Bispecific binding proteins containing anti-BAFF antibodies and uses thereof

Abstract

The present disclosure relates to bispecific binding proteins comprising, from amino to carboxy terminus: (a) a first portion that is an anti-BAFF antibody or antigen-binding fragment thereof, and (b) a second portion that is an anti-MASP2 scFv or a truncated TACI polypeptide. Additionally, the present invention relates to nucleic acid molecules encoding such bispecific binding proteins, and vectors and host cells comprising such nucleic acid molecules. Furthermore, the present invention relates to methods for producing the bispecific binding proteins and to uses of the bispecific binding proteins in the treatment of autoimmune diseases.

Description

[Technical Field]

[0001] The present invention relates generally to bispecific binding proteins that target B-cell and / or complement-mediated pathways. In addition, the present invention relates to nucleic acid molecules encoding such bispecific binding proteins, as well as vectors and host cells comprising such nucleic acid molecules. Furthermore, the present invention relates to methods of producing the bispecific binding proteins of the invention and methods of using these bispecific binding proteins to treat autoimmune diseases. [Background technology]

[0002] Systemic lupus erythematosus (SLE) is a disabling and potentially fatal autoimmune disease in which multiple cytokines, signaling pathways, and immune cells are dysregulated.

[0003] Treatment of SLE mainly relies on corticosteroids and immunosuppressants, but these drugs have a series of unavoidable side effects, so it is crucial to explore new therapeutic targets that are more effective and have fewer side effects.

[0004] B cell-activating factor (BAFF, also known as BLyS) is a member of the TNF family and exists in two forms: membrane-associated and soluble forms (Karpusas M, Cachero T, Qian F, Boriack-Sjodin A, Mullen C, Strauch K, et al. Crystal structure of extracellular human BAFF, a TNF family member that stimulates B lymphocytes. J Mol Biol. (2002) 315:1145-54. doi: 10.1006 / jmbi.2001.5296). BAFF plays an important role in the survival and differentiation of B cells by binding to three distinct receptors: BAFF-R, TACI, and BCMA (Smulski C, Eibel H. BAFF and BAFF-Receptor in B Cell Selection and Survival. Front Immunol. (2018) 9:2285. doi: 10.3389 / fimmu.2018.02285). Belimumab is a fully humanized IgG1 monoclonal antibody that binds to soluble BAFF and inhibits its binding to its three receptors, thereby reducing naive and transient B cells.

[0005] However, monospecific antibodies such as belimumab directed against a single target (i.e., BAFF) have several limitations in clinical application. Patients may become resistant or unresponsive to monospecific antibody therapy. Research on autoimmune diseases has revealed that multiple signaling pathways are involved in the onset and progression of the disease, and immunotherapy directed against a single target is usually insufficient for the treatment of autoimmune diseases.

[0006] Multispecific binding proteins, such as bispecific binding proteins, have the ability to simultaneously specifically bind to multiple different molecules involved in different signaling pathways. Because of this advantage, the applications of multispecific binding proteins, such as bispecific binding proteins, are expanding.

[0007] However, creating bispecific binding proteins suitable for autoimmune diseases such as SLE is not an easy task, and many challenges must be overcome, including the efficacy, toxicity, applicability, and productivity of the bispecific binding proteins.

[0008] Given the difficulties associated with bispecific binding proteins suitable for autoimmune diseases such as SLE, there remains a need for new and improved molecules. Summary of the Invention

[0009] The present invention provides bispecific binding proteins designed to target two important molecules in autoimmune diseases such as SLE, which combine good efficacy and manufacturability with low toxicity and good pharmacokinetic properties.

[0010] According to a first aspect, the present invention provides a bispecific binding protein comprising, from amino to carboxy terminus, a first portion and a second portion, (a) the first portion is an anti-BAFF antibody or antigen-binding fragment thereof comprising: (i) a heavy chain variable domain (VH) comprising CDR-H1, CDR-H2, and CDR-H3; and (ii) a light chain variable domain (VL) comprising CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence NNAIN (SEQ ID NO: 6); CDR-H2 comprises the sequence GIIPMFGTAKYSQNFQG (SEQ ID NO: 7); CDR-H3 comprises the sequence SRDLLLFPHHALSP (SEQ ID NO: 8); CDR-L1 comprises the sequence QGDSLRSYYAS (SEQ ID NO: 9); CDR-L2 comprises the sequence GKNNRPS (SEQ ID NO: 10); CDR-L3 comprises the sequence SSRDSSGNHWV (SEQ ID NO: 11); Herein, CDRs are defined according to Kabat numbering; (b) the second portion is an anti-MASP2 scFv or truncated TACI polypeptide comprising (i) a heavy chain variable domain (VH) comprising CDR-H1, CDR-H2, and CDR-H3, and (ii) a light chain variable domain (VL) comprising CDR-L1, CDR-L2, and CDR-L3, CDR-H1 comprises the sequence of DYYIN (SEQ ID NO: 16); CDR-H2 comprises the sequence WIFPGSESAYHSEKFKA (SEQ ID NO: 17); CDR-H3 comprises the sequence GDRSGPFAY (SEQ ID NO: 18); CDR-L1 comprises the sequence KSSQSLLYSNGKTYLN (SEQ ID NO: 19); CDR-L2 comprises the sequence LVSKLDS (SEQ ID NO: 20); CDR-L3 comprises the sequence VQVTHFPFT (SEQ ID NO: 21); Herein, CDRs are defined according to Kabat numbering; wherein the truncated TACI polypeptide is the extracellular domain of human TACI set forth in SEQ ID NO: 24 or a fragment or variant thereof; wherein the carboxy terminus of the heavy chain of the first portion is covalently linked to the amino terminus of the second portion, providing a bispecific binding protein.

[0011] According to one aspect, a bispecific binding protein of the invention comprises two heavy chains and two light chains, wherein each heavy chain comprises, from amino terminus to carboxy terminus, a heavy chain of the first moiety, a linker peptide, and the second moiety, and each light chain is a light chain of the first moiety.

[0012] According to one embodiment, the bispecific binding protein of the invention is a bispecific anti-BAFF x anti-MASP2 antibody, wherein The anti-BAFF antibody or antigen-binding fragment thereof comprises or consists of (a) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, and (b) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, The anti-MASP2 scFv comprises or consists of (a) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 14, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to said sequence, and (b) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 15, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to said sequence, or consists of said VH and VL.

[0013] In one aspect, the anti-BAFF antibody and / or the anti-MASP2 scFv within the bispecific anti-BAFF x anti-MASP2 antibody is a chimeric, humanized, or human anti-BAFF antibody and / or anti-MASP2 scFv.

[0014] According to one aspect, the bispecific anti-BAFF x anti-MASP2 antibody comprises two heavy chains and two light chains, wherein the constant region of each light chain is derived from a human kappa or lambda light chain constant region, and the constant region of each heavy chain is derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. Preferably, each heavy chain in the bispecific anti-BAFF x anti-MASP2 antibody comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, and each light chain in the bispecific anti-BAFF x anti-MASP2 antibody comprises or consists of the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto.

[0015] According to one aspect, the bispecific anti-BAFF x anti-MASP2 antibody of the invention has one or more properties selected from the following properties: (1) Binds to human BAFF and human MASP2. The KD of binding to human BAFF, as measured by biolayer interferometry, is approximately 10 × 10 -9 Less than M, 5 x 10 -9 Less than M, 1 x 10 -9 Less than M or 5 x 10 -10 M, and the KD for binding to human MASP2 is approximately 10 × 10 -8 Less than M, 5 x 10 -8 Less than M, 1 x 10 -8 Less than M or 5 x 10 -9 It is less than M. (2) Binds to human BAFF and human MASP2, wherein the EC50 for binding to human BAFF is about 0.1 nM or less, 0.08 nM or less, 0.06 nM or less, or 0.05 nM or less, and the EC50 for binding to human MASP2 is about 1 nM or less, 0.8 nM or less, 0.6 nM or less, or 0.4 nM or less, as measured by ELISA. (3) Neutralizes BAFF activity in BCMA-expressing cells with an IC50 of about 250 nM or less, 200 nM or less, 170 nM or less, or 140 nM or less. (4) Inhibiting the activation of complement factor C4 with an IC50 of about 10 nM or less, 8 nM or less, 6 nM or less, or 4 nM or less. (5) Depleting B cells in vivo.

[0016] In one aspect, a bispecific binding protein of the invention is a fusion protein comprising an anti-BAFF antibody or antigen-binding fragment thereof and a truncated TACI polypeptide or fragment or variant thereof, wherein the anti-BAFF antibody or antigen-binding fragment thereof comprises, or consists of, (a) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, and (b) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto. Furthermore, the truncated TACI polypeptide or its fragment or variant comprises the amino acid sequence set forth in SEQ ID NO: 25 or its fragment or variant (e.g., the fragment set forth in SEQ ID NO: 26), or consists of the amino acid sequence set forth in SEQ ID NO: 25 or its fragment or variant.

[0017] In one aspect, the anti-BAFF antibody in the fusion protein is a chimeric, humanized, or human anti-BAFF antibody, and the truncated TACI polypeptide in the fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25 or a fragment or variant thereof (e.g., the fragment set forth in SEQ ID NO: 26).

[0018] According to one embodiment, the fusion protein comprises two heavy chains and two light chains, wherein the constant region of each light chain is derived from a human kappa or lambda light chain constant region and the constant region of each heavy chain is derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. Preferably, each heavy chain in the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 22, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, and each light chain in the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto.

