a methionyl residue at the N-terminus. Other insertion variants of the antigen-binding moiety include polypeptides with an enzyme (e.g., ADEPT) fused to the N- or C-terminus of the antigen-binding moiety or those that increase the serum half-life of the antigen-binding moiety.

[0231] Fc variant

[0232] In some embodiments, the TACI fusion protein of this application comprises an Fc region, and one or more amino acid modifications are introduced into the Fc region to generate an Fc variant. In some embodiments, the Fc variant has enhanced ADCC efficacy, generally associated with receptors (FcRs) that bind to Fc. In some embodiments, the Fc variant has reduced ADCC efficacy. There are numerous examples of how alterations or mutations in the Fc sequence affect its efficacy; for example, WO 00 / 42072 and Shields et al. J Biol. Chem. 9(2):6591-6604 (2001) describe antibody variants with enhanced or weakened binding to FcRs. The contents of these publications are incorporated herein by reference.

[0233] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells (e.g., infected cells) when antigens on the surface of target cells are bound by specific antigen-binding domains (e.g., monoclonal antibodies that specifically bind to MASP3 or bispecific antibodies that specifically bind to MASP3 and MASP2). Typically, ADCC effects involve antibody-activated NK cells. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of antibody molecules that bind to the surface of target cells. The most common Fc receptors on the surface of NK cells are CD16 or FcγRIII. Binding of the Fc receptor to the Fc region of the antibody leads to NK cell activation, release of cytotoxic granules, and subsequent apoptosis of the target cells.

[0234] In some embodiments, the TACI fusion protein of this application comprises an Fc region having one or more effector functions. For example, the in vivo half-life of the TACI fusion protein is important, but certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. The reduction / elimination of CDC and / or ADCC activity is confirmed by performing in vitro and / or in vivo cytotoxicity assays. For example, Fc receptor (FcR) binding assays can be performed to confirm the lack of FcγR binding (and therefore possible lack of ADCC activity), but the retention of FcRn binding capacity. In the main cells mediating ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Table 3 on page 464 of Ravetch and Kinet Annu. Rev. Immunol. 9:457-492 (1991) summarizes the expression of FcR on hematopoietic cells. Non-limiting examples of in vitro assessment of ADCC activity of target molecules are described in US Pat. No. 5, 500, 362 (see, for example, Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); US Pat. No. 5, 821, 337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive detection methods can be used (see, for example, ACTI). TM Flow cytometry non-radioactive cytotoxicity assay (Cell Technology, Inc., Mountain View, Calif.) and CYTOTOX 96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wis.). Effector cells used in these assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the target molecule can be detected in vivo, for example in animal models, as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind to C1q, thus lacking CDC activity. See, for example, C1q and C3c binding ELISAs in WO2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC detection can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, for example, Petkova, S B et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0235] Antibodies with reduced effector function include those with one or more residue substitutions at positions 238, 265, 269, 270, 297, 327, and 329 in the Fc region (US Pat. No. 6,737,056). These Fc variants include those with two or more residue substitutions at positions 265, 269, 270, 297, and 327, including the Fc variant known as “DANA,” which has alanine substitutions at positions 265 and 297 (US Pat. No. 7,332,581).

[0236] Antibody variants with increased or decreased binding affinity to FcRs have been described (see, for example, US Pat. No. 6, 737, 056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0237] In some embodiments, changes in the Fc region lead to changes in the conditioning effect (i.e., enhancement or reduction), as described in Moore et al., MAbs.2(2):181–189 (2010).

[0238] In some embodiments, the TACI fusion protein of this application comprises an Fc variant with one or more amino acid substitutions that can prolong its half-life and / or enhance its binding to the Fc receptor (FcRn). Antibodies with prolonged half-life and improved FcRn binding are described in US2005 / 0014934A1 (Hinton et al.). These antibodies contain one or more amino acid substitutions in the Fc region that enhance the binding of the Fc region to FcRn. These Fc variants contain one or more substitutions of residues at positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 in the Fc region, for example, the substitution of residue 434 in the Fc region (USPat. No. 7,371,826).

[0239] See also Duncan & Winter, Nature 322:738-40 (1988); US Pat. No. 5, 648, 260; US Pat. No. 5, 624, 821 and WO 94 / 29351 for examples of other Fc variants.

[0240] TACI fusion proteins, including any Fc variant or combination thereof described in this application, are also considered.

[0241] Glycosylation variants

[0242] In some embodiments, the antigen-binding domains that specifically bind to MASP3 or MAPS2 in the TACI fusion protein of this application are modified to increase or decrease the degree of glycosylation of the antigen-binding domains that specifically bind to MASP3 or MASP2. By changing the amino acid sequence of the antigen-binding domains that specifically bind to MASP3 or MASP2 to add or remove one or more glycosylation sites, it is possible to conveniently add or delete glycosylation sites on the antigen-binding domains that specifically bind to MASP3 or MASP2.

[0243] The antigen-binding domain that specifically binds to MASP3 or MASP2 contains an Fc region, which can be altered to the sugars linked to it. Naturally occurring antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are usually linked to the Fc region via N-linkage. H2. The Asn297 domain is linked, see, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may comprise various sugars, such as mannose, N-acetylglucosinolate (GlcNAc), galactose, and sialic acid, as well as trehalose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the antibody or antigen-binding fragment specifically binding to MASP3 in the TACI fusion protein of this application may be modified with oligosaccharides to produce variants of the antibody or antigen-binding fragment specifically binding to MASP3 with certain improved properties.

[0244] C with Fc region H The N-glycans linked by the 2-domain are heterogeneous. Antibodies or Fc fusion proteins produced in CHO cells are fucosylated via fucosyltransferase activity (see Shoji-Hosaka et al., J. Biochem. 2006, 140:777-83). Typically, a small subset of naturally occurring non-fucosylated IgGs can be detected in human serum. N-glycosylation of the Fc region is important for its binding to FcγR; while non-fucosylated N-glycans enhance the binding affinity of Fc to FcγRIIIa. Enhanced binding affinity to FcγRIIIa leads to enhanced ADCC effects, which is advantageous in certain antibody therapeutic applications requiring cytotoxicity.

[0245] In some embodiments, enhanced effector function may be detrimental when Fc-mediated cytotoxicity is not required. In some embodiments, the Fc fragment or C H 2. The structural domain is non-glycosylated. In some embodiments, by adjusting C... H Mutate the N-glycosylation sites in the 2 domain to prevent glycosylation.

[0246] In some embodiments, the antigen-binding domain specifically binding to MASP3 or MASP2 included in the TACI fusion protein of this application comprises an Fc region, wherein the glycosylation structure linked to the Fc region has reduced or absent fucose, which may enhance ADCC function. Specifically, this application provides antigen-binding domains specifically binding to MASP3 or MASP2 that have reduced fucose compared to the same antigen-binding domains specifically binding to MASP3 or MASP2 produced by wild-type CHO cells. That is, they are characterized by having less fucose than antibodies produced by native CHO cells (e.g., CHO cells producing the native glycosylated form, CHO cells containing the native FUT8 gene). In some embodiments, the N-linked glycan of the antigen-binding domain specifically binding to MASP3 or MASP2 has less than 50%, 40%, 30%, 20%, 10%, or 5% fucose. For example, the fucose content of the antigen-binding domain that specifically binds to MASP3 or MASP2 may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. In some embodiments, the N-linked glycan of the antigen-binding domain that specifically binds to MASP3 or MASP2 does not contain fucose, i.e., the antigen-binding domain that specifically binds to MASP3 or MASP2 is completely fucose-free, or contains no fucose, or is defucosylated. The fucose content is determined by calculating the average fucose content within the sugar chain linked to Asn297 relative to the total amount of all sugar structures (such as complex, hybrid, or mannose structures) linked to Asn297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 of the Fc region (EU Fc region residue numbering system). However, due to minor sequence variations in the antibody, Asn297 can also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants may possess enhanced ADCC function. See, for example, US Patent Publication Nos. US2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to antibody variants that are “defucosylated” or “fucosylated” include US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US 2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. Cell lines capable of producing defucosylation antibodies include Lec13CHO cells lacking protein fucosylation function (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No. US 2003 / 0157108A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and gene knockout cell lines, such as CHO cells with α-1,6-fucosylation gene and FUT8 gene knockout (see Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003 / 085107).

[0247] Antigen-binding domain variants that specifically bind to MASP3 or MASP2 further provide bimeric oligosaccharides, for example, wherein biantennary oligosaccharides linked to the Fc region of the antigen-binding domain that specifically binds to MASP3 or MASP2 are bimeric by GlcNAc. Such antigen-binding domain variants that specifically bind to MASP3 or MASP2 may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described in WO 2003 / 011878 (Jean-Mairet et al.); US Pat. No. 6, 602, 684 (Umana et al.); US 2005 / 0123546 (Umana et al.); and Ferrara et al., Biotechnology and Bioengineering, 93(5): 851-861 (2006). Antigen-binding domain variants that specifically bind to MASP3 or MASP2 are also provided, which have at least one galactose residue in the oligosaccharide linked to the Fc region. These variants, which specifically bind to the antigen-binding domains of MASP3 or MASP2, may have enhanced CDC function. Such variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0248] In some embodiments, the antigen-binding domain variant that specifically binds to MASP3 or MASP2 includes an Fc region capable of binding to FcγRIII. In some embodiments, the antigen-binding domain variant that specifically binds to MASP3 or MASP2 and includes an Fc region has ADCC activity in the presence of human effector cells (e.g., T cells), or has enhanced ADCC activity in the presence of human effector cells compared to other identical antigen-binding domains that specifically bind to MASP3 or MASP2 and have a human wild-type IgG1 Fc region. In some embodiments, the antigen-binding domain that specifically binds to MASP3 or MASP2 described herein includes a monoclonal antibody that specifically binds to MASP3 or a bispecific antibody that specifically binds to both MASP3 and MASP2.

[0249] Cysteine ​​engineered variants

[0250] In some embodiments, the TACI fusion protein described in this application requires the preparation of a cysteine-engineered antigen-binding domain specifically binding to MASP3 or MASP2, in which one or more amino acid residues are replaced by cysteine ​​residues. In some embodiments, the substituted residues appear at accessible sites of the antigen-binding domain specifically binding to MASP3 or MASP2. By replacing those residues with cysteine, an active thiol group is located at an accessible site of the antigen-binding domain specifically binding to MASP3 or MASP2, which can be used to conjugate the antigen-binding domain specifically binding to MASP3 or MASP2 to other parts, such as a pharmaceutical part or a linker-pharmaceutical part, to prepare an immunoconjugate of the antigen-binding domain specifically binding to MASP3 or MASP2 as further described in this application. The cysteine-engineered antigen-binding domain specifically binding to MASP3 or MASP2 can be prepared according to, for example, US Pat. No. 7,521,541. In some embodiments, the antigen-binding domain specifically binding to MASP3 or MASP2 described in this application comprises a monoclonal antibody specifically binding to MASP3 or a bispecific antibody specifically binding to MASP3 and MASP2.

[0251] derivative

[0252] In some embodiments, the antigen-binding domain of the TACI fusion protein involved in this application may be further modified to include other non-protein portions known in the art and readily available. Suitable portions for derivatizing the antigen-binding domain that specifically binds to MASP3 or MASP2 include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde is advantageous in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antigen-binding domains that specifically bind to MASP3 or MASP2 can vary, and if more than one polymer is attached, they can be the same or different molecules. Typically, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the need to improve the properties or function of the antigen-binding domains that specifically bind to MASP3 or MASP2, and whether the antigen-binding domains that specifically bind to MASP3 or MASP2 are used for treatment under specific conditions. In some embodiments, the antigen-binding domains that specifically bind to MASP3 or MASP2 described in this application include monoclonal antibodies that specifically bind to MASP3 or bispecific antibodies that specifically bind to both MASP3 and MASP2.

[0253] Pharmaceutical Composition

[0254] This application also provides compositions comprising any of the TACI fusion proteins described above, nucleic acids encoding TACI fusion proteins, vectors comprising nucleic acids encoding TACI fusion proteins, or host cells comprising the nucleic acids or vectors described in this application (e.g., pharmaceutical compositions, also referred to herein as formulations). In some embodiments, a pharmaceutical composition is provided comprising any of the TACI fusion proteins described in this application and a pharmaceutically acceptable vector.

[0255] Suitable formulations of TACI fusion proteins can be obtained by mixing TACI fusion proteins of the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), and prepared as lyophilized or liquid formulations. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used, including buffers such as phosphates, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzyl chloride; phenol; butanol or benzyl alcohol; alkyl esters of parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than 1) (0 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG); exemplary formulations are described in WO98 / 56418, which is expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted into high-protein-concentration formulations with suitable diluents, and the reconstituted formulations can be administered subcutaneously to the individual to be treated in this application. Cationic liposomes or liposomes can be used to deliver the TACI fusion protein of this application into cells.

[0256] In addition to the TACI fusion protein, the formulations described in this application may also contain one or more other active substances necessary for treating a specific condition, preferably substances with complementary activities and no adverse reactions with each other. For example, in addition to the TACI fusion protein, it may be necessary to further include other substances with therapeutic activity, such as other drugs used to treat autoimmune diseases. These molecules are present in a combination of amounts effective for the intended purpose. The effective amount of other active substances depends on the content of the TACI fusion protein in the formulation, the disease or condition or mode of treatment, and other factors as described above. These drugs are typically used at the same dosage and route of administration as described in this application, or at 1% to 99% of the currently used dosage.

[0257] The TACI fusion protein can also be encapsulated in microcapsules, for example, prepared by coagulation techniques and interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or crude emulsions. Sustained-release formulations can be prepared.

[0258] Sustained-release formulations of TACI fusion proteins can be prepared. Suitable examples of sustained-release formulations include solid hydrophobic polymer semi-permeable matrices containing TACI fusion proteins, which are in the form of molded articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (US Pat. No. 3,773,919), L-glutamic acid and L-glutamic acid ethyl ester copolymers, non-degradable ethylene-vinyl acetate copolymers, and degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT. TM (Injectable microspheres composed of lactic-glycolic acid copolymer and leuprolide acetate) and poly-D(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic-glycolic acid can release molecules for more than 100 days, some hydrogels can release proteins in a shorter time. When encapsulated antibodies remain in vivo for extended periods, they may denature or aggregate due to exposure to a humid environment at 37°C, potentially leading to loss of biological activity or altered immunogenicity. Appropriate strategies can be designed to stabilize TACI fusion proteins based on the corresponding mechanisms. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds through thiodisulfide exchange, stabilization can be achieved by modifying thiol residues, lyophilizing in acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0259] In some embodiments, the TACI fusion protein is formulated in a buffer containing citrate, sodium chloride, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof. In some embodiments, the TACI fusion protein is formulated in a buffer with a pH between 4 and 9.

[0260] Formulations intended for internal administration must be sterile. This can be easily achieved, for example, by applying a sterile filter membrane.

[0261] Methods of preventing or treating diseases

[0262] In some embodiments, a method is provided for preventing or treating a desired individual disease or condition, the method comprising administering to the individual an effective amount of any TACI fusion protein described in this application or a composition comprising thereof.

[0263] In some embodiments, this application also provides the use of any of the TACI fusion proteins or compositions comprising them in the preparation of medicaments for the prevention or treatment of diseases or conditions desired by an individual.

[0264] In some embodiments, the disease or condition includes autoimmune diseases, transplant-related diseases, inflammatory diseases, blood diseases, coagulation disorders, angiogenesis-dependent diseases, and / or viral infections. In some embodiments, the diseases or conditions described are selected from, for example, ischemia-reperfusion injury, atherosclerosis, nephrotic syndrome, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, acute post-infectious glomerulonephritis, cryoglobulinemia glomerulonephritis, lupus nephritis, systemic lupus erythematosus (SLE), Henoch-Schönlein purpura nephritis, IgA nephropathy, ischemic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), transplant-associated TMA, Upshaw-Schulman syndrome, arthritis, traumatic brain injury, aspiration pneumonia. Neuromyelitis optica, multiple sclerosis, amyotrophic lateral sclerosis (ALS), chronic obstructive pulmonary disease (COPD), C3 glomerulonephritis, transplant rejection, graft-versus-host disease (GVHD), sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, myasthenia gravis, Degos disease, disseminated intravascular coagulation (DIC), angiogenesis-dependent cancer, hematopoietic stem cell transplantation, cold agglutinin disease, Sjögren's syndrome, age-related macular degeneration, retinopathy, proliferative diabetes mellitus, retinopathy secondary to vitreous hemorrhage, neovascular glaucoma, corneal neovascularization, retinopathy of prematurity, and respiratory distress syndrome or pneumonia caused by coronavirus infection.

[0265] Products and reagent kits

[0266] In some embodiments of this application, an article is provided comprising a substance capable of preventing or treating a desired individual disease or condition. The article may include a container and a label or packaging instructions on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be made of various materials, such as glass or plastic. Typically, the container contains a composition capable of effectively treating the disease or condition described in this application and has a sterile port (e.g., the container may be an intravenous infusion bag or a vial with a piercing cap via a hypodermic needle). At least one active substance in the composition is an antibody or antigen-binding fragment or multispecific antibody as described in this application. The label or packaging instructions indicate the specific condition for which the composition can be used to treat. The label or packaging instructions further include instructions for administering the multispecific antibody or pharmaceutical composition to a patient. Articles and kits comprising combination therapies as described in this application are within the scope of consideration.

[0267] Instructions for use are instructions typically included in the commercial packaging of a therapeutic product, containing information regarding indications, usage, dosage, administration, contraindications, and / or warnings related to the use of these therapeutic products. In some embodiments, the instructions for use state that the composition may be used to treat a desired individual disease or condition.

[0268] In addition, the article may include a second container containing a pharmaceutically acceptable buffer solution, such as bacteriostatic water for injection (BWFI), phosphate buffer, Green's solution, or glucose solution. It may also include other materials required from a commercial and user perspective, including additional buffer solutions, diluents, filters, needles, and syringes.

[0269] Kits are also provided for various purposes, such as for the prevention or treatment of desired individual diseases or conditions. The kits of this application include one or more containers containing the TACI fusion protein or a composition containing it (or a single-dose form and / or article of manufacture), and in some embodiments, further containing another agent (e.g., the agent described in this application) and / or instructions for use consistent with any of the methods described in this application. The kit may further include a description suitable for selecting the individual to be treated. Instructions for use accompanying the kits of this application are typically written instructions on a label or packaging (e.g., paper pages included within the kit), and machine-readable instructions (e.g., instructions on a magnetic or optical storage disc) are also acceptable.