[0019] In one embodiment, the fusion proteins of the present invention have one or more properties selected from the following properties: (1) Binds to human BAFF and human APRIL, where the KD for binding to human BAFF is approximately 1 × 10 as measured by biolayer interferometry. -12 M, and the KD of binding to human APRIL is approximately 10 × 10 -9 Less than M, 5 x 10 -9 Less than M, 1 x 10 -9 Less than M or 5 x 10 -10 It is less than M. (2) binds to human BAFF and human APRIL, wherein the EC50 for binding to human BAFF is about 0.1 nM or less, 0.08 nM or less, 0.06 nM or less, or 0.05 nM or less, and the EC50 for binding to human APRIL is about 1 nM or less, 0.8 nM or less, 0.6 nM or less, or 0.4 nM or less, as measured by ELISA. (3) Neutralizes BAFF activity in BCMA-expressing cells with an IC50 of about 50 nM or less, 35 nM or less, 20 nM or less, or 10 nM or less. (4) Depleting B cells in vivo.

[0020] In a second aspect, the invention provides polynucleotides encoding one or more polypeptide chains in the bispecific binding proteins of the invention.

[0021] In a third aspect, the invention provides a vector, preferably an expression vector, comprising a polynucleotide encoding one or more polypeptide chains in a bispecific binding protein of the invention.

[0022] In a fourth aspect, the present invention provides a host cell comprising a polynucleotide or vector of the present invention. For example, the host cell is a mammalian cell, preferably a CHO cell or a HEK293 cell. For example, the host cell is a prokaryotic cell, preferably an E. coli cell.

[0023] In a fifth aspect, the invention provides a method of producing a bispecific binding protein of the invention, the method comprising (i) culturing a host cell of the invention under conditions permissive for the production of said bispecific binding protein, and (ii) recovering said bispecific binding protein from said culture.

[0024] In a sixth aspect, the invention provides a pharmaceutical composition comprising both a bispecific binding protein of the first aspect and a nucleic acid as described above.

[0025] In a seventh aspect, the present invention provides a method for treating or preventing an autoimmune disease, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to the sixth aspect to a subject in need thereof, preferably the individual is a mammal, more preferably a human.

[0026] According to one embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), IgAN, rheumatoid arthritis (RA), neuromyelitis optica / neuromyelitis optica spectrum disorder (NOD / NMOD), multiple sclerosis (MS), neuromyelitis optica, Sjogren's syndrome, ANCA-associated vasculitis, myasthenia gravis, and Devic's disease. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a schematic diagram of the constructed bispecific antibody, designated Blm129. [Figure 2] FIG. 2 shows the purity of the bispecific antibody Blm129 by SEC-HPLC. [Figure 3] FIG. 3 is a schematic diagram of the constructed bifunctional fusion protein, designated BlmTAC. [Figure 4] FIG. 4 shows the purity of the bifunctional fusion protein BlmTAC by SEC-HPLC. [Figure 5] FIG. 5 shows the binding of the bispecific binding proteins Blm129 and BlmTAC to human BAFF by enzyme-linked immunosorbent assay (ELISA). [Figure 6] FIG. 6 shows the binding of the bispecific binding protein BlmTAC to human APRIL by ELISA. [Figure 7] FIG. 7 shows the binding of the bispecific binding protein Blm129 to human MASP2 by ELISA. [Figure 8] FIG. 8 shows simultaneous binding of the bispecific binding protein Blm129 to human BAFF and human MASP2 by ELISA. [Figure 9] Figure 9 shows that the bispecific binding proteins Blm129 and BlmTAC neutralize BAFF activity in a cell-based reporter assay, using anti-HEL-hIgG1 antibody as an isotype control. [Figure 10] FIG. 10 shows that the bispecific binding protein Blm129 inhibits the activation of complement factor C4 in a dose-dependent manner. [Figure 11] FIG. 11 shows the in vivo pharmacodynamic effects of the bispecific binding proteins Blm129 and BlmTAC on B cells. [Figure 12] FIG. 12 shows the in vivo depletion rate of B cells after Blm129 and BlmTAC administration, normalized to the control group. [Figure 13A] FIG. 13A shows the PD effect on blood B cells after intravenous administration of BlmTAC. [Figure 13B] FIG. 13B shows the PD effect on splenic B cells after intravenous administration of BlmTAC. [Figure 14A] FIG. 14A shows the depletion rate of blood B cells after BlmTAC administration normalized to the control group. [Figure 14B] FIG. 14B shows the depletion rate of splenic B cells after BlmTAC administration normalized to the control group. [Figure 15] FIG. 15 shows the mean plasma concentration-time curves of the drug in Balb / c mice. DETAILED DESCRIPTION OF THE INVENTION

[0028] I. Definition Unless otherwise defined in this disclosure, scientific and technical terms used in connection with this disclosure have the meanings commonly understood by those of ordinary skill in the art. In the event of any potential ambiguity between this disclosure and dictionary or external definitions, the definitions in this disclosure shall prevail. Furthermore, unless otherwise required by context, the singular shall include the plural and the plural shall include the singular. The use of "or" in this application means "and / or" unless otherwise indicated. Furthermore, the use of the term "including," as well as other variations such as "includes" and "included," is intended to be non-limiting. Additionally, terms such as "element" and "component" are intended to encompass both elements and components containing a single unit and elements and components containing two or more subunits, unless otherwise indicated.

[0029] The term "about" when used in conjunction with a numerical value is intended to include a range of values ​​up to 5% below that numerical value and up to 5% above that numerical value.

[0030] As used herein, the term "binding protein" refers in its broadest sense to a protein that specifically binds to a target molecule.

[0031] The term "bispecific" means that a binding protein is capable of specifically binding to at least two different target molecules. According to certain embodiments, a bispecific binding protein is capable of simultaneously binding to two different target molecules, particularly those involved in two different signaling pathways.

[0032] The terms "specific binding" or "specifically binding," with respect to the interaction of an antibody, binding protein, or peptide with a second chemical entity, mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the second chemical entity. For example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. Generally, if an antibody is specific for epitope "A," in a reaction involving labeled "A" and the antibody, the presence of other molecules containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" that binds to the antibody.

[0033] In this disclosure, the terms "first" and "second" with respect to moieties within a bispecific binding protein are used for convenience to distinguish between two or more moieties when they are present.

[0034] The BAFF receptor (BAFF-R) is an atypical member of the TNF receptor superfamily. Members of this family are typically characterized by multiple extracellular cysteine-rich domains (CRDs). These CRDs are involved in ligand binding and ligand-independent receptor aggregation from monomeric to dimeric, trimeric, or multimeric receptors. However, unlike most other TNF-R family members, BAFF-R only partially contains the CRDs involved in ligand binding and self-assembly. BAFF-R is expressed on the surface of all human peripheral B cell subsets except plasma cells and germinal center cells in the germinal center dark zone. BAFF-R binds the TNF-like molecule BAFF as its sole ligand.

[0035] "TACI" (T cell activator and calcium-modulating ligand interactor) is expressed by activated B cells, marginal zone B cells, switched memory B cells, and plasma cells. Compared with BAFF-R, TACI has a different function: on the one hand, it acts as a decoy receptor, and on the other hand, it induces immunoglobulin class switch recombination.

[0036] BCMA (B cell maturation antigen) is upregulated in activated B cells and is constitutively expressed in long-lived plasma cells, supporting their survival.

[0037] As used herein, amino acid positions in all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). This is referred to in the present disclosure as Kabat numbering. Specifically, the Kabat numbering system (see pages 647-660 of Kabat et al., 1991) is used for the light chain constant regions CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723 of Kabat et al., 1991) is used for the constant heavy chain domains (CH1, hinge, CH2, and CH3). For clarity, the latter is referred to in the present disclosure as the "Kabat EU index numbering system."

[0038] See also Kabat et al., 1991 for general information regarding human immunoglobulin light and heavy chain sequences.

[0039] An "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, by reason of its origin or derivation, is not associated with natural components with which it is associated in its natural state, or is substantially free of other proteins from the same species, or is expressed by cells of a different species, or is not naturally occurring. A chemically synthesized polypeptide or a polypeptide synthesized in a cellular system different from its natural source may be "isolated" from its naturally associated components. Proteins may also be isolated to substantially remove naturally associated components using protein purification techniques well known in the art.

[0040] The term "antibody" refers broadly to an immunoglobulin (Ig) molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, or a functional fragment, mutant, variant, or derivative thereof that retains the essential epitope-binding properties of an Ig molecule. Forms of such mutant, variant, or derivative antibodies are known in the art, and non-limiting embodiments are described below.

[0041] In a full-length antibody, each heavy chain comprises a heavy chain variable region (abbreviated as VH in this disclosure) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated as VL in this disclosure) and a light chain constant region. The light chain constant region is composed of a single domain, CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The first, second, and third CDRs of the VH domain are generally numbered CDR-H1, CDR-H2, and CDR-H3. Similarly, the first, second, and third CDRs of a VL domain are generally numbered CDR-L1, CDR-L2, CDR-L3, etc. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, IgY, etc.), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.), or subclass.

[0042] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. This region may be generated, for example, by digesting an intact antibody with papain. The Fc region may be a native-sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally contains two constant regions, a CH2 region and a CH3 region, and optionally contains a CH4 region, e.g., the Fc regions of IgM and IgE antibodies. The Fc regions of IgG, IgA, and IgD antibodies contain a hinge region, a CH2 domain, and a CH3 domain. In contrast, the Fc regions of IgM and IgE antibodies lack a hinge region and contain a CH2 domain, a CH3 domain, and a CH4 domain. Fc region mutants that change the effector functions of antibodies by substituting amino acid residues in the Fc region are known in the art (see, for example, Winter et al., U.S. Patent Nos. 5,648,260 and 5,624,821).