[0270] For example, in some embodiments, the kit comprises a TACI fusion protein or a composition comprising it. In some embodiments, the kit comprises: a) any of the TACI fusion proteins described in this application, and b) at least one effective amount of another agent that can enhance the effect of the TACI fusion protein (e.g., therapeutic effect, detection effect). In some embodiments, the kit comprises: a) any of the TACI fusion proteins described in this application, and b) instructions for use to administer the TACI fusion protein to an individual for the treatment of a desired individual disease or condition. In some embodiments, the kit comprises: a) any of the TACI fusion proteins described in this application, and b) at least one effective amount of another agent that can enhance the effect of the TACI fusion protein (e.g., therapeutic effect, detection effect), and c) instructions for use to administer the TACI fusion protein and other substances to an individual for the treatment of a desired individual disease or condition. The TACI fusion protein and other substances may be in separate containers or in the same container. For example, the kit may include one specific composition or two or more compositions, wherein one composition comprises a TACI fusion protein and another composition comprises another agent.

[0271] In some embodiments, the kit comprises one (or a group) of nucleic acids encoding a TACI fusion protein. In some embodiments, the kit comprises: a) one (or a group) of nucleic acids encoding a TACI fusion protein, and b) a host cell expressing the nucleic acid (or a group of nucleic acids). In some embodiments, the kit comprises: a) one (or a group) of nucleic acids encoding a TACI fusion protein, and b) instructions for use suitable for: i) expressing a TACI fusion protein in a host cell, ii) preparing a composition containing a TACI fusion protein, and iii) administering the composition containing a TACI fusion protein to an individual to prevent or treat a desired individual disease or condition. In some embodiments, the kit comprises: a) one (or a group) of nucleic acids encoding a TACI fusion protein, b) a host cell expressing the nucleic acid (or a group of nucleic acids), and c) instructions for use suitable for: i) expressing a TACI fusion protein in a host cell, ii) preparing a composition containing a TACI fusion protein, and iii) administering the composition containing a TACI fusion protein to an individual to prevent or treat a desired individual disease or condition.

[0272] The kit described in this application is packaged in a suitable form. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth flasks, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit may optionally include other components, such as buffer solutions and instruction information. This application therefore also provides articles of manufacture including vials (e.g., sealed vials), bottles, wide-mouth flasks, flexible packaging, etc.

[0273] Instructions for use regarding the TACI fusion protein composition typically include information such as dosage, dosing cycle, and route of administration. Containers may be single-dose, bulk (e.g., multi-dose packs), or subunit doses. For example, a kit may be provided containing sufficient doses of the TACI fusion protein as described in this application for long-term effective treatment of an individual, such as for one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or longer. The kit may also contain multiple unit doses of the TACI fusion protein, the pharmaceutical composition, and instructions for use, and be packaged in sufficient quantities for storage and use in pharmacies, such as hospital pharmacies and multi-functional pharmacies.

[0274] Those skilled in the art will recognize several possible embodiments within the scope and spirit of this application. The application will now be described in more detail with reference to the following non-limiting embodiments. These embodiments further illustrate the application but should not be construed as limiting its scope in any way. Detailed Implementation

[0275] In the embodiments disclosed below, the following abbreviations are used: MASP2 (Mannan-Binding Lectin Associated Serine Protease-2); MASP3 (Mannan-Binding Lectin Associated Serine Protease-3); MBL (Mannan-Binding Lectin).

[0276] Example 1: Preparation of recombinant protein

[0277] 1.1 Preparation of recombinant full-length MASP3 protein and derived peptides: cDNA encoding human MASP3 (GenBank ID: 5648), monkey MASP3 (GenBank ID: 708206), or mouse MASP3 (GenBank ID: 17174) was constructed into eukaryotic expression vectors via subcloning. cDNA encoding the CCP2-SP domain or SP domain of human MASP3 was also constructed into eukaryotic expression vectors via subcloning.

[0278] 1.2 Preparation of recombinant human MASP2 protein without autocleavage activity: First, the cDNA encoding the human MASP2 protein (synthesized by Shanghai Jereh Biotechnology Co., Ltd.) was constructed into an expression vector via subcloning. The arginine residue at position 444 of the protease domain (SP) was replaced with a glutamine residue using site-directed mutagenesis to construct a full-length MASP2 mutant without autocleavage activity, named MASP2(R444Q). Based on this mutant, the cDNA of the CCP1-CCP2 domain of human MASP2 was further subcloned to generate CCP1-CCP2-derived peptides of human MASP2.

[0279] 1.3 Preparation of recombinant human pro-factor D (pFD): The cDNA encoding human pro-factor D (GenBank ID: 1675) was constructed into a prokaryotic expression vector by subcloning.

[0280] 1.4 Preparation of recombinant human BAFF protein: To obtain high-yield and stable human BAFF antigen, the cDNA encoding human BAFF protein (Uniprot: Q9Y275-1, amino acid 134 of Ala to amino acid 285 of Leu) was first constructed into an expression vector by subcloning.

[0281] 1.5 Preparation of recombinant human APRIL protein: To obtain high-yield and stable human APRIL antigen, the cDNA encoding human April protein (Uniprot: O75888-1, amino acid Gln111-Leu250) was first constructed into an expression vector by subcloning.

[0282] A His tag and / or an Avi tag and / or an mFc2a tag and / or other tags commonly used by those skilled in the art were added to the end of the cDNA of the above proteins, wherein "Avi" represents an avidin tag. The above plasmids were then transfected into 293F cells to express the recombinant proteins.

[0283] Following the instructions for using a nickel column (Ni), a recombinant protein with a His tag was purified. Following the instructions for using a protein A chromatography column, a recombinant protein with an mFc2a tag was purified. Furthermore, following the instructions for using the biotinylated ligase B0101A (GeneCopoeia), a biotinylated human MASP3-Avi-His recombinant protein was successfully prepared and named human MASP3-Bavih.

[0284] Example 2: Screening for anti-MASP3 single-chain antibodies (scFv)

[0285] 2.1 Construction of a phage display library for anti-MASP3 scFv antibodies:

[0286] Six- to eight-week-old MASP1 / 3 gene knockout mice (purchased from Nanmo Biotechnology, NM-KO-200353) were immunized with human MASP3 CCP2-SP-His or human MASP3 SP-His protein as antigens. Serum from immunized mice was collected, and the antigen-specific IgG titer in the serum was detected by ELISA. After several rounds of immunization, a phage display library was established using mouse spleens. In short, RNA was extracted from the spleens of immunized mice, and cDNA was obtained through reverse transcription. A phage display library was then constructed using V... H and V K Specific primer amplification V H and V K The fragment, after gel recovery and purification, was ligated with V... H and V K scFv was constructed and cloned into the phage display plasmid pDAN3X. Subsequently, the plasmid was electroporated into E. coli TG1, and scFv antibody phage display library was obtained by infecting E. coli TG1 with phage.

[0287] 2.2 Screening for anti-MASP3 single-chain antibodies (scFv):

[0288] In multiple rounds of screening, two screening strategies were used to identify and enrich antigen-binding positive phages from the phage display library. Further ELISA analysis of individual positive clones yielded a series of positive scFv antibodies.

[0289] 2.3 Preparation of full-length chimeric antibodies against MASP3

[0290] The positive scFv samples enriched after multiple rounds of screening were sequenced, and V was amplified from the phage display vector pDAN3X. L and V H The plasmids expressing the light chain and the heavy chain were constructed and inserted into the eukaryotic expression vectors pTTa1-L (containing the κ constant region) and pTTa1-H4 (containing the IgG4 heavy chain constant region), respectively. The plasmids expressing the light chain and the heavy chain were co-transfected into 293F cells and cultured at 37°C, 8% CO2, and 120 rpm for 5 days. The culture medium was purified using a Protein A affinity chromatography column according to the manufacturer's instructions, and the IgG concentration was measured after ultrafiltration and concentration.

[0291] The affinity of the above-mentioned anti-MASP3 chimeric antibody for the MASP3 antigen was detected by ELISA, OD450 was measured, and a binding curve was generated using PRISM to calculate EC50. 50The affinity values ​​were used to analyze the affinity of anti-MASP3 antibodies for human, monkey, or mouse antigens. The application of the anti-MASP3 antibody OMS906 (used as a control antibody in the examples section of this application) is disclosed in Chinese patent application CN109715209A.

[0292] The results are shown in Table 11. Both anti-MASP3 antibodies M3-K1 and M3-K2 can bind to human, monkey, or mouse MASP3, and their binding activity is significantly better than that of the control antibody OMS906.

[0293] Table 11: Cross-binding ability of anti-MASP3 antibodies to MASP3 antigen

[0294] 2.4 Detection of the inhibitory activity of anti-MASP3 chimeric antibody on MASP3 enzyme activity:

[0295] MASP3 is a serine protease with the ability to hydrolyze specific amino acids, particularly those containing Ca. 2+ Under certain conditions, the amide bond of the fluorescent tripeptide substrate Boc-Val-Pro-Arg-AMC can be cleaved, causing it to hydrolyze and release AMC (7-amino-4-methylcoumarin, a highly fluorescent group). Fluorescence values ​​were detected at different durations under excitation conditions of 380 nm and emission conditions of 460 nm, and these values ​​were used to reflect the ability of the anti-MASP3 antibody to inhibit MASP3 enzyme activity.