[0043] In the context of antibodies, the terms "antigen-binding portion," "antigen-binding fragment," and "functional fragment" are used interchangeably and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, i.e., the same antigen (e.g., BAFF) as the full-length antibody from which the portion or fragment is derived. It has been shown that the antigen-binding function of an antibody can also be exhibited by fragments of the full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include the following: (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of the VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) dAb fragment (Ward et al., Nature, 341: 544-546 (1989); PCT International Publication No. WO90 / 05144) consisting of a single variable domain, and (vi) isolated complementarity-determining regions (CDRs). Furthermore, the two domains of an Fv fragment, VL and VH, are encoded by separate genes but can be linked by a synthetic linker, allowing the VL and VH domains to be synthesized recombinantly as a single protein chain that pairs to form a monovalent molecule (commonly known as a "single-chain Fv" (scFv)). The VL and VH domains in an scFv can be arranged in any order, such as VH-linker-VL or VL-linker-VH (see, for example, Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody and the equivalent terms used above.

[0044] The constant (C) region of an immunoglobulin refers to the constant region of the heavy (C) or light (C) chain. The amino acid sequences of the constant regions of mouse and human IgG heavy and light chains are known in the art.

[0045] The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies; that is, the individual antibodies comprising the population are identical except for natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant (epitope). Furthermore, whereas a polyclonal antibody preparation typically includes several different antibodies directed against several different antigenic determinants (epitopes), each mAb targets a single antigenic determinant on the antigen. The modifier "monoclonal" does not require the antibody to be produced by any particular method.

[0046] With respect to the light chain constant region CL, heavy chain constant region CH, and Fc region of an antibody or binding protein of the invention, the term "human sequence" means a human immunoglobulin sequence or a sequence derived from a human immunoglobulin sequence. The human sequences of the present disclosure can be naturally occurring human sequences or variants thereof that contain one or more (e.g., up to 20, 15, 10) amino acid residue mutations.

[0047] The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody in which murine heavy and light chain variable regions are linked to human constant regions.

[0048] A "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences has been modified to be more "human-like," i.e., more similar to human germline variant sequences. One type of humanized antibody is a CDR-grafted antibody, in which CDR sequences from a non-human species (e.g., mouse) are introduced into human VH and VL framework sequences. A humanized antibody is an antibody, variant, derivative, analog, or fragment thereof, that immunospecifically binds to an antigen of interest and comprises framework and constant regions that have substantially the amino acid sequences of a human antibody, and complementarity-determining regions (CDRs) that have substantially the amino acid sequences of a non-human antibody. In this disclosure, the term "substantially," as used in the context of CDRs, refers to a CDR that has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody is an antibody that comprises substantially all of at least one, and usually two, variable domains (Fab, Fab', F(ab')2, Fv), with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions being human immunoglobulin consensus sequences. In certain embodiments, the humanized antibody further comprises at least a portion of an immunoglobulin (usually a human immunoglobulin) constant region (Fc). In certain embodiments, the humanized antibody comprises a light chain and at least the variable region of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In certain embodiments, the humanized antibody comprises only a humanized light chain. In certain embodiments, the humanized antibody comprises only a humanized heavy chain. In certain embodiments, the humanized antibody comprises only a humanized variable region of the light chain and / or a humanized heavy chain.

[0049] Humanized antibodies can be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. Humanized antibodies may contain sequences from more than one class or isotype. Specific constant regions may also be selected to optimize desired actions and functions using techniques well known in the art.

[0050] The framework and CDR regions of a humanized antibody need not exactly match the parental sequences. For example, the donor antibody CDR or acceptor framework may be mutated by introducing at least one amino acid substitution, insertion, and / or deletion so that the CDR or framework residue at that site does not match either the donor antibody or the consensus framework. However, according to certain exemplary embodiments, such mutations will not be extensive. Typically, at least 80%, at least 85%, at least 90%, or at least 95% of the residues in a humanized antibody correspond to those in the parental FR and CDR sequences. Back mutations are often used at specific framework positions to restore the amino acid at the corresponding position in the donor antibody to preserve a particular loop structure or to properly orient the CDR sequences for contacting the target antigen.

[0051] The term "CDR" refers to the complementarity-determining region within an antibody variable region sequence. Each heavy and light chain variable region contains three CDRs, designated CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In this disclosure, the term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently in different systems. The system described by Kabat (National Institutes of Health, Bethesda, Maryland (1987) and (1991)) not only provides an unambiguous residue numbering system that can be applied to any antibody variable region, but also provides the precise residue boundaries that define the three CDRs.

[0052] The art-recognized term "Kabat numbering," in reference to the CDRs of antibody heavy and light chains, refers to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the variable regions of the heavy and light chains of an antibody or its antigen-binding site. See Kabat et al., Ann. NY Acad. Sci., 190: 382-391 (1971); and Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

[0053] Over the past 20 years, the growth and analysis of extensive public databases of amino acid sequences of variable heavy and light chain regions has led to an understanding of the typical boundaries between framework regions (FRs) and CDR sequences within variable region sequences, allowing those skilled in the art to accurately determine CDRs according to Kabat numbering, Chothia numbering, or other systems. See Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Kontermann and Duebel, eds., Antibody Engineering (Springer-Verlag, Berlin, 2001), chapter 31, pages 432-433.

[0054] In this disclosure, "k onThe term "k" (also known as "K on" or "k on"), as known in the art, refers to the binding rate constant at which a binding protein (e.g., an antibody) binds to an antigen to form a complex, such as an antibody / antigen complex. "k on" is also synonymous with the term "binding rate constant" or "k on", and these terms are used interchangeably in the present disclosure. This value represents the rate at which an antibody binds to its target antigen, or the rate of complex formation between an antibody and an antigen, and is expressed by the following formula: Antibody (“Ab”) + Antigen (“Ag”) → Ab-Ag.

[0055] In this disclosure, "k off The term "off-rate constant" (also known as "Koff" or "koff") refers to the off-rate constant, or "dissociation rate constant," for the dissociation of a binding protein (e.g., an antibody) from a binding complex (e.g., an antibody / antigen complex). This value indicates the rate at which an antibody dissociates from its target antigen, or the rate at which an Ab-Ag complex dissociates into free antibody and antigen over time, as shown in the following equation: Ab + Ag → Ab-Ag.

[0056] In this disclosure, the term "KD" (also "Kd") refers to the "equilibrium dissociation constant," which may be obtained by titration measurement at equilibrium or by dividing the dissociation rate constant (koff) by the association rate constant (k). off ), equilibrium dissociation constant (K D ) is used to express the binding affinity of an antibody to an antigen. Methods for measuring the association and dissociation rate constants are well known in the art. Fluorescence-based techniques are highly sensitive and can examine samples at equilibrium in physiological buffers. WAVE system (Grating Coupling Interferometry, GCI) assay (Creoptix AG, Switzerland), BIAcore (登録商標) Other experimental approaches and equipment, such as the Biomolecular Interaction Analysis (BIAcore) assay (BIAcore International AB, Uppsala, Sweden), can also be used. (登録商標)Biolayer interferometry (BLI) using the RED96 system (Pall Forte Bio LLC) is another affinity assay. (登録商標) A Kinetic Exclusion Assay (Kinetic Exclusion Assay) can also be used.

[0057] "Isolated nucleic acid" refers to a polynucleotide (e.g., of genomic, cDNA, synthetic origin, or a combination thereof) that is not associated by human intervention with all or part of a polynucleotide found in nature, or that is operably associated with a polynucleotide with which it is not associated in nature, or that is not found in nature as part of a larger sequence.

[0058] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as plasmids are the most commonly used term for vectors. However, the present disclosure is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0059] The term "operably linked" refers to a juxtaposition where the described components are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operatively linked" sequences include both expression control sequences contiguous with a gene of interest and expression control sequences that regulate the gene of interest in trans or at a distance. In this disclosure, the term "expression control sequence" refers to polynucleotide sequences necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, if necessary, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism. In prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences. In eukaryotes, generally, such control sequences include promoters and transcription termination sequences. As used herein, the term "control sequences" is intended to include components whose presence is essential for expression and processing, and also to include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0060] In this disclosure, "transformation" refers to any process by which exogenous DNA enters a host cell. Transformation can occur under natural or artificial conditions using a variety of methods well known in the art. Transformation can rely on any known method for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. The method is selected based on the host cell to be transformed and includes, but is not limited to, transfection, viral infection, electroporation, lipofection, particle bombardment, and the like. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replicating either as an autonomously replicating plasmid or as part of the host chromosome. Also included are cells that transiently express the inserted DNA or RNA for a period of time.

[0061] The term "recombinant host cell" (or simply "host cell") refers to a cell into which exogenous DNA has been introduced. In one embodiment, the host cell contains two or more (e.g., a plurality) nucleic acids encoding an antibody, such as the host cells described in U.S. Pat. No. 7,262,028. Such terms refer not only to the particular subject cell but also to the progeny of such a cell. Because certain variations may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" in this disclosure. In one embodiment, host cells include prokaryotic and eukaryotic cells selected from all kingdoms of life. In another embodiment, eukaryotic cells include protist, fungal, plant, and animal cells. In another embodiment, host cells include, but are not limited to, the prokaryotic cell line Escherichia coli, the mammalian cell lines CHO, HEK293, COS, NS0, SP2, and PER.C6, the insect cell line Sf9, and the fungal cell Saccharomyces cerevisiae.

[0062] In this disclosure, an "effective amount" refers to an amount of a therapeutic agent sufficient to reduce or ameliorate the severity and / or duration of a disease or one or more symptoms thereof; prevent the progression of a disease; cause regression of a disease; prevent the recurrence, onset, or progression of one or more symptoms associated with a disease; detect a disease; or enhance or improve the prophylactic or therapeutic effect of another treatment (e.g., a prophylactic or therapeutic agent).

[0063] Antibodies, functional fragments thereof, and binding proteins of the present invention can be purified (depending on the intended use) using one or more of the various methods and materials available to those skilled in the art for purifying antibodies and binding proteins. These methods and materials include affinity chromatography (e.g., using resins, particles, or membranes coupled with Protein A, Protein G, Protein L, or specific antibody ligands, functional fragments thereof, or binding proteins), ion exchange chromatography (e.g., using ion exchange particles or membranes), hydrophobic interaction chromatography ("HIC"; e.g., using hydrophobic particles or membranes), ultrafiltration, nanofiltration, diafiltration, size exclusion chromatography ("SEC"), low pH treatment (which inactivates contaminating viruses), and combinations thereof, which can be used to achieve purity acceptable for the intended use. A non-limiting example of a low pH treatment that inactivates contaminating viruses includes lowering the pH of a solution or suspension containing an antibody, functional fragment thereof, or binding protein of the present invention to 3.5 using 0.5 M phosphoric acid at 18°C ​​to 25°C for 60 to 70 minutes.

[0064] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture, and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or methods commonly practiced in the art or described in this disclosure. The techniques and procedures described above can generally be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and described herein. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0065] "Individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domestic animals (such as cattle, sheep, cats, dogs, and horses), primates (such as humans and non-human primates, such as monkeys), rabbits, and rodents (such as mice and rats). In particular, an individual or subject is a human.

[0066] A "pharmaceutical composition" refers to a formulation that contains an active ingredient in a form that effectively exerts its biological activity and does not contain any ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0067] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, preservatives, etc.

[0068] In this disclosure, "treatment" (and grammatical variations such as "treat" and "treating") refers to a clinical intervention that attempts to alter the natural history of a disease in the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of a disease, slowing the rate of disease progression, improving or mitigating the disease state, achieving remission, or improving prognosis. In one aspect, the bispecific binding proteins of the invention are used to delay the onset of a disease or slow the progression of a disease.

[0069] II. BAFF×MASP2 Bispecific Binding Protein The pathogenic role of B cells in autoimmunity includes the production of pathogenic autoantibodies and the regulation of immune responses through the production of cytokines and chemokines.

[0070] B cell-activating factor (BAFF), also known as B lymphocyte-stimulating factor (BLyS), plays an important role in the differentiation, maturation, and class switching of B cells. BAFF binds to three receptors: BAFF-R, TACI, and BCMA. Overexpression of BAFF promotes the survival and differentiation of B lymphocytes into Ig-producing plasma cells, and is involved in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE).

[0071] Belimumab is a fully humanized immunoglobulin G1 lambda (IgG1λ) monoclonal antibody (mAb) that binds to soluble BAFF and inhibits its binding to BAFF-R, TACI, and BCMA, thereby depleting naive and transient B cells. Belimumab is the only biologic approved for SLE and the first of a class of drugs known as B-lymphocyte stimulatory factor-specific inhibitors.

[0072] Autoimmune diseases are also recognized as conditions in which autoantibodies are expressed and fixed to autoantigens, leading to complement activation and consequent inflammation and tissue damage. Complement mediates the deposition of immune complexes, which further contributes to the involvement and damage of the deposition site. Blocking complement-mediated pathways and reducing the immune response is one way to reduce organ involvement in SLE (Trouw L, Pickering M, Blom A. The complement system as a potential therapeutic target in rheumatic disease. Nat Rev Rheumatol. (2017) 13:538-47. doi: 10.1038 / nrrheum.2017.125).

[0073] It is now widely accepted that the complement system is activated by three distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is typically triggered by a complex consisting of a host antibody bound to a foreign particle (i.e., an antigen), thus requiring prior exposure to the antigen for a specific antibody response to occur. Because activation of the classical pathway depends on a prior adaptive immune response by the host, the classical pathway is part of the adaptive immune system. In contrast, the lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune system.

[0074] In the lectin pathway, human mannan-binding lectin (MBL) interacts specifically and with high affinity through its collagen-like domain with unique C1r / C1s-like serine proteases called MBL-associated serine proteases (MASPs). To date, three types of MASPs have been reported: MASP-1, MASP-2, and MASP-3. However, it has been demonstrated that the MBL-MASP-2 complex alone is sufficient for complement activation (Vorup-Jensen et al., J. Immunol. 7(55:2093-2100, (2000)). Furthermore, only MASP-2 cleaved C2 and C4 at high rates (Ambrus et al., J. Immunol. 770: 1374-1382, (2003)). Therefore, MASP-2 is a protease that activates C4 and C2 to generate the C3 convertase, C4b2a, and is a potential drug target.

[0075] The MASP-2 inhibitor antibody (mAb129C10) invented by Transcenta has the effect of inhibiting MASP-2-dependent complement activation.

[0076] According to one aspect, the bispecific binding protein of the invention is capable of, on the one hand, specifically binding to human BAFF, i.e. inhibiting the binding of BAFF to its three receptors BAFF-R, TACI and BCMA, and, on the other hand, specifically binding to human MASP-2, i.e. inhibiting complement activation.

[0077] According to one aspect, the BAFF×MASP2 bispecific binding protein of the present application comprises: a) a first antigen-binding site that specifically binds to BAFF, and b) A second antigen-binding site that specifically binds to MASP2.

[0078] According to one embodiment, the bispecific binding protein described herein comprises six CDR pairs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any anti-BAFF antibody or antigen-binding fragment thereof, which form the BAFF-binding site of the bispecific binding protein. According to some further embodiments, the bispecific binding protein described herein comprises a VH / VL pair derived from any anti-BAFF antibody or antigen-binding fragment thereof, which form the BAFF-binding site of the bispecific binding protein.

[0079] According to one aspect, the bispecific binding protein described herein further comprises six CDR pairs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any anti-MASP2 antibody or antigen-binding fragment thereof, which form the MASP2-binding site of the bispecific binding protein. According to some further aspects, the bispecific binding protein described herein comprises a VH / VL pair derived from any anti-MASP2 antibody or antigen-binding fragment thereof, which form the MASP2-binding site of the bispecific binding protein.

[0080] In one aspect, the BAFF binding site and the MASP2 binding site of the bispecific BAFF / MASP2 binding protein of the present application are humanized and comprise humanized VH / VL sequences.

[0081] In one embodiment, the BAFF×MASP2 bispecific binding protein of the present application has the format shown in Figure 1, where the Fab fragment of the bispecific binding protein forms a first antigen-binding site that specifically binds to BAFF, and the scFv domain of the bispecific binding protein forms a second antigen-binding site that specifically binds to MASP2.

[0082] According to one embodiment, the bispecific binding protein of the present invention has a linker between the anti-BAFF monoclonal antibody (mAb) portion and the anti-MASP2 scFv portion. According to one embodiment, the anti-MASP2 scFv portion of the bispecific binding protein of the present invention comprises a pair of disulfide bonds between positions 44 and 100.

[0083] In one aspect, a bispecific binding protein of the invention has the ability to bind to BAFF and has an EC50, which reflects its binding strength, of about 0.1 nM or less, 0.08 nM or less, 0.06 nM or less, or 0.05 nM or less, as measured by ELISA.

[0084] In one aspect, the bispecific binding protein of the present invention has the ability to bind to MASP2 and has an EC50, which reflects its binding strength, of about 1 nM or less, 0.8 nM or less, 0.6 nM or less, or 0.4 nM or less, as measured by ELISA.

[0085] According to one embodiment, the bispecific binding protein of the present invention has the ability to bind to BAFF and has a KD, which reflects its binding strength, of about 10×10 as measured by biolayer interferometry. -9 Less than M, 5 x 10 -9 Less than M, 1 x 10 -9 Less than M or 5 x 10 -10 It is less than M.

[0086] According to one embodiment, the bispecific binding protein of the present invention has the ability to bind to MASP2 and has a KD, which reflects its binding strength, of about 10 x 10 as measured by biolayer interferometry. -8 Less than M, 5 x 10 -8 Less than M, 1 x 10 -8 Less than M or 5 x 10 -9 It is less than M.

[0087] In one aspect, the bispecific binding protein of the invention has the ability to bind to both BAFF and MASP2.

[0088] In one embodiment, the bispecific binding proteins of the invention, upon contact with the cell surface of a cell expressing BCMA, block the binding of BAFF to its receptor BCMA, inhibiting intracellular signaling. Inhibition of intracellular signaling can be confirmed by detecting levels of NF-κB. NF-κB can be detected, for example, using a reporter-based approach as described in Example 6.

[0089] In one aspect, the bispecific binding proteins of the invention have the ability to reduce the amount of NF-κB by more than 1-fold, for example >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, >10-fold, >20-fold, >30-fold, >40-fold, >50-fold, >60-fold, >70-fold, >80-fold, >90-fold, or >100-fold, in an equivalent assay, compared to the level of NF-κB detected at the cell surface following culture of cells expressing BCMA in the absence of the bispecific binding protein of the invention or in the presence of a control molecule (e.g., an isotype control).