[0296] The anti-MASP3 antibody was diluted to 2000 μg / ml with reaction buffer (containing 25 mM Tris-base, 10 mM CaCl2, and 0.15 M NaCl), followed by serial dilutions at a 1:5 ratio. Different concentrations of anti-MASP3 antibody were incubated with human MASP3-His (final concentration 0.25 μM) for 15 min at room temperature. A final concentration of 0.1 mM of the fluorescent tripeptide substrate Boc-Val-Pro-Arg-AMC (R&D system, ES011) was added to the above system, mixed, and incubated at 37°C using a microplate reader. Fluorescence intensity (RFU) was measured every 5 min under excitation light at 380 nm and emission light at 460 nm, for a total of 60 min. Inhibition curves were generated using PRISM, and the IC50 was calculated. 50 value.

[0297] The results are shown in Table 12. Anti-MASP3 antibodies M3-K1 and M3-K2 were able to inhibit the enzyme activity of MASP3, and their inhibitory activity was significantly better than that of the control antibody OMS906.

[0298] Table 12: Inhibition of MASP3 enzyme activity by anti-MASP3 chimeric antibodies

[0299] 2.5 Detection of the activity of anti-MASP3 chimeric antibody in inhibiting the alternative complement pathway:

[0300] In the alternative complement pathway, the serine protease activity of MASP3 can activate precomplementary factor D (pFD) in the blood into active complement factor D (FD). FD further cleaves complement factor B (FB) into Bb. Bb and C3b, formed by the hydrolysis of C3, form the alternative pathway C3 convertase (C3bBb). C3 convertase is one of the important central regulatory molecules in the complement system. It cleaves complement molecule C3 to form two highly pro-inflammatory molecules: anaphylatoxin C3a and the regulatory molecule C3b. C3b further combines with the alternative pathway C3 convertase (C3bBb) to form C5 convertase (C3bBb3b). C5 convertase is another important central regulatory molecule in the complement system. Its hydrolytic activity cleaves C5 to form anaphylatoxin C5a and C5b. Finally, C5b and C6-9 form the biological effector of complement—the membrane attack complex MAC (C5b-9).

[0301] Unsensitized rabbit or guinea pig erythrocytes can activate the complement alternative pathway in human serum. Once activated, complement triggers a series of protein cascade reactions, ultimately leading to rabbit cell lysis. The addition of anti-MASP3 antibody inhibits the alternative pathway-mediated hemolysis. The absorbance at 415 nm (OD415) was measured using an ELISA reader, and the anti-hemolytic activity of the anti-MASP3 antibody was calculated accordingly. Furthermore, ethylene glycol diaminotetraacetic acid (EGTA) was added to the reaction system, which reacts with calcium in plasma... 2+ Chelation, but with Mg2 + Its binding ability is very weak, so adding EGTA can block the classical pathway to eliminate its influence.

[0302] First use HBSS-Mg 2+ The anti-MASP3 antibody was diluted to 900 μg / ml in EGTA buffer (containing 5 mM MgCl2·6H2O and 10 mM EGTA), followed by serial dilutions at a 1:8 ratio. Different concentrations of anti-MASP3 antibody were incubated with depleted factor D human plasma for 15 min at room temperature, and pre-factor D prepared in Example 1 (final concentration 2.5 μg / ml) was added. Fresh rabbit or guinea pig red blood cells were washed three times with HBSS buffer (containing 0.1% gelatin) and then treated with HBSS-Mg... 2+Dilute washed red blood cells with EGTA buffer. Add the diluted red blood cells to the above system. Incubate at 37°C for 45 min, mix again, and incubate for another 20 min. After incubation, add 80 μl of pre-chilled stop buffer (containing 10 mM EDTA, 1×HBSS buffer, pH 7.4) to each well, and centrifuge at 4°C, 1200g for 5 min. Transfer 100 μl of supernatant to a 96-well plate and measure OD415. Generate an inhibition curve using PRISM and calculate IC50. 50 value.

[0303] The results are shown in Table 13. Anti-MASP3 antibodies M3-K1 and M3-K2 were able to inhibit hemolytic reactions mediated by complement alternative pathway activation, and their inhibitory activity was significantly better than that of the control antibody OMS906.

[0304] Table 13: Inhibitory hemolytic activity of anti-MASP3 chimeric antibodies

[0305] Example 3: Humanization of anti-MASP3 antibody and detection of its biological activity

[0306] Based on the typical structures of the light and heavy chain variable regions (CDRs) of the obtained mouse antibodies M3-K1 or M3-K2, the sequences of the light and heavy chain variable regions were compared with sequences in the antibody germline database to obtain highly homologous human germline templates. The CDR regions of the mouse antibodies were transplanted into the selected human germline templates to generate humanized variable regions, which were then recombined with the corresponding human IgG constant regions (preferably the IgG4 heavy chain and κ light chain). Based on the three-dimensional structure of the mouse antibodies, the embedded residues, residues that directly interact with the CDR regions, and residues that interact with V... L and V H By reversing mutations in residues that have a significant impact on the conformation of the molecules, different light and heavy chains are designed and combined to obtain a series of humanized molecules.

[0307] The biological activity of the M3-K2 humanized antibody was detected using the corresponding experimental protocol in Example 2 (such as the hemolysis assay mediated by the complement alternative pathway and affinity assay).

[0308] 3.1 Detection of the activity of anti-MASP3 humanized antibody in inhibiting the alternative complement pathway

[0309] Using the experimental method described in Example 2.5, a partially humanized antibody (reconstructed adult IgG4 form) of M3-K2 was selected as an example to detect its inhibitory activity in the complement substitution pathway-mediated hemolysis assay.

[0310] The results are shown in Figures 1A-1B. The exemplary humanized antibodies M3-K2-2, M3-K2-3, M3-K2-5, and M3-K2-6 to M3-K2-11 all inhibited hemolytic activity mediated by the complement substitution pathway, and their activity was essentially equivalent to that of the parental antibody molecules before humanization. For example, the inhibitory IC50 values ​​for M3-K2-2, M3-K2-3, and M3-K2-5 were significantly higher. 50 The affinity (nM) is approximately 1.2-1.5 nM, comparable to M3-K2. A subset of humanized antibodies was selected as an example for affinity testing, and the results showed affinity comparable to the parent molecule (data not shown).

[0311] Example 4: Screening of anti-MASP2 antibodies

[0312] Using the same method described above, MASP2 fusion protein immunization was performed, and an scFv phage library was constructed. Through screening and activity identification of antibody molecules, the anti-human MASP2 chimeric antibody M2mc51 was obtained. Subsequently, this antibody molecule was humanized, yielding a series of humanized antibody molecules. Finally, through a combination of appropriately designed biochemical and biological experiments (such as ELISA binding assays and complement molecule deposition assays), the anti-MASP2 antibody humM2mc51-1 was screened out. Based on this antibody, its V... L Some potential deamidation sites in CDR3 were modified to improve antibody stability, resulting in a series of mutant antibodies. The sequences of the aforementioned anti-MASP2 antibodies are detailed in Tables 4 and 5.

[0313] 4.1 Affinity assay of the mutated anti-MASP2 antibody

[0314] The binding activity of the mutated anti-MASP2 antibody (reconstructed adult IgG4 form) to MASP2 was detected using an ELISA assay. Absorbance at 450 nm was read using a microplate reader, and a binding curve was generated using PRISM to calculate the EC50. 50 The value was used to analyze the affinity between the mutated anti-MASP2 antibody and the antigen.

[0315] The results are shown in Table 14. Compared with the parental antibody humM2mc51-1, the binding activity of the mutated anti-MASP2 antibody humM2mc51-1-S93A to MASP2 was not significantly changed, and it still had high binding activity.

[0316] Table 14. Binding ability of anti-MASP2 antibodies to human MASP2.

[0317] 4.2 The mutated anti-MASP2 antibody inhibits the activity of the complement lectin pathway.

[0318] Mannan is a known lectin pathway activator, capable of binding to MBL and thereby activating the complement lectin pathway. Following activation, the proteolytic activity of MASP2 cleaves its complement substrate C4 into C4a and C4b, and C2 into C2a and C2b. C4b and C2a further form C3 convertase (C4b2a). C3 convertase is a crucial centrally regulatory molecule in the complement system, cleaving complement molecule C3 into two highly pro-inflammatory molecules: anaphylatoxin C3a and the regulatory molecule C3b. C3b further binds to C4b2a to form C5 convertase (C4b2a3b). C5 convertase is another important centrally regulatory molecule in the complement system; its hydrolytic activity cleaves C5 into anaphylatoxin C5a and C5b, ultimately forming the membrane attack complex MAC (C5b-9) with C6-9. C3b, C4b, and MAC (C5b-9) all contain highly reactive thioester groups. These thioester groups can form covalent bonds with hydroxyl or amino groups on macromolecules fixed at the bottom of the plastic wells via ester or amide bonds, allowing C3b, C4b, and MAC (C5b-9) to deposit at the bottom of the plastic ELISA plate. Therefore, the deposition of these molecules can be detected using the ELISA method.