[0090] According to certain aspects, the bispecific binding proteins of the invention have an IC50 of about 10 nM or less, 8 nM or less, 6 nM or less, or 4 nM or less for their ability to inhibit the activation of complement factor C4 as measured by ELISA.

[0091] In one aspect, the bispecific binding proteins of the invention are capable of depleting B cells in vivo, in particular the bispecific binding proteins of the invention are capable of depleting B cells that express CD19 and CD45, as well as depleting B cells that express CD19 and CD21.

[0092] In one aspect, a bispecific binding protein of the invention has the ability to reduce the number of B cells expressing CD19 and CD45 by more than 1-fold, e.g., >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, >10-fold, >20-fold, >30-fold, >40-fold, >50-fold, >60-fold, >70-fold, >80-fold, >90-fold, or >100-fold, compared to the number of B cells expressing CD19 and CD45 detected in an equivalent assay following administration of a bispecific binding protein of the invention or a control molecule (e.g., saline) in vivo.

[0093] III. BAFF x APRIL Bispecific Binding Protein The present invention further provides a BAFF / APRIL bispecific binding protein having the structure shown in Figure 3 and capable of binding to both BAFF and APRIL. As a dual-targeting protein, such a protein can simultaneously inhibit two cytokines, BAFF and APRIL, thereby more effectively suppressing immune responses and achieving the goal of treating autoimmune diseases.

[0094] B-cell activating factor (BAFF) is a member of the TNF family and binds to the receptors BAFF-R, TACI, and BCMA.

[0095] The proliferation-inducing ligand (APRIL) is also a member of the TNF family, has high homology to BAFF, and binds to receptors called TACI and BCMA.

[0096] BAFF and APRIL are cytokines expressed by antigen-presenting cells and play important roles in B lymphocyte differentiation and the pathogenesis of immune-mediated diseases.

[0097] In one embodiment, the BAFF×APRIL bispecific binding protein of the present application comprises: a) a first antigen-binding site that specifically binds to BAFF; and b) as a second portion, a truncated TACI polypeptide that specifically binds to the ligands BAFF and APRIL Includes:

[0098] According to one embodiment, the bispecific binding protein described herein comprises six CDR pairs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any anti-BAFF antibody or antigen-binding fragment thereof, which form the BAFF-binding site of the bispecific binding protein. According to some further embodiments, the bispecific binding protein described herein comprises a VH / VL pair derived from any anti-BAFF antibody or antigen-binding fragment thereof, which form the BAFF-binding site of the bispecific binding protein.

[0099] In one embodiment, the bispecific binding protein described herein further comprises a second portion derived from a truncated TACI polypeptide (TACI, Uniprot ID: O14836), wherein the second portion specifically binds to the TACI ligands BAFF and APRIL. In some further embodiments, the bispecific binding protein described herein comprises the amino acid sequence set forth in SEQ ID NO: 25, or a fragment or variant thereof, wherein the fragment or variant specifically binds to both BAFF and APRIL.

[0100] In one embodiment, the BAFF×APRIL bispecific binding protein of the present application has the format shown in Figure 3, where the Fab fragment of the bispecific binding protein forms a first antigen binding site that specifically binds BAFF, and the truncated TACI polypeptide of the bispecific binding protein forms a trap that specifically binds both BAFF and APRIL.

[0101] In some embodiments, the bispecific binding proteins of the invention comprise a linker between the anti-BAFF monoclonal antibody (mAb) portion and the truncated TACI polypeptide. In one embodiment, the truncated TACI polypeptide in the bispecific binding proteins of the invention comprises the second CRD domain (69 aa-108 aa) of TACI.

[0102] In one aspect, a bispecific binding protein of the invention has the ability to bind to BAFF and has an EC50, which reflects its binding strength, of about 0.1 nM or less, 0.08 nM or less, 0.06 nM or less, or 0.05 nM or less, as measured by ELISA.

[0103] According to one aspect, a bispecific binding protein of the invention has the ability to bind to APRIL, wherein the EC50, which reflects its binding strength, is about 1 nM or less, 0.8 nM or less, 0.6 nM or less, or 0.4 nM or less, as measured by ELISA.

[0104] According to one embodiment, the bispecific binding protein of the present invention has the ability to bind to BAFF and has a KD, which reflects its binding strength, of about 1 x 10 as measured by biolayer interferometry. -12 It is less than M.

[0105] According to one embodiment, the bispecific binding protein of the present invention has the ability to bind to APRIL, and wherein the KD, which reflects its binding strength, is about 10 x 10 as measured by biolayer interferometry. -9 Less than M, 5 x 10 -9 Less than M, 1 x 10 -9 Less than M or 5 x 10 -10 It is less than M.

[0106] In one embodiment, the bispecific binding protein of the invention is capable of binding to both BAFF and APRIL.

[0107] In one embodiment, the bispecific binding proteins of the invention, upon contact with the cell surface of a cell expressing BCMA, inhibit the binding of BAFF to its receptor BCMA and inhibit intracellular signaling. Inhibition of intracellular signaling can be detected by measuring levels of NF-κB. NF-κB can be detected, for example, by using a reporter-based method as described in Example 6.

[0108] According to one aspect, the ability of a bispecific binding protein of the invention to reduce the amount of NF-κB is by more than 1-fold, e.g., >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, >10-fold, >20-fold, >30-fold, >40-fold, >50-fold, >60-fold, >70-fold, >80-fold, >90-fold, or >100-fold, compared to the level of NF-κB detected at the cell surface following culturing of cells expressing BCMA in an equivalent assay in the absence of the bispecific binding protein of the invention or in the presence of a control molecule (e.g., an isotype control).

[0109] In one aspect, the bispecific binding proteins of the invention have the ability to deplete B cells in vivo, in particular, the bispecific binding proteins of the invention have the ability to reduce B cells expressing CD19 and CD45, and B cells expressing CD19 and CD21.

[0110] In one aspect, the ability of a bispecific binding protein of the invention to reduce the number of B cells expressing CD19 and CD45 is more than 1-fold, such as >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, >10-fold, >20-fold, >30-fold, >40-fold, >50-fold, >60-fold, >70-fold, >80-fold, >90-fold, or >100-fold, compared to the number of B cells expressing CD19 and CD45 detected in vivo in an equivalent assay following administration of a bispecific binding protein of the invention or a control molecule (e.g., saline).

[0111] IV. Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising a bispecific binding protein of the invention (i.e., a primary active ingredient) and a pharmaceutically acceptable carrier.

[0112] The pharmaceutical compositions of the present invention may further comprise at least one additional active ingredient, including, but not limited to, prophylactic and / or therapeutic agents, detection agents, etc., in certain embodiments.

[0113] In one embodiment, such pharmaceutical compositions comprise one or more additional prophylactic or therapeutic agents, i.e., agents other than an antibody or binding protein of the invention, for treating or ameliorating a disease. In one embodiment, such additional prophylactic or therapeutic agents are agents known to be useful for, or have been used, or are currently being used to prevent, treat, manage, or ameliorate a disorder or one or more symptoms thereof.

[0114] Pharmaceutical compositions comprising proteins of the invention are used, but are not limited to, in the diagnosis, detection, or monitoring of disease, the treatment, management, or amelioration of disease or one or more symptoms thereof, and / or research. In certain embodiments, such compositions may further comprise a carrier, diluent, or excipient. An excipient is generally a compound, or combination of two or more compounds, other than the primary active ingredient (i.e., other than the bispecific binding protein of the invention), that imparts desired properties to the composition.

[0115] V. Nucleic Acids, Vectors, and Host Cells In yet another aspect, the present disclosure provides isolated nucleic acids encoding one or more amino acid sequences of the bispecific binding proteins of the present invention. Such nucleic acids can be inserted into vectors to perform various genetic analyses or to express, characterize, or improve one or more properties of the antibodies or binding proteins described in this disclosure. The vectors comprise one or more nucleic acid molecules encoding one or more amino acid sequences of the bispecific binding proteins of the present invention, operably linked to appropriate transcription and / or translation sequences that enable expression of the bispecific binding protein in a particular host cell carrying the vector. In the present disclosure, examples of vectors for cloning or expressing nucleic acids encoding the amino acid sequences of binding proteins include, but are not limited to, pcDNA and its derivatives.

[0116] The present invention also provides host cells that express or are capable of expressing vectors containing nucleic acids encoding one or more amino acid sequences of the bispecific binding proteins of the present invention. Host cells useful in the present invention can be prokaryotic or eukaryotic. An example of a prokaryotic host cell is Escherichia coli. Eukaryotic cells useful as host cells of the present invention include protist cells, animal cells, plant cells, and fungal cells. An example of a fungal cell is a yeast cell, including Saccharomyces cerevisiae. Examples of animal cells useful as host cells of the present invention include, but are not limited to, mammalian cells, avian cells, and insect cells. Examples of mammalian cells include, but are not limited to, CHO cells, HEK cells, and COS cells.

[0117] VI. Manufacturing method According to another aspect, the invention provides a method of producing a bispecific binding protein of the invention, the method comprising culturing a host cell comprising an expression vector encoding the bispecific binding protein in a culture medium under conditions sufficient to cause the host cell to express the bispecific binding protein of the invention.

[0118] Bispecific binding proteins produced by the methods described in this disclosure can be isolated and used in the various compositions and methods described in this disclosure.

[0119] VII. Treatment Methods and Medical Uses According to one aspect, the present invention provides a method of treating an autoimmune disease, including diseases or conditions in which autoantibodies damage self-tissues, in a subject in need thereof, comprising administering to the subject a bispecific binding protein as described in this disclosure.

[0120] According to certain aspects, autoimmune diseases treated by the methods and bispecific binding proteins disclosed herein include systemic lupus erythematosus (SLE), IgAN, rheumatoid arthritis (RA), neuromyelitis optica / neuromyelitis optica spectrum disorder (NOD / NMOD), multiple sclerosis (MS), neuromyelitis optica, Sjogren's syndrome, ANCA-associated vasculitis, myasthenia gravis, Devic's disease, and the like.

[0121] In one embodiment, the autoimmune disease to be treated is SLE, which is resistant or refractory to conventional immunosuppressive therapy with compounds such as IFN-β-1a, IFN-β-1b, anti-CD52 antibodies (alemtuzumab, alemtuzumab), natalizumab, or anti-CD20 agents (rituximab, ocrelizumab, ofatumumab).

[0122] Therapeutic methods described herein can further comprise administering to a subject in need thereof an additional active ingredient, suitably present in combination with a bispecific binding protein described herein, for therapeutic purposes. In therapeutic methods of the present invention, the additional active ingredient can be formulated into a composition comprising a bispecific binding protein of the present invention, and the composition can be administered to a subject in need of treatment. In another aspect, therapeutic methods of the present invention can comprise administering to a subject in need of treatment a bispecific binding protein described herein, and administering the additional active ingredient to the subject before, simultaneously with, or after administering to the subject the bispecific binding protein of the present invention. [Example]

[0123] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0124] Example 1: Construction and expression of anti-BAFF / anti-MASP2 bispecific antibodies A bispecific antibody designated Blm129 targeting both B-cell activating factor (BAFF) and MBL-associated serine protease 2 (MASP2) was purified (Figure 1). The bispecific antibody comprises an anti-BAFF monoclonal antibody (mAb) portion and an anti-MASP2 scFv portion, with the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2. The sequence of belimumab (having a heavy chain set forth in SEQ ID NO: 3 and a light chain set forth in SEQ ID NO: 2) was used as the anti-BAFF mAb portion, and the sequence of 129C10, a humanized anti-MASP2 mAb with neutralizing activity developed by Transcenta, was used as the anti-MASP2 scFv portion.

[0125] Furthermore, to increase the stability of the anti-MASP2 scFv portion, the 44th amino acid of VH and the 100th amino acid of VL were substituted with cysteine ​​to form a pair of disulfide bonds.

[0126] DNA sequences encoding the heavy and light chains of Blm129 were synthesized and cloned into the expression vector pcDNA3.1(+) (Invitrogen, product number V79020) to obtain plasmids encoding the heavy and light chains of Blm129. The resulting two plasmids were then prepared in large quantities using Qiagen's Plasmid Maxi-prep system.

[0127] To express the bispecific antibody Blm129, two plasmids encoding the heavy and light chains of Blm129, respectively, were transfected with Invitrogen's ExpiFectamine™ according to the manufacturer's protocol. (登録商標) Expi-CHO cells were co-transfected with the CHO reagent. On day 10, the cell supernatant was collected and purified using Mabselect SuRe affinity chromatography and size-exclusion chromatography (SEC). The purity was 95.9% by SDS-PAGE and SEC-HPLC (Figure 2).

[0128] In addition, the monoclonal antibody 129C10 against MASP2 (i.e., anti-MASP2 monoclonal antibody) was expressed and used as a positive control.

[0129] Example 2. Construction and expression of anti-BAFF / TACI bifunctional molecules We generated a bifunctional fusion protein, BlmTAC, containing the BAFF mAb moiety and the second CRD domain (cysteine-rich domain) (aa 69-108) of transmembrane activator and CAML interactor (TACI, Uniprot ID: O14836) (Figure 3). Here, the second CRD domain (aa 69-108) of TACI functions as a trap for BAFF and proliferation-inducing ligand (APRIL). The sequence of the heavy chain of BlmTAC is shown as SEQ ID NO: 22, and the sequence of the light chain of BlmTAC is shown as SEQ ID NO: 2. The expression and purification procedures for BlmTAC were the same as those for Blm129. A purity of 98.4% was obtained by SDS-PAGE and SEC-HPLC (Figure 4).

[0130] Additionally, we expressed and purified two analogs of Blm129 and BlmTAC, belimumab (sometimes referred to as "belimumab analog") and telitacicept (sometimes referred to as "telitacicept analog"), as positive controls. The sequences of these two drugs were obtained from the IMGT / mAb database.

[0131] Example 3: ELISA binding of Blm129 and BlmTAC to BAFF, APRIL, and MASP2 The binding activity of the bispecific antibody was evaluated by ELISA.

[0132] First, MASP2-his protein was prepared. Specifically, the full-length human MASP-2 gene (Uniprot ID: O00187) fused to a C-terminal 6His tag was introduced into the pcDNA3.1(+) vector, and the construct was confirmed by sequencing. This expression construct was transfected into ExpiCHO-s cells using the ExpiFectamineCHO transfection kit. ExpiCHO-s cells were cultured in ExpiCHO expression medium. The supernatant was collected 14 days after transfection. After centrifugation and filtration, the supernatant was loaded onto a HisTrap column (Cytiva, part number 29048586) and purified using the GEAKTA purification system. After washing, the MASP2-his protein was eluted with 250 mM imidazole buffer, and the imidazole in the eluted protein was removed by dialysis.

[0133] Next, human BAFF-his (ACRO Biosystems, part number BAF-H5248), APRIL-his (ACRO Biosystems, part number APL-52D1), or prepared MASP2-his was immobilized on an ELISA plate. The bispecific binding proteins Blm129 and BlmTAC or control proteins (belimumab analogs or telitacicept analogs) were serially diluted in PBS and added to the ELISA plate for 1 hour. Next, goat pAb against human IgG-HRP (Abcam, part number GR3256019-10) and TMB were added, and binding activity was detected at OD 450 nm. Finally, the data were analyzed using GraphPad Prism.

[0134] As shown in Figure 5, Blm129 bound to human BAFF with high affinity, with an EC50 of approximately 0.04517 nM. BlmTAC also bound to human BAFF with high affinity, with an EC50 of approximately 0.02799 nM. In comparison, the EC50 for the binding of belimumab analogs to human BAFF was approximately 0.01847 nM, and the EC50 for the binding of telitacicept analogs to human BAFF was approximately 0.1000 nM.

[0135] As shown in Figure 6, BlmTAC bound to human APRIL with high affinity, with an EC50 of approximately 0.4067 nM. As a positive control, the EC50 of the binding of a telitacicept analog to human APRIL was approximately 0.5613 nM.

[0136] As shown in Figure 7, Blm129 bound to human MASP2 with high affinity, with an EC50 of approximately 0.2461 nM. As a positive control, the EC50 of 129C10 binding to human MASP2 was approximately 0.2493 nM.

[0137] Example 4: Dynamic binding of bispecific binding proteins to BAFF, APRIL, and MASP2 100 nM of the bispecific binding proteins Blm129 and BlmTAC or control proteins (belimumab analogs or telitacicept analogs) in 1x kinetics buffer (1x PBS, pH 7.4, 0.02% Tween 20, 0.1% BSA) were loaded onto four pre-wetted Protein A biosensors and incubated with various concentrations of human BAFF, APRIL, or MASP2 solutions. All binding data were collected at 30°C. Specifically, the experimental procedure included the following five steps. The following steps were performed: (1) baseline acquisition (60 s), (2) loading the protein A biosensor with the bispecific binding proteins Blm129 and BlmTAC or a control protein (belimumab analog or telitacicept analog) (60 s), (3) a second baseline acquisition (60 s), (4) antigen binding for k measurement (90 s), and (5) antigen dissociation for k measurement (150 s). Four concentrations of antigen (100 nM, 33.3 nM, 11.1 nM, and 0 nM) diluted in 1x kinetics buffer were used. The baseline and dissociation steps were performed in 1x kinetics buffer. KD was determined from the ratio of k to k. The biosensor was regenerated in regeneration buffer (10 mM glycine-HCl, pH 1.7) for 5 s and then neutralized in neutralization buffer (1x PBS, pH 7.4, 0.02% Tween 20, 0.1% BSA) for 5 s. The experiment was repeated three times.

[0138] As shown in Table 1 below, Blm129 binds with high affinity to human BAFF and MASP2, and BlmTAC binds with high affinity to human BAFF and APRIL.

[0139] [Table 1]

[0140] Example 5: Simultaneous binding of the bispecific binding protein Blm129 to human BAFF and MASP2 ELISA plates were coated overnight at 4°C with 1 μg / ml human MASP2-his prepared in Example 3. Next, 300 μl of blocking buffer was added and blocked for 1 hour at room temperature. After 1 hour, 100 μl of Blm129 or control mAb (belimumab analogs and 129C10) at concentrations ranging from 200 nM to 0.01 nM (5-fold serial dilutions) was added and incubated for 1 hour at room temperature. After washing three times with 0.5% PBS + Tween-20, 0.5 μg / ml human BAFF-Fc-biotin (Acro Biosystems, Cat: BAF-H82F3) was added to each well. After 1 hour, 100 μl of HRP-conjugated streptavidin (1:5000) was added. After incubation at room temperature for 1 hour, TMB substrate reagent mixture (InnoReagents, TMB-S-003) was added and incubated at room temperature for 5 minutes. The reaction was stopped by adding 0.1 M H 2 SO 4 . OD450nm was measured using a microplate reader.