[0319] Based on the above principle, ELISA was used to detect the deposition level of C4b to reflect the activity of anti-MASP2 antibody in inhibiting the cleavage of its direct substrate C4 by MASP2. First, mannan (Sigma, M7504) was diluted to 100 μg / ml using alkaline coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.8), with 100 μl coated per well, and incubated overnight at 4°C. The next day, the wells were washed three times with PBST buffer. 100 μl of blocking buffer (1% human serum albumin HSA) was added to each well, and the wells were incubated at 37°C for 1 hour. The wells were then washed three times with PBST buffer. The deposition level was then measured using Ca... 2+ Mg 2+ Normal human plasma was diluted to 2% using a buffer solution (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 mM MgCl2, 2 mM CaCl2, 0.1% gelatin, 0.05% Tween-20, pH 7.4). CaCl2 was then used to dilute the plasma. 2+ Mg 2+The anti-MASP2 antibody (reconstructed human IgG4 form) at an initial concentration of 66.67 μg / ml was diluted 1:10 with buffer. Equal volumes of the diluted plasma and serially diluted anti-MASP2 antibody were mixed (to achieve a final concentration of 1% human plasma and a maximum final concentration of 33.335 μg / ml anti-MASP2 antibody in the reaction system), and incubated at 37°C for 2 hours. The reaction was terminated by washing the wells with PBST buffer. Chicken anti-human C4b antibody (Abcam, ab48582) was added to the sample wells as a capture antibody, and goat anti-chicken antibody (Southern Biotech, 6100-05) was added as a detection antibody to detect C4b deposition in human plasma. The absorbance at 450 nm was read using a microplate reader, and a binding curve was generated using Graphpad Prism to calculate the IC50. 50 value.

[0320] The results are shown in Table 15. The mutated anti-MASP2 antibodies humM2mc51-1-S93A, humM2mc51-1-S93E, humM2mc51-1-N92Q, humM2mc51-1-N92S, and humM2mc51-1-ES93DA can effectively inhibit the lectin pathway, and their inhibitory activity is not significantly different from that of the parent antibody humM2mc51-1.

[0321] Table 15. Anti-MASP2 antibody activity against complement C4b deposition

[0322] Furthermore, the changes in charge heterogeneity and aggregates of the mutated anti-MASP2 antibodies after pressurization were evaluated using capillary isoelectric focusing and size exclusion chromatography. The results showed that the aggregate content of the exemplary anti-MASP2 antibodies humM2mc51-1-S93A, humM2mc51-1-N92Q, and humM2mc51-1-ES93DA all decreased significantly; and the charge heterogeneity was also improved to some extent after pressurization (results not shown).

[0323] Example 5: Preparation of bispecific antibodies that specifically bind to MASP2 and MASP3

[0324] In the following sequence design: V H and V L These represent the heavy chain variable region and the light chain variable region of the antibody, respectively; C H Represents the constant region of the antibody heavy chain, including C H 1. C H 2 and C H 3. Structural domain; scFv is composed of the antibody's V... H and V LAntibodies linked by linker peptides; IgG1Fc represents the Fc region of the IgG1 subclass antibody, which contains C H 2 and C H 3. Structural domains; IgG4Fc represents the Fc region of the IgG4 subclass antibody, which contains C H 2 and C H 3. Structural domains; C L This represents the constant region of the light chain.

[0325] 5.1 Construction of bispecific antibodies with Hetero H and CrossMab structures:

[0326] This application uses CrossMab C H 1-C L The structure is described in detail, and a schematic diagram of the structure is shown in Figure 2. Table 16 shows the compositional design of the exemplary Hetero H,CrossMab structure bispecific antibody used in the examples, and Table 8 shows the specific amino acid sequences of its heavy and light chains. GenScript Biotech was commissioned to synthesize the encoding genes for the light chain (L chain) and heavy chain (H chain) of the bispecific antibody, and the encoding genes were optimized for expression in 293F cells.

[0327] 5.2 Expression and purification of bispecific antibodies:

[0328] After restriction endonuclease digestion, the light and heavy chain genes encoding the bispecific antibody were subcloned into the pTTα1 vector. Recombinant plasmids expressing the light and heavy chains of the antibody were extracted and co-transfected into 293F cells. Cells were cultured at 37℃, 5% CO2, and 120 rpm for 7 days, and the culture medium was purified using a protein A affinity chromatography column. After endotoxin removal using an endotoxin removal kit (SA031K, Changzhou Tiandi Renhe Biotechnology Co., Ltd.), the concentration of IgG protein and the content of endotoxin were measured.

[0329] Table 16: Compositional Design of Heavy and Light Chains of Bispecific Antibodies with Hetero H and CrossMab Structure-Specific Binding to MASP3 and MASP2

[0330] Example 6: Activity characterization of bispecific antibodies that specifically bind to MASP2 and MASP3

[0331] The affinity of the bispecific antibody for human, monkey, and mouse MASP2 and MASP3 antigens was characterized using a Biacore 8K (GE) assay. The results showed that the bispecific antibody bound well to both human and monkey MASP2 antigens, as well as human, mouse, and monkey MASP3 antigens, with affinity constants on the order of 10. -9 M~10 -11M (Data not displayed)

[0332] 6.1 Inhibitory activity of bispecific antibodies in the lectin pathway:

[0333] Using the experimental principles and methods described in Example 4.2, the deposition of complement molecule C4b in human plasma was detected by ELISA to reflect the biological activity of bispecific antibodies in the lectin pathway.

[0334] The results are shown in Table 17. The exemplary bispecific antibodies humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1, humM2mc51-1-S93A+M3-K2-6-CrossMab-IgG1, humM2mc51-1-S93A+M3-K2-8-CrossMab-IgG1 and humM2mc51-1-S93A+M3-K2-11-CrossMab-IgG1 can all inhibit C4b deposition in the lectin pathway, and their inhibitory activity is significantly better than that of the anti-MASP2 monoclonal antibody OMS721.

[0335] Table 17: Bispecific antibody activity against complement C4b deposition

[0336] 6.2 Inhibitory activity of bispecific antibodies in alternative pathways:

[0337] Using the experimental principles and methods described in Example 2.5, a hemolysis assay was employed to detect the biological activity of the bispecific antibody in the alternative pathway.

[0338] The results are shown in Table 18. The exemplary bispecific antibodies humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1, humM2mc51-1-S93A+M3-K2-6-CrossMab-IgG1, humM2mc51-1-S93A+M3-K2-8-CrossMab-IgG1 and humM2mc51-1-S93A+M3-K2-11-CrossMab-IgG1 can all inhibit the alternative pathway-mediated hemolysis, and their inhibitory activity is also superior to that of the anti-MASP3 monoclonal antibody OMS906.

[0339] Table 18: Inhibitory activity of bispecific antibodies against hemolysis

[0340] The above results indicate that the exemplary bispecific antibodies humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1, humM2mc51-1-S93A+M3-K2-8-CrossMab-IgG1, humM2mc51-1-S93A+M3-K2-8-CrossMab-IgG1, and humM2mc51-1-S93A+M3-K2-11-CrossMab-IgG1 can simultaneously inhibit the biological activities of MASP2 and MASP3, i.e., they have the ability to simultaneously inhibit the lectin pathway and the alternative pathway.

[0341] Example 7: Construction, expression, and purification of TACI fusion protein

[0342] 7.1 Construction of the TACI fusion protein:

[0343] The aforementioned anti-MASP3 humanized antibody or a bispecific antibody that specifically binds to MASP3 and MASP2 is fused with the TACI peptide to construct a TACI fusion protein. Exemplarily, the anti-MASP3 humanized antibody is M3-K2-5, and the bispecific antibody that specifically binds to MASP3 and MASP2 is humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1. The extracellular domain of TACI or a variant thereof capable of binding BlyS and / or APRIL can be any adapted TACI peptide; exemplaryly, the TACI peptide adopts the amino acid sequence (NCBI Reference Sequence: NP_036584.1, Ser13-Leu118, as shown in SEQ ID NO: 64). Preferably, a linker peptide (e.g., SEQ ID NO: 63) can be used to link the TACI peptide to the heavy chain of the antibody. In some embodiments, the Fc region of the TACI fusion protein contains a KIH mutation. In some embodiments, the Fc region of the TACI fusion protein further includes YTE mutations (M252Y, S254T, T256E). YTE mutations involve replacing methionine (Met), serine (Ser), and threonine (Thr) at positions 252, 254, and 256 of the Fc region with tyrosine (Tyr), threonine (Thr), and glutamate (Glu), respectively, where the numbering follows the EU numbering system. This enhances the binding affinity to the FcRn receptor, thereby reducing lysosomal degradation and prolonging the drug's circulation time in vivo. The specific compositional design of the TACI fusion protein is shown in Tables 19-20. Genscript Biotech Co., Ltd. was commissioned to synthesize the coding genes for each polypeptide chain of the TACI fusion protein and optimize the coding genes for expression in 293F cells. The TACI fusion proteins shown in structural forms 3 and 4 are alternative molecules to the TACI fusion proteins shown in structural forms 1 and 2, respectively.

[0344] Since the antigen-binding domain of the bispecific antibody that specifically binds to MASP2 does not cross-link with that of mice, a corresponding alternative antibody, h16B11, was prepared to conduct in vivo experiments in a mouse model. The amino acid sequence of this fusion protein alternative molecule is shown in Table 10. The alternative antibody to the bispecific antibody humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1 is h16B11+M3-K2-5-CrossMab-IgG1. The amino acid sequence of this bispecific antibody alternative molecule is shown in Table 10. Its four polypeptide chains form the heavy chain h16B11 V. H -C L -IgG1 Fc-hole and M3-K2-5 V H -IgG1 C H-knob and light chain h16B11 V L -C H 1 and M3-K2-5 V L -C L .