[0141] As shown in Figure 8, Blm129, which is composed of an anti-BAFF arm and an anti-MASP2 arm, was able to simultaneously bind to human BAFF and MASP2.

[0142] Example 6: Bispecific binding proteins neutralize the activity of BAFF in a cell-based reporter assay For this assay, HEK-293-BCMA-NFkB-luciferase cells were purchased from Cobior (product number CBP74072). These cells were generated by stably transfecting HEK293 cells with the human BCMA gene and an NF-κB-inducible luciferase construct. Binding of BAFF to its receptor, BCMA, triggers a cascade that leads to NF-κB activation and luciferase production, which can be monitored by ONE-Glo. Therefore, luciferase production by BAFF can be inhibited using a neutralizing antibody.

[0143] Briefly, a 50 nM human BAFF protein solution was prepared using DMEM medium containing 10% FBS, and 25 μl was added to a 96-well plate. The bispecific binding proteins Blm129 and BlmTAC or control proteins (belimumab analogs or telitacicept analogs) were used as samples. Sample dilutions were prepared, and 25 μl of each dilution was added to a 96-well plate. The plate containing BAFF and competitor protein samples was preincubated at 37°C for 30 minutes. 3 × 10 HEK-293-BCMA-NFkB-Luciferase cells in logarithmic growth phase were cultured. 4 100 μl of ONE-Glo was added to each well. (登録商標) Reagents are added and the contents are mixed. The plate is incubated at room temperature for 10 minutes to allow the luminescent signal to stabilize. Luminescence is measured using a spectrophotometer. Data are analyzed using GraphPad Prism9.

[0144] As shown in Figure 9, Blm129 and BlmTAC inhibited the BAFF-induced NFκB signaling pathway in a dose-dependent manner. BlmTAC most effectively inhibited the BAFF-induced NFκB signaling pathway with an IC50 of 8.740 nM. It was unexpected that Blm129 was significantly more effective in inhibiting the BAFF-induced NFκB signaling pathway than its belimumab analogues.

[0145] Example 7: Evaluation of the effect of Blm129 on the activity of complement factor C4 MASP-2 is a key component of the lectin pathway, cleaving complement factors C4 and C2 to generate the C3 convertase C4bC2a. Activation of C3 ultimately leads to the formation of the membrane attack complex (MAC). To test whether antibodies that block MASP-2 suppress activation of the lectin pathway, we assessed the activity of C4 in the presence of Blm129 and 129C10.

[0146] ELISA plates were coated overnight at 4°C with 100 μl / well of 10 μg / ml mannan. After washing three times with PBS + 0.1% Tween 20, the plates were blocked for 1 hour with blocking buffer (10 mM Tris-HCl + 0.1% human serum albumin + 140 mM NaCl). Blm129 or 129C10 was serially diluted in assay buffer (0.1% human serum albumin + 20 mM Tris-HCl + 2 mM CaCl2 + 140 mM NaCl + 1 mM MgCl2 + 0.05% Tween 20) containing 2% human serum (Quidel, A113) and incubated on ice for 45 minutes. The blocking buffer was removed from the mannan-coated plates, and the antibody and serum mixture was added. The plates were then incubated for 1 hour at 37°C. Activated complement components precipitated to the bottom of the plate, while inactivated components remained dissolved in the buffer. After washing the plate three times with washing buffer, the activity of complement factor C4 was measured using an HRP-conjugated anti-C4c antibody (Quidel-A211).

[0147] As shown in FIG. 10, Blm129 and 129C10 inhibited the activation of complement factor C4 in a dose-dependent manner, with IC50 values ​​of 2.474 nM and 0.1558, respectively.

[0148] Example 8: In vivo pharmacodynamic (PD) effects of Blm129 and BlmTAC Effect of B-cell depletion after treatment with subcutaneous injections three times a week To better understand the in vivo efficacy of Blm129 and BlmTAC, female BALB / c mice were purchased from Hangzhou Ziyuan Laboratory Animal Co., Ltd. and housed under controlled conditions (temperature: 20-26°C, relative humidity: 40-70%, light / dark cycle: 12 h). On the first day of the experiment (D0), mice were randomly divided into five groups (12 mice per group) and subcutaneously injected with saline, 10 mg / kg Blm129, BlmTAC, belimumab analogs, or telitacicept analogs three times a week. Blood samples were collected via the orthotopic vein on days 7, 14, and 18 after treatment. On days 7, 14, and 18, four mice per group were sacrificed and their spleens were collected and dissociated using a gentleMACS Dissociator (Miltenyi Biotec, 130-093-235) according to the manufacturer's instructions. Next, red blood cells were removed by adding lysis buffer (eBioscience, 00-4300-54). Single splenocytes and peripheral blood lymphocytes were isolated and stained for CD45, CD19, and CD21 for B cell phenotype determination by flow cytometry using a CytoFLEX (Beckman Coulter). Data analysis software (Cytoflex and GraphPad Prism) was used to calculate the percentage of CD19+ B cells among CD45+ immune cells and the percentage of CD21+CD19+ B cells in blood and spleen samples. Comparisons between the Blm129-treated group, the BlmTAC-treated group, and the control group were performed by statistical analysis (Student's t-test). Differences were considered significant when p values ​​were less than 0.05 (*), 0.01 (**), 0.001 (***), or 0.0001 (#).

[0149] As shown in Table 2, Figures 11 and 12, treatment with Blm129, BlmTAC, and Benchmark (belimumab analog or telitacicept analog) significantly reduced the percentage of CD19+ / CD45+ B cells, which are involved in B cell activation and maturation, and the percentage of CD19+ B cells expressing CD21+ (also known as the complement C3d receptor), in blood and spleen samples during the treatment period compared to the saline treatment group. Compared to the belimumab analog and telitacicept analog groups, Blm129 showed a more potent effect on B cell depletion, and BlmTAC also showed similar activity.

[0150] [Table 2]

[0151] B cell subgroup and phenotype analysis after weekly intravenous treatment B cell differentiation occurs in the bone marrow, where B cells differentiate and mature from pro-B cells to pre-B cells, immature B cells, and mature B cells through the expression of a series of surface biomarkers, and then migrate to the periphery. Therefore, the effects of CD19 (total B cells), IgM (immature B cells), IgD (mature B cells), CD27 (memory B cells), CD138 (plasmocytes / plasmocytes), CD21 (C3d receptor-expressing B cells), and CD23 (Fc epsilon RII-expressing B cells) were further investigated using BALB / c mice as described in Example 8.1. Briefly, mice were randomly divided into three groups and administered saline or 10 mg / kg BlmTAC, a telitacicept analog, intravenously once a week. Fresh blood samples were collected from the orthotopic vein on days 7, 14, and 21 after treatment. Animals from each group (n = 4) were then sacrificed, and spleens were collected. The spleens were dissociated using a gentleMACS Dissociator (Miltenyi Biotec, 130-093-235) according to the manufacturer's instructions. Lysis buffer (eBioscience, 00-4300-54) was then added to remove red blood cells. To determine B cell phenotypes, single splenocytes and peripheral blood lymphocytes were isolated and subjected to flow cytometry staining for CD19, IgM, IgD, CD138, CD21, and CD23 using a CytoFLEX (Beckman Coulter). Using data analysis software (Cytoflex and GraphPadPrism), the percentages of CD19+ B cells, IgM+ / CD19+, IgD+ / CD19+, CD138+ / CD19+, CD23+ / CD19+, and CD21+CD19+ B cells among immune cells were calculated for blood and spleen samples, respectively. Comparisons between the BlmTAC or telitacicept-analog group and the control group were performed by statistical analysis (Student's t-test). Differences were considered significant when p values ​​were <0.05 (* / #), <0.01 (** / ##), or <0.001 (*** / ###).

[0152] As shown in Figures 13A-13B and 14A-14B, treatment with BlmTAC and Benchmark (a telitacicept analog) significantly reduced the percentages of CD19+ total B cells, IgD+-expressing CD19+ mature B cells, CD23+-expressing CD19+ B cells, an Fc epsilon RII biomarker that regulates IgE levels involved in allergies and autoimmune diseases, and CD21+ (also known as the complement C3d receptor)-expressing CD19+ B cells compared to the saline-treated group. Meanwhile, no suppressive effect on IgM+ / CD19+ immature B cells or CD138+ / CD19+ preplasmocytes / plasmocytes was observed in blood and spleen samples during the treatment period. Furthermore, a decrease in CD27+ / CD19+ memory B cells was observed in blood samples after administration of BlmTAC and a telitacicept analog. Compared with the telitacicept analogue group, BlmTAC demonstrated a more potent and sustained effect on the depletion of mature B cells.

[0153] Example 9: Single-dose pharmacokinetic (PK) study of Blm129 and BlmTAC in Balb / c mice We characterized and directly compared the pharmacokinetic (PK) profiles of Blm129, BlmTAC, and the benchmark belimumab analog, telitacicept, after a single intravenous administration in Balb / c mice. Sixteen female mice were randomized into four groups (four mice per group) and received a single intravenous bolus injection of 13.5 mg / kg Blm129, 10 mg / kg BlmTAC, 10 mg / kg belimumab analog, or 5 mg / kg telitacicept analog at a dose of 10 mL / kg. Plasma samples from each group were collected pre-dose (0 min), 5 min, 30 min, 2 h, 8 h, 24 h, 48 h, and at D4, D7, D10, D14, and D21 for PK analysis using a partially validated ELISA assay. Microplate wells were precoated with a human IgG-specific anti-IgG antibody [R10z8e6] (Abcam, ab124055). After blocking, standards (STDs), quality control (QC) samples, matrix blank samples, and test samples were added to the wells. After washing, goat anti-human IgG (HRP) (Abcam, ab98624) was added to the microplate wells. Tetramethylbenzidine (TMB) was added to the microplate wells to develop a colorimetric signal (blue) in the presence of HRP. After color development, the reaction was terminated by adding stop solution to each well. Optical density (OD) was measured using a microplate reader set at 450 nm and 620 nm. The OD values ​​of the QC and test samples were converted to concentrations by comparing them to a standard curve regressed with a four-parameter logistic model analyzed simultaneously. The mean plasma concentrations are shown in Table 3, and the mean plasma concentration-time curves are shown in Figure 15. Relevant PK parameters (Table 4) were calculated and evaluated by non-compartmental analysis (NCA) using Phoenix software. The results showed that the half-lives (T1 / 2) of Blm129 and BlmTAC were similar and longer than those of the telitacicept-analog.

[0154] [Table 3]

[0155] [Table 4]

[0156] equivalent While certain embodiments that are the subject of the present invention have been described, the above details are illustrative and not restrictive. Numerous variations of the present invention will become apparent to those skilled in the art upon consideration of this specification and the appended claims. The full scope of the present invention should be determined by reference to the claims, their full scope of equivalents, and such variations, in addition to the specification. [Sequence List Free Text]

[0157] [Table SL1] [Table SL2] [Table SL3] [Table SL4]

Claims

1. 1. A bispecific binding protein comprising, from the amino terminus to the carboxy terminus, a first portion and a second portion, (a) the first portion is an anti-BAFF antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof comprises (i) a heavy chain variable domain (VH) comprising CDR-H1, CDR-H2, and CDR-H3, and (ii) a light chain variable domain (VL) comprising CDR-L1, CDR-L2, and CDR-L3, CDR-H1 comprises the sequence NNAIN (SEQ ID NO: 6); CDR-H2 comprises the sequence GIIPMFGTAKYSQNFQG (SEQ ID NO: 7); CDR-H3 comprises the sequence SRDLLLFPHHALSP (SEQ ID NO: 8); CDR-L1 comprises the sequence QGDSLRSYYAS (SEQ ID NO: 9); CDR-L2 comprises the sequence GKNNRPS (SEQ ID NO: 10); CDR-L3 comprises the sequence SSRDSSGNHWV (SEQ ID NO: 11); wherein CDRs are defined according to Kabat numbering; (b) the second portion is an anti-MASP2 scFv or a truncated TACI polypeptide, wherein the anti-MASP2 scFv comprises (i) a heavy chain variable domain (VH) comprising CDR-H1, CDR-H2, and CDR-H3, and (ii) a light chain variable domain (VL) comprising CDR-L1, CDR-L2, and CDR-L3, CDR-H1 comprises the sequence DYYIN (SEQ ID NO: 16); CDR-H2 comprises the sequence WIFPGSESAYHSEKFKA (SEQ ID NO: 17); CDR-H3 comprises the sequence GDRSGPFAY (SEQ ID NO: 18); CDR-L1 comprises the sequence KSSQSLLYSNGKTYLN (SEQ ID NO: 19); CDR-L2 comprises the sequence LVSKLDS (SEQ ID NO: 20); CDR-L3 comprises the sequence VQVTHFPFT (SEQ ID NO: 21); wherein CDRs are defined according to Kabat numbering; wherein the truncated TACI polypeptide is the extracellular domain of human TACI set forth in SEQ ID NO: 24 or a fragment or variant thereof; A bispecific binding protein, wherein the carboxy terminus of the heavy chain of said first portion is covalently linked to the amino terminus of said second portion.

2. 2. The bispecific binding protein of claim 1, comprising two heavy chains and two light chains, wherein each heavy chain comprises, from amino terminus to carboxy terminus, a heavy chain of the first portion, a linker peptide, and the second portion, and each light chain is a light chain of the first portion.

3. 3. The bispecific binding protein of claim 1 or 2, wherein the anti-BAFF antibody and / or the anti-MASP2 scFv is a chimeric, humanized, or human anti-BAFF antibody and / or anti-MASP2 scFv, and wherein the truncated TACI polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25 or a fragment or variant thereof (e.g., the fragment set forth in SEQ ID NO: 26).

4. the bispecific binding protein is a bispecific anti-BAFF x anti-MASP2 antibody; The anti-BAFF antibody or antigen-binding fragment thereof comprises, or consists of, (a) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, and (b) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto; 4. The bispecific binding protein of any one of claims 1 to 3, wherein the anti-MASP2 scFv comprises, or consists of, (a) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 14, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, and (b) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 15, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto.

5. the bispecific binding protein comprises two heavy chains and two light chains, wherein the constant region of each light chain is derived from a human kappa or lambda light chain constant region and the constant region of each heavy chain is derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region; 5. The bispecific binding protein of claim 4, wherein each heavy chain preferably comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, and each light chain comprises or consists of the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto.

6. the bispecific binding protein is a fusion protein comprising an anti-BAFF antibody or antigen-binding fragment thereof and a truncated TACI polypeptide or a fragment or variant thereof; wherein the anti-BAFF antibody or antigen-binding fragment thereof comprises, or consists of, (a) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, and (b) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto; 4. The bispecific binding protein of any one of claims 1 to 3, wherein the truncated TACI polypeptide or fragment or variant thereof comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25 or a fragment or variant thereof (e.g. the fragment set forth in SEQ ID NO: 26).

7. the bispecific binding protein comprises two heavy chains and two light chains, wherein the constant region of each light chain is derived from a human kappa or lambda light chain constant region and the constant region of each heavy chain is derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region; 7. The bispecific binding protein of claim 6, wherein each heavy chain preferably comprises or consists of the amino acid sequence of SEQ ID NO: 22, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, and each light chain preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto.

8. The bispecific binding protein has the following properties: (1) binds to human BAFF and human MASP2, with a KD of approximately 10 x 10 for binding to human BAFF as measured by biolayer interferometry. －９ M, 5 x 10 －９ M, 1 x 10 －９ M, or 5 x 10 －１０ M or less, and the KD for binding to human MASP2 is approximately 10 x 10 －８ M, 5 x 10 －８ M, 1 x 10 －８ M, or 5 x 10 －９ is less than M; (2) binds to human BAFF and human MASP2, wherein the EC50 for binding to human BAFF is about 0.1 nM or less, 0.08 nM or less, 0.06 nM or less, or 0.05 nM or less, and the EC50 for binding to human MASP2 is about 1 nM or less, 0.8 nM or less, 0.6 nM or less, or 0.4 nM or less, as measured by ELISA; (3) neutralizes BAFF activity in cells expressing BCMA with an IC50 of about 250 nM or less, 200 nM or less, 170 nM or less, or 140 nM or less; (4) inhibiting the activation of complement factor C4 with an IC50 of about 10 nM or less, 8 nM or less, 6 nM or less, or 4 nM or less; (5) depleting B cells in vivo; 6. The bispecific binding protein of claim 1, wherein the bispecific binding protein has one or more properties selected from the group consisting of:

9. The bispecific binding protein has the following properties: (1) binds to human BAFF and human APRIL, and the KD of binding to human BAFF is approximately 1 x 10 as measured by biolayer interferometry. －１２ M or less, and the KD of binding to human APRIL is approximately 10×10 －９ Less than M, 5 x 10 －９ Less than M, 1 x 10 －９ Less than M or 5 x 10 －１０ is less than M; (2) binds to human BAFF and human APRIL, wherein the EC50 for binding to human BAFF is about 0.1 nM or less, 0.08 nM or less, 0.06 nM or less, or 0.05 nM or less, and the EC50 for binding to human APRIL is about 1 nM or less, 0.8 nM or less, 0.6 nM or less, or 0.4 nM or less, as measured by ELISA; (3) neutralizes BAFF activity in cells expressing BCMA with an IC50 of about 50 nM or less, 35 nM or less, 20 nM or less, or 10 nM or less; (4) depleting B cells in vivo; 8. The bispecific binding protein of any one of claims 1 to 3, 6, and 7, having one or more properties selected from:

10. A nucleic acid molecule encoding the bispecific binding protein of any one of claims 1 to 9.

11. A vector comprising the nucleic acid molecule of claim 10.

12. 12. A host cell comprising the nucleic acid molecule of claim 10 or the vector of claim 11.

13. A method for preparing a bispecific binding protein according to any one of claims 1 to 9, comprising: Culturing the host cell of claim 12 under conditions that allow for the production of said bispecific binding protein; Recovering the bispecific binding protein from the culture. The method includes:

14. 13. A pharmaceutical composition comprising the bispecific binding protein of any one of claims 1 to 9, the nucleic acid of claim 10, the vector of claim 11, or the host cell of claim 12.

15. 15. A method for treating or preventing an autoimmune disease, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 14 to a subject in need thereof, preferably wherein the subject is a human.

16. 16. The method of claim 15, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), IgAN, rheumatoid arthritis (RA), neuromyelitis optica / neuromyelitis optica spectrum disorder (NOD / NMOD), multiple sclerosis (MS), neuromyelitis optica, Sjogren's syndrome, ANCA-associated vasculitis, myasthenia gravis, and Devic's disease.

Images