[0345] Table 19: Compositional Design of Heavy and Light Chains of the Hetero H, CrossMab Bispecific Antibody-TACI Fusion Protein

[0346] Table 20: Compositional Design of Heavy and Light Chains in Monoclonal Antibody-TACI Fusion Protein

[0347] 7.2 Expression and purification of TACI fusion protein:

[0348] After restriction endonuclease digestion, the light and heavy chain genes encoding the TACI fusion protein were subcloned into the pTTα1 vector. Recombinant plasmids expressing the light and heavy chains of the antibodies were extracted and co-transfected into 293F cells. Cells were cultured at 37°C, 5% CO2, and 120 rpm for 6 days, and the culture medium was purified using a protein A affinity chromatography column. In short, the protein A column was first equilibrated with 6 column volumes of 50 mM PBS buffer (containing 0.15 M NaCl, pH 7.4) at a flow rate of 150 cm / h. The pH of the culture supernatant was adjusted to 7.4, and the sample was loaded at a flow rate of 150 cm / h. After loading, the column was reequilibrated, and finally eluted with a buffer containing 0.1 M glycine and 0.15 M sodium chloride (pH 3.2), and the eluent was collected. The protein was concentrated to 5 ml using a 30 kDa ultrafiltration tube and then further purified by gel filtration chromatography (Superdex 200 pg) to remove aggregates. The TACI fusion protein was then replaced in PBS solution, and the concentration of the TACI fusion protein was measured.

[0349] Example 8: Biological activity assay of TACI fusion protein

[0350] 8.1 Affinity of TACI fusion protein to MASP3 antigen

[0351] First, the binding activity of the TACI fusion protein to human MASP3 antigen was verified using an ELISA method. In short, human MASP3 antigen was coated onto a 96-well plate, blocked, and washed three times with PBST solution. Each TACI fusion protein sample was serially diluted and added to a 96-well plate (100 μl per well), incubated at 37°C for 1 hour. After washing with PBST solution, 100 μl of Goat Anti-Human Kappa HRP (1:5000 dilution) was added to each well, and incubated at 37°C for 1 hour. After washing with PBST solution, 100 μl of TMB was added to each well, and the reaction was incubated at room temperature in the dark. The reaction was terminated with 2M H2SO4. OD450 was measured, and a binding curve was generated using PRISM. EC50 was calculated. 50 The affinity of the TACI fusion protein to the human MASP3 antigen was analyzed using this value.

[0352] The results are shown in Table 21. Different TACI fusion proteins can bind to human MASP3.

[0353] 8.2 Affinity of TACI fusion protein to MASP2 antigen

[0354] Similarly, based on the method described in 8.1, human MASP2 antigen was coated in 96-well plates, and the binding activity of TACI fusion protein to human MASP2 antigen was detected.

[0355] The results are shown in Table 22. Different TACI fusion proteins can bind to human MASP2.

[0356] 8.3 Affinity of TACI fusion protein to APRIL and BAFF antigens, respectively

[0357] The binding activity of the TACI fusion protein with human APRIL and BAFF antigens was detected separately. In short, different TACI fusion proteins were coated onto 96-well plates, blocked, and washed three times with PBST solution. Serially diluted APRIL and BAFF antigens were added, and the plates were incubated at 37°C for 1 hour. After washing with PBST solution, 100 μl of Goat Anti-Mouse IgG-AP (1:1000 dilution) was added to each well, and the plates were incubated at 37°C for 1 hour. After washing with PBST solution, 100 μl of PNPP was added to each well, and the plates were incubated at room temperature in the dark. The OD450 was measured, and a binding curve was generated using PRISM. The EC50 value was calculated, and this was used to analyze the affinity of the antibody for human APRIL and BAFF antigens. The TACI-Te-Fc fusion protein used in the following experiments, whose TACI portion is identical to that of teltascept, was synthesized internally and used as a teltascept control. Its sequence is shown in Table 6.

[0358] The results are shown in Table 23. Different TACI fusion proteins can bind to human APRIL and BAFF.

[0359] 8.4 Detection of the inhibitory activity of TACI fusion protein on MASP3 enzyme activity:

[0360] Using the experimental principles and methods described in step 2.4, the inhibitory effects of different TACI fusion proteins on MASP3 enzyme activity were detected. The results are shown in Table 24, indicating that all different TACI fusion proteins could inhibit MASP3 enzyme activity.

[0361] 8.5 Inhibitory activity of TACI fusion protein in the lectin pathway:

[0362] Applying the experimental principle described in step 4.2, ELISA was used to detect the deposition level of MAC to reflect the biological activity of the TACI fusion protein in the lectin pathway. First, mannan (Sigma, M7504) was diluted to 100 μg / ml using alkaline coating buffer, with 100 μl coated per well, and incubated overnight at 4°C. The next day, the wells were washed three times with PBST buffer. 200 μl of blocking buffer (2% human serum albumin HSA) was added to each well, and the wells were incubated at 37°C for 1 hour. The wells were then washed three times with PBST buffer. The solution was then applied using Ca... 2+ Mg 2+ Normal human plasma was diluted to 2% using a buffer solution (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 mM MgCl2, 2 mM CaCl2, 0.1% gelatin, 0.05% Tween-20, pH 7.4). CaCl2 was then used to dilute the plasma. 2+ Mg 2+The TACI fusion protein was diluted to 1000 nM with buffer, followed by 4-fold serial dilutions. Equal volumes of diluted plasma and serially diluted TACI fusion protein were mixed and incubated at room temperature with shaking for 15 min. The incubated TACI fusion protein and plasma mixture was then added to a mannan-coated plate and incubated at 37°C for 2 h. The reaction wells were washed with PBST buffer to terminate the reaction. Rabbit-anti-C5b-9 was added to the sample wells as a capture antibody, and HRP goat anti-rabbit-IgG (H+L) was added as a detection antibody to detect MAC deposition. The absorbance at 450 nm was read using a microplate reader, and binding curves were generated using Graphpad Prism to calculate the IC50. 50 value.

[0363] The results are shown in Table 25. Different TACI fusion proteins can inhibit the activation of the complement lectin pathway.

[0364] Example 9: Biological activity experiment of TACI fusion protein: inhibition of BAFF biological activity

[0365] Inhibition of BAFF-induced reporter gene activation in cell lines: HeLa / NF-κB cells, which carry the NF-κB regulatory element of the luciferase reporter gene and stably express TACI protein on their cell surface, were transduced with a virus and named TACI / HeLa / NF-κB cells. BAFF antigen was diluted to a final concentration of 10 μg / mL. An equal volume of BAFF antigen and TACI fusion protein were mixed and incubated at 37°C for 20-30 min. After adjusting the density, TACI / HeLa / NF-κB cells were added to 96-well plates, and the mixture of BAFF antigen and TACI fusion protein was added to each well. The plates were then cultured at 37°C for 24 h. After culture, Promega ONE-Glo was applied. TM The luciferase reporter gene assay system (catalog number E6120) was used to detect and calculate the inhibition rate of reporter gene activation by the TACI fusion protein. The fusion protein TACI-Te-Fc was used as a control for teltascept.

[0366] The results are shown in Table 26. Different TACI fusion proteins can inhibit BAFF-induced reporter gene cell line activation.

[0367] Example 10: IgA Nephropathy Model

[0368] The in vivo biological activity of the TACI fusion protein was evaluated using a mouse IgA nephropathy model.

[0369] Model establishment: Mice were administered BSA by gavage once a day for 5 days a week, and CCL4 (carbon tetrachloride) and LPS (lipopolysaccharide) were injected intraperitoneally once a week for 10-14 weeks.

[0370] Administration: Along with model construction, mice in the treatment group were subcutaneously injected with equimolar concentrations of the test substance: a substitute molecule for the TACI fusion protein humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1-2-(TACI)2, or TACI-M3-K2-5-KIH, or a bispecific antibody specifically binding to MASP3 and MASP2, or telitacicept control TACI-Te-Fc, once weekly for 10-14 weeks. Mice in the model group were subcutaneously injected with an equal volume of physiological saline. The bispecific antibody humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1 was used in in vivo with the substitute antibody h16B11+M3-K2-5-CrossMab-IgG1, the amino sequence of which is shown in Table 10. The substitution molecule for humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1-2-(TACI)2 is h16B11+M3-K2-5-CrossMab-IgG1-(TACI)2.

[0371] Testing: Serum was collected to detect serum creatinine and blood urea nitrogen. Mice urine was collected to detect urinary microalbumin and creatinine. Finally, kidneys were harvested for complement deposition detection and pathological scoring.

[0372] The above experiments fully demonstrate that the TACI fusion protein prepared in this application can exert a positive and effective therapeutic effect in a mouse IgA nephropathy model and has excellent biological activity.

[0373] Example 11: SLE Nephropathy Model

[0374] The in vivo biological activity of the TACI fusion protein was evaluated using a mouse SLE model experiment.

[0375] Model Construction 1: Pristane-induced lupus nephritis model in mice. Each mouse was injected intraperitoneally with 0.5 mL of Pristane, while the control group was injected intraperitoneally with 0.5 mL of physiological saline. Mice that developed proteinuria after about six months were selected for further treatment.

[0376] Administration: Mice in the treatment group were subcutaneously injected with equimolar concentrations of the test substance: a substitute molecule for the TACI fusion protein humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1-2-(TACI)2, or TACI-M3-K2-5-KIH, or a bispecific antibody specifically binding to MASP3 and MASP2, or telitacicept control TACI-Te-Fc, once weekly for 4-8 weeks. Mice in the model group were subcutaneously injected with an equal volume of physiological saline. The bispecific antibody humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1 was used in in vivo with the substitute antibody h16B11+M3-K2-5-CrossMab-IgG1, the amino sequence of which is shown in Table 10. The substitution molecule for humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1-2-(TACI)2 is h16B11+M3-K2-5-CrossMab-IgG1-(TACI)2.

[0377] Detection: Serum samples were collected to detect anti-dsDNA, serum creatinine, and blood urea nitrogen. Mice urine was collected for the detection of urinary microalbumin and urinary creatinine. Kidneys were harvested at the end of the test, washed with 0.9% NaCl, blotted dry with filter paper, and the weight of the kidneys in each group was measured. The corresponding organ-to-body ratio was calculated.

[0378] The urinary microalbumin-to-creatinine ratio (UACR) of each group of lupus nephritis mice is shown in Figure 4. The UACR level in the treatment group treated with the TACI fusion protein humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1-2-(TACI)2 was lower than that in other treatment groups and the model group at weeks 2, 3, and 4, indicating that this exemplary TACI fusion protein molecule has a superior ability to reduce proteinuria in lupus nephritis mice compared to other treatment groups. The kidney weight ratio of each group of lupus nephritis mice is shown in Figure 5. Compared to the model group of lupus nephritis mice, the TACI fusion protein humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1-2-(TACI)2 molecule effectively reduced the elevated kidney organ-to-body weight ratio in lupus nephritis mice, with a statistically significant difference, indicating that the treatment effect of this TACI fusion protein was superior to that of other treatment groups.

[0379] Similarly, the TACI fusion protein TACI-M3-K2-5-KIH, as an example, showed superior ability to reduce proteinuria in lupus nephritis mice compared to other treatment groups in this lupus nephritis model (results not shown).

[0380] Model Construction 2: R848-induced mouse lupus nephritis model

[0381] Eight-week-old Balb / c mice were treated with 100 μg of R848 (Resiquimod) three times a week for seven consecutive weeks to induce lupus nephritis. During the modeling process, the treatment groups were simultaneously injected subcutaneously with alternative molecules of the TACI fusion protein humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1-2-(TACI)2, or TACI-M3-K2-5-KIH, or anti-MASP3 monoclonal antibody M3-K2-5, or telitacicept control TACI-Te-Fc, or a combination of anti-MASP3 monoclonal antibody M3-K2-5 and telitacicept control TACI-Te-Fc. All treatment groups received the drug 4 hours before the first modeling session of the week, at a dose of 15 mg / kg, once a week for seven consecutive weeks. During the experiment, the growth and weight of the mice were observed regularly. Surrounding blood and urine were collected from the mice for testing of indicators such as urinary creatinine, urinary microalbumin, and blood urea nitrogen. At the end of the 7th week after drug administration, the weight of each mouse was measured, and then the mice were euthanized. The spleen and kidneys were removed, washed with 0.9% NaCl, blotted dry with filter paper, and the weight of the spleen and kidneys in each group was measured. The corresponding organ-to-body ratio was calculated.

[0382] The changes in serum urea levels in lupus nephritis mice are shown in Figure 6. The TACI fusion protein TACI-M3-K2-5-KIH effectively reduced the elevated serum urea (URE) levels in lupus nephritis mice and improved renal function in systemic lupus erythematosus (SLE) mice. Compared with other treatment groups, the TACI fusion protein TACI-M3-K2-5-KIH treatment group showed the best effect.

[0383] The urinary microalbumin-to-creatinine ratio (UACR) of each group of lupus nephritis mice is shown in Figure 7. The UACR level in the TACI fusion protein TACI-M3-K2-5-KIH treatment group showed a significant decreasing trend, indicating that this molecule has the ability to reduce proteinuria in lupus nephritis mice. Furthermore, compared with other treatment groups, the UACR of the TACI fusion protein TACI-M3-K2-5-KIH treatment group was significantly lower than that of the anti-MASP3 monoclonal antibody M3-K2-5 treatment group and the TACI-Te-Fc treatment group receiving both anti-MASP3 monoclonal antibody M3-K2-5 and teltasicipeptide, demonstrating its superior ability to improve renal function in mice with lupus nephritis.

[0384] Similarly, an alternative molecule to the TACI fusion protein humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1-2-(TACI)2, as exemplarily demonstrated, effectively reduced the level of UACR in lupus nephritis mice in this lupus nephritis model (results not shown).

[0385] The kidney-to-body weight ratios of the lupus nephritis mice in each group are shown in Figure 8A, and the spleen-to-body weight ratios are shown in Figure 8B. These results indicate that the kidney-to-body weight ratio and spleen-to-body weight ratio were significantly increased in R848-induced lupus nephritis mice. The TACI fusion protein TACI-M3-K2-5-KIH treatment group effectively reduced the elevated organ-to-body weight ratios in lupus nephritis mice, showing a statistically significant difference and demonstrating superior therapeutic efficacy compared to other treatment groups.

[0386] The above experiments fully demonstrate that the TACI fusion protein prepared in this application can exert a positive and effective therapeutic effect in a mouse SLE nephropathy model and has excellent biological activity.

[0387] Example 12: Mouse ANCA-associated vasculitis model

[0388] The in vivo biological activity of the TACI fusion protein was evaluated using a mouse ANCA-associated vasculitis model.

[0389] Model Construction: Systemic necrotizing vasculitis and crescentic glomerulonephritis were induced in mice using IgG against myeloperoxidase (MPO), the most common antigenic target of ANCA (MPA). The specific modeling steps were as follows: On Day 2, anti-MPO antibody 0.8 mg / mL was administered intravenously. Approximately 1 hour after administration of anti-MPO antibody, lipopolysaccharide (LPS) 0.05 mg / mL was administered intraperitoneally. On Day 3, anti-MPO antibody 4 mg / mL was administered subcutaneously. Approximately 1 hour after administration of anti-MPO antibody, LPS 0.05 mg / mL was administered intraperitoneally. On Day 5, anti-MPO antibody 2 mg / mL was administered subcutaneously. Approximately 1 hour after administration of anti-MPO antibody, LPS 0.05 mg / mL was administered intraperitoneally.

[0390] Administration: On Day 1 and Day 5, mice in the treatment group were intravenously injected with equimolar concentrations of the test substance: a substitute molecule for the TACI fusion protein humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1-2-(TACI)2, or TACI-M3-K2-5-KIH, or a bispecific antibody specifically binding to MASP3 and MASP2, or telitacicept control TACI-Te-Fc. Mice in the model group were subcutaneously injected with an equal volume of physiological saline. The bispecific antibody humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1 was used in in vivo with the substitute antibody h16B11+M3-K2-5-CrossMab-IgG1, the amino sequence of which is shown in Table 10. The substitution molecule for humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1-2-(TACI)2 is h16B11+M3-K2-5-CrossMab-IgG1-(TACI)2.

[0391] Detection: Mouse urine was collected to detect urinary microalbumin and urinary creatinine.

[0392] The exemplary TACI fusion protein humM2mc51-1-S93A+M3-K2-5-CrossMab-IgG1-2-(TACI)2 alternative molecular therapy group and the TACI-M3-K2-5-KIH treatment group can significantly reduce the level of proteinuria in ANCA-associated vasculitis mice, demonstrating excellent biological activity.

Claims

1. A TACI fusion protein comprising: (i) an extracellular domain of TACI or a variant thereof capable of binding BAFF and / or APRIL; and (ii) an antigen-binding domain specifically binding MASP3.

2. The fusion protein according to claim 1, wherein the antigen-binding domain that specifically binds to MASP3 comprises: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:2; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:5; LC-CDR2, which contains the amino acid sequence SEQ ID NO:6; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:

8.

3. The fusion protein according to claim 1 or 2, wherein the antigen-binding domain that specifically binds to MASP3 comprises: (i)V H It comprises the amino acid sequence SEQ ID NO:14 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:14; and V L It contains the amino acid sequence SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:20; (ii)V H It comprises the amino acid sequence SEQ ID NO:13 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:13; and V L It comprises the amino acid sequence SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:21; or (iii)V H It comprises the amino acid sequence SEQ ID NO:14 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:14; and V L It contains the amino acid sequence SEQ ID NO:22 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:

22.

4. The fusion protein according to any one of claims 1-3 further comprises an antigen-binding domain that specifically binds to MASP2.

5. The fusion protein according to claim 4, wherein the antigen-binding domain that specifically binds to MASP2 comprises: (i)V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:23; HC-CDR2, which contains the amino acid sequence SEQ ID NO:24; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:25; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:26; LC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:28; or (ii)V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:23; HC-CDR2, which contains the amino acid sequence SEQ ID NO:24; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:25; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:26; LC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:

29.

6. The fusion protein of claim 5, wherein the antigen-binding domain that specifically binds to MASP2 comprises: (i)V H It comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V L It comprises the amino acid sequence shown in SEQ ID NO:36 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:36; or (ii)V H It comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V L It contains the amino acid sequence shown in SEQ ID NO:37 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:

37.

7. The fusion protein according to any one of claims 1-6, wherein: The antigen-binding domain that specifically binds to MASP3 includes: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:2; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:5; LC-CDR2, which contains the amino acid sequence SEQ ID NO:6; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:

8. And the antigen-binding domain that specifically binds to MASP2 includes: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:23; HC-CDR2, which contains the amino acid sequence SEQ ID NO:24; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:25; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:26; LC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:

29.

8. The fusion protein according to claim 7, wherein: a) The antigen-binding domain that specifically binds to MASP3 includes: V H It comprises the amino acid sequence shown in SEQ ID NO:14 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:14; and V L It comprises the amino acid sequence shown in SEQ ID NO:20 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:20; and an antigen-binding domain specifically binding to MASP2 comprising: V H It comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V L It comprises the amino acid sequence shown in SEQ ID NO:37 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:37; or b) The antigen-binding domain that specifically binds to MASP3 includes: V H It comprises the amino acid sequence shown in SEQ ID NO:13 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:13; and V L It comprises the amino acid sequence shown in SEQ ID NO:21 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:21; and an antigen-binding domain specifically binding to MASP2 comprising: V H It comprises the amino acid sequence shown in SEQ ID NO:34 or a variant thereof, said variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:34; and V L It contains the amino acid sequence shown in SEQ ID NO:37 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence shown in SEQ ID NO:

37.

9. The fusion protein according to any one of claims 1-8, wherein the antigen-binding domain that specifically binds MASP3 or MASP2 comprises an Fc fragment.

10. The fusion protein according to claim 9, wherein the antigen-binding domain that specifically binds to MASP3 or MASP2 is a full-length IgG antibody.

11. The fusion protein according to any one of claims 1-10, wherein the extracellular domain of TACI or a variant thereof capable of binding BAFF and / or APRIL comprises the amino acid sequence SEQ ID NO:64 or a fragment or variant thereof.

12. The fusion protein according to any one of claims 1-11, comprising one, two, three or four extracellular domains of TACI or variants thereof capable of binding BAFF and / or APRIL.

13. The fusion protein according to any one of claims 1-12, wherein the extracellular domain of TACI or a variant thereof capable of binding BAFF and / or APRIL is linked to the heavy chain variable region (V) of the antigen-binding domain specifically binding MASP3 or MASP2. H ) or light chain variable region (V L The N-terminus or C-terminus of ).

14. The fusion protein according to any one of claims 1-12, wherein the extracellular domain of TACI or a variant thereof capable of binding BAFF and / or APRIL is attached to the N-terminus or C-terminus of the Fc fragment.

15. The fusion protein according to any one of claims 1-14, comprising: (i) Four polypeptide chains, two of which contain V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure, with the other two polypeptide chains containing V from the N-terminus to the C-terminus. L -C L Structure, where V H 1 and V L These are the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domains; or (ii) Four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H 1-C H The 1-Fc structure, with the other two polypeptide chains containing V from the N-terminus to the C-terminus. L -C L Structure, where V H 1 and V L These are the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domains, preferably, the Fc further contains a KIH (Knob-into-hole) mutation; or (iii) Four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H 2-C L -Fc-TACI structure, a polypeptide chain containing V from N-terminus to C-terminus L 1-C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L1 These are the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domains, preferably, the Fc further contains a KIH (Knob-into-hole) mutation; or (iv) Four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H 2-C L -Fc-TACI structure, a polypeptide chain containing V from N-terminus to C-terminus L1 -C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively; V H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domains. Preferably, the Fc further contains a KIH (Knob-into-hole) mutation; or (v) Four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H 2-C L -Fc structure, a polypeptide chain containing V from N-terminus to C-terminus. L 1-C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L1 These are the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively. H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domains. Preferably, the Fc further contains a KIH (Knob-into-hole) mutation; or (vi) Four polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc-TACI structure consists of a polypeptide chain containing V from the N-terminus to the C-terminus. H 2-C L -Fc structure, a polypeptide chain containing V from N-terminus to C-terminus. L 1-C L The structure, and a polypeptide chain containing V from the N-terminus to the C-terminus. L 2-C H 1 structure, where V H 1 and V L1 These are the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. H 2 and V L 2 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP2 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domains. Preferably, the Fc further contains a KIH (Knob-into-hole) mutation; or (vii) Three polypeptide chains, one of which contains V from the N-terminus to the C-terminus. H 1-C H The 1-Fc structure is a polypeptide chain containing V from the N-terminus to the C-terminus. L 1-C L The structure, and another polypeptide chain containing a TACI-Fc structure from the N-terminus to the C-terminus, wherein V H 1 and V L 1 represents the heavy chain variable region and light chain variable region that specifically bind to the MASP3 antigen-binding domain, respectively. Fc contains C H 2 and C H 3. Structural domain, preferably, the Fc further contains a KIH (Knob-into-hole) mutation.

16. The fusion protein of claim 15, comprising: (i) the amino acid sequence SEQ ID NO:70 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:70; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:54; or (ii) an amino acid sequence SEQ ID NO:71 or a variant thereof, the variant having at least about 80% sequence homology with amino acid sequence SEQ ID NO:71; and an amino acid sequence SEQ ID NO:67 or a variant thereof, the variant having at least about 80% sequence homology with amino acid sequence SEQ ID NO:67; and an amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with amino acid sequence SEQ ID NO:54; (iii) an amino acid sequence SEQ ID NO:66 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:66; and an amino acid sequence SEQ ID NO:52 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:52; and an amino acid sequence SEQ ID NO:67 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:67; and an amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:54; (iv) The amino acid sequence SEQ ID NO:66 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:66; and the amino acid sequence SEQ ID NO:52 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:52; and the amino acid sequence SEQ ID NO:60 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:60; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:54; (v) an amino acid sequence SEQ ID NO:71 or a variant thereof, the variant having at least about 80% sequence homology with amino acid sequence SEQ ID NO:71; and an amino acid sequence SEQ ID NO:72 or a variant thereof, the variant having at least about 80% sequence homology with amino acid sequence SEQ ID NO:72; and an amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with amino acid sequence SEQ ID NO:54; (vi) The amino acid sequence SEQ ID NO:74 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:74; and the amino acid sequence SEQ ID NO:52 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:52; and the amino acid sequence SEQ ID NO:75 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:75; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:54; or (vii) The amino acid sequence SEQ ID NO:76 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:76; and the amino acid sequence SEQ ID NO:77 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:77; and the amino acid sequence SEQ ID NO:54 or a variant thereof, the variant having at least about 80% sequence homology with the amino acid sequence SEQ ID NO:

54.

17. A nucleic acid molecule encoding a fusion protein according to any one of claims 1-16.

18. A carrier comprising the nucleic acid molecule of claim 17.

19. An isolated host cell comprising the fusion protein of any one of claims 1-16, the nucleic acid molecule of claim 17, or the vector of claim 18.

20. A method for preparing the fusion protein according to any one of claims 1-16, comprising: a) Culturing the host cells of claim 19 under conditions that enable efficient expression of the fusion protein; and b) Obtain the expressed fusion protein from the host cell.

21. A pharmaceutical composition comprising a fusion protein according to any one of claims 1-16, a nucleic acid molecule according to claim 17, a carrier according to claim 18, an isolated host cell according to claim 19, or a fusion protein prepared by the method according to claim 20, and a pharmaceutically acceptable carrier or excipient.

22. A method for preventing and / or treating a desired individual disease or symptom, the method comprising administering to the individual an effective amount of a fusion protein as described in any one of claims 1-16, a nucleic acid molecule as described in claim 17, a carrier as described in claim 18, an isolated host cell as described in claim 19, a fusion protein prepared by the method of claim 20, or a pharmaceutical composition as described in claim 21.

23. The method of claim 22, wherein the disease or condition includes autoimmune diseases, transplant-related diseases, inflammatory diseases, blood diseases, coagulation disorders, angiogenesis-dependent diseases and / or viral infectious diseases.

24. The method of claim 23, wherein the disease or condition is selected from ischemia-reperfusion injury, atherosclerosis, nephrotic syndrome, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, acute post-infectious glomerulonephritis, cryoglobulinemia glomerulonephritis, lupus nephritis, systemic lupus erythematosus (SLE), Henlein-Schönlein purpura nephritis, IgA nephropathy, ischemic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), transplant-associated TMA, Upshaw-Schulman syndrome, arthritis, traumatic brain injury, and inhalation. Pneumonia, neuromyelitis optica, multiple sclerosis, amyotrophic lateral sclerosis (ALS), chronic obstructive pulmonary disease (COPD), C3 glomerulonephropathy, transplant rejection, graft-versus-host disease (GVHD), sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, myasthenia gravis, Degos disease, disseminated intravascular coagulation (DIC), angiogenesis-dependent cancer, hematopoietic stem cell transplantation, cold agglutinin disease, Sjögren's syndrome, age-related macular degeneration, retinopathy, proliferative diabetes mellitus, retinopathy secondary to vitreous hemorrhage, neovascular glaucoma, corneal neovascularization, retinopathy of prematurity, and respiratory distress syndrome or pneumonia caused by coronavirus infection.

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